ALTERNATIVES TO ANIMALS
0
Hitchman N, Leaver M, George S. ALTERNATIVES TO WHOLE
ANIMAL TESTING USE OF CDNA PROBES FOR STUDIES OF PHASE I AND II ENZYME
INDUCTION IN ISOLATED PLAICE HEPATOCYTES.  Seventh International Symposium on
Responses of Marine Organisms to Pollutants (Primo 7), Goteborg, Sweden, April
20-22, 1993. Marine Environ Res 1994;39(1-4):289-92. 

1
Reinhardt CA. THE SIAT RESEARCH TEACHING AND CONSULTING PROGRAM IN THE AREA OF
IN VITRO TOXICOLOGY EXPERIMENTAL RESEARCH SCREENING AND VALIDATION. In:
Reinhardt CA, editor. Alternatives to Animal Testing: New Ways in the
Biomedical Sciences, Trends and Progress; Symposium; 1992  Nov 30; Zurich,
Switzerland. New York: VCH Publishers, Inc: 1994. p. 89-98.

2
Spielmann H. HET-CAM TEST. Methods Mol Biol 1995;43:199-204.

3
Atterwill CK. ALTERNATIVE METHOD OF ASSESSING TOXICITY. Methods Mol Biol
1995;43:1-9. 

4
Alarie Y, Nielsen GD, Andonian-Haftvan J, Abraham MH. PHYSICOCHEMICAL
PROPERTIES OF NONREACTIVE VOLATILE ORGANIC CHEMICALS TO ESTIMATE RD50:
ALTERNATIVES TO ANIMAL STUDIES. Toxicol Appl Pharmacol 1995;34(1):92-9.

This article presents the correlations obtained between the results on the
potency of nonreactive airborne chemicals as sensory irritants and several of
their physicochemical properties. The potency of airborne sensory irritants
obtained from a reflexively induced decrease in respiratory frequency has been
measured in the past using mice. Typically, their potency has been expressed
as the exposure concentration necessary to decrease respiratory frequency by
50% (RD50). A large database of RD50 values is now available and such values
are highly correlated with occupational exposure guidelines such as threshold
limit values (TLVs). We used the nonreactive volatile organic chemicals from
this database, for which relevant physicochemical variables are available or
can be calculated. These variables were vapor pressure (P) or Ostwald
gas-liquid partition coefficients (L). The liquids used for L values were
n-hexadecane, octanol, N-formylmorpholine, tri-(2-ethylhexyl)phosphate, and
olive oil. Excellent correlations were found between log RD50 and log P, as
well as between log RD50 and log L16, log L(Oct), log L(NFM), log L(EHP), or
log L(Oil). It follows that as an alternative to the bioassay, these
physicochemical variables can be used to estimate RD50 of       nonreactive
volatile organic chemicals. Appropriate exceptions to general estimation of
RD50 values from physicochemical variables are also presented, as well as the
most appropriate estimates which can be obtained within homologous series. 

5
Ehrich M. USING NEUROBLASTOMA CELL LINES TO ADDRESS DIFFERENTIAL SPECIFICITY
TO ORGANOPHOSPHATES. Clin Exp Pharmacol Physiol 1995;2(4): 291-2. 

1. Organophosphates can cause acute toxicity, which follows inhibition of
acetylcholinesterase (AChE), or delayed neuropathy, which follows inhibition
of neuropathy target esterase (NTE). 2. Human neuroblastoma SH-SY5Y cells
contain AChE and NTE. 3. Organophosphates actively able to inhibit AChE in
animal models inhibited AChE in neuroblastoma cells. 4. Inhibition of NTE in
neuroblastoma cells could identify active organophosphates capable of causing
delayed neuropathy in animal models and distinguish these organophosphates
from those that do not cause delayed neuropathy in animal models. 

6
Sina JF, Galer DM, Sussman RG, Gautheron PD, Sargent EV, Leong B, Shah PV,
Curren RD, Miller K. A COLLABORATIVE EVALUATION OF SEVEN ALTERNATIVES TO THE
DRAIZE EYE IRRITATION TEST USING PHARMACEUTICAL INTERMEDIATES. Fundam Appl
Toxicol 1995;26(1):20-31.

Much of the data which have been generated on in vitro alternatives to the
Draize eye irritation test have dealt with compounds within a specific
chemical class or product category. However, in the pharmaceutical industry,
it is often necessary to evaluate materials which are not related in structure
or properties. It was thus decided to evaluate a diverse series of chemicals
in seven in vitro methods for estimating ocular irritation. Thirty-seven test
materials were chosen to represent a broad range of pH, solubility, and in
vivo irritation potential. Assays were chosen to include as many different
types of end points as practical. The group of assays was composed of  TOPKAT
(assessing structure-activity relationships), bovine corneal
opacity-permeability (BCO-P; corneal opacity/toxicity), Eytex (protein
coagulation), neutral red uptake (cytotoxicity), MTT in living dermal
equivalent (cytotoxicity), Microtox (cytotoxicity in bacteria), and CAMVA
(inflammation/toxicity). The results of the study indicated that, in general,
the      cytotoxicity end points did not correlate well with the in vivo data.
The BCO-P, CAMVA, and Eytex assays had the best overall concordance (88.9,
75.8, and 75.0%, respectively) with this set of compounds. Estimation of
irritation potential based on structure-activity (TOPKAT) was possible for
only approximately 50% of the compounds; however, the assay showed 100%
sensitivity (i.e., no false negatives), but low specificity (i.e., negatives
correctly identified only 54.5% of the time). These data suggest that for
screening of chemicals of diverse structure and properties, the more
mechanism-based assays, as opposed to general cytotoxicity assays, hold more
promise and should be further evaluated. 

7
Lang L. MOUSE OR MOLECULE? MECHANISM-BASED TOXICOLOGY IN CANCER RISK
ASSESSMENT [NEWS]. Environ Health Perspect 1995;103(4):334-6.
Balls M. IN VITRO TESTS IN REGULATORY TOXICOLOGY: SYMPOSIUM CHAIRMAN'S
SUMMING-UP. Arch Toxicol Suppl 1995;17:205-8. 

8
Bass R.  IN VITRO METHODS IN REGULATORY TOXICOLOGY. Arch Toxicol Suppl
1995;17:192-204. 

9
Balls M. IN VITRO METHODS IN REGULATORY TOXICOLOGY: THE CRUCIAL SIGNIFICANCE
OF VALIDATION. Arch Toxicol Suppl 1995;17:155-62. 

10
Herzinger T, Korting HC,Maibach HI. ASSESSMENT OF CUTANIOUS AND OCULAR
IRRITANCY: A DECADE OF RESEARCH ON ALTERNATIVES TO ANIMAL EXPERIMENTATION.
Fundam Appl Toxicol 1995;24(1):29-41.

Over the past decade increasing societal and scientific pressure has promoted
the development of alternatives to local tolerance testing in laboratory
animals. The use of isolated organs, fertilized hen's eggs, and cell culture
systems has been proposed as well as the study of a toxicant's biochemical
effects and structure-activity relationships. This paper critically reviews
the current status of these approaches and discusses the preliminaries for the
establishment of alternative methods in the process of safety assessment.

11
Prati M, Giavini E, Menegola E. ALTERNATIVES TO IN VIVO TESTS FOR TERATOLOGIC
SCREENING. Ann Ist Super Sanita 1993;29(1):41-6.

During the last decade there has been a tremendous increase in publications
describing methods for in vitro toxicological research and emphasizing their
advantages, suitability and necessity, rather than the classical in vivo
studies. In this review we shall look at and consider only a few short term
tests, chosen on the basis of the particular relevance that they have in the
evaluation of the substances: rodent whole embryo culture (for both pre- and
post-implantation embryos), non-mammalian vertebrate embryo and invertebrate
embryo cultures, organ culture, and at last cell culture. We have seen however
that sometimes some of the developed methods suit neither the necessity of
saving time, money, and animals, nor the consistency of the results in
xenobiotic screening. The concomitant application of both in vivo and in vitro
methodologies will improve the quality of teratological research, and
therefore will contribute to a critical evaluation of developmental hazards.

12
Diener B, Abdel-Latif H, Arand M, Oesch F. XENOBIOTIC METABOLIZING ENZYME
ACTIVITIES AND VIABILITY ARE WELL PRESERVED IN EDTA-ISOLATED RAT LIVER
PARENCHYLAM CELLS AFTER CRYOPRESERVATION.  Toxicol Appl Pharmacol
1995;130(1):149-53. 

Rat liver parenchymal cells (PC) were isolated by EDTA perfusion and were
purified by a subsequent Percoll centrifugation. The isolated PC had a
viability of 95%, as judged by trypan blue exclusion. Freshly isolated PC were
cryopreserved with an optimized protocol in a computer-controlled freezer.
After thawing, the PC still retained a viability of 89%. The activities of
representative xenobiotic metabolizing enzymes were compared between freshly
isolated and cryopreserved PC after thawing. The cytochrome P450 content and
the cytochrome P450 2C11 isoenzyme activity, determined by hydroxylation of
testosterone in intact cells, were not affected by the cryopreservation. The
following phase II enzyme activities were also well maintained after
cryopreservation: Phenol sulfotransferase (92%), 1-naphthol UDP-glucuronosyl
transferase (95%), soluble epoxide hydrolase (87%), and glutathione
S-transferase (88%), determined with broad spectrum substrate
1-chloro-2,4-dinitrobenzene. However, there was a significant decrease in
plating efficiency between freshly isolated (86%) and cryopreserved (57%) PC
when they were cultured. The initial quality of the freshly isolated PC is
decisive for the success of cryopreservation. These results support the use of
cryopreserved PC in pharmacology and toxicology with the aim to reduce the
number of experimental animals used.


CARCINOGENESIS
13
Spitz MR, Hsu TC, Wu X, Fueger JJ, Amos CI, Roth JA.  MUTAGEN SENSITIVITY AS A
BIOLOGICAL MARKER OF LUNG CANCER RISK IN AFRICAN AMERICANS.  Cancer Epidemiol
Biomarkers Prev 1995;4(2):99-103.

Cigarette smoking is the major determinant of lung cancer. However, only a
fraction of smokers develops lung cancer; genetically determined
susceptibility factors seem to play an important role also. Previous
case-control studies have shown that in vitro bleomycin-induced mutagen
sensitivity is an independent risk factor for head-and-neck cancers, and
preliminary data suggest a similar association with lung cancer. However,
these studies were almost exclusively performed on Caucasian populations. To
test whether ethnic differences in cancer risk are due to differences in
mutagen sensitivity, we are using the in vitro mutagen sensitivity assay to
conduct a case-control study of mutagen sensitivity and lung cancer risk in
low-risk (Mexican-American) and high-risk (African-American) groups. Here we
report the results of our ongoing study of 209 African-Americans (90 cases and
119 controls) in the Houston-Galveston area. Mexican-American data will be
reported separately as case accrual increases. Predictably, all measures of
cigarette smoking status (including intensity, duration, tar content, depth of
inhalation, and type of cigarette) were significant predictors of risk. In
addition, 55.3% of the cases were mutagen sensitive (defined as > or = 1
break/cell), compared with 24.6% of the controls, with an age-, sex-, and
smoking-adjusted odds ratio (OR) of 3.7 (95% confidence limits = 1.4, 9.4). Of
interest, higher risks were noted for former smokers (OR = 5.4) compared with
current smokers (OR = 3.1) and especially for younger former smokers (< 55
years). By histologic-specific analysis, mutagen sensitivity was significantly
associated with risk for adenocarcinoma (OR = 4.8) and squamous cell carcinoma
(OR = 8.5).(ABSTRACT TRUNCATED AT 250 WORDS)

14
Jones TD.  TOXICOLOGICAL POTENCY OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN
RELATIVE TO 100 OTHER COMPOUNDS: A RELATIVE POTENCY ANALYSIS OF IN VITRO AND
IN VIVO TEST DATA. Arch Environ Contam  Toxicol 1995;29(1):77-85.

A common definition of relative potency is the dose of a reference compound
required to cause a particular incidence of a specific toxic response divided
by the dose of a test compound needed to cause an equal incidence of that same
effect. In this simple manner, toxicological assessments for a chemical of
concern can be made in terms of another compound about which much is known
from a human health perspective. Relative potency factors were used to compare
2,3,7,8-tetrachlorodibenzo-p-dioxin  CAS # 1746-01-6 (TCDD) with 100 other
compounds both individually and collectively. All results were standardized to
a common scale that spanned many orders of magnitude and was indexed to an
arbitrary potency of unity for benzo(a)pyrene (B(a)P). From comparisons
between 2,771 pairs of bioassay results (i.e., matched experimental design
conditions) for TCDD compared with the 100 other compounds, it was found that
TCDD is about 600 times as toxic as B(a)P (interquartile range of 130 to 
1,900). The distribution of relative potency values is fitted accurately with
a log-normal distribution function having an untransformed  ean of 550 and an
untransformed slope (i.e., the inverse of the standard deviation of the
distribution) of 140. These factors combined with (a) a  reference lifetime
carcinogenic risk level of 1/100,000 and (b) a universal, potency-dependent
risk coefficient (estimated from the collection of epidemiologically-based
carcinogens) yielded estimates that equally  toxic concentrations for TCDD
should be in the range of 13 pg/m3 and 7 pg/L in air and water, respectively.

15
Lichtenberg G, Nowak C, Gleier K, Meckert C, Richter-Reichhelm HB.  ANCHORAGE
INDEPENDENT COLONY GROWTH OF FETAL HAMSTER LUNG EPITHELIAL CELLS AFTER
TREATMENT WITH DIEPOXYBUTANE. Toxicol Lett 1995;75(1-3): 193-9.

To test the reliability of a new cell transformation assay, a cloned fetal
Syrian hamster lung epithelial cell line (M3E3/C3) was used. The target cells
originating from the respiratory tract were treated in vitro over a
concentration range of 0-10-5 M/l with diepoxybutane, cultured during the
expression period of 28 or 35 days and then transferred into soft agar.
Anchorage independent colony growth in soft agar occurs only if cells are
transformed. Growth and number of colonies were taken as  a score of the
carcinogenic potential of the test substance. Under the conditions of this
cell transformation assay it was possible to detect the carcinogenic potential
of diepoxybutane unequivocally.

16
Miyasaka K, Ohtake K, Nomura K, Kanda H, Kominami R, Miyashita N,  Kitagawa T.
FREQUENT LOSS OF HETEROZYGOSITY ON CHROMOSOME 4 IN DIETHYLNITROSAMINE-INDUCED
C3H/MSM MOUSE HEPATOCELLULAR CARCINOMAS IN CULTURE. Mol Carcinog
1995;13(1):37-43.

Genetic changes, in particular the loss of heterozygosity (LOH) and the
presence of c-Ha-ras codon 61 point mutations, were investigated in
diethylnitrosamine-induced hepatocellular carcinomas (HCCs) in C3H/MSM F1
mice. (MSM are wild mice.) LOH analysis of 48 primary tumors with
microsatellite probes covering at least one proximal and one distal site of
each autosome revealed no obvious positive results for LOH. Analysis of 23
cell lines established from seven of these HCCs, however, showed  LOH on
chromosome 4 in all (seven of seven), even in early passages (G2-G3). With
regard to other chromosomes, LOH was observed only rarely on chromosomes 16
and 19. These allelotype features were maintained in later passages (G11-G14),
with only a few additional occurrences of LOH appearing on chromosomes 1, 6,
and 8. Extensive analyses with multiple microsatellite probes from chromosome
4 and with 52 cell lines established from 24 HCCs of 18 mice revealed LOH in
22 of the tumors (92%), with  the shortest region about 10 cM distal to the
alpha-interferon gene. No c-Ha-ras oncogene activation in codon 61 was
observed. These data indicate that loss of tumor suppressor genes on
chromosome 4 may play an important role in mouse hepatocarcinogenesis in
progression in vivo or in immortalization in vitro or both.

17
Cunningham ML, Elwell MR, Matthews HM.  CORRECTION OF PREVIEWS 97552935.
RELATIONSHIP OF CARCINOGENICITY AND CELLULAR PROLIFERATION INDUCED BY
MUTAGENIC NONCARCINOGENS VS CARCINOGENS: III. ORGANOPHOSPHATE PESTICIDES VS
TRIS(2,3-DIBROMOPROPYL)PHOSPHATE. CORRECTION OF TITLE FROM RELATIONSHIP OF
CARCINOGENICITY AND CELLULAR PROLIFERATION INDUCED BY MUTAGENIC NONCARCINOGENS
VS CARCINOGENS: III.       ORGANOPHOSPHATE PESTICIDES VS
TRIS(2,3-DIBROMOPROFYL)-PHOSPHATE.  Fundam Appl Toxicol 1994;23(3):363-9.

Our laboratory has been examining the mechanisms whereby chemicals produce
mutagenicity in short-term in vitro assays yet fail to produce carcinogenesis
in 2-year rodent bioassays. Previous studies indicated that some mutagenic
hepatocarcinogens increased cell proliferation in the target organ, the liver,
while other structurally related mutagens that were noncarcinogenic failed to
do so. We demonstrate in this report that another mutagenic carcinogen,
tris(2,3-dibromopropyl)phosphate, increased cell proliferation that was
localized in the outer medulla of the kidney. This was also the target site
for carcinogenesis in a 2-year bioassay and is another example of the
association between chemically induced cell proliferation and carcinogenesis.
This study also reports the absence of increased cell proliferation in the
liver or kidney after exposure in the diet to the mutagenic organophosphate
insecticides dimethoate, dioxathion, and dichlorvos following dietary exposure
for 2  weeks at the same dose levels and routes of exposure that did not
increase the tumor incidence in either organ in 2-year carcinogenesis assays.
The present studies support the tenet that chemically induced cell
proliferation may be a necessary prerequisite for chemical carcinogenesis,
since in rat liver and kidney there was neither cell proliferation after 2
weeks nor tumor development after 2 years dietary exposure to the mutagenic
organophosphate insecticides dimethoate, dioxathion, and  dichlorvos. 

18
Silverman JA, Hill BA.   CHARACTERIZATION OF THE BASAL AND CARCINOGEN
REGULATORY ELELMENTS OF THE RAT MDR1B PROMOTER.  Mol Carcinog 1995;13(1):50-9.

In this report we characterized the transcriptional regulation of the rat
mdr1b gene by xenobiotics. The expression of this gene was increased in
primary rat  hepatocytes and in the H4-II-E hepatoma cell line by exposure to
carcinogens such as aflatoxin B1, N-acetoxy-2-acetylaminofluorene, and methyl
methanesulfonate. Nuclear run-on experiments indicated that the higher
steady-state levels of mdr1b mRNA were due to an increase in transcription.
The 5'-flanking region of the mdr1b gene was  isolated, sequenced, and
functionally characterized in transient and stable transfection assays. A
single transcription start site was identified for this gene; no alternate
start sites were used after induction with aflatoxin B1. Deletion analysis of
this promoter demonstrated that the  sequence between nt -214 and -178 was
critical for basal promoter activity. This region did not contain any
consensus-binding sites for previously identified  transcription factors. A
negative regulatory region  was also identified between nt -940 and -250. No
specific carcinogen-responsive element was identified; the xenobiotic response
required a large part of the promoter. These data suggest that the carcinogen
induction of mdr1b expression is mediated through sequences that overlap or
that are identical to the basal promoter element. 

19
Pintao AM, Pais MS, Coley H, Kelland LR, Judson IR.  IN VITRO AND IN VIVO
ANTITUMOR ACTIVITY OF BENZYL ISOTHIOCYANATE: A NATURAL PRODUCT FROM TROPAEOLUM
MAJUS. Planta Med 1995;61(3):233-6.

Cultured cells of Tropaeolum majus produce significant amounts of benzyl
glucosinolate which, through enzymatic hydrolysis, results in the production
of benzyl isothiocyanate (BITC). This study reports on the in vitro anticancer
properties of BITC against a variety of human and murine tumor cell lines by
four independent methods; SRB, MTT, cell counting, and clonogenic assays.
Regardless of the assay used, BITC showed promising cytotoxicity in the low
micromolar range (0.86 to 9.4 microM) against four human ovarian carcinoma
cell lines (SKOV-3, 41-M, CHl, CHlcisR), a human lung tumor (H-69), a murine
leukemia (L-1210), and a murine plasmacytoma (PC6/sens). The L1210 cells were
most sensitive. BITC administered to mice bearing the ADJ/PC6 plasmacytoma
subcutaneous tumor showed toxic effects at a dose of 200 mg/kg (within 24 h of
drug administration) but no reduction in tumor mass. However, the growth
inhibitory properties of BITC against a range of tumor cell types warrant
further in vivo anti-tumor evaluation as well as its biotechnological
production.

20
Segerback D, Calleman CJ, Schroeder JL, Costa LG, Faustman EM.  FORMATION OF
N-7-(2-CARBAMOYL-2-HYDROXYETHYL)GUANINE IN DNA OF THE MOUSE AND THE RAT
FOLLOWING INTRAPERITONEAL ADMINISTRATION OF [14C]ACRYLAMIDE. Carcinogenesis
1995;16(5):1161-5.

Acrylamide is an alkylating agent which reacts very slowly in direct reactions
with DNA and is negative in the Ames test, but is carcinogenic in mice and
rats. In order to explain the cancer-initiating properties of acrylamide we
have studied DNA adduct formation in vitro with a metabolizing system and in
vivo in mice and rats following i.p. administration of 14C-labeled acrylamide.
A major adduct found in both species was
N-7-(2-carbamoyl-2-hydroxy-ethyl)guanine, formed by reaction of the DNA with
the epoxide metabolite glycidamide. The levels of this adduct were similar in
the different organs of the two rodent species, which supports the notion that
glycidamide is relatively evenly distributed among tissues and that the
organ-specificity in acrylamide carcinogenesis cannot be explained by a
selective accumulation of the DNA-reactive metabolite in target organs.

21
Hiraga S, Arita N, Ohnishi T, Izumoto S, Taki T, Yamamoto H, Higuchi M,
Hayakawa T.  TRANSFORMATION OF TYPE 1 ASTROCYTES WITH N-ETHYL-N-NITROSOUREA:
ESTABLISHMENT OF AN IN VITRO SYSTEM AND THE ROLE OF THE p53 GENE. Glia
1995;13(1):51-63.

N-ethyl-N-nitrosourea (ENU)-induced gliomas, animal models of human gliomas,
are most frequently oligodendrocytic, while human gliomas tend to be
astrocytic. To facilitate a detailed study of human glial carcinogenesis, we
developed an in vitro system using type 1 astrocyte transformation with ENU.
Type 1 astrocytes from fetal Wistar rat brain were treated by a single dose of
ENU. Transformed colonies appeared 50 days after exposure to single doses of
ENU greater than 150 micrograms/mL. Cloned cells from these colonies retained
the immunohistochemical characteristics of type 1 astrocytes. They showed
rapid growth and high saturation densities, colony formation in low (2%) serum
medium and gave rise to tumors when injected into nude mice. When p53
expression was studied at each passage, a single cell positive for mutant p53
protein emerged 40 days after ENU treatment. In the next 1-3 passages, the
mutant p53 positive cell formed piled-up colonies and exhibited dominant
growth. Northern blot analysis showed markedly increased accumulations of p53
mRNA in transformed cells. This in vitro transformation system of type 1
astrocytes provides a valuable tool for further investigations of astrocyte
carcinogenesis.

22
Swanson SM, Guzman RC, Collins G, Tafoya P, Thordarson G, Talamantes F, Nandi
S.   REFRACTORINESS TO MAMMARY CARCINOGENESIS IN THE PAROUS MOUSE IS
REVERSIBLE BY HORMONAL STIMULAITON INDUCED BY PITUITARY ISOGRAFTS. Cancer Lett
1995;90(2):171-81.

We have previously reported that mouse mammary epithelial cells transformed in
vitro yield tumors which vary qualitatively and quantitatively as a function
of the mitogenic environment in which the cells are propagated at the time of
carcinogen treatment. One milieu supportive of transformation in vitro was
medium supplemented with progesterone and prolactin as the mitogens. We have
performed parallel studies in which virgin mice were isografted with
pituitaries resulting in elevated serum titers of progesterone and prolactin.
After carcinogen treatment, these mice developed mammary tumors which included
those identical genotypically and phenotypically to tumors induced in vitro in
cells grown in progesterone and prolactin during carcinogen exposure. Our
current working hypothesis is that the mitogenic environment around the time
of carcinogen       administration can modulate the incidence and phenotype of
the resultant tumors. To further test this hypothesis, we have evaluated the
susceptibility of hormonally-stimulated parous mice to chemically induced
mammary carcinogenesis since parity is known to significantly reduce the
susceptibility of the mouse mammary gland to carcinogenesis. Virgin or
multiparous BALB/c mice were isografted with two pituitaries. Five weeks after
surgery, the mice were injected with N-methyl-N-nitrosourea (MNU; 50
micrograms/g i.v.). Mammary carcinomas arose in 85% (11/13) with a median
latency of 22.8 weeks and 1.9 tumors per virgin mouse and 80% (24/30) with a
median latency of 22.1 weeks at a frequency of 1.9 tumors per parous mouse.
Only 14% (2/14) of the non-isografted, age-matched parous controls developed
tumors when injected with MNU. Fourteen parous mice receiving only pituitary
isografts (no MNU), did not develop mammary carcinomas within the 7-month
period of the study. These results demonstrate that parous BALB/c mice are
refractory to MNU-induced mammary carcinogenesis and that this refractoriness
is not permanent, but can be overcome by hormonal stimulation mediated by
pituitary isografts. 

23
Park NH, Gujuluva CN, Baek JH, Cherrick HM, Shin KH, Min BM. COMBINED ORAL
CARCINOGENICITY OF HPV-16 AND BENZO(A)PYRENE: AN IN VITRO MULTISTEP
CARCINOGENESIS MODEL. Oncogene 1995;10(11):2145-53.

We previously immortalized normal human oral keratinocytes by transfection
with recombinant HPV-16 DNA and subsequently exposed the cells to
benzo(a)pyrene for 7 days. The exposure to benzo(a)pyrene modified the
immortalized cells: the modified cells (HOK-16B-BaP) proliferated in an
ordinary culture medium containing physiological calcium level (1.5 mM), but
demonstrated only enhanced proliferation capacity without tumor formation in
nude mice and failed to show in vitro anchorage-independency. In this study,
we further modified the HOK-16B-BaP cells by subculturing the cells in a
medium containing benzo(a)pyrene for 6 months. The cells were further modified
with a chronic benzo(a)pyrene exposure and were termed HOK-16B-BaP-T cells (1)
demonstrated a malignant phenotype in organotypic 'raft' culture, (2) showed
in vitro anchorage-independency, (3) developed  tumors in nude mice when
injected subcutaneously, (4) contained a significantly higher copy number of
intact and integrated  HPV-16 DNA; (5) contained higher level of HPV-16 E6/E7
messages and E7 protein, (6) were more resistant to transforming growth
factor-beta1 and (7) secreted higher level of vascular endothelial growth
factor with molecular weight of 56 kd than parental HOK-16B-BaP cells.
However, the levels of p53 and ras proteins and the levels of p53, c-myc and
c-fos transcripts in the HOK-16B-BaP-T cells were not different from those in
the HOK-16B-BaP cells. The highly conserved coding regions of the p53, 
c-Ha-ras1, and c-Ki-ras2 genes of the tumor cells were not mutated. These data
indicate that the HPV-immortalized human oral keratinocytes can convert to
tumorigenic cells by chronic exposure to benzo(a)pyrene. The tumorigenic
conversion seems to be associated with (1) the overexpression of viral
oncogenes such as E6 and E7 genes, (2) the higher resistance of cells to
transforming growth factor-beta1 and (3) the high secretion of 56 kd vascular
endothelial growth factor from the cells. 

CELL, TISSUE AND ORGAN TOXICITY
24
Murphy JE, Janszen DB, Gargas ML.  AN IN VITRO METHOD FOR DETERMINATION OF
TISSUE PARTITION  COEFFICIENTS OF NON-VOLATILE CHEMICALS SUCH AS
2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN AND ESTRADIOL. J Appl Toxicol
1995;15(2):147-52. 

The development of an in vitro vial equilibration technique for determining
tissue and liquid partition coefficients for non-volatile chemicals is
described. Radiolabeled chemical dissolved in propylene carbonate is
equilibrated with tissues or liquid at 37 degrees C in a vial system. The
solvent must be essentially immiscible with the test material. The amount  of
chemical movement to the tissue or liquid is compared to an appropriate
reference vial, and tissue or liquid:solvent partition coefficients are
calculated. Tissue:solvent values divided by blood:solvent values provide
tissue:blood partition coefficients required for developing physiologically
based pharmacokinetic models for chemicals. These models are useful for
estimating internal tissue doses to assess human risk from exposure to
chemicals. Partition coefficients for various rat tissues, 0.9% saline
solution and olive oil were determined in this study for radiolabeled
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and for the less fat-soluble
compound, estradiol. The TCDD tissue:propylene carbonate partition
coefficients were found to be: blood, 0.091; fat, 17.02; liver, 0.419; brain,
0.632; kidney, 0.305; muscle, 0.408. For estradiol, the tissue:propylene
carbonate partition coefficients were: blood, 0.286; fat, 0.169; liver, 1.032;
brain, 0.554. The TCDD results compared well with values reported and
estimated from a more protracted in vivo approach. Thus, this current
technique offers a simpler and time-saving alternative to in vivo approaches
for determining the partition coefficients of non-volatile compounds. 

25
Clothier RH.  THE FRAME CYTOTOXICITY TEST KENACID BLUE. Methods Mol Biol
1995;43:109-18. 

26
Cheung RC, Gray C, Boyde A, Jones SJ.  EFFECTS OF ETHANOL ON BONE CELLS IN
VITRO RESULTING IN INCREASED RESORPTION. Bone 1995;16(1):143-7.

Abuse of alcohol has been found to be an important risk factor for fractures
and osteoporosis, and tissue culture experiments have indicated that low
concentrations of ethanol can affect bone formation and resorption. This study
investigated direct effects of ethanol on bone cells using an in vitro
resorption assay. Osteoclasts from long bones of 19-day prehatch chicks were
seeded onto slices of dentine and cultured with control medium alone, or
medium containing 0.001%, 0.01% or 0.1% ethanol, at 37 degrees C with 5% CO2
for 24 h before being removed. The volumes and areas of resorption pits made
in the dentine were measured using confocal laser reflection microscopy
(Lasertech 1LM21 system) and the pits counted. An increase in the pit numbers
and mean pit areas, volumes and volume/area ratios was observed with 0.001%
and 0.01% ethanol, with a dose-related, bell-shaped curve of resorption.
Greatest mean volume resorbed per pit (p < 0.05), mean area resorbed per pit
(p < 0.01) and number of pits was at 0.01% ethanol. Volume/area (mean depth)
per pit was greatest at 0.001% ethanol (p < 0.05). This study has shown that
ethanol, even at blood concentrations experienced by the social drinker, has
an immediate direct effect on bone cells in vitro, resulting in increased
resorption by osteoclasts. 

27
Zwahlen RD, Holden WJ, Wyder-Walther M, Holub M, Moiola F.  INFLUENCE OF
ANTI-INFLAMMATORY DRUGS ON ADHESION OF NEUTROPHILS TO ENDOTHELIAL CELLS
CUTLURED ON MICROCARRIERS: A NOVEL IN VITRO SYSTEM AS AN ALTERNATIVE TO ANIMAL
EXPERIMENTATION. Zentralbl Veterinarmed A 1994;41(9):671-82.

Pharmacological control of inflammation by steroidal (SAIDs) and nonsteroidal
(NSAIDs) antiinflammatory drugs is of substantial clinical importance. To
reduce the number of animals used in pharmacological and toxicological
evaluation of these drugs we developed a novel assay to determine adhesion of
bovine neutrophils (PMN) to bovine aortic endothelial cells (BAEC) cultured on
microcarriers in a flow-through system. Pretreatment of BAEC with thrombin
(10(-7)-10(-4) M) led to a dose-dependent increase of PMN-adhesion
(10(-6)-10(-4) M:P < 0.05); platelet-activating factor (10(-9) M) and 1:200
diluted zymosan-activated serum (ZAS) had similar effects (P < 0.001). 
Pretreatment of PMN with SAIDs (50.9 and 509 microM dexamethasone, 12.2 and
24.4 microM flumethasone) did inhibit adhesion to ZAS-treated BAEC
dose-dependently. Pretreatment of PMN with NSAIDs had a less consistent
influence on adhesion to ZAS-stimulated BAEC. While phenylbutazone (0.33 and
3.3 mM), diclofenac (0.392 and 0.574 mM), indomethacine (0.436 and 0.872 mM),
and acetylsalicylic acid (3.47 and 16.94 mM) induced dose-dependent inhibition
of PMN-adhesion to ZAS-treated BAEC,       piroxicam (0.377 and 0.754 mM)
inhibited PMN-adhesion strongly (P < 0.001) but not dose-dependently, and
ketoprofene (0.614 and 1.228 mM) had no effect on PMN-adhesion. The method
presented here is efficient for evaluating the pharmacological modulation of
PMN interaction with endothelial cells, and useful for studying further
aspects of endothelial cell biology.

28
Kuehn U, Empertz U, Knop J, Becker D.  A NEW METHOD FOR PHENOTYPING
PROLIFERATING CELL NUCLEAR ANTIGEN POSITIVE CELLS USING FLOW CYTOMETRY:
IMPLICATIONS FOR ANALYSIS OF THE IMMUNE RESPONSE IN VIVO.  J Immunol Methods
1995;179(2):215-22.

The incorporation of radioactive nucleotides into newly synthesized DNA has
been established as a standard method for the detection of proliferation in
eucaryotic cells. Unfortunately the use of this method makes it harder to
obtain information on the phenotype of proliferating cells in mixed cell
populations. For this reason we established a flow-cytometric approach
employing a monoclonal antibody specific for murine as well as human
proliferating cell nuclear antigen (PCNA) and a double labeling technique for
detection of cell membrane-expressed phenotypic markers. The efficiency of
this immunostaining procedure was confirmed by simultaneous and highly
specific detection of PCNA in nuclear structures as well as cell
membrane-expressed antigens using cytological techniques. In vitro 
experiments with mitogen- and alloantigen-stimulated murine lymph node cells
(LNC) and human peripheral blood mononuclear leukocytes (PBML) revealed a good
correlation of total (3H)thymidine incorporation into DNA and expression of
PCNA. For the analysis of proliferating cells activated in vivo the method was
employed to evaluate the local lymph node assay which assesses the
allergenicity of small chemicals. LNC prepared from the cervical lymph nodes
of mice treated on 4 consecutive days with sensitizing concentrations of the
contact allergens oxazolone, TNCB and DNFB as well as the irritants benzoic
acid and SLS in comparison to the solvent control showed a dramatic increase
in  the total amount of proliferating cells for contact allergen-treated
animals in comparison to the solvent control and irritant-treated mice. In
addition a detailed phenotyping of the proliferating cell populations was
possible. This approach offers an easy to perform, non-radioactive method for
the assessment of proliferation of murine as well as human leukocytes in vitro
and especially in vivo and will be of great advantage for situations where the
phenotype of proliferating cellular subsets in heterogeneous populations is of
interest.

29
Marinovich M,  Guizzetti M, Ghilardi F, Paoletti R, Galli CL.  EFFECT OF
25-HYDROXYCHOLESTEROL, 26-HYDROXYCHOLESTEROL AND ITS ANALOGUE,
26-AMINOCHOLESTEROL, ON PROTEIN CONTENT, PROTEIN SYNTHESIS AND LDH LEAKAGE IN
MOUSE EPIDERMAL CELLS.  Toxicology 1995;99(1-2):125-34.

The cytotoxicity of 25-hydroxycholesterol, 26-hydroxycholesterol and its
analogue,       26-aminocholesterol was investigated in murine epidermal cell
line, HEL-30. Lactate dehydrogenase (LDH) leakage, protein synthesis and
protein content were determined after exposure of cell monolayers to the
compounds ranging from 0.1-200 muM for 2, 6 or 24 h. 26-Aminocholesterol
affected all the parameters studied time and concentration dependently;
25hydroxycholesterol and 26-hydroxycholesterol were not toxic  for HEL-30
cells. The cellular target for 26-aminocholesterol was primarily the membrane,
since the LDH leakage was already detectable 10 min after exposure. On the
other hand, for the other two oxysterols a protective role on this structure
might be postulated. In fact 25- hydroxycholesterol and 26-hydroxycholesterol
decreased the natural LDH leakage due to the ageing of the culture. In
addition, 20 AM 25-hydroxycholesterol reversed the effect of moderately lytic
doses of 26-aminocholesterol and Triton X-100, but not of sodium dodecyl
sulfate.

30
Vogel C, Abel J.  EFFECT OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN ON GROWTH
FACTOR EXPRESSION IN THE HUMAN BREAST CANCER CELL LINE MCF-7.  Arch Toxicol
1995;69(4):259-65. 

The aim of this study was to examine whether changes in growth factor or
cytokine expression could be responsible for the growth inhibitory effect of 
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the human breast cancer MCF-7
cell line. Treatment of MCF-7 cells with 10 nM TCDD  for 7 days reduced the
cell growth to 60% of control; this effect was partly abolished by cotreatment
of the cells with 100 nM 17beta-estradiol (E2). The inhibition of cell growth
by TCDD was accompanied by an enhanced  secretion of transforming growth
factor-beta (TGF-beta) and the TGF-beta content in cell culture supernatants
was 2-fold higher than in controls. Using reverse transcription polymerase
chain reaction (RT-PCR), the effect of  TCDD on the expression of TGF-beta
isoforms, transforming growth factor-alpha (TGF-alpha), tumor necrosis
factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was investigated. It
was demonstrated that incubation with 1, 10 and 100 nM TCDD for 24 h increased
mRNA levels  IL-1beta. The strongest effect was found on IL-1beta, the mRNA
level of which was dose-dependently increased. TCDD had a minor effect on
TGF-alpha and TNF-alpha mRNA. The mRNA levels were significantly increased
after treatment with 10 and 100 nM TCDD. The mRNA expression of TGF-beta1 and
TGF-beta2 was unchanged, whereas the TGF-beta3 mRNA level was enhanced 2 to
3-fold after TCDD treatment. From the results, we suggest that TCDD-induced
growth inhibition in MCF-7 cells is related to the growth  action of a set of
growth factors and cytokines which have a contextual action on MCF-7 cell
proliferation. 

31
Lee J , Green MH, Amiel D.  SYNERGISTIC EFFECT OF GROWTH FACTORS ON CELL
OUTGROWTH FROM EXPLANTS OF RABBIT ANTERIOR CRUCIATE AND MEDIAL COLLATERAL
LIGAMENTS.  J Orthop Res 1995;13(3):435-41.

Cellular migration and proliferation are integral aspects of wound healing. An
in vitro assay for cellular migration and proliferation using explants of
rabbit anterior cruciate and medial collateral ligaments was developed
previously. This study presents the effects of serum-free culture medium
supplemented with basic fibroblast growth factor, bovine insulin, transforming
growth factor-beta 1, and platelet-derived growth factor-B, added either
individually or in combination, on cell outgrowth in explants of rabbit
anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3
and 6 days by counting the number of cells surrounding the tissue explants.
For explants of both ligaments, cell outgrowth was dependent on the presence
of 10% fetal bovine serum or the combination of growth factors.  Little
outgrowth occurred in explants of either ligament in the presence of basic
fibroblast growth factor, transforming growth  factor-beta 1, or bovine
insulin. Platelet-derived growth factor-B at concentrations of 20 and 100
ng/ml seemed to increase cell outgrowth from medial collateral ligament
explants at 6 days. The outgrowth from the explants of both ligaments was much
greater in the presence of all four growth factors than the sum of the
outgrowth with the individual factors. This synergistic effect was as much as
10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and
6, respectively, but no more than 3-fold for the medial collateral ligament
explants at these times. Medial collateral ligament explants exhibited greater
cell outgrowth than anterior cruciate ligament explants in 10% serum and in
the presence of the four growth factors.

32
Chakradeo PP, Nair J, Bhide SV.   METABOLISM OF N'-NITROSONORNICOTINE BY ADULT
AND FETAL HUMAN OESOPHAGEAL CULTURES. Cell Biol Int 1995; 19(1):53-8. 

The metabolism of (3H) labelled N-Nitrosonomicotine a major constituent of the
class of Tobacco Specific Nitrosamines was studied in adult and fetal human
oesophageal cultures. The metabolites were separated by HPLC and were
identified when compared to the standards as OH-acid from 5'-hydroxylation,
NNN-1-N-oxide formed via the pyridine N-oxidation pathway and Keto acid from
2'-hydroxylation in both the adult and fetal cultures. These results indicate
that hydroxylation which leads to  electrophilic diazohyroxides is the major
pathway of NNN metabolism in cultured human oesophagus. In the adult cultures
levels of metabolites formed were 20.32 pmoles/mug DNA of OH acid 11.08
pmoles/mug DNA of NNN-1-N-oxide and 7.6 7 pmoles/mug DNA of Keto acid. In the
fetal cultures levels were 10.85 pmoles/mug DNA of OH acid, 9.40  pmoles/mug
DNA of NNN-1-N-oxide and 7.91 pmoles/mug DNA of Keto acid. These results
indicate that alpha-hydroxylation is the key step in the metabolic activation
of  NNN in human oesophagus. 

33
Datta K, Joseph P, Roy SK, Srinivasan SN, Kulkarni AP. PEROXIDATIVE XENOBIOTIC
OXIDATION BY PARTIALLY PURIFIED PEROXIDASE AND LIPOXYGENASE FROM HUMAN FETAL
TISSUES AT 10 WEEKS OF GESTATION. Gen Pharmacol 1995;26(1):107-12.

1. Present study reports the ability of partially purified peroxidase and
lipoxygenase from human fetal tissues at 10 weeks of gestation to oxidize
selected xenobiotics in vitro. 2. Peroxidase was found to oxidize four
different chemicals in the presence of H2O2. Sodium azide and potassium
cyanide inhibited peroxidase activity towards guaiacol in a
concentration-dependent manner. 3. The dioxygenase and co-oxidase activities
of lipoxygenase towards linoleic acid and four model xenobiotics,
respectively, were observed. Both the catalytic activities of lipoxygenase
were significantly inhibited by < 1.0 muM nordihydroguaiaretic acid. 4. These
findings suggest that peroxidase and lipoxygenase may be important pathways
for peroxidative xenobiotic oxidation in human fetal tissues.

34
Grote JJ, Hesseling SC, Tjebbes GJ, Van Blitterswijk CA. EFFECT OF HA-1A
MONOCLONAL IGM ANTIBODY ON ENDOTOXIN-INDUCED PROLIFERATION OF CULTURED RAT
MIDDLE EAR EPITHELIUM. Ann Otol Rhinol Laryngol 1995; 104(3):226-30.

The effect of human monoclonal antibody HA-1A (Centoxin) on the effect of
endotoxin on cultured rat middle ear epithelium was investigated. The addition
of endotoxin to the standard culture medium revealed a concentration-related
proliferative effect on cultured mt middle car epithelium, leading to
cobblestone cells, cell tracks, and stratification of epithelium, whereas rat
middle ear epithelium cultured in standard medium grew as a monolayer composed
of flat polygonal cells. Addition of HA-1A  to standard medium supplemented
with endotoxin gave rise to a statistically significant suppression of the
proliferative effects of endotoxin on these cells. The morphology of rat
middle ear epithelium cultured in the presence of HA-1A and endotoxin showed
that these cells still had a tendency to form cobblestone-like cells and cell
tracks, but to a substantially lower degree. The present results support the
hypothesis that HA-1A suppresses the proliferative and morphological effects
of  endotoxin on rat middle ear epithelium and may play an important role in
the pathogenesis of otitis media.

35
Muraki Y, Yamada M, Nii S, Kumon H, Ohmori H.  [IN VITRO ASSESSMENT OF
RELATIVE PHOTOTOXICITY OF QUINOLONE ANTIBACTERIAL AGENTS BY REDUCTION OF
NEUTRAL RED UPTAKE IN CULTURED CELLS.] Nippon Kagaku Ryoho Gakkai Zasshi
1995;43(3):357-60.  (Jpn)

The relative phototoxicity of 10 antibacterial drugs in the quinolone group
was detd. by an in vitro assay, in which redn. of neutral red uptake was used
as a marker of cell injury.  Human embryonal lung fibroblasts or Vero cells
derived from a green monkey kidney were incubated with potential phototoxins. 
The cell cultures were irradiated with long wave-length UV, and the capacity
for neutral red uptake was detd.  The phototoxicity of 10 quinolones was much
lower than that of doxycycline, a known photosensitizer.  Among them,
enoxacin, lomefloxacin, ciprofloxacin, ofloxacin, and nalidixic acid
demonstrated higher phototoxicity, suggesting a good correlation with the
clin. occurrence of photosensitivity. Norfloxacin, balofloxacin (Q-35),
AM-1155, and T-3761 had less       potent phototoxicity.  This methodol. may
provide a useful rapid method to quantitate the phototoxic potential of newly
developed drugs.

36
Dumitrescu M, Jucu V, Zaharia CN, Belu O, Rojanschi D, Diaconu C, Talos D, 
Panaitescu M. [ACTION OF SOME AMPHIPHILIC DRUGS ON HUMAN FIBROBLASTS IN VITRO.
POSSIBLE ANTIVIRAL AND ANTITUMOR USES.]  Rev Roum Virol 1993;44(3-4):211-21.
(Fre)

The release of lactic dehydrogenase (LDH) from human embryo fibroblasts in
vitro was used as a test of membrane stability. Two amphiphilic drugs,
metomidate and timolol, stabilized the membrane, as shown by a reduced release
of LDH. Another drug, Ca dobesylate, had the opposite effect, making the
membrane less stable. The use of the LDH test for the selection of some
natural complexes or synthetic drugs with membrane-stabilizing and hence
potential antiviral activity (by inhibiting virus penetration into cells) is
proposed. Furthermore, LDH activity was inhibited by metomidate, causing  the
intracellular accumulation of lactate and consequent lowering of intracellular
pH. The use of metomidate for potentiating the action of classical antitumor
drugs, lowering tumor cell pH, is proposed.

37
O'Hare S, Atterwill CK, editors. METHODS IN MOLECULAR BIOLOGY. Vol 43, IN
VITRO TOXICITY TESTING PROTOCOLS. Totowa (NJ): Humana Press Inc; 1995. 332 p.

38
Langner A, Melzig MF, Kempa S, Krause A. [THE USE OF LYMPHOCYTE CULTURES FOR
INVESTIGATING THE BIOTRANSFORMATION OF DRUGS.] Pharmazie 1995;50(2):130-8.
(Ger)

Rat lymphocyte and mouse myeloma cell cultures were used as in vitro test
systems for investigating the biotransformation of drugs. The biochem.
properties of both kinds of cells were qual. comparable. No reductive or
conjugating activities were present in the cultures. The established and
characterized systems were used to study the biotransformation of 4 potential
drugs. The Trapidil deriv. AR 12463
(5-piperidino-7-[N-pentyl-N-(beta-hydroxyethyl)]-amino-s-triazolo-
[1,5-a]pyrimidine) was transformed into 2 metabolites in both the lymphocyte
and myeloma cell cultures. These substances were characterized as the
hydroxypentyl- and the hydroxypyrimidine derivs. Both products are the initial
metabolites for further degrdn. reactions in vivo in the rat. The
immunostimulator AWD 100-041 (3-(2-mercaptoethyl)quinazoline-2,4-(1
H,3H)-dione) was metabolized in both lymphocyte and myeloma cell cultures to
the disulfide of the parent compd. After incubation of the S-Me analog of AWD
100-041, itself a metabolite of the drug, sulfoxidized metabolites occurred,
which were also detectable in vivo. After incubation of the anticonvulsant AWD
140-076 (4-chlorophenylpyrrole-3-morpholino-2-carboxylic acid Me ester) in the
cell cultures 2 metabolites were formed which were oxidized at the morpholine
N as well as at the pyrrole  skeleton. Both compds. are the main metabolites
in metab. in vivo. The biotransformation of the lipoxygenase inhibitor FLM
5011 (2-hydroxy-5-methyllaurophenone oxime) in lymphocyte and myeloma cell
cultures was characterized by the formation of the omega-hydroxy deriv. This
compd. is the initial metabolite for the further degrdn. of the lauryl side
chain. All these       substances were tested for cytotoxicity in myeloma
cells. The corresponding IC50 values were 4.5 .times. 10-6M for AR 12463, 1.4
.times. 10-5M for AWD 100-041, 1.3 .times. 10-4M for AWD 140-076 and 1.2
.times. 10-4M for FLM 5011. No relationship was found between cytotoxicity and
the degree of metab. 

39
Kim YS, Park KH. [SCREENING METHOD FOR ANTIHEPATOTOXIC ACTIVITY USING
CCl4-INDUCED CYTOTOXICITY IN PRIMARY CULTURED RAT HEPATOCYTES]. Saengyak
Hakhoechi 1995;26(1):51-6. (Kor)

To devise an in vitro screening method for antihepatotoxic activity,
CCl4-induced cytotoxicities in primary cultured rat hepatocytes were examd.
When rat hepatocytes were intoxicated with 0.5, 1.0 or 1.5 mM CCl4 for 1.5, 3
or 19 h, release of LDH, GOT, GPT from hepatocytes was increased in a
dose-dependent manner. Treatment with 1.5 mM Ccl4 for 1.5 h showed max.
increase in activity of LDH, GOT or GPT released in the medium compared with
the control. At this exptl. condition, well known antihepatotoxic substances,
glycyrrhizin and silybin markedly inhibited CCl4-induced cytotoxicities. These
results demonstrated that the screening method using CCl4-induced injury in
primary cultured rat hepatocytes might be suitable in vitro assay for
antihepatotoxic activity.

40
Ushimura H, Matsudo Y, Tanaka Y, Tsutsui T. [QUANTITATIVE COMPARISON OF
CYTOTOXICITY OF DENTAL OINTMENTS FOR PERIODONTAL DISEASES TO NORMAL HUMAN
GINGIVAL KERATINOCYTES IN VITRO.] Nippon Shishubyo Gakkai Kaishi
1995;37(1):76-83. (Jpn)

The cytotoxicities of dental ointments utilized for periodontal diseases were
examd. with normal human keratinocytes from gingival tissues by the uptake of
neutral red (NR assay).  The NR assay is a quant. in vitro assay that
distinguishes between viable, damaged or dead cells. Cultures from different
individuals were established, and secondary cultures in serum-free medium were
used. The cytotoxicity of ointments used on mucous membranes in the oral
cavity was detd. from the dose-response curves of inhibition of NR uptake in
cells treated with ointments for 2 days. As a quant. measure of cytotoxicity,
NR 50, i.e. the concn. of ointments that resulted in a 50% decrease in NR
uptake relative to untreated controls,  was extrapolated from dose-response
curves. The rank-order of cytotoxicities (NR 50) of injection type of
ointments applied to periodontal pockets was Hinoporon .mchgt. Tetracortisone
ointment > Periocline .apprxeq. Terracortrill ointment. The cytotoxicity of
Hinoporon was approx. 1300-fold that of Terracortrill ointment. The rank-order
of cytotoxicities of spread type of ointments was Dexaltin ointment > Despa
KOWA > Tetracycline presteron .apprxeq. Presteron .apprxeq. Kenalog .apprxeq.
Aphtasolone. If the same amt. of ointments was applied to the oral mucosa for
the same time, the lower-ranking ointments would be less cytotoxicity. These
results not only provide useful ests. of relative toxicities of dental
ointments to human cells, but also can be useful as a std. for screening of
toxicities of the new products.

41
Kaji T, Mishima A, Machida M, Yabusaki K,Suzuki M, Yamamoto C, Fujiwara Y,
Sakamoto M, Kozuka H. COMPARATIVE CYTOTOXICITY OF EXOGENOUS
CADMIUM-METALLOTHIONEIN AND CADMIUM ION IN CULTURED VASCULAR ENDOTHELIAL
CELLS. Bull Environ Contam Toxicol 1995;54(4):
501-6. 

42
Iida T, Tang GQ, Suttikulpitug S, Yamamoto K, Miwatani T, Honda T. ISOLATION
OF MUTANT TOXINS OF VIBRIO PARAHAEMOLYTICUS HEMOLYSIN BY IN VITRO MUTAGENESIS.
Toxicon 1995;33(2):209-16.

Thermostable direct hemolysin produced by Vibrio parahaemolyticus is a major
virulence factor of the organism. The hemolysin has a variety of biological
activities such as lethality to mice, cytotoxicity to cultured cells,
cardiotoxicity, and fluid accumulating activity in rabbit ileal loop test. In
this study, we attempted to isolate less hemolytic mutant toxins of the
thermostable direct hemolysin to use them for analysis of mode of action of
the hemolysin. Six mutant toxins were obtained  by in vitro mutagenesis of the
cloned gene for the hemolysin. Characterization of the mutant toxins
demonstrated that single amino acid substitutions at Gly62, Trp65, Thr67,
Gly86, Glu116 and Glu138 resulted in a loss or lowering of the hemolytic
activity. Two of the mutant toxins inhibited hemolysis by wild-type toxin on
rabbit blood agar plates, while their hemolytic activity was below the
detectable level. These mutant toxins would be useful for identifying the as
yet unknown  receptor for the hemolysin on the target cell membrane.

43
Coldham NG, Moore AS, Dave M, Graham PJ, Sivapathasundaram S, Lake BG, Sauer
MJ. [IMIDOCARB RESIDUES IN EDIBLE BOVINE TISSUES AND IN VITRO ASSESSMENT OF
IMIDOCARB METABOLISM AND CYTOTOXICITY.] Drug Metab Dispos 1995;23(4):501-5.

Imidocarb residues in liver and muscle were measured by HPLC after a single
therapeutic dose to cattle (3 mg imidocarb dipropionate kg-1). Imidocarb and
7-ethoxycoumarin metabolism were compared in three different in vitro systems
prepared from bovine liver: cultures of hepatocyte monolayers, precision-cut
liver slices, and microsomes. The potential hepatotoxicity of imidocarb
residues was tested on hepatocyte monolayers and assessed using the neutral
red and lactate dehydrogenase leakage assays. The concentration of imidocarb
(mean +/- SD) decreased between days 14 and 224 after treatment from 5.40 +/-
0.61 to 0.12 +/- 0.01 and from 1.05 +/- 0.31 to 0.06 +/- 0.02 microgram g-1 in
liver and muscle, respectively. The depletion kinetics of imidocarb fitted a
two-compartment model with alpha- and beta-phase half-lives of 31.7 and 48.5
days in liver and 34.9 and 120.7 days in muscle, respectively. Imidocarb
metabolites were not detected in any in vitro system. 7-Ethoxycoumarin
metabolism was found in all in vitro systems; the predominant metabolite
produced by hepatocyte and liver slice cultures was umbelliferone glucuronide.
Cytotoxicity of imidocarb (100 microM) to hepatocyte monolayers was maximal
after 72 hr treatment and dose-dependent above 10 microM imidocarb. It is most
likely that the hepatotoxicity of imidocarb is caused by the parent compound,
because no evidence for imidocarb metabolism was found. 

44
Flouriot G, Monod G, Valotaire Y, Devaux A, Cravedi JP.  XENOBIOTIC
METABOLIZING ENZYME ACTIVITIES IN AGGREGATE CULTURE OF RAINBOW TROUT
HEPATOCYTES. Seventh International Symposium on Responses of Marine Organisms
to Pollutants (Primo 7), Goteborg, Sweden, April 20-22, 1993. Marine Environ
Res 1995;39(1-4):293-7. 

45
Kleene KC, Wang M, Cutler M, Hall C, Shih D. DEVELOPMENTAL EXPRESSION OF
POLY(A) BINDING PROTEIN mRNAs DURING SPERMATOGENESIS IN THE MOUSE. Mol Reprod
Dev 1994;39(4):355-64.

The poly(A) binding protein (PABP), a conserved protein that binds to the 3'
poly(A) tail on mRNAs in eukaryotic cells, has been implicated in the
regulation of mRNA stability and translation. Two PABP cDNAs with different
sequences were isoalted from mouse testis cDNA libraries. The predicted amino
acid seuqence of one, PABP1, is nearly identical (98.9%) to human liver PABP,
while 80% of the amino acids of the second, PABPt, are identical to mouse and
human PABPs. Northern blots reveal that there is one major PABP mRNA species
in liver, muscle, kidney, and brain, two in spleen, and at least four in
testis. The levels of PABP mRNA in testis are 5-10-fold higher than in these
somatic tissues, but surprisingly the vast majority of all PABP mRNA size
variants sediment more slowly than single ribosomes, indicating strong
translational repression.  Reverse transcriptase-polymerase chain reaction
assays demonstrate that PABPt mRNAs are abundant only in testis. Northern
blots of RNAs purified from highly enriched spermatogenic cells show that
thehigh levels, multiple sizes of PABP mRNAs, and the PABPt mRNA are present
in meiotic and early haploid spermatogenic cells, and are sharply reduced in
late haploid cells. Comparison of the binding of PABP1 and PABPt to poly(A)
Sepharose in vitro revealed subtle differences, even though PABPt contains
substitutions for highly conserved arom. amino acids that are thought to be
necessary for binding to poly(A). The existence of two PABP isoforms in mouse
spermatogenic cells could influence cytoplasmic gene expressino during
spermatogenesis.

46
Delmas F, Trocheris V, Murat JC. EXPRESSION OF STRESS PROTEINS IN CULTURED
HT29 HUMAN CELL-LINE; A MODEL FOR STUDYING ENVIRONMENTAL AGGRESSION. Int J
Biochem Cell Biol 1995;27(4):385-91.
 The current study was undertaken to investigate the expression of stress
proteins (HSP) in cultured human HT29 cells submitted to stressing events
under in vitro conditions. Heat shocks (45~C, for 15-60 min) or cold shocks
(+1~C for 4 hr) were found to modify cell growth (growth curves) and to
enhance HSP expression. In most cases, changes in HSP expression are much more
pronounced than changes in cell growth. Exposure to 8% ethanol for 15 min
resulted in both growth inhibition and HSP overexpression. Propanol-1 was
found to be more toxic since 5% concentration given for 15 min stops cell
growth. 2.5% propanol-1 for 15 min induces a slight reduction of cell growth
but a clear-cut overexpression of stress proteins. We conclude that expression
of stress proteins, especially those of the HSP68/70 family, constitutes a
more sensitive response than changes in growth rate in case of external
aggression. This could make our model an interesting biological sensor to
environmental physical or chemical pollutants. 

47
Schilderman PA, Rhijnsburger E, Zwingmann I, Kleinjans JC. INDUCTION OF
OXIDATIVE DNA DAMAGE AND ENHANCEMENT OF CELL PROLIFERATION IN HUMAN
LYMPHOCYTES IN VITRO BY BUTYLATED HYDROXYANISOLE. Carcinogenesis
1995;16(3):507-12.

The food additive butylated hydroxyanisole (BHA) has been shown to induce
gastrointestinal hyperplasia in rodents by an unknown mechanism. The relevance
of this observation for human risk assessment is not clear. We therefore
analysed the effect of BHA and its primary metabolites tert-butylhydroquinone
(TBHQ) and tert-butylquinone (TBQ) on  8-oxo-deoxyguanosine formation and
labelling indices in human lymphocytes in vitro. Analysis of culture medium
and cell lysate fractions after administration of BHA or metabolites of BHA
revealed that BHA and TBHQ undergo biotransformation in whole blood cultures.
Moreover, TBQ can be reduced to TBHQ. While in cultures treated with BHA
50-60% of the dose administered was recovered, a much lower dose recovery was
found in cultures treated with either TBHQ or TBQ. This indicates a
considerable binding of these compounds to macromolecules. BHA and TBHQ, as
well as TBQ, induced a dose-dependent increase in cell proliferation of 
phytohaemagglutinin-stimulated lymphocytes, 50 muM being the optimal dose.
Since BHA is metabolized to TBHQ, it is not clear which compound is
responsible for the proliferation enhancing effects observed in culture.
Inhibition of TBHQ metabolism to its semiquinone radical by acetylsalicylic
acid (ASA) reduced the increase in labelling indices induced by TBHQ. This
indicates that this metabolic pathway is involved in the       enhancement of
cell proliferation induced by the hydroquinone. HPLC-ECD analysis of oxidative
DNA damage in lymphocytes exposed to 10, 50 and 100 muM BHA, TBHQ or TBQ
respectively showed that BHA was not capable of inducing oxidative DNA damage
to a significant degree. TBQ and, in particular, TBHQ at a dose of 50 muM (the
optimal dose for induction of cell proliferation), however, increased
lymphocyte 7-hydroxy-8-oxo-2'-deoxyguanosine formation by 320 and 680%
respectively. Inhibition of prostaglandin H synthase by ASA in cultures
treated with TBHQ decreased the oxidation ratio significantly, confirming the
significance of this enzyme system in the mechanism of toxicity of BHA.

48
Hilton J, Kimber I. THE MURINE LOCAL LYMPH NODE ASSAY. Methods Mol Biol
1995;43:227-35.

49
Gillies GE, Buckingham JC. THE APPLICATION OF IN VITRO MODELS OF HYPOTHALAMIC
FUNCTION IN TOXICITY TESTING. Methods Mol Biol 1995;43: 95-107. 

50
Gillies GE, Buckingham JC.  THE APPLICATION OF IN VITRO MODELS OF ANTERIOR
PITUITARY FUNCTION IN TOXICITY TESTING. Methods Mol Biol 1995;43:81-93.

51
 Hodgson E. CHEMICAL AND ENVIRONMENTAL FACTORS AFFECTING METABOLISM OF
XENOBIOTICS. In: Hodgson E and Levi  PE, editors. Introduction to Biochemical
Toxicology. 2nd ed. East Norwalk (CT): Appleton and Lange; 1994. p. 153-75.

52
Jiang Y, Moller G. IN VITRO EFFECTS OF HGCl2 ON MURINE LYMPHOCYTES: I.
PREFERABLE ACTIVATION OF CD4+ T CELLS IN A RESPONDER STRAIN. 
J Immunol 1995;154(7):3138-46.

Mercury-induced autoimmune disorders have been demonstrated in rats and mice
injected with HgCl2. We have studied the ability of HgCl2 to activate murine
lymphocytes in vitro and found that it induced increased DNA synthesis, which
peaked at days 4 to 6. Other metal ions, such as Mg2+ and Zn2+, had no or much
less effect. Consistent with the in vivo studies, there were strain
differences, and the most significant increase in thymidine uptake was induced
in A.SW and BALB/c spleen cells. Both T lymphocytes and adherent cells were
required for activation, and anti-CD4 Ab completely abrogated HgCl2-induced
proliferation, suggesting the involvement of T CD4+ and CD8+ T cells from
BALB/c mice (responder strain). In contrast, only CD8+ T cells from the
nonresponder DBA/2 mice were transformed. These findings indicate that helper
T cells play a crucial role for the immunologic effects caused by HgCl2 and
determine the ability of different mouse strains to respond to HgCl2.

53
Hwang DF, Lin JF, Jengs SS. STUDIES ON SUSPENDING CONDITIONS FOR ISOLATED EEL
HEPATOCYTES. J Fish Soc Taiwan 1994;21(3):273-80.

To establish fish hepatocytes as experimental materials for in vitro system of
toxicological and metabolic studies, the livers of Japanese eel Auguilla
japonica were used to isolate hepatocytes by using perfusion method. Based on
the viability of hepatocytes by staining with trypan blue the optimal
suspending conditions for eel hepatocytes were determined. It was found that
the optimal suspending temperature, time, cell concentration and pH value were
37~ C, 2 hr, 207 cells/ml and 7.5, respectively. It was also found that the
viability of isolated eel hepatocytes was improved when the suspending buffer
was added with either 5.6 mM glucose or 2.5 mM Ca++.

54
Kristen U, Kappler R. THE POLLEN TUBE GROWTH TEST. Methods Mol Biol
1995;43:189-98.

55
Blein-Sella O, Adolphe M. RABBIT ARTICULAR CHONDROCYTE FUNCTIONAL TOXICITY
TEST. Methods Mol Biol 1995;43:169-75.

56
Bidey SP. THYROID FOLLICULAR CELLS IN MONOLAYER CULTURE IN VITRO MODELS FOR
THYROID TOXICITY TESTING. Methods Mol Biol 1995;43:33-42.

57
Becerro MA, Uriz MJ, Turon X. MEASURING TOXICITY IN MARINE ENVIRONMENTS:
CRITICAL APPRAISAL OF THREE COMMONLY USED METHODS. Experientia
1995;51(4):414-8.

Toxicity quantification is important in environmental monitoring, in the field
of natural products, and in chemical ecology. The sensitivity and precision of
three commonly used methods detecting toxicity in marine environments were
compared, using the toxic marine sponge Crambe crambe as a test organism. The
paper disk diffusion method (run  with marine bacteria) showed the least
sensitivity and did not permit toxicity levels to be quantified. The sea
urchin and the MICROTOX tests showed greater sensitivity, and the latter had
the higher precision. The relative performance of these methods is discussed.
It is concluded that the MICROTOX bioassay displays the best characteristics
for toxicity quantification.

58
Blanquart C, Giuliani I, Houcine O, Jeulin C, Guennou C, Marano F. IN VITRO
EXPOSURE OF RABBIT TRACHEAL EPITHELIUM TO SO2: EFFECTS ON MORPHOLOGY AND
CILIARY BEATING. Toxicol In Vitro 1995;9(2):123-32.

The aim of this in vitro study was to characterize the direct effects of
short-term exposure to low concentrations of sulfur dioxide (SO2) on both the
morphology and the physiology of rabbit tracheal primary cultures. Scanning
electron microscopy (SEM) studies revealed that ciliated cells exposed for 1
hr to 10 ppm or 30 ppm SO2 exhibited aggregated cilia. Transmission electron
microscopy revealed numerous swollen mitochondria in cells exposed to 30 ppm
SO2 for 1 hr. This morphological damage to cells was coupled with
physiological alterations. A 25% decrease in ciliary beat frequency (CBF) was
measured in cells exposed to 30 ppm SO2. This inhibition was partially
reversible within 24 hr. This SO2 concentration also induced a significant
depletion of cellular ATP content which was completely restored after a 24-hr
recovery period. A correlation was found between cellular ATP level depletion
and CBF decrease. 

59
Krautschick I, Krugmann J, Neuenfeld M. THE EFFECT OF PEROXIDES ON THE
VASCULAR ENDOTHELIUM OF ISOLATED PIG AORTA IN VITRO. Exp Toxicol Pathol
1995;47(1):51-61.

The effect of peroxide on endothelial cells (perfused pig aorta) was examined
using an in vitro perfusion model. Hydrogen peroxide was added to the
perfusion medium (pig serum together with a buffer solution) which was
expected to lead to an increased oxidation of lipids and lipoproteins.
Oxidation processes of this type play a decisive role in the pathogenesis and
progression of arteriosclerosis. The aim of the present investigation was to
demonstrate by introducing hydrogen peroxide (H2O2)  in varying concentrations
(0.5-1.5%), the destructive impact of peroxides on the endothelium, while
these cells are believed to play a key role in the pathogenesis of
arteriosclerosis. The extent of endothelial cell impairment was assessed by
means of silver staining visualization of endothelial cell borders as well as
light- and scanning-electronmicroscopic investigation. It was discovered that
the endothelial cells show increasing impairment after 10 h of perfusion due
to the effect of  peroxide (hydrogen peroxide).

60
Argese E, Bettiol C, Ghelli A, Todeschini R, Miana P. SUBMITOCHONDRIAL
PARTICLES AS TOXICITY BIOSENSORS OF CHLOROPHENOLS. Environ Toxicol Chem
1995;14(3):363-8.

An in vitro bioassay procedure was used to investigate the toxic action of
chlorophenols on mitochondrial respiratory parameters. The toxicity of these
compounds was evaluated by determining their effects on the energy-coupled
reverse electron transfer (RET) in submitochondrial particles (SMPs) from beef
heart mitochondria. The bioassay procedure is based on the spectrophotometric
recording of the effects of toxicants on the rate of NAD+ reduction, induced
by ATP and succinate at the first  site level of the respiratory chain. The
toxicity end point was expressed as the toxicant concentration that causes 50%
inhibition of NAD+ reduction rate (EC50). The EC50 values determined for the
14    tested chlorophenols ranged from 17 mg/L for 2-chlorophenol to 0.081
mg/L for pentachlorophenol, indicating a general trend of increasing toxicity
with increasing chlorine substitution. Among chlorophenol isomers, which have
the same number of chlorine atoms, a lesser toxicity was associated with
ortho-substituted chlorophenols, whereas meta-substituted chlorophenols were
much more toxic. The EC50 values were compared with the toxicity data for a
variety of bioassays, by means of linear regression analysis. High degrees of
correlation obtained with toxicity tests involving different freshwater
species demonstrate the ability of SMPs to reproduce the toxic effects of the
tested compounds upon aquatic organisms. This supports the assessment that the
respiratory chain is the main target of  this class of toxicants. Results
obtained with chlorophenols and, in previous studies, with other environmental
contaminants confirm the suitability of the SMP bioassay as a prescreening or
complementary short-term test for monitoring aquatic toxicity. 

61
Celander M, Broman D, Forlin L, Naf C. EFFECTS OF PETROLEUM HYDROCARBONS ON
THE HEPATIC CYTOCHROME P450 1A1 SYSTEM IN RAINBOW TROUT. Seventh International
Symposium on Responses of Marine Organisms to Pollutants (Primo 7), Goteborg,
Sweden, April 20-22, 1993. Marine Environ Res 1995;39(1-4):61-5.


Burke MD, Brown D, Mayer RT, Houlihan DF. ALKOXYQUINOLINE O-DEALKYLATION A NEW
FLUORIMETRIC ASSAY FOR HYDROCARBON-INDUCED CYTOCHROME P450 IN FISH.  Seventh
International Symposium on Responses of Marine Organisms to Pollutants (Primo
7), Goteborg, Sweden, April 20-22, 1993. Marine Environ Res
1995;39(1-4):57-60.

62
Bylander JE, Li SL, Sens MA, Sens DA. EXPOSURE OF HUMAN PROXIMAL TUBULE CELLS
TO CYTOTOXIC LEVELS OF CdCl2 INDUCES THE ADDITIONAL EXPRESSION OF
METALLOTHIONEIN 1A mRNA. Toxicol Lett  1995;76(3):208-17.

Humans, in contrast to animals, have a complex expression of metallothionein
(MT) genes which involves many MT isoforms encoded by a family of genes
containing an upper limit of 12 possible functional genes. It is unknown if
these human isoforms of MT have distinct functions or if they simply represent
a non-essential duplication of gene function. In the present study, MT protein
and mRNA for the MT-2A, MT-1A, B, E, F, and G genes was determined for 3
isolates of human proximal tubule (HPT) cells having distinct sensitivities to
cadmium. For all 3 HPT isolates, the expression of MT protein and mRNA for the
MT-2A, MT-1E, MT-1F and MT-1G isoforms was similar among the isolates and
demonstrated no correlation to lethality. However, each isoform mRNA was
expressed at different levels when compared to one another. In contrast, the
expression of MT-1A mRNA differed in expression and correlated with the
differing lethalities displayed by each isolate. The finding of different
profiles of mRNA expression provides evidence that the MT isoforms may have
unique functions and that mRNA for the MT-1A gene could be a potential marker
for heavy metal exposure and/or toxicity.

63
Champion AR, Hanson JA, Court JB, Venables SE. THE MICRONUCLEUS ASSAY: AN
EVALUATION OF ITS USE IN DETERMINING RADIOSENSITIVITY IN VITRO. Mutagenesis
1995;10(3):203-8.

The cytokinesis-block micronucleus assay was used to measure radiosensitivity
in vitro in a panel of seven cell lines. Six of these cell lines were used to
study the major parameters of this assay. We observed varying sensitivities
following cytochalasin-B exposure. Treatment with 1 mug/ml cytochalasin-B for
24 h reduced cell survival in four of the six cell lines by > 60%.
Cytochalasin-B concentration and post-irradiation culture time were both found
to influence cell-response. In three cell  lines (V39, V134 and HX142), a
decrease in cytochalasin-B concentration (2-0.5 mug/ml) resulted in an
increase in the frequency of radiation-induced micronuclei per binucleate
cell. In other cell lines, either the opposite (V7M, CHO-K1) or no effect
(WiDr) was seen. A linear dose-response was observed between induced damage
expressed as the frequency of micronuclei and radiation dose in all but one
melanoma (V39) cell line. Evidence for radiation-induced division-delay, with
the maximum  frequency of binucleation in irradiated cultures occurring 24-48
h after that of controls, was only seen in two cell lines. Of particular note,
and in contrast to some other published reports, was the lack of a general
correlation between cell-response measured in the clonogenic and the
cytokinesis-block micronucleus assays. Consideration of lethal lesions,
determined from the clonogenic dose-response curve, with respect to
micronucleus frequency showed a complex relationship, with one micronucleus
per binucleate cell corresponding to a wide range of lethal lesions depending
on the cell line. It has been postulated that the binucleate cell with no
micronuclei may represent the surviving cell; however, we found no correlation
between the slope of the frequency of these cells with respect to radiation
dose and the clonogenic alpha slope. These observations should be considered
prior to attempting to use the cytokinesis-block micronucleus assay to measure
in vitro  radiosensitivity in human tumour cells. 

64
Clare C. MUTATION ASSAYS IN BACTERIA. Methods Mol Biol 1995;43:297-306.

65
Dean S. MEASUREMENT OF UNSCHEDULED DNA SYNTHESIS IN VITRO USING PRIMARY RAT
HEPATOCYTE CULTURES. Methods Mol Biol 1995;43: 267-76.

66
Bryan FL. PROCEDURES TO USE DURING OUTBREAKS OF FOOD-BORNE DISEASE. In: Murray
PR, et al, editors. Manual of Clinical Microbiology. 6th ed. Washington DC:
American Society for Microbiology; 1995: p. 209-26.

67
Edwards AJ, Anderson D, Phillips BJ. INDUCTION OF POLYPLOIDY IN HUMAN
LYMPHOCYTES IN VITRO BY EXCESS ADENINE, BUT NOT BY ADENOSINE.  Environ Mol
Mutagen 1995;25(3):197-201.

It is known that high levels of DNA precursors can be both clastogenic and
mutagenic in cultured cell lines and in vivo. The purpose of the present study
was to examine at an observational level the cytogenetic effects of adenine
and adenosine in primary human cell cultures. Human peripheral blood
lymphocytes from four donors were cultured and treated with a range of
concentrations of adenine and adenosine. Although no increase in sister
chromatid exchange (SCE) frequency was observed with either compound, there
was a statistically significant, dose-related increase in the proportion of
polyploid cells in cultures treated with adenine, but not in those treated
with adenosine. Some of the polyploid metaphases found after adenine treatment
contained diplochromosomes, suggesting that endoreduplicotion might have been
involved in polyploid formation in these cells. It is concluded that a high
level of adenine can cause genetic changes in human lymphocytes by interfering
with mitosis, perhaps by disturbing the balance of DNA precursor pools.

68
Dinjus U, Haenel I. CULTIVATION OF SALMONELLA IN CONTACT WITH EPITHELIAL
CELLS. Microbiol Res 1995;150(1):99-102.

An in vitro cultivation model for Salmonella having contact to epithelial
cells was developed, which led to an increase in the production of toxic
substances. The toxin assay on CHO-K1 cells was used for the determination of
the toxic activities. Salmonella strains cultivated in contact with a
monolayer of the intestinal cell line IEC-6 produced considerably more toxin
than Salmonella strains cultivated on VERO cells. The toxin formed was
heat-labile.

69
Falk PM, Sabater RT, Carballo DD Jr. RESPONSE OF THE HUMAN HEMATIC TISSUE
CULTURES HEP-G2 AND WRL-68 TO COCAINE. J Pharmacol Toxicol Methods
1995;33(2):113-20.

The hydrolytic metabolism of cocaine into benzoylecgonine, ecgonine methyl
ester, and ecgonine was studied in the human hepatoma cell line Hep-G2 and in
the nontumorigenic fetal hepatic cell line WRL-68. Also, the toxicological
response of these cells to cocaine was compared to previously published
results obtained with perfused liver cells and in vivo systems. Our
experiments indicated that Hep-G2 appear to have similar metabolic and
toxicological patterns to in vivo and perfused cell systems. The WRL-68 tissue
culture system was found to be less similar. These results suggest Hep-G2
cells can be utilized to study cocaine metabolism and toxicology, and possibly
in studies involving other xenobiotic compounds. 

70
Fiskesjo G. ALLIUM TEST. Methods Mol Biol 1995;43:119-27.

71
Male KB, Brown RS, Luong J HT. ENZYMATIC OXIDATION OF WATER-SOLUBLE
CYCLODEXTRIN-POLYNUCLEAR AROMATIC HYDROCARBON INCLUSION COMPLEXES, USING
LIGNIN PEROXIDASE. Enzyme Microb Technol 1995;17(7):607-14.

alpha-, beta-, gamma-, and 2-hydroxypropyl-beta-cyclodextrins were capable of
forming water-soluble inclusion complexes with several polynuclear aromatic
hydrocarbons (PAHs). The highest solubilities were noted for beta-cyclodextrin
and 2-hydroxypropyl-beta-cyclodextrin (hpbetaCD). The solubility of PAHs in
hpbetaCD was enhanced 224-fold and 7,500fold for naphthalene and
benzo(a)pyrene, respectively, with other PAHs yielding values between these
limits. The ability of lignin peroxidase (LiP) to cyclodextrin-included
substrates was similar to that previously reported for mixed solvent systems.
The enzyme oxidized anthracene, pyrene, and benzo(a)pyrene but not
naphthalene, phenanthrene, chrysene, and benzo(e)pyrene. The lignin peroxidase
exhibited a preference for oxidizing either anthracene or benzo(a)pyrene when
mixed with pyrene. On the basis of fluorescence measurement, anthracene and
benzo(a)pyrene were easily distinguished by exciting at 250 nm for anthracene
and 295 nm  for benzo(a)pyrene. Veratryl alcohol severely inhibited the pyrene
assay, with 50% inhibition noted at 0.3 mm while veratryl alcohol activated
the reactions between LiP and either anthracene or benzo(a)pyrene. Maximal
activation was obtained at 1.5 mm veratryl alcohol and no inhibitory effect
was detected up to 4.0 mm. Under identical conditions, the rate of reaction
with veratryl alcohol (4.0 mm) was 11- and 14-fold faster for benzo(a)pyrene
and anthracene, respectively, when compared to the assays in the absence of
veratryl alcohol. 

72
Fisher RL, Hasal SJ, Sipes IG, Gandolfi AJ, Brendel K. COMPARATIVE METABOLISM
AND TOXICITY OF DICHLOROBENZENES IN SPRAGUE-DAWLEY, FISCHER-344 AND HUMAN
LIVER SLICES. Hum Exp Toxicol 1995;14(5):414-21.

1 Precision-cut liver slices, prepared from Sprague-Dawley and Fischer-344
rats and donated human liver tissue, were used to identify differences in
1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB) and
1,4-dichlorobenzene (1,4-DCB) metabolism and how it may relate to toxicity. 2
Rat and human liver slices were incubated with 1 mM of either dichlorobenzene
to determine metabolism and toxicity, at 2 and 6 h of organ culture. 3 The
human liver slices metabolised the dichlorobenzenes to a greater extent than
those from either of the rat strains. Liver slices from the Fischer-344 strain
had a higher metabolic rate than the slices from the Sprague-Dawley rat
strain. 4 The metabolic rate of dichlorobenzene isomers did not consistently
correlate with its toxicity. For example, human slices did not exhibit any
hepatotoxicity, even though they metabolised these compounds to a greater
extent than either rat strain. 5 Cross species covalent binding did not
correlate with toxicity  endpoints measured in this study. 6 The phase two
metabolite profiles for each of the isomers in human and rat slices were
similar in that the glutathione-cysteine conjugate was the major metabolite. 7
The use of an in vitro system which utilises human liver slices might provide
an important bridge between animal derived data and the human situation.

73
Na MR, Koo SK, Kim DY, Park SD, Rhee SK, Kang KW, Joe CO. IN VITRO INHIBITION
OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION BY CHEMICAL CARCINOGENS.
Toxicology 1995;98(1-3):199-206.

This study was conducted to assess the effects of chemical carcinogens on the
gap junction-mediated intercellular communication in cultured mammalian cells.
The       method of scrape-loading dye transfer of lucifer yellow was adapted
as a measure of gap junctional communication. Clone 9 cells derived from rat
liver were treated with a model chemical carcinogen,
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the gap junctional
communication was assessed by measuring the transfer of scrape-loaded  lucifer
yellow dye. When cells were treated with the carcinogen at 0.3 mg/ml, the
fluorescent dye transfer was inhibited by 90% in 60 min. Other chemical
agents, which include direct or indirect carcinogens and antitumor drugs, were
also examined for their effects on the gap junctional communication. Direct
carcinogens, such as MNNG, hydroxylamine and ethidium bromide, exhibited
strong inhibition of intercellular communication, while indirect carcinogens,
such as aflatoxin B1 and ethionine, exerted minor effects. Effects of test
chemicals on the cell communication through gap junctions were readily
quantitated by counting the number of cells stained with the fluorescent dye.

74
Naughton BA, Sibanda B, San Roman J, Naughton GK. CHARACTERIZATION AND USE OF
LONG-TERM LIVER CULTURES TO EVALUATE THE TOXICITY OF CYCLOPHOSPHAMIDE OR
BENZENE TO BONE MARROW CULTURES.  In: Reinhardt CA, editor. Alternatives to
Animal Testing: New Ways in the Biomedical Sciences, Trends and Progress;
Symposium; 1992 Nov 30; Zurich, Switzerland. New York: VCH Publishers, Inc:
1994. p. 147-57.


Flint O. A TIMETABLE FOR REPLACING REDUCING AND REFINING ANIMAL USE WITH THE
HELP OF IN VITRO TESTS THE LIMULUS AMOEBOCYTE LYSATE TEST LAL AS AN EXAMPLE.
In: Reinhardt, CA, editor. Alternatives to Animal Testing: New Ways in the
Biomedical Sciences, Trends and Progress; Symposium; 1992 Nov 30; Zurich,
Switzerland. New York: VCH Publishers, Inc; 1994: p. 27-43.

75
Berg OH, Henriksen RN, Steinsvag SK. THE EFFECT OF A BENZALKONIUM
CHLORIDE-CONTAINING NASAL SPRAY ON HUMAN RESPIRATORY MUCOSA IN VITRO AS A
FUNCTION OF CONCENTRATION AND TIME OF ACTION.  Pharmacol Toxicol
1995;76(4):245-9.

Human respiratory mucosa was exposed to oxymetazoline nasal spray in varying
concentrations and for varying periods of time in vitro. The drug destroyed
the tissue in a concentration- and time-dependent manner. In the experiments
with various concentrations of the spray, some tissue fragments retained their
viability throughout the experiment. This number increased parallel to a
decrease in concentrations of the test substance. All the tissue fragments
exposed to undiluted nose spray underwent severe destructive alterations
during the exposure period. These alterations appeared first and were most
extensive in those exposed for the longest periods of time. It has previously
been demonstrated that the toxic effect of oxymetazoline nasal spray in vitro
is probably due to the preservative benzalkonium chloride. The apparent lack
of consistency between the toxic effects of benzalkonium chloride in vitro and
in vivo is discussed, with special reference to protective systems absent in
vitro but present in vivo.

76
Potgieter FJ, Torronen R, Wilke PI. THE IN VITRO ENZYME-INDUCING AND CYTOTOXIC
PROPERTIES OF SOUTH AFRICAN LABORATORY ANIMAL CONTACT BEDDING AND NESTING
MATERIALS. Lab Anim 1995;29(2):163-71.

Enzyme-inducing and cytotoxic effects of South African bedding materials were
investigated using a mouse hepatoma cell line, Hepa-1, cell culture system.
This cell culture system is a convenient and sensitive method for the
screening of bedding materials for the presence of compounds that could be
potentially harmful to animals and thus the experimental outcome. Cells were
exposed to acetone extracts of the different materials or their components.
Corn cobs displayed very little or no CYP1A1-inducing or cytotoxic effects,
whilst vermiculite and unbleached pulp from pine and eucalyptus showed greater
induction and cytotoxic properties. The latter properties were lower than
those produced by the different recycled paper extracts. Pine shavings (Pinus
elliottii) and the different wood components making up industrial sawdust
expressed the highest cytotoxic and CYP1A1-inducing properties.

77
Jontell M, Hanks CT, Bratel J, Bergenholtz G. EFFECTS OF UNPOLYMERIZED RESIN
COMPONENTS ON THE FUNCTION OF ACCESSORY CELLS DERIVED FROM THE RAT INCISOR
PULP. J Dent Res 1995;74(5):1162-7.

Monomeric resin components from dental composites are toxic to fibroblasts in
culture and thus may interfere with the local immune system of the pulp,
reducing its effective defense potential, either by cytotoxicity or by a more
specific immune mechanism. Therefore, the present study was undertaken to
observe the cytotoxic effects elicited by certain unpolymerized components of
resin composites upon the function of accessory pulp cells in mitogen-induced
proliferation of T-lymphocytes. Accessory cells from the rat incisor pulp were
released following enzymatic digestion with collagenase. The assay included
incubation of these cells with purified T-lymphocytes from cervical lymph
nodes for 72 h in the presence of different concentrations of the resin
components. The proliferative T-lymphocyte response was monitored by
3H-thymidine incorporation. Initially, we conducted experiments on spleen
cells to determine the proper concentration intervals for suitable testing of
the resin components. To assess the individual susceptibility of accessory
cells and T-lymphocytes, we pre-treated each of these cells with some of the
test materials prior to assay. At low concentrations, urethane      
dimethacrylate (UDMA), bisglycidyl methacrylate (bis-GMA), triethylene glycol
dimethacrylate (TEGDMA), and bis-phenol A (BPA) increased spleen cell
proliferation to concanavalin A (con A). Purified T-lymphocytes stimulated by
pulpal cells did not show enhanced responses to UDMA, bis-GMA, glycidyl
mehtacrylate (GMA), or N,N,-dihydroxyethyl-p-toluidine (DHEpT). At higher
concentrations, all substances except camphoroquinone (CAMP) showed inhibitory
effects in both test systems. The in vitro study shows that resin components
can evoke either immunosuppression or immunostimulation on mitogen-driven
proliferation of purified T-lyumphocytes and spleen cells. 

78
Chiao C, Zhang Y, Kaufman DG, Kaufmann WK. DERIVATION OF
PHENOBARBITAL-RESPONSIVE IMMORTAL RAT HEPATOCYTES.  Am J Pathol
1995;146(5):1248-59.

Two lines of rat hepatocytes, designated 6/15 and 6/27, were obtained from
carcinogen-treated livers by cultivation in medium containing the liver tumor
promoter, phenobarbital (PB). Both lines appeared to be PB-responsive and to
have an unlimited in vitro proliferative lifespan, i.e., immortality. The
ability of pure 6/27 hepatocytes to form colonies from single cells was
strictly dependent upon PB; it was reduced by 97 to 99% in the absence of PB.
These hepatocytes were not tumorigenic. For 6/27 hepatocytes in early passages
where cultures contained fibroblast contaminants and later when they were a
pure culture, PB was able to enhance colony growth from single cells and
facilitated       population expansion by sustaining DNA synthesis and by
inhibiting cell lysis. The 6/15 line displayed PB-dependent colony formation
and was not tumorigenic at early passages. At later passages 6/15 hepatocytes
were less dependent on PB for colony formation, and they formed hepatocellular
carcinoma when transplanted into livers of syngeneic rats. The demonstration
that PB sustained the proliferation and viability of hepatocytes with enhanced
growth capacity and indefinite proliferative lifespan suggests that PB may be
necessary for progression of these chemically initiated hepatocytes to
immortal and tumorigenic lines in vitro.

79
Pelin K, Kivipensas P, Linnainmaa K. EFFECTS OF ASBESTOS AND MAN-MADE VITREOUS
FIBERS ON CELL DIVISION IN CULTURED HUMAN MESOTHELIAL CELLS IN COMPARISON TO
RODENT CELLS.  Environ Mol Mutagen 1995; 25(2):118-25.

We report the effects of chrysotile and crocidolite asbestos, and glass and
rock wool fibers (man-made vitreous fibers, MMVF) on the duction of binucleate
cells in vitro. The response of human mesothelial cells (target cells in fiber
carcinogenesis) and rodent cells was compared. Human primary mesothelial
cells, MeT-5A cells (an immortalized human mesothelial cell line), and rat
liver epithelial (RLE) cells were exposed to asbestos and MMVF samples of
similar size range. Milled glass wool, milled rock wool, and titanium dioxide
were used as non-fibrous particle controls. All four fiber types caused
statistically significant increases in the amount of binucleate cells in human
primary mesothelial cells and MeT-5A cells (in the dose range 0.5-5.0
micrograms/cm2). Chrysotile and crocidolite asbestos were more effective
(1.3-3.0-fold increases) than thin glass wool and thin rock wool fibers
(1.3-2.2-fold increases). However, when the fiber doses were expressed as the
number of fibers per culture area, the asbestos and MMVF appeared equally
effective in human mesothelial cells. In RLE cells, chrysotile was the most
potent inducer of binucleation (2.9-5.0-fold increases), but the response of
the RLE cells to crocidolite, thin glass wool, and thin rock wool fibers was
similar to the response of the human mesothelial cells. No statistically
significant increases in the number of bi- or multinucleate cells were
observed in human primary mesothelial cells or RLE cells exposed to the
non-fibrous dusts. In MeT-5A cells exposed to 5 micrograms/cm2 of milled glass
wool and milled rock wool, as well as in cultures exposed to 2 and 5
micrograms/cm2 of TiO2, significant increases were, however, observed. Our
results show that rodent cells respond differently to mineral fibers than
human cells. The results also add evidence to the suggested importance of
disturbed cell division in fiber carcinogenesis.

80
Rat P, Korwin-Zmijowska C, Warnet JM, Adolphe M. NEW IN VITRO FLUORIMETRIC
MICROTITRATION ASSAYS FOR TOXICOLOGICAL SCREENING OF DRUGS. Cell Biol Toxicol
1994;10(5-6):329-37.

Flow cytometry has been widely used to quantify fluorescent probes in cell
culture. However, FCM is not adapted to toxicological screenings due to the
cost, the length and the poor reproducibility of this technique. Moreover,
several multicenter studies have preferred microtitration methodologies for
drug screening. A new fluorimetric technology has been designed that is
sensitive and adapted to direct screening in 96-well microplates. This
fluorimeter uses cold light technology (CLF) with chemical and physical
modifications of the lighting system (Rat et al., 1995). CLF allows reading of
UV, visible and near infrared fluorescence by increasing light energy (from
1000 to 2300 lumens) and reducing the calorific part of light (IR > 900 nm,
Joule effect). It induces a decrease in background and a 500- to 1000-fold
improvement of detection limit of probes in comparison with classical
fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy
evaluation of cell injury induced by physical agents (UVA) or chemical toxins
(CCl4). Four biological endpoints for cytotoxicity evaluation have been tested
with several probes: proliferation (H33258); viability (fluorescent Neutral
Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects
(Rhodamine 123). Rh123 assay appeared more sensitive than fluorimetric or
photometric detection of Neutral Red assay. Cold light fluorimetry (CLF)
permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF
is shown to improve classical cytotoxicity assays and, owing to its
adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri
dishes), it is thus a promising alternative to flow cytometry for drug
cytotoxicity screening. 

81
Ruotsalainen M, Savolainen KM. EFFECTS OF A PROTEIN KINASE C INHIBITOR, Ro
31-7549, ON THE ACTIVATION OF HUMAN LEUKOCYTES BY PARTICULATE STIMULI.  Hum
Exp Toxicol 1995;14(3):266-72.

1. A new specific protein kinase C (PKC) inhibitor, Ro 31-7549, was used to
explore the mechanisms by which particulate stimuli, quartz and chrysotile,
stimulate human polymorphonuclear leukocytes (PMNL) to produce reactive oxygen
metabolites (ROM). Also soluble stimuli, formyl-Methionyl-Leucyl-Phenylalanine
(fMLP) and phorbol myristate acetate (PMA) were used. 2. Ro 31-7549 inhibited
chrysotile-induced free intracellular calcium ((Ca2+)i) elevations but did not
have an effect on quartz-induced elevations of (Ca2+)i. Both quartz and
chrysotile induced production of ROM were partially inhibited by Ro 31-7549.
fMLP-induced elevation of (Ca2+)i was inhibited by Ro 31-7549 whereas PMA did
not affect (Ca2+)i. Ro 31-7549 strongly inhibited  fMLP-induced ROM
production, and completely abolished that induced by PMA. 3. These results
suggest that PKC may have an important role in the activation of PMNL to
produce ROM by particulate and soluble stimuli. However, the inhibition of
chrysotile-, but not of quartz-induced (Ca2+)i elevations by Ro 31-7549
provides evidence that both PKC-dependent and -independent mechanisms may play
a role in the activation of human leukocytes to produce ROM.

82
Ogawa N, Hirose T, Fukushima K, Suwa T, Satoh T. METABOLISM OF A NITRATE
ESTER, DIHYDROPYRIDINE DERIVATIVE IN RABBIT HEPATIC MICROSOMES AND CYTOSOL.
Xenobiotica 1995;25(3):283-90.

1. The metabolism of a nitrate ester-substituted dihydropyridine derivative
(NND) in vitro was characterized with rabbit hepatic microsomes and cytosol.
2. Denitration activity was located in both the microsomal and cytosolic
fractions, whereas oxidation to the pyridine analogue was solely located in
the microsomal fraction. 3. Oxidation to the pyridine analogue required NADPH
and was inhibited by carbon monoxide, miconazole and SKF-525A, suggesting that
oxidation was catalysed by P450. 4.  Denitration activity in the microsomes
required either NADPH or GSH. Together with these results, responses to
various inhibitors indicate participation of both P450 and glutathione
S-transferase (GST). 5. Denitration activity in cytosol was activated by
glutathione (GSH), and by       dithiothreitol (DTT) to a greater extent.
GSH-dependent denitration was inhibited by S-hexyl GSH, an inhibitor of GST,
but DTT-dependent denitration was not. Moreover, the formation patterns of the
mono-denitrated  metabolites, M1 and M2, were shown to be different in each
incubation condition. 6. These results suggest that the denitration of NND in
cytosol could be catalysed by a GSH-independent enzyme as well as the
GSH-dependent enzyme, GST. 


83
Link CJ Jr, Evans MK, Cook JA, Muldoon R, Stevnsner T, Bohr VA. CAFFEINE
INHIBITS GENE-SPECIFIC REPAIR OF UV-INDUCED DNA DAMAGE IN HAMSTER CELLS AND IN
HUMAN XERODERMA PIGMENTOSUM GROUP C CELLS.  Carcinogenesis 1995;16(5):1149-55.

We have studied the effect of caffeine on gene- and strand-specific DNA repair
after exposure of Chinese hamster ovary cells and human xeroderma pigmentosum
complementation group C (XPC) cells to ultraviolet irradiation (UV). In
hamster cells, caffeine inhibited the repair of cyclobutane dimers (CPDs) in
the dihydrofolate reductase (DHFR) gene by up to 66% after 8 h of repair
incubation. This effect was dose-dependent, with more inhibition at 10 than at
1.5 mM caffeine. The inhibition was due  to decreased repair in the
transcribed strand of the hamster DHFR gene. This decrease in repair of CPDs
in the DHFR gene correlated with an enhancement of UV-induced cell killing by
caffeine. DNA repair was also measured in the overall genome by
repair-replication analysis. In hamster cells, caffeine caused a modest
enhancement of repair. Caffeine did not produce a significant effect on cell
cycle progression up to 8 h after UV irradiation, but it caused a distinct
block in early S phase during  the 24 h post-irradiation period. In XPC cells,
10 mM caffeine inhibited the removal of CPDs from the transcribed strand of
the DHFR gene by 92%. The removal of all photoproducts from the overall genome
was inhibited by 26% in these cells. Since the residual repair in XPC cells is
thought to occur in active genomic regions, we propose that caffeine
preferentially inhibits gene-specific repair.

84
Zastawny TH, Altman SA, Randers-Eichhorn L, Madurawe R, Lumpkin JA, 
Dizdaroglu M, Rao G. DNA BASE MODIFICATIONS AND MEMBRANE DAMAGE IN CULTURED
MAMMALIAN CELLS TREATED WITH IRON IONS. Free Radic Biol Med
1995;18(6):1013-22.

We investigated DNA base damage in mammalian cells exposed to exogenous iron
ions in culture. Murine hybridoma cells were treated with Fe(II) ions at
concentrations of 10 muM, 100 muM, and 1 mM. Chromatin was isolated from
treated and control cells and analyzed by gas chromatography/mass spectrometry
for DNA base damage. Ten modified DNA bases were identified in both
Fe(II)-treated and control cells. The quantification of modified bases was
achieved by isotope-dilution mass spectrometry. In  Fe(II)-treated cells, the
amounts of modified bases were increased significantly above the background
levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M
in the culture medium did not significantly inhibit the formation of modified
DNA bases. A mathematical simulation used to evaluate the plausibility of DNA
damage upon Fe(II) treatment predicted a dose-dependent response, which agreed
with the experimental results. In addition, Fe(II) treatment of cells
increased the  cell membrane permeability and caused production of lipid
peroxides. The nature of DNA base lesions suggests the involvement of the
hydroxyl radical in their formation. The failure of dimethyl sulfoxide to
inhibit their formation indicates a site-specific mechanism for DNA damage
with involvement of DNA-bound metal ions. Fe(II) treatment of cells may
increase the intracellular iron ion concentration and/or cause oxidative
stress releasing metal ions from their storage sites with subsequent  binding
to DNA. Identified DNA base lesions may be promutagenic and play a role in
pathologic processes associated with iron ions. 

85
Warren WG. ESTIMATION WITH VARYING DETECTION LIMITS. In: Markert B, editor.
Environmental Sampling for Trace Analysis. New York: VCH Publishers, Inc:
1994. p. 109-21.

86
Shumyantesva VV, Meshkov SV, Ivanov YU D, Alexandrova OV, Uvarov V YU, 
Archakov AI. INTERACTION OF ORGANOPHOSPHORUS ANALOGUES OF AMINO ACIDS WITH
P450. Xenobiotica 1995;25(3):219-27.

1. This study deals with the oxidation of organophosphorus amino acid
analogues by phenobarbital-induced rabbit liver microsomes. It has been shown
that 1-aminoalkylphosphonous and 1 -aminoalkylthiophosphonic acids are
converted by P450 to 1-aminoalkylphosphonic acids. 2. Phosphonous analogues of
amino acids cause type I spectral changes, and thiophosphonic analogues
produce reverse type I changes in difference spectra. 3. In the presence of
NADPH, the 1-aminoalkylphosphonous acids form the corresponding
1-aminoalkylphosphonic acids by the reaction P H - P OH, as monitored using 1H
nmr spectroscopy. 4. Aminoalkylthiophosphonic acids have also proven to be the
substrates for the NADPH-dependent monoxygenase system. During the course of
oxidative desulphuration 1-aminoalkylphosphonic acids were formed by the
reaction P S - P O, as monitored by 31P-nmr spectroscopy. 5. Using resonance
Raman (RR) spectroscopy, the interaction of 1-aminoisobutylphosphonous acid
with P450 was investigated, and characteristic changes in spectral frequencies
in the region between 1370 and 1700 cm-1 were demonstrated. These latter
changes indicate that substrate binding of organophosphorus compounds leads to
alterations in haem conformation and to redistribution of the electron
density.

87
Shackleton GL, Gibson GG, Sharma RK, Howes D, Orrenius S, Kass GE. DIVERSE
MECHANISMS OF CALCIUM MOBILIZATION BY PEROXISOME       PROLIFERATORS IN RAT
HEPATOCYTES. Toxicol Appl Pharmacol 1995; 130(2):294-303.

The ability of six peroxisome proliferators to modulate Ca2+ homeostasis was
studied in freshly isolated rat hepatocytes. Clofibrate and bifonazole (0.5
mM) caused a transient increase in cytosolic-free Ca2+ concentration ((Ca2+)i)
by releasing the intracellular inositol 1,4,5-trisphosphate-sensitive Ca2+
pool. However, the mobilization of this pool by clofibrate was only transient;
a subsequent exposure of the cells to the endoplasmic reticulum Ca2+-ATPase
inhibitor thapsigargin  resulted in a second release of the same Ca2+ store,
indicating that this pool could refill from the cytosol, independently of
extracellular Ca2+. By contrast, bifonazole-exposed hepatocytes no longer
responded to a stimulation by thapsigargin. Bifonazole also strongly inhibited
Ca2+ influx. Ciprofibrate and nafenopin (0.5 mM) produced increases in (Ca2+)i
that were sustained, even in the absence of extracellular Ca2+. The (Ca2+)i
response was not due to release of the inositol 1,4,5-trisphosphate-sensitive
Ca2+ pool and was not inhibited by prior treatment with the protonophore
carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, but was slightly
antagonized by prior exposure to the Ca2+ ionophore ionomycin. Pretreating the
cells with nafenopin completely abolished the response elicited by
ciprofibrate, and vice versa. By contrast to the other peroxisome
proliferators, WY-14,643 and bezafibrate (1 mM) increased cytosolic free Ca2+
only by approximately 30 nM. In conclusion, the structurally diverse
peroxisome proliferators tested in this study all produced changes in (Ca2+)i
in hepatocytes but through the redistribution of different internal Ca2+
pools. Further studies are needed to determine whether any of the observed
Ca2+ changes have a role in the pleiotropic effects elicited by peroxisome
proliferators. 

88
Tanaka T, Tanigawa T, Nose T, Imai S, Hayashi Y. IN VITRO CYTOTOXICITY OF
SILICIC ACID IN COMPARISON WITH THAT OF SELENIOUS ACID. J Trace Elem Exp Med
1994-1995;7(3):101-11.

Cytotoxic effects of silicic acid were investigated in two mouse cell line
systems of a macrophage cell line (A640-BB-2 cells) and a fibroblast cell line
(3T6 cells), in comparison with those of selenious acid. Phagocytic activity
of the A640-BB-2 cells incubated for 48 h was significantly decreased at 2.0
mmol/l of silicic acid or 0.01       mmol/l of selenious acid in the culture
medium, as compared to control culture with medium alone. During 48 h
incubation, proliferation of the 3T6 cells was  depressed at 2.0 mmol/l of
silicic acid or 0.04 mmol/l of selenious acid in the culture medium.
Comparison between silicon and selenium contents of cells incubated at the
minimum toxic level of medium Si(OH)4 or H2SeO3 indicated the silicon content
to be 500 times higher in A640-BB-2 cells and 300 times higher in 3T6 cells
than the selenium content. These results indicate that the cytotoxicity of
silicic acid on macrophages and fibroblasts is far weaker than that of
selenious acid.

89
Petite H, Duval J, Frei V, Abdul-Malak N, Sigot-Luizard M, Herbage D. 
CYTOCOMPATIBILITY OF CALF PERICARDIUM TREATED BY GLUTARALDEHYDE AND BY THE
ACYL AZIDE METHODS IN AN ORGANOTYPIC CULTURE MODEL. Biomaterials
1995;16(13):1003-8.

Glutaraldehyde (GTA) is used to crosslink collagen-based biomaterials, but
these materials are often cytotoxic. In order to overcome this problem, the
use of the acyl azide methods with either hydrazine or diphenylphosphoryl
azide (DPPA) as reagents was proposed. The cytocompatibility of acyl azide-
and GTA-treated pericardium was evaluated in vitro by an organotypic chick
aorta culture technique developed for the evaluation of the propensity of
vascular cells (both endothelial and smooth muscle cells) to migrate and
growth on the surface of biomaterials. Pericardium stabilization was examd. as
a function of GTA concn. and time, so that the residual GTA mols. in the
material were minimized. Treatment for 72 h with 0.95% GTA was optimal for
thermal stabilization of the pericardium with a denaturation temp. (Td) of
86.8.degree., providing similar results to treatment wit 0.6%  GTA for 4 h (Td
-85.1.degree.). Pericardium treated in this way was, however, poorly
cytocompatible with little vascular cell migrating and growth when compared
with tissues treated by the acyl azide methods. The best results were obtained
with 0.5% DPPA; treated tissues showed a high level of crosslinking (Td =
82.4.degree.) and three-fold increases in cell growth and migration over those
in a non-toxic control. 

90
Sakai A, Yamakoshi YN, Miyata N. THE EFFECTS OF FULLERENES ON THE INITIATION
AND PROMOTION STAGES OF BALB/3T3 CELL TRANSFORMATION. Fullerene Sci Technol
1995;3(4):377-88.

Fullerene C60 and a mixt. of fullerenes C60 and C70 were solubilized in a
culture medium with poly(vinylpyrrolidone) and examd. for the effects on
two-stage cell transformation in vitro, a model of carcinogenesis. They showed
neither initiating nor promoting activity in the assays. 

91
Attawia MA, Devin JE, Laurencin CT. IMMUNOFLUORESCENCE AND CONFOCAL LASER
SCANNING MICROSCOPY STUDIES OF OSTEOBLAST GROWTH AND PHENOTYPIC EXPRESSION IN
THREE-DIMENSIONAL DEGRADABLE SYNTHETIC MATRIXES. J Biomed Mater Res
1995;29(7):843-8.

In the development of three-dimensional cell-polymer synthetic matrixes for
tissue       regeneration, visualization of cells growing in these porous
structures can be difficult. The focus of this study was the development and
use of a novel method that would allow for visualization of osteoblasts inside
opaque matrixes. The morphol. responses and phenotypic characterization of
osteoblasts as they attach, spread, and migrate through a porous
three-dimensional biodegradable polymer-ceramic matrix in vitro were studied
using immunofluorescence and confocal laser scanning microscopy (CLSM). CLSM
offers several advantages over the most commonly used imaging methods
(traditional light microscopy and SEM). CLSM filters out-of-focus background
and provides more structural details of cells. In addn., CLSM does not require
extensive sample prepn. as does SEM. When used in conjunction with
fluorescence-labeled antibodies to identify cells and their products, it can
characterize morphol. of growing cells and successfully det. phenotypic
function. Using monoclonal antibody to osteocalcin, a bone cell-specific
protein, cells throughout the matrix were found to have preserved
osteoblast-like phenotype with growth. The morphol. of cells throughout the
matrix was found to be similar to osteoblast cells grown on tissue culture
polystyrene and consisted of spread polygonal forms. Using the technique of
CLSM with immunofluorescent antibodies, it was demonstrated for the first time
that these three-dimensional degradable polymer matrixes can support
osteoblast growth and phenotypic expression throughout its structure. 

92
Smith CG, Lee SJ, Marquardt DL. EFFECTS OF TUBERCIDIN AND ITS 5'-O-METHYL
ETHER ON ADENOSINE RECEPTORS AND MEDIATOR RELEASE FUNCTIONS IN MAST CELLS. J
Med Chem 1995;38(12):2259-62.

Tubercidin (7-deazaadenosine, Tu) is a highly cytotoxic nucleoside xenobiotic
that, as the nucleoside or nucleotide derivs., closely mimics the actions of
adenosine (or its corresponding nucleotides) in a wide variety of
biochem./biol. systems. In light of its acceptance in these test systems as an
adenosine (Ado) surrogate, it was postulated that the compd. might interact
with adenosine receptors. To test this  hypothesis, a nonphosphorylatable
deriv. (5'-O-Me tubercidin, MeTu) was prepd. and evaluated in comparison with
tubercidin and Ado in a variety of biol. systems. In a cell culture assay
using Chinese hamster ovary cells, MeTu is approx. one-third as cytotoxic as
is Ado and 105-fold less cytotoxic than Tu. Both Tu and MeTu inhibited the
antigen-stimulated release of beta-hexosaminidase from mouse bone marrow
derived mast cells in vitro, but only Tu was active in the in vivo PCA test.
The inhibitory effect of MeTu on mast cell mediator release does not appear to
involve interaction with adenosine receptors or to be the result of conversion
to Tu per se.

93
Zhang BT. [EFFECTS OF MITOGENS ON ADULT SCHWANN CELLS ISOLATED FROM MOUSE
SCIATIC NERVE.] Yokohama Igaku 1995;46(2):111-7. (Jpn)

We developed a new method of extg. adult Schawann cell (SCs) from the mouse
sciatic nerve and investigated the effects of mitogens on adult SCs in vitro.
This culture system provided 99.5% pure SCs populations at cell yields of
greater than 3 .times. 103 cell/mg of starting nerve wet wt. at 5 culture days
by the following method (1) culture the dissocd. adult SCs for 24 h in 10%
FCS-F12 medium, (2) culture these cells in serum-free medium for 48 h, (3)
differential adhesion step. We examd. proliferating response of adult SCs to 3
identified neonatal SCs mitogens: glial growth factor (GGF), platelet-derived
growth factor BB (PDGF-BB), and basic fibroblastic growth factor (bFGF) in
serum-free medium. GGF alone had mitogenicity for adult SCs in a
dose-dependent manner, and synergistic activation coupling with forskolin was
not obsd. Neither PDGF-BB nor bFGF was mitogenic for adult SCS when used alone
or with forskolin. GGF and bFGF did not change the cAMP levels in SCs.
However, PDGF-BB induced one half lower level than basal cAMP level. These
results indicate that cAMP was not related with DNA synthesis of SCs in GGF
response, and GGF activate adult SCs through a signal transduction pathway
sep. from the cAMP-dependent system.

94
Rigano L, Cavalletti T, Benetti S, Traniello S. EVALUATION OF PREDICTABLE
IRRITATIVE POWER OF SURFACTANT MIXTURES BY HUMAN FIBROBLAST CULTURE AND ITS
CORRELATION TO PHYSICO-CHEMICAL PARAMETERS. Int J Cosmet Sci 1995;17(1):27-43.

The predictable toxic effects of some surfactants, their blends and some
preserving agents on human fibroblast cultures were investigated with in vitro
tests, with the aim of finding a possible correlation between the biol.
evaluations and some phys. characteristics of detergent solns. Lactate
dehydrogenase release into the medium was used as a marker of the plasma
membrane integrity, while the amt. of 3H-radiolabeled proteins in the
fibroblasts was measured in order to assess the cell biosynthetic machinery
function.  Disodium alkyl semisulfosuccinate induced membrane damage in the
lactate dehydrogenase test and decreased the protein synthesis, with an EC 50
around 1 mM, while sodium lauryl ether sulfate had an EC 50 at about 100 muM,
indicating that this compd. is ten-fold more toxic, when measured by this
method. An ethoxylated glyceride, on the contrary, was completely harmless on
the plasma membrane and, surprisingly, activated fibroblast protein synthesis
in a dose-response way up to two-fold.  Mixts. of the three surfactants
evidenced the protective effect of the non-ionic against the cellular
functionality damage. Parabens do not influence this type of evaluation, while
some influence was shown by the formaldehyde releaser
2-bromo-2-nitropropanediol at the highest concn.  The comparison between crit.
micellar concn. measures of the different surfactants and their in vitro
detected irritative power shows, for the two anionics, that in vitro toxicity
is proportionally bound to the amt. of micelles even if the structural
differences between the two types of mols. are reflected into different damage
values, while the non-ionic compd. shows a not defined CMC and a very low
toxicity profile. Blends of anionics with the non-ionic show an increased CMC
and a reduced toxicity profile. Toxicity evaluations of complex finished
foaming formulations, carried out with human fibroblast cultures evaluation
show that a relationship between micelles amt. and cell toxicity seems to
exist, mainly when multiple surfactants blends are tested. 

95
Carlson SL, McGillis JP. MODULATION OF LEUKOCYTE ADHESION, MIGRATION, AND
HOMING BY NEOROTRANSMITTERS AND NEUROPEPTIDES. Methods Neurosci
1995;24:335-54. 

 A review describing cell culture methods for endothelial cells (from human
umbilical vein, mouse lymphoid microvessels, and rat heart microvessels) and
in vitro adhesion assays using these endothelial cells, in vivo assays of
leukocyte homing to lymphoid tissues, and tissue-specific lymphocyte migration
assays. These methods are used to det. the effects of catecholamines,
neuropeptide Y, substance P, calcitonin gene-related peptide, and other
neurotransmitters found in nerve terminals in lymphoid tissue and surrounding
blood vessels.

96
Lindl T. CELL SYSTEMS ON THE ADVANCE. PART 1. CLB. Chem Labor Biotech
1994;45(5):258-61.


CYTOTOXICITY
97
Liebsch HM, Spielmann H.  BALB-C 3T3 CYTOTOXICITY TEST. Methods Mol Biol
1995;43:177-87.

98
Guigand M, Pellen P,  Mouton C, Bonnaure-Mallet M.  CYTOTOXIC EFFECT OF
VESICLES PRODUCED BY PORPHYROMONAS GINGIVALIS ON FIBROBLASTS IN CULTURE.  J
Periodont Res 1995;30(2):141-3.

It has been shown that the vesicles produced by Porphyromonas gingivalis under
certain growth conditions contribute to its pathogenicity. In this study, we
demonstrate the cytotoxic effect of the vesicles using two methods: one
quantitative (the colorimetric cytotoxicity test using sulforhodamine B) and
the other qualitative (flow cytometry).

99
Brambilla G, Marteili A. CYTOTOXICITY DNA FRAGMENTATION AND DNA REPAIR
SYNTHESIS IN PRIMARY HUMAN HEPATOCYTES. Methods Mol Biol 1995;43:59-66.

100
Zeromski J, Jezewska E.  FUNCTIONAL ALTERATIONS OF HUMAN BLOOD MONOCYTES AFTER
EXPOSURE TO VARIOUS NICKEL COMPOUNDS IN VITRO: AN EFFECT ON THE PRODUCTION OF
HYDROGEN PEROXIDE. Immunol Lett 1995;45(1-2):117-21.

It is generally known that nickel, a metal with distinct carcinogenic
properties, can significantly alter the functioning of host defense mechanisms
and impair various components of the immune system. In the present study the
influence of 3 nickel salts on the production of hydrogen peroxide (H2O2) by
human monocytes was examined in in vitro culture. Highly purified, resting and
PMA-stimulated normal human monocytes were cultured with subtoxic
concentrations of nickel subsulfide, nickel  sulfate, nickel acetate and
manganese chloride. A portion of the cells was cultured with nickel-manganese
salt mixture. Following culture cells were tested in an in vitro functional
assay for H2O2 production. It has been shown that all nickel salts, used in
micromole concentrations, suppressed H2O2 formation both in resting and
PMA-stimulated monocytes, while it was not the case when manganese chloride
was used for cell cultures. The strongest suppressive effect was manifested by
nickel  sulfate. The cells subjected to nickel-manganese mixture displayed
H2O2 production similar to that of control ones. These results show that
nickel salts in micromole concentrations exert a suppressive effect on
oxygen-dependent antimicrobial system of human monocytes and manganese
prevents this effect. 

101
Nichols WK, Terry CM, Cutler NS, Appleton ML, Jesthi PK, Yost GS.  OXIDATION
AT C-1 CONTROL THE CYTOTOXICITY OF 1,1-DICHLORO-2,2-BIS(P-CHLOROPHYNYL)ETHANE
BY RABBIT AND HUMAN LUNG CELLS.  Drug Metab Dispos 1995;23(5):595-9.

Isolated rabbit Clara cells and a transformed human bronchial epithelial cell
line, BEAS-2B, were used to investigate the mechanism of cytotoxicity of
1,1-dichloro-2,2-bis(p-chlorophenyl)ethane DDD), a persistent insecticide and
stable metabolite of 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane. Both
BEAS-2B cells and rabbit Clara cells were highly susceptible to DDD toxicity
and were partially protected by 1-aminobenzotriazole, a suicide substrate
inhibitor of cytochrome P450 enzymes. DDD  (0.05 mM) killed 47 : 1.8% of
rabbit Clara cells and 42 : 7.9% of  BEAS-2B cells after 3 hr and 84 : 3.0% of
rabbit Clara cells and 80 : 14% of BEAS-2B cells after 6 hr. Consequently, DDD
is the most potent Clara cell toxicant recognized to date. The cytotoxicity of
DDD to these cells was decreased by deuterium substitution at the C-1
position. Rabbit Clara cells and pulmonary microsomes incubated with 14C-DDD
produced the fully oxidized acetic acid metabolite 
2,2'-bis(p-chlorophenyl)acetic acid (DDA), but DDA was not formed by Clara
cells when DDD was coincubated with 1-aminobenzotriazole. These results
support the       hypothesis that the cytotoxicity of DDD to susceptible
subpopulations of rabbit and human lung cells is, at least in part, caused by
cytochrome P450-mediated oxidation of DDD at C-1. A required step for the
production of the cytotoxic intermediate is proposed to be the formation of a
highly reactive acyl halide intermediate that is readily hydrolyzed to a
stable, nontoxic metabolite, DDA.

102
Tu B, Wallin A,  Moldeus P, Cotgreaveia.  CYTOTOXICITY  OF NO2 GAS TO CULTURED
HUMAN AND MURINE CELLS IN AN INVERTED MONOLAYER EXPOSURE SYSTEM. Toxicology
1995;96(1):7-18.

We report the development of an optimised exposure system for the exposure of
inverted cell cultures to NO2, which presents several advantages over
conventional, right-side-up exposure systems. Firstly, the cells may be
directly exposed to NO2 in the gas phase for up to 1 h, without the
interposition of an aqueous layer. Secondly, the chamber system allows simple
and precise control of the gas concentration during the exposure. Finally, the
system allows the simultaneous exposure of large  numbers of cells under
sterile conditions, facilitating further culture of the cells after the
exposure period. We report the application of this system to a comparative
study of the toxicity of NO2 in three different cell types involved in the
circuit of the inflammatory response, the IC-21 murine macrophage line, the
A-549 human pulmonary type II-like epithelial cell line and human umbilical
vein endothelial cells. As little as 2 ppm NO2 for 20 min reduced
colony-forming efficiency of HUVE  cells and A-549 cells to 35% and 78% of
their air controls, respectively. Exposure to 5 ppm NO2 for 1 h increased
lactate dehydrogenase release of HUVE cells, IC-21 macrophages and A-549 cells
from 7.9% to 21.6%, 5.7% to 10.9% and 2.0% to 3.4%, respectively, whilst 10
ppm NO2 for 1 h lowered cellular glutathione in HUVE cells, IC-21 cells and
A-549 cells from 35.2 nmol/mg to 23.3 nmol/mg, from 45.0 nmol/mg to 31.0
nmol/mg and from 86.4 nmol/mg to 69.2 nmol/mg, respectively. Of the cell types

tested it was shown that HUVE cells and IC-21 cells were equally sensitive to
the toxicity of NO2, whilst A-549 cells displayed considerable resistance,
perhaps due to the considerably higher levels of glutathione in this cell
line. Further, a comparison of the sensitivity of HUVE cells to NO2, using
several modes of exposure (inverted and right-side-up (either rocked or
static)) and the assay of lactate dehydrogenase and (3H)deoxyglucose release,
revealed that the present inverted exposure technique potentiated the acute
cytotoxicity of the gas.

103
Rath NC, Huff WE, Bayyari GR, Balog JM. EFFECT OF THIRAM ON CHICK CHONDROCYTES
IN CULTURE.  J Toxicol Environ Health 1995;44(3):369-76.

The effect of thiram, a fungicide that increases the incidence of tibial
dyschondroplasia (TD) in poultry, was studied in vitro using growth plate
chondrocyte culture. Thiram caused a significant reduction in alkaline
phosphatase, acid phosphatase, and lactate dehydrogenase (LDH) activities at
concentrations of 5 muM and above. It was highly cytotoxic to chondrocytes at
and above this concentration as determined by their ability to reduce
3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium  bromide (triazolyl blue,
MTT), a marker of cellular viability. An increase in the leakage of LDH into
culture media was evident at concentration as low as 1 muM. Very few
differences were noticed in the electrophoretic migration profiles of
cell-extract proteins at any treatment level relative to control. The
cytotoxic effect of thiram is possibly due to its damaging effect on the cell
membrane, which may be responsible for chondrocyte death.

104
Dunn K LR, Virji M, Moxon ER.  INVESTIGATIONS INTO THE MOLECULAR BASIS OF
MENINGOCOCCAL TOXICITY FOR HUMAN ENTOTHELIAL AND EPITHELIAL CELLS: THE
SYNERGISTIC EFFECT OF LPS AND PILI. Microb Pathog 1995;18(2):81-96.

Using human umbilical vein endothelial cells as an in vitro model of toxicity,
it was found that Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria
lactamica and Neisseria sicca caused damage to these cells, in contrast to the
lack of cytotoxicity exhibited by Haemophilus influenzae type b. N.
meningitidis was also found to be toxic for human epithelial cells. The major
toxic factor of N. meningitidis was found to be a heat-stable component of
outer membrane vesicles, and  could be inhibited by polymyxin B, suggesting
that lipopolysaccharide plays a major role in toxicity. However, the toxicity
mediated by lipopolysaccharide was modulated significantly by pilus-dependent
adherence. Intra-strain variants expressing altered pilins which exhibited
different levels of       adherence to epithelial and endothelial cells were
used to study the role of pilus. The degree of toxicity observed correlated
with their relative level of adherence to cultured cells. In contrast,
Opc-dependent increased adherence did not result in  increased toxicity for
endothelial cells, suggesting that pili have a synergistic effect,
contributing to the overall damage. 

105
de Arruda M, Cocchiaro CA, Nelson CM, Grinnell CM,  Janssen B, Haupt A, 
Barlozzari T.  LU103793 (NSC D-669356): A SYNTHETIC PEPTIDE THAT INTERACTS
WITH MICROTUBULES AND INHIBITS MITOSIS. Cancer Res 1995;55(14):3085-92.

LU103793 (NSC D-669356) is a new synthetic derivative of Dolastatin 15, an
antiproliferative compound which was isolated from the mollusk Dolabella
auricularia. Like Dolastatin 15, LU103793 is highly cytotoxic in vitro (IC50 =
0.1 nM). To investigate the mechanism of action of LU103793, we used a
combination of biochemical and cellular methods. Turbidity assays with bovine
brain microtubules demonstrated that LU103793 inhibits microtubule
polymerization in a concentration-dependent manner (IC50 = 7 microM).
Treatment with this compound also induced depolymerization of preassembled
microtubules. Cell cycle analysis of tumor cell lines treated with LU103793
indicated a block in the G2-M phase. At the cellular level, it induced
depolymerization of microtubules in interphase cells and development of
abnormal spindles and chromosome distribution in mitotic cells. Although these
effects are very similar to the cellular alterations caused by vinblastine,
LU103793 does not inhibit vinblastine binding to unpolymerized tubulin in
vitro. Our results suggest that LU103793 exerts its cytotoxic activity
primarily through disruption of microtubule organization.

106
Fridborg H, Nygren P, Larsson R. RELATIONSHIP BETWEEN PHARMACOKINETIC
PARAMETERS IN PATIENTS AND CYTOTOXICITY IN VITRO OF STANDARD AND
INVESTIGATIONAL ANITCANCER DRUGS. Anticancer Drugs 1995;6(1):64-9.

The selection of the starting dose for initial clinical trials of anticancer
agents is mostly determined by toxicological endpoints in mice (LD10). So far,
very few attempts have been made to evaluate the potential value of
cytotoxicity assays for this purpose. The present study was undertaken as a
first attempt to investigate the relationship between cytotoxicity of
anticancer drugs in vitro and pharmacokinetic parameters in vivo in patients,
at suggested maximum tolerated doses. Using the fluorometric microculture
cytotoxicity assay (FMCA), we determined the concentration giving 50% cell
survival (IC50) in vitro, for 25 cytotoxic drugs in fresh preparations of
normal peripheral blood mononuclear cells (PBMC) and of tumor cells from
patients with acute or chronic lymphocytic leukemia (ALL or CLL). Using linear
regression, we investigated the relationship between the IC50s and clinically
achievable peak plasma concentrations (Cmax) or concentration-time products (C
x T) in humans. The clinical data was obtained from the literature. Based on
all drugs tested, good correlations were obtained between IC50s for CLL cells,
and both Cmax and C x T (R approximately 0.7, p < 0.0002), and for ALL cells
and normal PBMC between IC50 and Cmax, while the two latter cell types showed
somewhat weaker relationships to C x T. Using the IC50 data of CLL cells,
predictions of Cmax and C x T exceeded 1 log for only four drugs. No
tendencies to under- or overprediction within different classes of drugs were
noted. The results demonstrate a significant relationship between toxicity in
vitro and achievable systemic exposure of anticancer drugs in humans, which
suggests that non-clonogenic in vitro assays for drug sensitivity testing may
provide pharmacokinetic information useful in the development of
investigational cytotoxic drugs.

107
Fischel JL, Barbe V, Berlion M, Formento P, Berrile J, Bizzari JP, Milano G. 
[TAMOXIFEN INCREASES CYTOTOXIC EFFECTS OF FOTEMUSTINE. EXPERIMENTAL RESULTS ON
CELL LINES OF HUMAN MELANOMA.] Bull Cancer 1994; 81(7):599-604. (Fre)

Fotemustine (Fote) is a new aminoacid linked chloroethyl nitrosourea which has
been shown to be useful in disseminated malignant melanoma. The aim of the
present study was to analyze the cytotoxic effects resulting from the
combination of antiestrogens and Fote on human melanoma cell lines. The
antiestrogens tested were tamoxifen (TMX 5.10(-7) M and 5.10(-6) M) and 40H
TMX (5.10(-8) M and 5.10(-7) M). As a preliminary step, a series of nine human
melanoma cell lines was screened in order to identify and quantify the
presence of estradiol receptors (ER) in these cell lines. This led to
selecting an ER positive (+) cell line. The drugs alone or in combination were
then tested against CAL 1 ER (+) and CAL 7 ER (-) melanoma cell lines.
Different sequences of drug combinations were tested using clinically
compatible drug concentrations. For CAL 1 cells there was a growth inhibitory
effect induced by the antiestrogens given alone. Overall, the presence of the
antiestrogens resulted in higher cytotoxic effects than when cells were
exposed to Fote alone. The lowest IC50 Fote values as compared to Fote alone
were generated by the sequences with the antiestrogens administered before
Fote. Significantly, these associations with antiestrogens enabled the IC50
values of Fote to be reduced up to 80%. Globally TMX and 40H TMX had similar
synergistic effects. TMX and 40H TMX had a modest influence on Fote cytotoxic
effects against CAL 7 ER (-) cells. These data may be useful for optimal
planning of future clinical trials for malignant melanoma using antiestrogens
and nitrosoureas. 

108
Copaceanu ML, Coucke PA, Cottin E, Paschoud N, Mirimanoff RO.  AZIDOTHYMIDINE
(AZT) AS A POTENTIAL MODIFIER OF RADIATION RESPONSE IN VITRO. Acta Oncol
1995;34(2):213-8. 

The potential effect of AZT as a thymidine analogue on radiation response in
vitro was investigated. Two human cell lines (WiDr and HeLa) were used. The
effect of 10 microM AZT on exponentially growing cells was studied after
different exposure times (24, 48 and 72 h). The surviving fraction (clonogenic
assay) or metabolic activity (MTT assay) after irradiation of AZT-exposed
cells, was compared to unexposed irradiated controls. Flow cytometry was used
to assess the cell-cycle effect of pre-exposure of exponentially growing cells
to AZT. AZT had a radioprotective effect for all experimental time points as
far as WiDr was concerned. For HeLa the effect was significant at 24 h.
Cell-cycle analysis showed a significant accumulation in S-phase at 72 h for
WiDr. For HeLa there was a significant accumulation in S-phase at 48 h. We
conclude that under the reported       experimental conditions, AZT as a
thymidine analogue seems to reduce the cytotoxic effect of irradiation. 

109
Yourtee DM, Tong PY, Rose LA, Eick JD, Chappelow CC, Bean TA. THE EFFECT OF
SPIROORTHOCARBONATE VOLUME MODIFIER CO-MONOMERS ON THE IN VITRO TOXICOLOGY OF
TRIAL NON-SHRINKING DENTAL EPOXY CO-POLYMERS. Res Commun Mol Pathol Pharmacol
1994;86(3):347-60. 

A major improvement in dental restoratives is possible through the development
of biomaterials that do not shrink upon polymerization, hence, avoid leakage
and subsequent breakdown. Polymers containing spiroorthocarbonates (SOCs) show
promise in this respect, but their toxicology in copolymerized materials has
not been explored. In this study, the in vitro toxicology of these materials
in homopolymer form and in two trial non-shrinking epoxy co-polymers was
evaluated for cytotoxicity and mutagenicity. Cytotoxicity was determined by
the MTT test to measure the lethality effect on mouse L929 cells. Mutagenicity
was evaluated using the Ames-Salmonella Test. For comparison, commercial
composite and adhesive materials as well as several other materials of current
interest in dentistry were also evaluated. Epoxy resin samples containing 5%
of either T/T SOC or Dp SOC reduced the cytotoxicity (TC50) from approximately
400 to 800 micrograms/200 microliters. The epoxy-spiro copolymers had more
favorable TC50 values than the commercial product Super-Bond. They showed TC50
values on the order of 35% greater than Super-Bond and 45% less than
Scotchbond 2, the latter two being materials currently used in the clinic.
These two comparatives demonstrated dose response curves with lower doses at
maximum cell kill values than the spiro materials. The epoxy formulations all
showed weak mutagenesis, but this is attributed to the epoxy formulation and
not the SOCs. Although considerable toxicology is yet be conducted, these in
vitro results suggest that biocompatible copolymer formulations for
spiroorthocarbonates are a developmental reality.

110
Fos E, Suesa N, Borras L, Lobato C, Banfi P, Gambetta RA, Zunino F, Mauleon D,
Carganico G.  SNYTHESIS OF ALKYL CHAIN-MODIFIED ETHER LIPIDS AND EVALUATION OF
THEIR IN VITRO CYTOTOXICITY.  J Med Chem 1995;38(7): 1216-28.

A series of alkyl lysophospholipid (ALP) analogs of ET-18-OCH3
(1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) containing
modifications in the long C-1 chain has been synthesized and evaluated in
human tumor cell line cytotoxicity assays. The compounds have also been
evaluated in platelet activating factor (PAF) receptor agonism and hemolysis
tests. Two modifications have been studied, introduction of a carbonyl group
at different positions of the C-1 chain and branching of this chain, in some
compounds with incorporation of a phenyl group. Several compounds showed a
cytotoxic potency comparable to that of the reference compound ET-18-OCH3,
associated with reduced proaggregating and hemolytic effects. The two
enantiomers of 1-O-(7-oxooctadecyl)-2-O-methyl-rac-glycero-3-phosphocholine
(2) showed the same level of cytotoxicity or antiproliferative activity, with
the PAF-agonistic effect confined to R-2. The very low stereoselectivity found
in the in vitro cytotoxicity confirms earlier results and indicates a lack of
stereospecific interactions with a macromolecular target.

111
DeCesare SL, Michelini-Norris B, Blanchard DK, Barton DP, Cavanagh D, Roberts
WS, Fiorica JV, Hoffman MS, Djeu JY. INTERLEUKIN-12-MEDIATED TUMORICIDAL
ACTIVITY OF PATIENT LYMPHOCYTES IN AN AUTOLOGOUS IN VITRO OVARIAN CANCER ASSAY
SYSTEM.  Gynecol Oncol 1995;57(1):86-95.

This study was designed to examine if interleukin-12 (IL-12) can induce
cytolytic function of lymphocytes from ovarian cancer patients against either
an ovarian cancer cell line or their own autologous tumor cells. Lymphocytes
were obtained from the peripheral blood or ascites of ovarian cancer patients
and activated with IL-12 alone or concomitantly with interleukin 2 (IL-2) for
2 to 3 days. Activation of lymphocytes and assessment of tumoricidal function
by a chromium release assay were performed directly in a standard control
medium (RPMI 1640 containing 2 mM glutamine, 100 micrograms/ml streptomycin,
100 units penicillin, 5% heat-inactivated human AB serum, and 5 mM
4-(2-hydroxyethyl)-1-piperazinesulfonic acid) and in 50% ascitic fluid (50% by
volume filter-sterilized ascites with 50% of the above-mentioned control
medium). Target cells were added directly into the medium in which the
lymphocytes were activated in order to more closely mimic in vivo conditions.
Lymphocytes, activated by IL-12 in 50% ascitic fluid, were able to lyse
autologous tumor cells in 3 of 6 assays and were able to lyse SKOV3 cells (an
ovarian cancer cell line) in 5 of 7 assays. The results were not significantly
different in the control medium. When both IL-2 and IL-12 were used to
activate lymphocytes in 50% ascitic fluid, significant cytotoxicity was
generated in 6 of 6 autologous assays and in all 7 patient assays using SKOV3
as a target (P <  0.05). Synergy between the two cytokines was seen in all 13
patient assays in ascitic medium compared to only 5 of 13 assays in control
medium. Additionally, when lymphocytes were stimulated with both IL-2 and
IL-12, significantly greater cytotoxicity was seen in the ascitic fluid medium
compared to the control medium in 13 of 14 assays (P < 0.05). No significant
tumoricidal activity was seen by lymphocytes maintained in either medium
without the addition of IL-2 or IL-12. Ascitic fluid consistently potentiates
the synergy between IL-2 and IL-12 in   generating cytotoxicity against
ovarian cancer cells but does not increase       cytotoxicity induced by IL-12
alone. IL-12 by itself activates tumoricidal activity of lymphocytes in
ascitic fluid; however, the addition of IL-2 increases the degree and
consistency of this effect. These data support the possibility that IL-12 may
warrant       further investigation as a potential therapeutic agent in the
treatment of advanced ovarian cancer. 

112
Kaspers GJ, Veerman AJ, Pieters R, Van Zantwijk I, Hahlen K, Van Wering ER. 
DRUG COMBINATION TESTING IN ACUTE LYMPHOBLASTIC LEUKEMIA USING      THE MTT
ASSAY. Leuk Res 1995;19(3):175-81.

Drug resistance assays may be useful to identify drug interactions. For this
purpose, we studied three drug combinations, each at 8-12 concentrations, with
the MTT assay in acute lymphoblastic leukemia (ALL) samples from 34 children 
btained at initial diagnosis. This resulted in a total of 518 comparisons
between expected and observed leukemic cell survivals. The combinations
prednisolone (PRD) with vincristine (VCR), PRD with mafosfamide (MAF), and PRD
with daunorubicin (DNR) were tested without technical difficulties, and
without an increased assay variation as compared to single drugs. We observed
a marked heterogeneity in drug interactions between patients, between
combinations, and between different concentrations within one specific
combination. Between PRD+VCR, synergism was found in 46%, antagonism in 18%,
and additivity in       36% of the 228 observations. Between PRD+MAF,
synergism was found in 51%, antagonism in 20%, and additivity in 29% of the
140 observations. Between PRD+DNR, synergism was found in 35%, antagonism in
31%, and additivity in 34% of the 150 observations. PRD+VCR and PRD+MAF showed
more often synergism than PRD+DNR, while antagonism was observed more
frequently between PRD+DNR (p < 0.05). However, the magnitude of antagonism
was not much different between the three drug combinations, nor was there a
significant antagonistic interaction in any of the drug combinations tested,
if all samples were considered together. We conclude that the MTT assay can be
used to study drug interactions in vitro in ALL samples. The type of
interaction was different between patients, and depends on the drug
combination and concentrations. The combinations PRD+VCR and PRD+MAF generally
showed additive and even synergistic interactions. The cytotoxicity of PRD+DNR
was generally not markedly higher than that of the most active single drug. 

113
Timbrell JA, Seabra V, Waterfield CJ.  THE IN VIVO AND IN VITRO PROTECTIVE
PROPERTIES OF TAURINE. Gen Pharmacol 1995;26(3):453-62.

1. Taurine is a ubiquitous, free amino acid found in mammalian systems. 2. The
biological functions of taurine are unclear. 3. Various in vivo data suggest
that taurine has a variety of protective functions and deficiency leads to
pathological changes. 4. Depletion in rats of taurine increases susceptibility
to liver damage from carbon tetrachloride. 5. Susceptibility to a variety of
hepatotoxicants correlates with the estimated hepatic taurine level. 6. In
vitro data suggest that taurine can  protect cells against toxic damage. 7.
Taurine protects isolated hepatocytes against carbon tetrachloride, hydrazine
and 1,4-naphtho- quinone but not against allyl alcohol,
alpha-naphthylisothiocyanate (ANIT) or diaminodiphenyl methane (DAPM)
cytotoxicity. 8. The mechanisms of  protection are unclear but may include
modulation of calcium levels, osmoregulation and membrane stabilization. 

114
Sinensky MC, Leiser AL, Babich H. OXIDATIVE STRESS ASPECTS OF THE CYTOTOXICITY
OF CARBAMIDE PEROXIDE: IN VITRO STUDIES. Toxicol Lett  1995;75(1-3):101-9.

Carbamide peroxide is the active ingredient in many at-home patient-applied
tooth whiteners. The cytotoxicity of carbamide peroxide, as related to
oxidative stress, was evaluated in vitro with several human cell lines,
including Smulow-Glickman (S-G) gingival epithelial cells. The potency of
carbamide peroxide was related to its hydrogen peroxide component rather than
to carbamide, was eliminated in the presence of exogenous catalase, and was
enhanced in the presence of aminotriazole, an  inhibitor of cellular catalase.
The intracellular level of glutathione, a scavenger of toxic oxygen
metabolites, was decreased in cells exposed to carbamide peroxide; at higher
concentrations of carbamide peroxide, leakage of lactic acid dehydrogenase was
also evident. Cells pretreated with the glutathione-depleting agents,
buthionine sulfoximine, chlorodinitrobenzene, and bis(chloroethyl)
nitrosourea, were hypersensitive to subsequent challenge with carbamide
peroxide. Conversely, pretreatment  with the iron chelator, deferoxamine,
protected the cells against subsequent exposure to carbamide peroxide.

115
Wieslander AP, Deppisch R, Svensson E, Forsback G, Speidel R, Rippe B.  IN
VITRO BIOCOMPATIBILITY OF A HEAT-STERILIZED, LOW-TOXIC, AND LESS ACIDIC FLUID
FOR PERITONEAL DIALYSIS. Perit Dial Int 1995;15(2):158-64.

The aim of this study was to investigate a peritoneal dialysis (PD) fluid
(PD-Bio), produced with the intention of reducing the amount of glucose
degradation products and to increase the final pH. The heat sterilization of
the fluid was  performed with the glucose separated from the electrolytes.
After sterilization the two solutions were combined. METHODS: The in vitro
biocompatibility of PD-Bio was measured as the inhibition of cell growth of a
cultured fibroblast cell line and as the stimulated release of interleukin-1
beta from cultured human mononuclear cells. The glucose degradation products
were measured as UV absorbance at 228 nm or 284 nm and the concentration of
aldehydes was estimated with high-performance liquid chromatography and gas
chromatography. RESULTS: Our results demonstrate that in comparison to
conventional PD fluids the pH of PD-Bio was increased, to about 6.5. Due to
less contaminating glucose degradation products in PD-Bio, basal cytotoxicity
was significantly decreased for both 1.5% and 4% glucose-containing fluids,
and the stimulated release of interleukin-1 beta was normalized compared to
sterile filtered controls with the same pH. UV absorbance measured at 228 nm
was decreased, whereas the absorbance at 284 nm was equal to that of a
conventional fluid. In PD-Bio the concentrations of formaldehyde,
acetaldehyde, methylglyoxal, and 2-furaldehyde were found to be below the
detection limit, whereas glyoxal was present in the same and
5-hydroxymethylfurfural (5-HMF) in higher concentrations than in      
conventionally produced PD fluid. CONCLUSIONS: The results demonstrate that it
is possible to improve biocompatibility of PD fluids by simply changing the
way the fluid is produced. 

116
Kumar G, Ray S, Walle T, Huang Y, Willingham M, Self S, Bhalla K.  COMPARATIVE
IN VITRO CYTOTOXIC EFFECTS OF TAXOL AND ITS MAJOR HUMAN METABOLITE 6
ALPHA-HYDROXYTAXOL. Cancer Chemother Pharmacol 1995; 36(2):129-35. 

Taxol is metabolized by the liver microsomal cytochrome P450 enzyme system
into its principal metabolite 6 alpha-hydroxytaxol (6HT). In the present in
vitro studies 6HT was compared to taxol with respect to its effects on tubulin
depolymerization, mitotic arrest, clonogenic survival and apoptosis in HL-60
cells. 6HT was generated by incubating taxol with human liver microsomes in a
NADPH-generating system. HL-60 cells were incubated for 24 h with either taxol
or 6HT, washed and placed in drug-free suspension or cultured for colony
growth in agarose. For the suspension and colony culture growth of the cells,
the IC50 concentrations of 6HT were 500 +/- 46 and 350 +/- 37 nM, while those
of taxol were 3.2 +/- 0.2 and 2.8 +/- 0.5 nM, respectively. Immediately after
a 24-h exposure of HL-60 cells to 50 nM taxol, electrophoresis of genomic DNA
from HL-60 cells revealed an internucleosomal DNA fragmentation 'ladder'. In
addition, 39% of the cells were arrested in mitosis and 16% showed the
morphologic features of  apoptosis. In contrast, an identical treatment with
6HT resulted in the mitotic arrest of only 2.8% of the cells, with 4.0%
displaying apoptosis (P < 0.01); internucleosomal DNA fragmentation was not
observed. 6HT was also significantly less effective than taxol in inhibiting
the temperature-induced depolymerization of microtubules in a cell-free
system. However, at equipotent concentrations, the effect of 6HT on tubulin
depolymerization, mitotic arrest or apoptosis was similar to that of taxol. In
addition, at concentrations of taxol or 6HT at or below their IC50, there was
little tubulin depolymerization, mitotic arrest or apoptosis. The results
presented here show that the biotransformation of taxol to 6HT substantially
detoxifies taxol.

117
Leteurtre F, Sackett DL, Madalengoitia J, Kohlhagen G, MacDonald T, Hamel E, 
Paull KD, Pommier Y. AZATOXIN DERIVATIVES WITH POTENT AND SELECTIVE ACTION ON
TOPOISOMERASE II. Biochem Pharmacol 1995;49(9):1283-90.

Azatoxin was rationally designed as a DNA topoisomerase II (top2) inhibitor
[Leteurtre et al., Cancer Res 52: 4478-4483, 1992] and was also found to
inhibit tubulin polymerization. Its cytotoxicity is due to action on tubulin
at lower concentrations and on top2 at higher concentrations. At intermediate
concentrations, the combination of the two mechanisms appears antagonistic
[Solary et al., Biochem Pharmacol 45: 2449-2456, 1993]. The aim of this study
was to design azatoxin derivatives that would act only on tubulin or on top2.
Selective targeting of top2 or tubulin was tested using top2-mediated DNA
cleavage assays, and tubulin polymerization and tubulin proteolysis assays, as
well as COMPARE analyses of cytotoxicity assays in the National Cancer
Institute in vitro Drug Screening Program. Selective inhibitors of top2 and
tubulin polymerization have been obtained. Top2 inhibition, abolished by
methylation at position 4', was enhanced by the addition of a bulky group at
position 11. Bulky substitution at position 11 determined different patterns
of top2 cleavage sites and suppressed the action on tubulin. Selective
inhibition of tubulin was obtained with 4'-methylazatoxin that was found to
bind to the colchicine site. These results are consistent with those obtained
in the podophyllotoxin family to which azatoxin is structurally related. Some
azatoxin derivatives are under consideration for further preclinical
development.

118
Schmalz G, Schweikl H. CHARACTERIZATION OF AN IN VITRO DENTIN BARRIER TEST
USING A STANDARD TOXICANT.  J Endod 1994;20(12):592-4. 

To characterize a new artificial pulp chamber using bovine dentin, the
influence of different phenol concentrations on the cell reaction was
evaluated. Bovine dentin slices of 100, 200, 300, 500, and 700 microns were
cleaned, sterilized, and mounted between two 5-mm-thick glass plates with a
borehole of 5 mm in diameter. L-929 mouse fibroblasts were seeded into the
"pulpal" part of the device and incubated for 24 h. Different concentrations
of a phenol solution as a standard toxicant were applied to a sterile cotton
pellet in the "cavity" side of the device. After incubation for 24 h, the
cytotoxic reaction was evaluated by cell counting after vital staining with
fluorescein diacetate and related to the negative control. A dependency of the
cytotoxic reaction on the concentration of the standard toxicant and on the
thickness of the dentin slice was demonstrated. The simple dentin barrier test
using bovine dentin may be a suitable alternative to other more complicated
procedures using human dentin.

119
Wataha JC, Hanks CT, Sun Z. EFFECT OF CELL LINE ON IN VITRO METAL ION
CYTOTOXICITY. Dent Mater 1994;10(3):156-61.

The choice of cell line for in vitro biological tests which assess the
cytotoxicity of dental materials remains controversial, yet this issue is
important because these tests are widely used to rate the biocompatibility of
new and existing materials, and many different cell lines are commonly used.
The purpose of the current study was to quantify the responses of four cell
lines (Balb/c 3T3, L929, ROS 17/2.8 and WI-38) to 14 metal ions which are
released from dental materials, and relate these responses to the metabolic
activity and population doubling times of these cells. METHODS. Succinic
dehydrogenase (SDH) activity was used to monitor metabolic activity and
cytotoxic response. RESULTS. The cell lines responded differently to most
metal ions. In general, the Balb/c 3T3 line was the most sensitive, and the
WI-38 line was the least sensitive. However, there were many exceptions
depending on the metal ion. The passage number of the cells also affected the
cytotoxic response. It was concluded that the cytotoxicity of materials which
release metal ions will be significantly different depending on which cell
line is selected and its passage number. SIGNIFICANCE. Based on the findings
that cell lines ranked the toxicities of the metal ions similarly, it seems
reasonable to use these types of in vitro tests to rank the cytotoxicities of
materials. However, if these types of tests are used to predict in vivo
cytotoxicity, care should be taken to choose conditions and cells which are
relevant.

120
Zoli W, Flamigni A, Frassineti GL, Bajorko P, De Paola F, Milandri C, Amadori
D,  Gasperi-Campani A. IN VITRO ACTIVITY OF TAXOL AND TAXOTERE IN COMPARISON
WITH DOXORUBICIN AND CISPLATIN ON PRIMARY CELL CULTURES OF HUMAN BREAST
CANCERS. Breast Cancer Res Treat 1995; 34(1):63-9.

The in vitro activities of taxol and taxotere in comparison with cisplatin and
doxorubicin were assessed in 30 primary tumor cultures from human breast
cancers. Both taxanes were much more potent than cisplatin and doxorubicin.
Taxotere was 3.1; 296, and 9.6-fold more cytotoxic than taxol, cisplatin, and
doxorubicin respectively. The cytotoxic activity observed in our experiments
confirms the potential clinical relevance of the two taxanes in the management
of breast cancer. 

121
Sami SM, Dorr RT, Solyom AM, Alberts DS, Remers WA.  AMINO-SUBSTITUTED
2-[2'-(DIMETHYLAMINO)ETHYL]-1,2-DIHYDRO-3H-DIBENZ[DE,H]-ISOQUINOLINE-1,3-DIONE
S. SYNTHESIS, ANTITUMOR ACTIVITY, AND QUANTITATIVE STRUCTURE--ACTIVITY
RELATIONSHIP.  J Med Chem 1995;38(6):983-93.

Sets of 2-[2-(dimethylamino)ethyl]-1,2-dihydro-3H-
dibenz[de,h]isoquinoline-1,3-diones with amino and actylamino groups at each
of the eight positions on the anthracene nucleus were synthesized from
appropriately substituted anthracenes. Their evaluation in in vitro antitumor
and cardiotoxicity assays revealed a very strong dependence of potency on the
position of substitution. Certain compounds, including the 4-, 5-, 7-, and
9-amino derivatives, showed significantly higher potency than the
unsubstituted parent compound, azonafide. Among them, 7-aminoazonafide had low
cardiotoxicity relative to cytotoxicity. In general, the acetylamino analogues
were less potent than the amino derivatives against tumor cells and neonatal
rat heart myocytes; however, 5-(acetylamino)azonafide was highly cardiotoxic.
9-Aminoazonafide was more efficacious than azonafide or amonafide against P388
leukemia in mice. Statistically significant correlations were made between the
ability of amino analogues to increase the transition melt temperature (delta
Tm) of DNA and their potency against solid tumors, leukemia  cells, or cardiac
myocytes.

122
Da Silva CP, De Oliveira CR, De Lima MC.  IN VITRO ASSAY OF THE EFFECTS OF
ANTICANCER DRUGS ON LEUKEMIC T-CELLS BY FLOW MICRO-CALORIMETRY. Int J Pharm
1994;111(Oct 6):25-30. 

The effects of some antineoplastic agents on leukemic T-cells were studied in
vitro using flow microcalorimetry and conventional cell proliferation assays.
Alterations in the metabolic heat power of cell suspensions due to drug
treatment were measured as a function of time and dose of exposure and
correlated with cell growth inhibition data. While no clear correlation was
found for vincristine, a good correlation between calorimetric and
cytotoxicity data was obtained for the other drugs. It was concluded that flow
microcalorimetry may be used to examine the effects of antineoplastic agents
on leukemic T-cells.

123
Morrison C, Macnair R, MacDonald C, Wykman A, Goldie I, Grant MH. IN VITRO
BIOCOMPATIBILITY TESTING OF POLYMERS FOR ORTHOPEDIC IMPLANTS USING CULTURED
FIBROBALSTS AND OSTEOBLASTS.  Biomaterials 1995; 16(13):987-92.

The biocompatibility of 2 polymers for potential use as orthopedic implant
materials in an isoelestic hip prosthesis was investigated. The interactions
of polyether ketone (PEEK) and epoxy resin polymers (with and without carbon
fiber reinforcement) with both fibroblasts and osteoblasts were tested using
cell protein, intracellular reduced glutathione (GSH), leakage of lactate
dehydrogenase and the MTT assay as indexes of cellular cytotoxicity. The epoxy
resin was slightly cytotoxic and inhibited the growth rate of fibroblasts (as
assessed by total cell protein), and depleted GSH in both cell types. In
contrast, the PEEK material did not display over signs of cytotoxicity and, in
fact, increased osteoblast cell protein content. Of these 2 materials, PEEK
would be the one of choice for development of an isoelastic implant and, in
view of its stimulatory effect on osteoblast protein content, it may encourage
ingrowth of bone around the prosthesis and thus minimize joint loosening.

124
Fulda S, Honer M, Menke-Moellers I, Berthold F. ANTIPROLIFERATIVE POTENTIAL OF
CYTOSTATIC DRUGS ON NEUROBLASTOMA CELLS IN VITRO.  Eur J Cancer
1995;31A(4):616-21.

The role of single drugs in the treatment of neuroblastoma is poorly defined.
Therefore, a monolayer proliferation assay was used to test neuroblastoma cell
survival after a 72-h exposure to each of 20 cytostatic drugs. 6 Cell lines
were selected on the basis of MYCN amplification and PGY1 overexpression. EC50
values were related to plasma levels achievable in patients during
chemotherapy. The more effective substances were mitoxantrone, doxorubicin,
hydroxyurea, bleomycin, dactinomycin, cisplatin, thiotepa, melphalan,
carboplatin, etoposide, vincristine, cytarabine, 6-thioguanine,
cyclophosphamide, ifosfamide and zilascorb. Parental drugs (e.g.,
cyclophosphamide, cisplatin) appeared to be more cytotoxic on a molar basis
than derived drugs (e.g., ifosfamide, carboplatin). The least effective drugs
were 5-fluorouracil, 6-mercaptopurine, CCNU and procarbazine. Fractional addn.
of a given dose of cyclophosphamide, ifosfamide and cisplatin was more
efficient than a single dose. The various neuroblastoma cell lines showed
distinct individual sensitivities to the cytostatic drugs. Cell lines with
MYCN amplification were more sensitive than PGY1-overexpressing cells. Thus,
comparative in vitro testing of cytostatic drugs may provide a rationale for
their clin. evaluation. Investigation of drug combinations and application of
the monolayer proliferation assay to tumor biopsy material for preclin.
chemosensitivity testing are clearly warranted.

125
Brueschweiler BJ,  Wuergler FE, Fent K. CYTOTOXICITY IN VITRO OF ORGANOTIN
COMPOUNDS TO FISH HEPATOMA CELLS PLHC-1 (POECILIOPSIS LUCIDA). Aquat Toxicol
1995;32(2,3):143-60.

Cytotoxicity in vitro to fish hepatoma cells PLHC-1 has been analyzed for a
series of 21 organotin compds. consisting of all degrees of alkylation and
arylation. The sensitivity of the neutral red (NR) assay and the tetrazolium
salt redn. (MTT) assay was similar for most of the organotin compds. Cytotoxic
effects were found at concns. between 10-8M and 10-2M For various
trisubstituted organotin compds., including tributyltin and triphenyltin,
which are used in antifouling paints and as pesticides, resp., cytotoxic
concns. in the range of 10-8M to 10-6M were obsd.  Based on the concn.
reducing NR uptake by 50% (NR50), the sequence of cytotoxicity amongst
butyltins was tributyltin > bis(tributyl)- tin > dibutyltin > tetrabutyltin >
butyltin > tin(IV).  Tributyltin induced effects on cell functions quickly, as
a redn. in NR uptake by 30% was recorded after 30 min exposure to
4.cntdot.10-7M.  The ranking order in cytotoxicity in the MTT assay of
phenylated organotins was tri- phenyltin > diphenyltin > phenyltin > tin(IV).
Cytotoxic concns. of tributyltin and triphenyltin measured in the
bromodeoxyuridine (BrdU) assay and with the crystal violet (CV) staining
method do not differ significantly from those detd. in the NR and MTT assay. 
Tri- and disubstituted organotin compds. exhibit a significant correlation
between the n-octanol/water partition coeff. (logKow) and the NR50 (r = 0.86)
and the MTT50 values (r = 0.85), resp.  In addn., good qual. correlations
between in vitro cytotoxicity data and in vivo fish toxicity   data were found
(for NR assay, r = 0.86,; for MTT assay, r = 0.80). The results indicate that
the in vitro cytotoxicity assays using PLHC-1 cells are useful tools for the
estn. of the acute toxicity to fish of organotins and possibly other compds. 

126
Kim HM, Oh GT, Han SB, Hong DH, Hwang BY, Kim YH, Lee JJ. COMPARATIVE EFFECTS
OF ADRIAMYCIN AND 28-DEACETYL SENDANIN ON IN VITRO GROWTH INHIBITION OF HUMAN
CANCER CELL LINES.  Arch Pharmacal Res 1994;17(2):100-3.

The limonoid compd. (28-deacetyl sendanin, I) isolated from the fruit of Melia
toosendan SIEB. et ZUCC. was evaluated on anticancer activity. According to a
std. in vitro cytotoxicity assay, eight human cancer cell lines and SRB assay
were introduced for present evaluation. As a pos. std., adriamycin was tested
in parallel. The cell lines were originated from six different organs. In view
of dose-response profiles to 28-deacetyl sendanin, the most sensitive cells
were SF-539 and PC-3 which were derived from CNS and prostate, resp. In
contrast, all the cell lines responded similarly to adriamycin to give rise to
nearly identical dose-response profiles. By comparison of GI50 between
28-deacetyl sendanin and adriamycin,  six cell lines were more sensitive to
28-deacetyl sendanin and two were more resistant. As a result, 28-deacetyl
sendanin had more sensitive and selective inhibitory effects on in vitro
growth of human cancer cell lines in a comparison with adriamycin.

127
Lee MH, Kim CD, Ahn HJ. EVALUATION OF SURFACTANT CYTOTOXICITY POTENTIAL BY
NEUTRAL RED UPTAKE ASSAY, MTT ASSAY AND CELL PROTEIN ASSAY. Korean J Toxicol
1994;10(2):215-20.

Ten surfactants were compared and ranked according to their cytotoxic
potential.  Cytotoxicity was measured by assessing lysosomal neutral red
uptake, MTT redn. and protein measurement. The comparative cytotoxicity of
surfactants, in decreasing order, shown by all test was cationics > anionics =
amphoterics > nonionics. This is in agreement with in vivo observation.
Surfactants also showed very similar rank order of toxicity with each of the
asssay systems. Thus, these in vitro test methods may be considered reliable
alternatives in the assessment of surfactant toxicity.

128
Gabor F, Wollmann K, Theyer G, Haberl I, Hamilton G.  IN VITRO
ANTIPROLIFERATIVE EFFECTS OF ALBUMIN-DOXORUBICIN CONJUGATES AGAINST EWING'S
SARCOMA AND PERIPHERAL NEUROECTODERMAL TUMOR CELLS.  Anticancer Res
1994;14(5A):1943-50.

The 3-5 yr survival rates of patients with disseminated Ewing's sarcoma (ES)
or the closely related peripheral primitive neuroectodermal tumors (PNET)
remain low,  even under aggressive treatment involving highly toxic multidrug 
chemotherapeutic regimens. ES and PNET are sensitive to doxorubicin, but may
escape treatment by expression of the multidrug-resistant phenotype and/or
other mechanisms. In this study, we have identified albumin as growth
supporting factor for ES and PNET cells in IGF-I-supplemented serum-free
tissue culture medium. To investigate the specificity and toxicity of
albumin-based drug conjugates, doxorubicin was coupled to bovine serum albumin
(BSA) by either a two step glutaraldehyde or carbodiimide-C4-spacer technique,
yielding monomeric DOX-albumin conjugates with conjugation nos. ranging from 3
- 20 mol DOX/mol       BSA.  Cellular uptake of
fluorescein-isothiocyanate-(FITC)-labeled albumin and DOX-albumin conjugates
could be demonstrated by flow cytometric measurements of cell-assocd.
fluorescence and confocal microscopy. The cytostatic activity of these
conjugates against ES/PNET cell lines, a neuroblastoma (LAN-1) and prostate
cancer carcinoma cell line (PC-3) and normal lymphoblasts was tested in
short-term proliferation assays (48 h).  The results show a high selectivity
of the DOX-albumin conjugates for ES/PNET cell lines, with highest growth
inhibition by conjugates with low DOX conjugation nos. (n = 3) in
serum-supplemented medium (17 - 32 fold loss of activity compared to free
DOX), followed by 20-DOX-C4-albumin in serum-free medium and low activity of
the other conjugates.  In conclusion, DOX-albumin conjugates inhibit the
growth of ES/PNET cell lines selectively, showing low activity against the
unrelated carcinoma line PC-3 and sparing normal lymphoblasts. The inverse
correlation of activity and conjugation no. demonstrates a low cytotoxic
activity of DOX in acid-stable binding to monomeric albumin, pointing to a
selective cytostatic activity of the modified albumin against ES and PNET
cells, even in the presence of a 100 fold excess of unmodified serum albumin. 

129
Multhoff G, Meier T, Botzler C, Wiesnet M, Allenbacher A, Wilmanns W, Issels
RD.   DIFFERENTIAL EFFECTS OF IFOSFAMIDE ON THE CAPACITY OF CYTOTOXIC T
LYMPHOCYTES AND NATURAL KILLER CELLS TO LYSE THEIR TARGET CELLS CORRELATE WITH
INTRACELLULAR GLUTATHIONE LEVELS. Blood 1995;85(8):2124-31.

We established an in vitro model to study the influence of ifosfamide
treatment on intracellular glutathione (GSH) levels in activated human
effector cells with specific phenotypes and immunol. functions. Besides its
role as the major intracellular reductant, GSH has been shown to affect the
initiation and progression of lymphocyte activation after stimulation with
lectins. An incubation of activated human peripheral blood lymphocytes (PBL)
with 4-hydroxyifosfamide,  the activated form of ifosfamide (4-OH-IF),
resulted in a depletion of the intracellular GSH levels and a significant
inhibition of the proliferative capacity in a dose-dependent manner. The
cytotoxic activity of sepd. CD3- natural killer (NK) cells and CD3+
allospecific, cytotoxic T lymphocytes (CTL), either untreated or treated with
4-OH-IF at different concns., was compared in a std. 51chromium release assay
(CML). There were three major findings. (1) the capacity of CD3+ major
histocompatibility complex (MHC)-restricted CTL to lyse their specific
allogeneic target cells was substantially reduced by preincubation of the
effector cells with 4-OH-IF. This inhibition of the lytic activity in CD3+ CTL
correlated with a substantial depletion of the intracellular GSH levels in
this population. Rapid reconstitution of depleted GSH levels and restoration
of cytotoxic activity of CTL was achieved by incubation of the effector cells
with thiols eg, glutathione ester (GSH-ester) or 2-mercapto-ethanesulfonate
(mesna).  (2) In contrast, the lytic activity in CD3- NK cells was not
substantially affected (up to 100 mumol/L 4-OH-IF). This result correlates
with the capacity of NK cells to maintain their intracellular GSH levels after
an ifosfamide treatment.  (3) In comparison with CD3+ CTL, CD3- NK cells are
more resistant to ifosfamide treatment because they have higher initial GSH
levels and a more than fourfold higher relative rate of GSH synthesis. 


DERMAL TOXICITY
130
Joller PW, Coquette A, Noben J, Pirovano R, Southlee JA, Logemann PK. EUROPEAN
INTERLABORATORY EVALUATION OF AN IN VITRO OCULAR IRRITATION MODEL SKIN-2 MODEL
ZK1100 USING 18 CHEMICALS AND FORMULATED PRODUCTS. In: Reinhardt, CA, editor.
Alternatives to Animal Testing: New Ways in the Biomedical Sciences, Trends
and Progress: Symposium; 1992 Nov 30; Zurich, Switzerland. New York, VCH
Publishers, Inc; 1994. p. 159-64.

131
Berkers J AM, Hassing I, Spenkelink B, Brouwer A, Blaauboer BJ.  INTERACTIVE
EFFECTS OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN AND RETINOIDS ON PROLIFERATION
AND DIFFERENTIATION IN CULTURED HUMAN KERATINOCYTES: QUANTIFICATION OF
CROSS-LINKED ENVELOPE FORMATION. Arch Toxicol 1995;69(6):368-78.

Dioxins are potent inducers of chloracne in humans. This skin aberration can
be interpreted as an altered differentiation pattern of acinar sebaceous base
cells and a change in the rate of terminal differentiation of the
keratinocytes. We measured this rate induced by
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in primary cultures of human
keratinocytes. As parameters for differentiation, we quantified the
35S-methionine incorporation into cross-linked envelopes (revealing the total
CLE  biomass), as well as the number of microscopically visible CLEs. It was
shown that TCDD is a very potent inducer of both CLE biomass and number with a
half-maximal effect concentration (EC50) of 1.4 nM. CLE biomass was maximally
increased 10-fold and the number of cells in culture producing a CLE was
increased from 15% in control cultures to maximally 75% of the cells in
TCDD-treated cultures. Both effects were Ca2+-dependent and increased with
elevated cell density, being optimal in  post-confluent cultures. Retinoic
acid dose-dependently decreased the effect of 10-8 M TCDD, 10-6 M having a
nearly complete antagonistic action. This interaction of retinoic acid with
TCDD-induced differentiation was non-competitive. Retinol was equally potent
as an antagonist of the TCDD-induced elevation of CLE formation as compared
with retinoic acid. Retinyl palmitate and etretinate were not very effective
as TCDD antagonists. Supplementation of hydrocortisone suppressed the
TCDD-induced keratinocyte differentiation. It was concluded that CLE biomass
quantification provides a reliable and sensitive parameter for keratinocyte
differentiation. In this in vitro system it is shown that TCDD strongly
induces a switch from proliferation to terminal differentiation and that this
effect can be antagonized effectively by retinoic acid and retinol. 

132
Frankild S,  Anderson KE, Nielsen GD. EFFECT OF SODIUM LAURYL SULFATE (SLS) ON
IN VITRO PERCUTANEOUS PENETRATION OF WATER, HYDROCORTISONE AND NICKEL. Contact
Dermatitis 1995;32(6):338-45.

The dose- and time-related effect of sodium lauryl sulfate (SLS) on in vitro
percutaneous penetration was studied using 3 radiolabeled tracer compounds
with different physicochemical properties: tritiated water, hydrocortisone and
nickel. Human cadaver abdominal skin from Caucasian women was used as membrane
in static in vitro penetration cells. Simultaneous application of SLS together
with 1 of the tracer compounds showed, after 48 h, a significant dose-effect
relationship between SLS concentration (0.25%, 2% and 10%) and penetration of
tritiated water or nickel (P<0.001, Spearman), whereas SLS had no significant
effect on penetration of hydrocortisone. When 4% SLS was applied as
pretreatment, a significant time-effect relationship, after 48 h, was found
between pretreatment time (0.5, 2 and 8 h) and penetration of tritiated water.
A similar relationship was not found for penetration of nickel or
hydrocortisone. Pretreatment of the skin with SLS for 2 h using 3 
concentrations (0.25%, 4% and 10%) showed, after 48 h, a significant
dose-effect relationship between SLS treatment and penetration of tritiated
water or nickel (P<0.001, Spearman). Pretreatment had no effect on penetration
of hydrocortisone. Pretreatment simulates a cleaning-washing situation. The
present in vitro skin penetration model, using human cadaver skin, described
the dose-effect and time-effect relationships for SLS on the penetration
profiles of 3 different compounds. The model may be extended to other
compounds with suspected irritant/damaging effect on the skin barrier. It
should be kept in mind that the model uses a dead skin membrane without the
barrier repair mechanisms of live skin. 

133
Chang SK, Brooks JD, Monteiro-Riviere NA, Riviere JE.  ENHANCING OR BLOCKING
EFFECT OF FENVALERATE ON THE SUBSEQUENT PERCUTANEOUS ABSORBTION OF PESTICIDES
IN VITRO. Pestic Biochem Physiol 1995;51(3):214-9.

The percutaneous absorption of pesticides has been receiving much research
attention. However, most work is conducted with single exposures and potential
interactions of previous pesticide exposure have received little attention. In
the present study, the effect of in vivo pretreatment of the skin with a 3%
fenvalerate in ethanol or a 3% parathion in ethanol solution on carbaryl,
fenvalerate, lindane, and parathion absorption was studied in vitro using
weanling pig skin in a flowthrough  diffusion cell system. Concentrations of
40 or 400 mug/cm2 of carbaryl, fenvalerate, lindane, and parathion in ethanol
were applied topically. Environmental conditions of air and perfusate
temperature (37~C), relative humidity (60%), flow rate (4 ml/hr), and
Kreb's-Ringer bicarbonate buffer with 4.5% bovine serum albumin medium were
controlled. The total absorption of these pesticides, both ethanol control and
fenvalerate or parathion pretreated, increased proportionally with the dose; 
however, the absorption efficiency (fraction of applied dose absorbed)
decreased as the dose increased. At both doses, fenvalerate pretreatment had
little or no effect on carbaryl and fenvalerate absorption; however, parathion
absorption was significantly decreased in fenvalerate-pretreated skin (P <
0.05). There was no significant difference (P : 0.05) of parathion absorption
between the parathion pretreatment and the fenvalerate pretreatment. Lindane
absorption increased at the 40-mug  dose and significantly increased at the
400-mug dose (P < 0.05) following fenvalerate pretreatment. Carbaryl
absorption was higher than other pesticides at the dose of 40 mug/cm2.
Furthermore, comparing ethanol control data with previous results indicates
that prolonged skin treatment with ethanol significantly increases parathion
absorption (P < 0.05). This study suggests that the absorption data from a
single parent compound alone were not adequate to determine the rate of
absorption of  pesticide under mixture exposure conditions. Pesticide
interactions may significantly affect the percutaneous absorption,
interpretation, and assessment of risk. 

134
Alemohammad MM, Alki J, Foley TJ.  DETECTION OF IgE ANTIBODIES TO LATEX
ALLERGENS IN HUMAN SERUM.  Contact Dermatitis 1995;32(5):298-302.

Reports indicate increasing incidence of Type I allergic reactions to latex
allergens. The proteins that act as allergens and produce such allergic
reactions are found in the natural latex sap of Hevea brasiliensis. All those
who are exposed to latex products,  particularly healthcare workers, are
potentially at risk. The lack of qualified allergen extracts makes it
difficult to perform skin testing on individuals who are at high risk.
Therefore, a reliable in vitro test system for the  detection of IgE
antibodies to latex would be of considerable utility. We have developed a
serological test for the qualitative determination of specific IgE antibodies
to latex. In our study, 75 sera from individuals with a history of latex
allergy and 29 serum samples from healthcare workers were tested by both
radioimmunoassay (RIA) and enzyme immunoassay (EIA). The allergen bound to
paper discs was the same solid phase for the RIA and EIA assays. The allergen
preparation used for coating  the paper discs was a mixture of proteins
obtained from raw latex. The data show a good correlation between the results
of RIA and EIA methods with data obtained using an RIA assay at an independent
laboratory and by skin prick testing. Comparing the performance of our test
using our latex material with that of the latex material obtained from the
Food and Drug Administration (FDA), along with results of other tests
including skin tests, we found that the specificity and sensitivity of our 
assay method approaches 100%. The data show no significant cross-reactivity
between latex and banana. A low level of cross-reactivity between latex and
avocado was observed. We conclude that, by using correct selection of
proteins, the detection of specific IgE to latex may be a valuable assay
method for screening individuals and for the diagnosis of allergy to latex.

135
Bonifer R, Neumann C,  Meuer S, Schulze G, Herrmann F.  INTERLEUKIN 5
EXPRESSING ALLERGEN-SPECIFIC T-LYMPHOCYTES IN PATIENTS WITH HOUSE DUST MITE
SENSITIZATION: ANALYSIS AT A CLONAL LEVEL.  J Mol Med 1995;73(2):79-83.

Interleukin 5 (IL-5) is a T-cell lymphokine known to stimulate development,
functional activity, and in vitro survival of eosinophils. Tissue and blood
eosinophilia occurring during allergic responses of the immune system are
potentially mediated by IL-5 secreting T-cells. To test this hypothesis a
series of allergen-specific T-cell clones were established from peripheral
blood and skin lymphocytes of patients with atopic dermatitis and house dust
mite sensitization. In addition, alloreactive  T-cell clones were also
prepared from peripheral blood lymphocytes of healthy donors. Cloned T-cells
were analyzed for IL-5 mRNA expression and IL-5 secretion by means of in vitro
gene amplification using the reverse transcriptase polymerase chain reaction
and IL-5 specific oligonucleotide hybridization, as well as IL-5-specific
ELISA. A majority of allergen-specific long-term cultured T-cell clones (84%)
of different donors and of either phenotype (CD8+ or CD4+) disclosed IL-5
transcripts on  stimulation with lectins. Almost all clones exhibiting IL-5
transcripts also released immunoreactive IL-5 protein into their culture
supernatants. In contrast, only 2% of alloreactive T-cell clones obtained from
healthy donors and none of alloreactive T-cell clones of one atopic patient
investigated expressed detectable amounts of IL-5 mRNA in response to lectin
stimulation, all of whom were CD4+.  These results suggest that eosinophilia
observed in allergic responses in the peripheral blood  and in tissues at the
site of induced late-phase cutaneous reaction may be associated with IL-5
release by allergen-specific T-cells.

136
Dick D, Ng K ME, Sauder DN, Chu I.   IN VITRO AND IN VIVO PERCUTANEOUS
ABSORPTION OF 14C-CHLOROFORM IN HUMANS.  Hum Exp Toxicol 1995; 14(3):260-5.

Chloroform has been found in potable water and there is concern that
significant dermal absorption may arise from daily bathing and other
activities. The present study examines percutaneous absorption of
14C-chloroform in vivo using human volunteers and in vitro using fresh,
excised human skin in a flow-through diffusion cell system. Fifty microlitre
doses of either 1000 mug ml-1 chloroform in distilled water, (16.1 mug cm-2)
or 5000 mug ml-1 of chloroform in ethanol,  (80.6 mug cm-1)  were applied to
the forearm of volunteers with exhaled air and urine being collected for
analysis. Single doses of either 0.4 mug ml-1 chloroform in distilled water
(low dose, 0.62 mug cm-2, 1.0 ml dosed) or 900 mug ml-1 chloroform in
distilled water (high dose, 70.3 mug cm-2, 50 mul dosed) were applied to discs
of the excised abdominal skin placed in flow-through diffusion cells and
perfused with Hepes buffered Hank's balanced salt solution, with a wash at 4
h. In vivo absorption was 7.8 : 1.4% (water as vehicle) and 1.6 : 0.3%
(ethanol as vehicle). Of the dose absorbed in vivo, more than 95% was excreted
via the lungs (over 88% of which was CO2), and the maximum pulmonary excretion
occurred between 15 min and 2 h after dosing. The percentage of dose absorbed
in vitro (skin + perfusate) was 5.6 : 2.7% (low dose) and 7.1 : 1.4% (high
dose).  The above data demonstrate that a significant amount of the dissolved
chloroform penetrates through the human skin, and that a higher percentage of
the applied dose was absorbed using water  as vehicle. In addition, the in
vitro method offers a good estimate for in vivo data.

137
Chung JH, Youn JI.  EFFECT OF ULTRAVIOLET A ON IL-1 PRODUCTION BY ULTRAVIOLET
B IN CULTURED HUMAN KERATINOCYTES. J Dermatol Sci 1995;9(2):87-93.

Human skin is exposed to significant amounts of UVA and UVB radiation
simultaneously. The effects of UVA and UVB interactions have been examined in
many aspects such as erythema response. The effects of UVA on the production
of cytokines by UVB have not been studied yet. The purpose of this study is to
observe the effect of UVB and UVA on the production of IL-1 in cultured human
keratinocytes and to determine whether UVA can modify the effects of UVB on
the IL-1 production. Human keratinocytes  derived from normal foreskin were
exposed to UVA (0-30 J/cm2) and subsequently to UVB (0-50 mJ/cm2). After 48 h
incubation, IL-1 levels in the culture supernatants and cell extracts of the
cultured keratinocytes were measured by thymocyte proliferation assay. We have
observed that UVB increased the production of IL-1 in cultured keratinocytes. 
However, UVA suppressed the production of IL-1 and also the stimulatory effect
of UVB on the IL-1 production. We think that the opposite effects of UVB and
UVA on the IL-1 production in human keratinocytes might explain the different
action mechanisms of UVB and UVA in many cutaneous responses. 

138
Edwards SM, Donnelly TA, Sayre RM, Rheins LA, Spielmann H,  Liebsch M. 
CORRECTION OF PREVIEWS 97425447. QUANTITATIVE IN VITRO ASSESSMENT OF
PHOTOTOXICITY USING A HUMAN SKIN MODEL, SKIN 2. ADDITION OF AUTHOR NAMES.
Erratum published in Photodermatol Photoimmunol Photomed 1994;10(5):226.
Photodermatol Photoimmunol Photomed 1994;10(3):111-7.

The ability to accurately predict the phototoxic potential of personal and
skin care products remains a key element in assessing the safety of
premarketed products. To find a reliable in vitro alternative test for
photoirritancy, the European Commission and the European Cosmetic Association
are conducting a 3-year, European validation study.  Based on the results of
this study, an in vitro photoirritancy method will be selected for
incorporation into new international guidelines for photoirritancy testing. As
a part of this study, Skin2, a cultured human skin system, was used to
evaluate the phototoxic potential of chemicals with known photoirritative
properties. The Skin2 ZH1351, a 3-dimensional co-culture system, consists of
dermal fibroblasts and a multilayered epidermis       comprising
differentiated keratinocytes. This product line has previously been used to
evaluate the irritative potential of topically applied ingredients and
products. In this study, various concentrations of the test chemicals were
applied to the epidermal side of the Skin2 tissue for contact times of 1 h or
24 h and then the tissue was exposed to 2.9 J/cm2 of ultraviolet A (UVA)
radiation. Treated but nonirradiated tissues were also assayed to predict the
cytotoxic potential of the test chemicals, which could mask the phototoxic
reaction. After exposure, the tissue substrates were rinsed free of test
chemicals and allowed to recover for 24 h. Following this incubation, the MTT
reduction assay was used to assess cytotoxicity. The results of tests using
this model skin substrate (Skin2) showed a high degree of correlation with
data from human and animal models now used to evaluate the phototoxic
potential of chemicals.

139
French JE, Libbus BL, Hansen L, Spalding J, Tice RR, Mahler J, Tennant RW. 
CYTOGENETIC ANALYSIS OF MALIGNANT SKIN TUMORS INDUCED IN CHEMICALLY TREATED TG
AC TRANSGENIC MICE. Mol Carcinog 1994;11(4): 215-26.

TG AC mice (which carry a v-Ha-ras transgene) rapidly develop papillomas in
response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Approximately 30% of
the papillomas are associated with subsequent development of malignancies.
Early-passage spindle-shaped tumor cells arising from explant cultures of
TPA-induced tumors in TG AC mice were tumorigenic when transplanted to
syngeneic recipients. The v-Ha-ras transgene in the transplanted tumors was
expressed at a high level. To identify possible genetic changes associated
with the development of malignant tumors, explanted cells were cultured in
vitro and assessed for karyotypic changes between the second and third
passages by analyzing C-banded metaphase chromosomes. For comparison, skin
malignancies were induced in nontransgenic FVB/N mice (parent strain) by
7,12-dimethylbenz(a)anthracene (DMBA) initiation and TPA promotion, and their
G-banded metaphase chromosomes were analyzed. Trisomy (in at least 50% of
about 30 metaphases) of  chromosome 15 (in five of 15 tumors) and chromosome 6
(four of 15 tumors) was observed in TGpendent of chemical treatment or tumor
type. Of six tumors from DMBA/TPA-treated FVB/N mice, three had trisomy 10 or
15 (or both), and two appeared normal. The absence of trisomy 7 is notable
because c-Ha-ras maps to that chromosome. The absence of trisomy 7 in the six
FVB/N DMBA/TPA-induced skin malignancies contrasts with DMBA/TPA-induced
karyotypic effects in SENCAR mice. Expression of the v-Ha-ras transgene may
have precluded the requirement for endogenous mutant ras and allelic imbalance
involving-chromosome 7 in TG/N mice. These results suggest the possibility
that the observed trisomies are  consequential, rather than causal, events in
the development of TGgenetic analysis will be required to understand the
changes associated with tumorigenesis in this transgenic line as well as in
the  parent mouse line. 

140
Hilton J, Kimber I.   THE MURINE LOCAL LYMPH NODE ASSAY. Methods Mol Biol
1995;43:227-35.

141
Supino R. MTT ASSAYS.  Methods Mol Biol 1995;43:137-49.

142
Guan YB,  Guo QZ, Zhang BZ. [TOXICOKINETICS OF UNSYMMETRICAL DIMETHYLHYDRAZINE
ABSORBED PERCUTANEOUSLY IN RABBITS.]  Zhongguo Yaolixue Yu Dulixue Zazhi
1995;9(2):137-39. (Chi)

Blood concentration-time relationship was studied after percutaneous
application (pc) of 78.2 and 156 mgbbit. The data were best fitted to a
one-compartment open model with a first order absorption process.  The
half-lives (t1/2 ka) were 0.08 : 0.09 h and 0.19 : 0.12 h, respectively, while
the fractions of absorption (f) were 0.11 :  0.04 and 0.17 : 0.09,
respectively. Following subcutaneous injection (sc) of 78.2 mg   kg-1  UDMH,
its blood levels were much higher than those after percutaneous application
with an absorption rate f = 0.992, indicating almost complete absorption of
the compound following sc. The t1/2 ka after sc was 0.68 : 0.19 h, which was
significantly longer than those after pc. In vitro application of UDMH on
rabbit skin demonstrated that 84.2 : 5.5% of the compound was evaporated from
the skin, which might account for the low absorption rate of UDMH after pc in
vivo. The elimination of UDMH was rapid, with a terminal elimination
half-lives of 0.3-1.5 h. The amount of UDMH excreted in urine was 2.7%-18.9%
of the total elimination, indicating that there might be elimination pathways
of UDMH other than renal in rabbits.

143
Van de Sandt JJ, Rutten AA.  DIFFERENTIAL EFFECTS OF CHEMICAL IRRITANTS IN
RABBIT AND HUMAN SKIN ORGAN CULTURES.  Toxicol In Vitro 1995; 9(2):157-68.

The toxicity of well known irritants was investigated in rabbit and human skin
organ cultures. Test chemicals were selected from various categories of
irritants and included both water-soluble and water-insoluble compounds. Using
a highly standardized protocol, test chemicals were applied topically at
concentrations relevant to the in vivo situation. Toxicity was assessed by
histomorphological examination, inhibition of conversion of the tetrazolium
salt MTT, inhibition of epidermal cell  proliferation and release of
pro-inflammatory hydroxy fatty acids. Chemicals that are known to induce
irritation in vivo invariably caused histopathological changes and inhibition
of cell proliferation, whereas non-irritating chemicals did not; however,
inhibition of MTT conversion and release of hydroxy fatty acids occurred with
only a limited number of irritants. The response to the chemical irritants was
different in rabbit and human skin cultures. Rabbit skin was slightly more
sensitive to  sodium dodecyl sulfate and benzalkonium chloride than human
skin. Moreover, mineral oil enhanced epidermal cell proliferation in rabbit
skin, but not in human skin. This study demonstrates that chemical irritants
cause substance- and species-specific effects in skin organ cultures. It is
therefore unlikely that irritant potential of chemicals from all irritant
categories can be detected in vitro using one single parameter. A multiple
endpoint approach and the inclusion of human tissue are recommended for
optimal in vitro irritancy testing of chemicals.

144
Van de Sandt JJ,  Maas WJ,  Doornink PC,  Rutten AA. RELEASE OF ARACHIDONIC
AND LINOLEIC ACID METABOLITES IN SKIN ORGAN CULTURES AS CHARACTERISTICS OF IN
VITRO SKIN IRRITANCY. Fundam Appl Toxicol 1995; 25(1):20-8.

In vitro techniques make a major contribution to the development of
alternatives to the in vivo "Draize" skin irritation test, and the development
of sensitive and generally applicable in vitro endpoints of cutaneous toxicity
is an area of intensive research. To investigate in vitro characteristics of
cutaneous irritation, skin explants of rabbit and human origin were topically
exposed to chemical irritants, after which the culture medium was analyzed for
the presence of metabolites of both  arachidonic and linoleic acid. In rabbits
exposed to the potent irritant benzalkonium chloride, a direct relation was
established between clinical signs of irritation and in vitro release of the
proinflammatory mediator 12-hydroxyeicosatetraenoic acid (12-HETE) by the
exposed skin. Histological examination revealed varying degrees of epidermal
damage. 12-HETE was also the predominant hydroxy fatty acid released in a
dose-dependent way by rabbit skin cultures after in vitro exposure to sodium
dodecyl sulfate (SDS), benzalkonium  chloride (BC), and formaldehyde (FA).
Human skin cultures released, in addition to 12-HETE, predominantly 15-HETE
and 13-hydroxyoctadecadienoic acid (13-HODE), omega-6 oxygenase products of
arachidonic acid and linoleic acid, respectively. The irritant-induced release
of hydroxy fatty acids was strongly inhibited by the lipoxygenase inhibitor
eicosatetraynoic acid, indicating enzyme-mediated generation of these
bioactive lipids. Comparison of hydroxy fatty aci release to more established
markers of cytotoxicity (leakage of the cellular enzymes, such as aspartate
aminotransferase (AST), alanine aminotransferase (ALT), and lactate
dehydrogenase (LDH)) revealed that increased levels of 13-HODE, 9-HODE,
12-HETE, and ALT were specific markers of cutaneous irritancy in rabbit skin
cultures. AST and LDH were more sensitive endpoints compared to ALT or hydroxy
fatty acids, but failed to identify the difference in irritation capacity
between SDS and BC  reported in vivo. In human skin cultures, release of
12-HETE or 15-HETE occurred at irritant concentrations that did not result in
enzyme leakage into the culture medium. In vitro effect levels of irritants
were found comparable to the in vivo situation. We conclude that release of
hydroxy fatty acids, in particular of 12-HETE, offers a mechanism-based
characteristic of skin irritation which may be applicable to in vitro toxicity
testing.

145
Pu Y, Bernstein IA. USE OF HUMAN PSEUDO-EPIDERMIS TO EVALUATE THE TOXICITY OF
BIS-(2-CHOLROETHYL)SULFIDE (BCES) ON WATER PERMEATION BARRIER FORMATION AND
FUNCTION.  Toxicol Let 1995; 76(1):85-91.

Human pseudo-epidermis was used to investigate the effects of
bis-(2-chloroethyl)sulfide (BCES) on the formation and function of the water
permeation barrier. To generate the culture, 2 million viable basal cells
derived from human skin were plated on a Puropore nylon microporous membrane
pre-coated with calf skin collagen. Addition of bovine pituitary extract and
epidermal growth factor to the medium favored the formation of homogeneous
cultures and better barrier function. The water  permeation constant (Kp) was
shown to decrease significantly and reached 25 : 6 from 71% of the cultures
prepared. The effects of topically applied BCES on the incorporation of
(14C)linoleic acid, as a marker for lipid synthesis, and Kp, as a measure of
water permeation, were studied.  Compared with untreated cultures, there was
no difference in the Kp immediately after exposure to 1-10 nmol BCES/cm2 for
30 min. On the other hand, (14C)linoleic acid incorporation was
dose-dependently decreased immediately after exposure and then returned to
normal by 48 h later. These data suggest that BCES produces no direct damage
to the water  permeation barrier but may affect barrier formation by
inhibiting lipid synthesis.

146
Norton JN, Rylander LA, Richards JL.  IN VITRO ORAL MUCOSA IRRITATION TESTING
WITH HUMAN CELL CULTURES. Toxicol In Vitro 1995;9(1):67-74.

Cell cultures are a potentially useful model to predict in vivo oral mucosa
irritation. To this end, oral mucosa organ equivalent cultures (OMOEC) and
skin equivalent cultures (SEC), both derived from human tissue, were evaluated
for their responsiveness to test dentifrices with graded degrees of irritation
potential. OMOEC and SEC were treated with test dentifrices and responses were
evaluated by histopathology, cell viability (MTT incorporation), and
cytotoxicity (release of aspartate  aminotransferase (AST)). Cell viability in
OMOEC and SEC was reduced in a dose- and time-dependent manner in response to
the test dentifrices. Correspondingly, AST release was increased in a dose-
and time-dependent manner in response to the test dentifrices. These results
demonstrate that OMOEC and SEC systems respond linearly to graded degrees of
irritation potential as represented by generic dentifrices. Such results in an
in vitro model of oral mucosa irritation allow direct comparison of  in vitro
responses with those obtained in an in vivo model, thus providing the
groundwork for a tiered approach to assessment of irritation potential of oral
care products. 

147
Shieh HL,  Hansen H,  Zhu J, Riedel H.  DIFFERENTIAL PROTEIN KINASE C LIGAND
REGULATION DETECTED IN VIVO BY A PHENOTYPIC YEAST ASSAY. Mol Carcinog
1995;12(3):166-76.

The molecular dissection of protein kinase C (PKC) action has been based in
part on time-consuming functional assays such as the mouse skin model for
testing the tumor promoter activity of phorbol esters and related PKC
activators. To help overcome the limitations imposed by the complexity of such
assays, we developed the yeast Saccharomyces cerevisiae as an alternative,
rapid, and simple experimental system. This model has a specific phenotype, an
increase in the cell doubling time, that is  proportional to the level of
enzymatic activity of expressed mammalian PKC isoforms. We used this phenotype
to assay and compare the regulation of native bovine PKCalpha and mutants in
the conserved regulatory region C1 in vivo by various activators: two
diterpenes, the phorbol ester phorbol-12-myristate-13-acetate (PMA) and
mezerein, and the indole alkaloid indolactam V. We found that PMA activated
PKC mutants lacking either Cys-rich, zinc finger-like repeat of the conserved
region C1 to comparably reduced levels, whereas indolactam V activated native
PKCalpha but none of the mutants at normal doses. In contrast, mezerein
activated native PKCalpha and a mutant lacking the second Cys repeat equally
well but mutants lacking the first Cys repeat of C1 at a greatly reduced
level. These differential responses were supported by the observed in vitro
PKC catalytic activities. Therefore, PMA regulates PKCalpha activity
comparably well via either Cys repeat, whereas mezerein regulation 
predominantly occurs via the first Cys repeat of C1. Indolactam V activation
was less potent, it was greatly reduced in the absence of either Cys repeat,
and displayed no preference. We introduce this phenotypic assay as a rapid and
general screen for the PKC-activating or possibly inhibitory potential of drug
candidates and to identify the PKC regulatory sites involved in these
interactions.

148
Vile GF,  Tyrrell RM.  UVA RADIATION-INDUCED OXIDATIVE DAMAGE TO LIPIDS AND
PROTEINS IN VITRO AND IN HUMAN SKIN FIBROBLASTS IS DEPENDENT ON IRON AND
SINGLET OXYGEN. Free Radic Biol Med 1995; 18(4):721-30. 

This study describes the damage that occurs to lipids and proteins that have
been irradiated in vitro or in human skin fibroblasts with physiological doses
of UVA radiation. Thiobarbituric acid-reactive species were formed from
hosphatidylcholine after UVA radiation in vitro. By using iron chelators, this
process was shown to involve iron. Ferric iron associated with potential
physiological chelators was reduced by UVA radiation, but iron within ferritin
was not. By enhancing the half life-time with deuterium oxide or by using 
scavengers, singlet oxygen was also shown to be involved in the UVA
radiation-dependent peroxidation of phosphatidylcholine. UVA
radiation-generated singlet oxygen reacted with phosphatidylcholine to form
lipid hydroperoxides, and the breakdown of these hydroperoxides to
thiobarbituric acid-reactive species was dependent on iron. We have shown that
iron and singlet oxygen are also involved in the UVA radiation-dependent
formation of thiobarbituric acid-reactive species in human skin fibroblasts,
and we propose that a similar concerted effect of iron and singlet oxygen is
involved in UVA radiation-dependent damage to fibroblast lipids. Sulphydryl
groups of bovine serum albumin and human gamma-globulin were oxidised upon UVA
irradiation in vitro. The use of scavengers and deuterium oxide showed that
UVA radiation-dependent sulphydryl oxidation was  dependent on singlet oxygen.
By adding or chelating iron, UVA radiation-dependent oxidation of sulphydryl
groups of bovine serum albumin and human gamma-globulin was shown to be
iron-dependent. The use of catalase and hydroxyl radical scavengers
demonstrated that hydrogen peroxide, but not the hydroxyl radical, was
involved. The oxidation of sulphydryl groups of proteins in human skin
fibroblasts that occurs as a result of UVA irradiation was also shown to
involve iron, singlet oxygen, and hydrogen peroxide. We conclude that iron,
singlet oxygen, and hydrogen peroxide are important redox active species
involved in the deleterious effects of UVA radiation on lipids and proteins of
human skin cells. 

149
Hertl M, Merk HF.  LYMPHOCYTE ACTIVATION IN CUTANEOUS DRUG REACTIONS. J Invest
Dermatol 1995;105(1 Suppl):95S-98S.

Peripheral blood lymphocytes from both drug-induced immediate and delayed
cutaneous hypersensitivity reactions frequently can be stimulated in vitro
with the particular culprit drug. Immunohistochemical analysis has identified
CD8+ T cells as the predominant epidermal T-cell subset in drug-induced
maculopapular and bullous eruptions and in patch-test reactions to beta-lactam
antibiotics. Beta-lactam-specific peripheral and epidermal T lymphocytes from
bullous exanthems were predominantly T-cell receptor alpha/beta+, CD8+, CD4-.
Three CD8+ epidermal T-cell clones from penicillin-induced bullous exanthems
displayed a TH1-like cytokine pattern and proliferated in an antigen- and
major histocompatibility complex-specific manner. These epidermal T-cell
clones were cytotoxic against autologous B cells upon stimulation through the
T-cell receptor and against epidermal keratinocytes in lectin-induced
cytotoxicity assays. In contrast, peripheral T-cell lines from patients with
penicillin-induced urticarial exanthems were predominantly T-cell receptor
alpha/beta+, CD4+, CD8- and displayed a Th2-like cytokine pattern. CD8+ dermal
T cells from a sulfamethoxazole-induced bullous exanthem proliferated in vitro
in response to sulfamethoxazole. This T-cell proliferation was significantly
increased in the presence of microsomes, which suggests that microsomal
enzymes, such as cytochrome P450 enzymes, generate highly reactive metabolites
which are the nominal antigens for T-cell activation. In summary, drugs may be
processed and  presented in different ways, which is reflected by the
observation that Th1-like CD8+ T cells are primarily activated in delayed
cutaneous hypersensitivity reactions, whereas Th2-like T-cell responses are
present in patients with drug-induced urticarial exanthems.

150
Brenner S,  Ruocco V, Wolf R, de Angelis E, Lombardi ML. PEMPHIGUS AND DIETARY
FACTORS. IN VITRO ACANTHOLYSIS BY ALLYL COMPOUNDS OF THE GENUS ALLIUM.
Dermatology 1995;190(3):197-202. 

Today it is generally accepted that every drug that possesses an active thiol
group in its molecule is capable of inducing pemphigus in vivo and provoking
acantholysis in vitro. We therefore suggested that plants, in particular those
belonging to the Allium group, that contain several active compounds with
stable disulfide and thiol groups in their molecule may cause the same.
OBJECTIVE: To verify this hypothesis by investigating the in vitro
acantholytic effect of three compounds of garlic. METHODS: Skin samples from
donors were cultured in the presence of three compounds of garlic (i.e.
allylmercaptan, allylmethylsulfide and allylsulfide) for 3 days. The skin
samples were then processed for microscopic control for acantholysis. RESULTS:
Results indicate that, indeed, the three garlic compounds tested are capable
of inducing acantholysis in vitro. Focal and diffuse acantholysis was observed
in the specimens from 4 out of 7 donors cultured in the presence of 6 and 9 mM
of each of the allyl compounds for 3 days. Interestingly, tissues from a DR4+
donor proved to be more acantholysis prone than others, showing large
blistering due to diffuse acantholysis, thus indicating that individual
susceptibility plays a crucial role also in vitro. CONCLUSION: Garlic
compounds with stable disulfide and thiol groups in their molecule are capable
of inducing acantholysis in vitro. These findings lend further support to the
theory that 'harmless' nutritional factors are capable of  inducing
acantholysis in vitro and possibly also in vivo. In view of these findings, it
is suggested that nutritional factors should be added to the ever-growing list
of exogenous factors capable of inducing pemphigus. 

151
Dominguez MC, Sole E,  Goni C, Ballabriga A.  EFFECT OF ALUMINUM AND LEAD
SALTS ON LIPID PEROXIDATION AND CELL SURVIVAL IN HUMAN SKIN FIBROBLASTS.  Biol
Trace Elem Res 1995;47(1-3):57-67.

The aim of this study was to see whether aluminum (Al) and lead (Pb) salts are
toxic for cultured human fibroblasts under different experimental conditions,
in the controllable situation offered by cell cultures. Cell survival and
membrane lipid peroxidation served as markers of Al and Pb toxicity.
Evaluation of the living cells was carried out using a colorimetric method,
the mitochondrial reduction of 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT). Lipoperoxidation assay was performed on whole cell
homogenates by measuring thiobarbituric acid-reactive substances (TBARS)
produced after incubation with ascorbic acid-ferrous sulfate. Al(III) and
Pb(II) salts (300 microM) produce a considerable decrease in cell survival
after an exposure period of 4d, evident with the three fetal calf serum
concentrations in the culture media: 2, 5, and 10%. Taking into account in
vitro cell aging, the cytotoxic effects of Al(III) and Pb(II) are greater in
senescent fibroblasts than in young cells. Lead-induced cytotoxicity is higher
than Al-induced cytotoxicity. A mechanism that contributes to cellular
toxicity is membrane       lipid peroxidation; our results demonstrate that
Al(III) and Pb(II) ions, 400 microM, exert an antioxidant-like effect or a
pro-oxidant action on cell membranes depending on exposure time.  We describe
significant increases in TBARS formation associated with the presence of 400
microM Al(III) or Pb(II) salts in the culture media. Our study also revealed
that these heavy metals induce a cell age-dependent action on membrane
lipoperoxidation that is greater in senescent fibroblasts and this could have
severe consequences for maintenance of cellular integrity. 

152
Fullerton A, Menne T.  IN VITRO AND IN VIVO EVALUATION OF THE EFFECT OF
BARRIER GELS IN NICKEL CONTACT ALLERGY.  Contact Dermatitis 1995;32(2):100-6. 

The protective effect of various ethylenediaminetetraacetate (EDTA) barrier
gels on nickel skin penetration was investigated in an in vitro model using
human skin. Application of the gels seemed to cause an increased release of
nickel from nickel alloys. This nickel did not penetrate the skin barrier but
was found to be immobilized on the skin surface. This emphasized the
importance of washing the skin surface to remove any surplus of barrier
formulation after use, since considerable amounts of nickel will be bound in
this formulation. It was found that application of the barrier gels beneath
the nickel alloy in contact with the skin significantly reduced the amount of
nickel found in the epidermal skin layer. In vivo patch testing with a disc of
nickel alloy, with and without use of barrier gel, was performed in 21
nickel-sensitive patients. Patch testing with the nickel alloy without use of
barrier gel resulted in positive patch test reactions in 11/21 (52.4%) of the
patients tested. Application of a Carbopol gel with 10% CaNa2-EDTA beneath the
nickel disc completely abrogated the allergic contact response in  all 21/21
(100%) patients. A Carbopol gel without CaNa2-EDTA was less effective,
inhibiting the response in 15/21 (71.4%). A high concordance was found between
epidermal nickel levels found in vitro and the in vivo patch test. 

153
Farriol M, Mourelle M, Schwartz S.  EFFECT OF VITAMIN C AND VITAMIN E ANALOG
ON AGED FIBROBLASTS. Rev Esp Fisiol 1994;50(4):253-7.

Human dermal fibroblasts were cultured and aged in vitro. Survival of young
and aged fibroblasts was determined in the presence and absence of different
concentrations of two vitamins. Vit C at doses of 5, 12.5, 25 and 50 mumol/L
and water-soluble Vit E (Trolox) at 1, 5, 10 and 50 mg/L, were added 30
minutes before oxidative stress, consisting of exposure to 5 mM hydrogen
peroxide for 30 minutes. A non-radioactive cell proliferation cytotoxicity
assay (MTT) was used to determine the protective effect of the vitamins
studied. Vit C produced a clear cytoprotective effect on aged cells over the
entire range of doses applied. The protection provided by Vit E, was less
pronounced. 

154
Fabreguette A, Zhi Hua S, Lasne F, Damour O. [EVALUATION OF THE CYTOTOXICITY
OF ANTISEPTICS USED IN CURRENT PRACTICE ON CULTURES OF FIBROBLASTS AND
KERATINOCYTES.] Pathol Biol 1994; 42(9):888-92. (Fre)

Infection is the greatest problem in burn patients and topical antiseptics
must be chosen with great care especially when cultured skin is grafted. We
examined the cytotoxicity of 6 antiseptics commonly used on cultured human
fibroblasts and keratinocytes. Cultured cells were exposed for 15 min to
Hibitane (chlorhexidine), Biseptine (chlorhexidine + benzalkonium chloride +
benzylic alcool), dermic Betadine (polvidone iodine + nonoxinol), scrub
Betadine (polyvidone iodine + quaternary ammonium) and gynecologic Betadine
(polyvidone iodine). The cell viability was determined using the MTT test. At
therapeutic concentration all the antiseptics were cytotoxic for fibroblasts
and keratinocytes. The data suggest that the antiseptics must be used in
function of the time of the grafting of the cultured epithelium.

155
Moody RP, Nadeau B, Chu I.  IN VIVO AND IN VITRO DERMAL ABSORPTION OF
BENZO[A]PYRENE IN RAT, GUINEA PIG, HUMAN AND TISSUE-CULTURED SKIN.  J Dermatol
Sci 1995;9(1):48-58. 

 Cross-species in vitro dermal absorption tests were conducted with
14C-labelled benzo[a]pyrene dissolved in acetone and applied to dermatomed
skin (0.5 mm thickness) at comparable dose rates (8-13 micrograms/cm2). Skin
absorption was determined using the Bronaugh in vitro flow-through procedure.
The  percentage (%) dermal absorption included the % 14C-activity detected
persisting in the skin added to that detected in the receiver solution. Listed
in decreasing order, total % in vitro dermal absorption obtained by 48 h
postexposure was: 95 +/- 9.6% (rat), 51 +/- 3.0% (hairless guinea pig), 43 +/-
8.7% (human; 50-year-old), 34 +/-  12.4% (Testskin) and 23 +/- 5.3% (human;
32-year-old). Comparative in vivo studies demonstrated urinary recovery of 8
+/- 1.8% and 25 +/- 5.0% for rats (dose rate: 6 micrograms/cm2) and hairless
guinea pigs (dose rate: 9 micrograms/cm2),  respectively. Total faecal
recovery was 61 +/- 6.0% and 43 +/- 6.1% for rats and guinea pigs,
respectively. Necropsies conducted at 14 days postexposure demonstrated total
14C-activity tissue recoveries of 0.5 +/- 0.13% and 0.6 +/- 0.17% in rats and
guinea pigs, respectively. Including the 14C-activity extracted from the skin
removed from the dose site at 14 days postexposure, the total % in vivo dermal
absorbtion was 70 +/- 7.6% and 68 +/- 9.3% for rats and guinea pigs,
respectively. In summary, the in vitro data was consistent with the in vivo
data in demonstrating that 14C-benzo[a]pyrene was well absorbed through skin. 

156
Silvennoinen-Kassinen S, Vainio O, Karvonen J, Kauppinen M,  Kallioinen M. 
ADHESION MOLECULES IN THE NICKEL ALLERGIC REACTION. Int Arch Allergy Immunol
1995;106(4):345-50.

Nickel is the major cause of allergic contact dermatitis, and to increase our
understanding of this immune reaction we studied changes in the expression of
adhesion molecules on mononuclear cells during nickel stimulation in vivo and
in vitro. Nickel-induced lymphocyte cultures were used in vitro, the cells
being examined with monoclonal antibodies (Mabs) and by flow cytometry.
Mononuclear cells from skin biopsies of in vivo cutaneous nickel reactions
were studied with Mabs and immunohistochemistry. The expression of adhesion
molecules in vitro was differential: the number of cells carrying CD11c, CD29,
CDw49b, CDw49d, CDw49e, CDw49f, CD54, CD56 and ELAM-1 being      significantly
overrepresented among the nickel-induced lymphoblasts whereas the number of
blasts carrying CD44 was underrepresented and those of CD11a, CD18, CD58 and
LAM-1 remained unchanged. CD4+ cells gained adhesion molecules during
nickel-induced blast transformation whereas CD8+ cells lost most of their
adhesion molecules. The in vivo results were in agreement with the in vitro
ones except that CDw49b, CDw49f, CD56 and ELAM-1 could not be detected in a
96-hour nickel reaction in vivo. In conclusion, the nickel allergic reaction
favors the expression of certain adhesion molecules, and this expression is
induced on CD4+ cells while CD8+ cells tend to lose such molecules. The
changes were more sensitively detected with the in vitro method.

157
Burnet NG,  Nyman J,  Turesson I,  Wurm R, Yarnold JR,  Peacock JH.  THE
RELATIONSHIP BETWEEN CELLULAR RADIATION SENSITIVITY AND TISSUE RESPONSE MAY
PROVIDE THE BASIS FOR INDIVIDUALISING RADIOTHERAPY SCHEDULES. Radiother Oncol
1994;33(3):228-38. 

There is a wide variation in normal tissue reactions to radiotherapy and in
many situations the severity of these reactions limits radiotherapy dose.
Clinical fractionation studies carried out in Gothenburg have demonstrated
that a large part of the spectrum of normal tissue reactions is due to
differences in individual normal tissue sensitivity. If this variation in
normal tissue reactions is due to differences in intrinsic cellular
radiosensitivity, it should be possible to predict tissue response based on
measurement of cellular sensitivity. Here we report the initial results of a
study aimed at establishing whether a direct relationship exists between
cellular radiosensitivity and tissue response. Ten fibroblasts strains,
including four duplicates, were established from a group of patients in the
Gothenburg fractionation trials who had received radiotherapy following
mastectomy. Skin doses were measured and both acute and late skin changes were
observed following radiotherapy. Right and left parasternal areas were treated
with different dose fractionation schedules. Clonogenic assays were used to
assess intrinsic cellular radiosensitivity, and all experiments were carried
out without prior knowledge of the clinical response, or which strains were
duplicates. Irradiation was carried o t using 60Co gamma-rays at high
dose-rate (HDR) of 1-2 Gy/min and low dose-rate (LDR) of 1 cGy/min. A spectrum
of sensitivity was seen, with SF2 values of 0.17-0.28 at HDR and 0.25-0.34 at
LDR, and values of D0.01 of  5.07-6.38 Gy at HDR and 6.43-8.12 Gy at LDR.
Comparison of the in vitro results with the clinical normal tissue effects
shows a correlation between cellular  sensitivity and late tissue reactions,
which is highly significant with p = 0.02. A       correlation between
cellular sensitivity and acute effects was noted in the left-sided parasternal
fields, but not the right. This is thought to be coincidental, and without
biological significance. Our results suggest that cellular sensitivity might
form the basis for the development of an assay system capable of predicting
late normal tissue effects to curative radiotherapy, which might allow dose
escalation in some patients. Increased local control and cure, with unchanged
or improved normal  issue complications, could result from such individualised
radiotherapy prescriptions. 

158
Chang SK,  Brownie C,  Riviere JE.  PERCUTANEOUS ABSORPTION OF TOPICAL
PARATHION THROUGH PORCINE SKIN: IN VITRO STUDIES ON THE EFFECT OF
ENVIRONMENTAL PERTURBATIONS.  J Vet Pharmacol Ther 1994;17(6):434-9.

Topical use of pesticides in domestic animals such as swine is a common
practice; however, the effect of environmental factors on the extent of
absorption has not  ceived attention. Since no single factor can exert its
effects alone in the natural environment, the interaction of environmental
factors on the percutaneous absorption of pesticides must be understood before
potential toxicity of dermal absorption of pesticides can be effectively
estimated. In the present studies, the effects of air temperature (Ta),
perfusate temperature (Tp), perfusate flow (F) and relative humidity (%RH) on
absorption of parathion were studied in vitro in porcine skin. Parathion
absorption was determined by measuring radiolabel  appearing in the perfusate
over time. Three main environmental parameters were found to have a
significant effect on parathion penetration. Increasing Ta from       37
degrees C to 42 degrees C, %RH from 60% to 90% or F from 4 ml/h to 8 ml/h each
produced a significant increase in penetration. The following significantly
positive two-way interactions among test parameters were seen: Ta x F and %RH
x F at the 4 micrograms dose, %RH x F at the 40 micrograms dose and Ta x %RH,
Ta x F and %RH x F at the 400 micrograms dose. There were no three-way 
interactions at any of the three doses tested.  These results suggest that the
factors tested are not independent variables and must be considered
interactive when used in assessing pesticide percutaneous absorption.

159
Vock EH, Cantoreggi S, Gupta RC, Lutz WK.  32P-POSTLABELING ANALYSIS OF DNA
ADDUCTS FORMED IN VITRO AND IN RAT SKIN BY METHYLENEDIPHENYL-4,4'-DIISOCYANATE
(MDI). Toxicol Lett 1995;76(1):
17-26. 

The 32P-postlabeling method was adapted for the detection of DNA adducts
formed by methylenediphenyl-4,4'-diisocyanate (MDI). Incubation of the
3'-phosphates of the deoxyribosides of cytosine (C), adenine (A), guanine (G)
and thymine (T) with MDI in Tris buffer resulted in the formation of 5, 7, 8,
and 2 reaction products, respectively. Incubation of DNA with MDI resulted in
detectable levels of 5, 2, and 1 adducts attributable to C, A, and G. Analysis
of DNA isolated from the epidermis of rats treated dermally with 9 mg MDI
showed an adduct pattern similar to the one seen in the in vitro DNA
incubation. A total adduct level of 7 per 10(8) nucleotides was measured, the
limit of detection was 2 adducts per 10(10) nucleotides. The data indicate
that a minute fraction of MDI can reach DNA in vivo in a chemically reactive
form. In comparison with the genotoxic skin carcinogen
7,12-dimethylbenz[a]anthracene on the other hand, the DNA-binding potency of
MDI was more than 1000-fold lower.

160
Liu C, Ho H, Hsieh M, Sokoloski TD, Sheu M.   STUDIES ON THE IN VITRO
PERCUTANEOUS PENETRATION OF INDOMETHACIN FROM GEL SYSTEMS IN HAIRLESS MICE.  J
Pharm Pharmacol 1995;47(5):365-72. 

The influence of co-solvents on the in-vitro percutaneous penetration of
indomethacin from gel systems was studied using a simplex lattice exptl.
design. Gel formulations were prepd. by gelling the vehicle mixt. of water,
either alc. or isopropanol and either propylene glycol or PEG 400 with 1%
wt./wt. Carbomer 940. Hairless mouse skin was employed as the barrier in a
Franz-type diffusion cell. The penetration rates at steady state for seven
formulations were fitted to a polynomial equation based on this simple lattice
method and a three-dimensional plot was constructed. The formulation having
the maximal penetration rate was detd. to be the vehicle with a solvent ratio
of water/alc./propylene glycol equal to 15:33:52, and which possessed a soly.
parameter. For those vehicles with a soly. parameter <15, both the drug soly.
and the penetration rate decreased with a decrease in the soly. parameter.
There was shown to be an approx. 20-fold  increase in the relative enhancement
factor when using both alc. and isopropanol, but only a threefold increase for
both propylene glycol and PEG 400, when compared with water. 

161
Carter WG, Gil SG, Ryan MC. IN VITRO MODEL SYSTEM FOR STUDY OF BASAL (STEM)
CELL FUNCTION INVOLVING FUNCTIONAL EPILIGRIN DETECTION.   PCT Int. Appl.
PATENT NO. 95 06660 03/09/95 (Fred Huchinson Cancer Research Center).

An in vitro epithelial model system where basal (stem) cells may be studied is
presented.  Previous to this work, studies of basal cell interactions with
basement membranes has been complicated due to alterations in structure,
shape, and compn. of basement membranes during development and acquisition of
specialized cellular functions. The assay presented here circumvents these
problems. Epiligrin is the major adhesion ligand present in the epidermal
basement membrane and mediates basal cell adhesion through integrin
alpha3beta1 in focal adhesions and alpha6beta4 in hemidesmosome adhesion      
structures.  CDNA sequences encoding the E170 epithelial ligand are presented
and may be used in the above assay. These sequences are useful in expressions
systems and may be used as diagnostic and therapeutic agents in identifying
and treating patients with the gravis form of junctional epidermolysis bullosa
and other epithelial diseases, including inflammation. Further, methods are
provided for the purifn. and utilization of this glycoprotein and for raising
antibodies against of this complex. Methods for identification of functional
epiligrin in tissues are also given. 

162
Ahmed S, Imai T, Otagiri M.  STEREOSELECTIVE HYDROLYSIS AND PENETRATION OF
PROPRANOLOL PRODRUGS: IN VITRO EVALUATION USING HAIRLESS MOUSE SKIN.  J Pharm
Sci 1995;84(7):877-83.

Stereoselective hydrolysis of two ester prodrugs of propranolol, isovaleryl
propranolol (IV-PL) and cyclopropanoyl propranolol (CP-PL), was studied in
Tris-HCl buffer (pH 7.4) contg. 0.15 M KCl, skin and liver homogenates, 5%
plasma in Tris-HCl buffer, skin cytosol and microsomes, and liver cytosol and
microsomes. The hydrolysis rate consts. of (R)-isomers of the prodrugs were
1.1-30.3 times greater than those of the resp. (S)-isomers in tissue prepns.
Skin showed considerable metabolic activity and very high stereoselectivity
(R/S ratio: 7.3-30.3). The hydrolyzing capacities of buffer and different
tissue prepns. per mg of protein content were in the following increasing
order: buffer < skin homogenate < plasma < liver homogenate. The studies with
microsomes and cytosols       indicated that the esterases, which are
responsible for the hydrolysis of prodrugs, were mainly present in the cytosic
and microsomal fractions of skin and liver, resp. There was a good correlation
between the octanol-buffer partition coeffs. of propranolol and its prodrugs
and the skin partition coeff. In vitro stereoselective penetration of
propranolol and the prodrugs through full-thickness hairless mouse skin was
evaluated with flow-through diffusion cells. Although the concn. of
propranolol was 14-22 times greater than those of the prodrugs in the donor
chamber, the steady-state flux of propranolol isomers [10.72 and 10.64 
ug/cm2.cntdot.h for (R)- and (S)-isomers, resp.] were similar to those of
CP-PL [10.80 and 10.78 mug/cm2.cntdot.h for (R)- and (S)-isomers, resp.] and
even lower than those of IV-PL [14.51 and 14.33 mug/cm2.cntdot.h for (R)- and
(S)-isomers,       resp.].  Moreover, the permeability coeffs. of IV-PL [2.82
.times. 10-3 and 2.78 .times. 10-3 cm/h for (R)- and (S)-isomers, resp.] and
CP-PL (1.29 .times. 10-3 cm/h for each isomer) were 14-30-fold greater than
those of propranolol isomers (0.09 .times. 10-3 cm/h for each isomer). The
diffusion coeffs. of all the compds. were similar, but their solvent membrane
distribution coeffs. differed greatly and proved that the higher permeability
coeffs. of the prodrugs were due to the higher affinity of the prodrugs for
skin. Neither propranolol nor the prodrugs showed stereoselective penetration.
However, highly stereoselective hydrolysis occurred during penetration of the
prodrugs, and the R/S ratios of the cumulative amt. of delivered propranolol
in 12 h were 11 and 13 for IV-PL and CP-PL, resp. A skin irritation test was
performed in Japanese white male rabbits and no irritation was obsd.  In
conclusion, the hairless mouse skin possesses highly stereoselective esterase
activity, and IV-PL and CP-PL might be promising prodrugs for transdermal
delivery of higher amts. of drug from a much lower initial concn. compared
with propranolol.

163
Plate H.  MAGNESIUM SURFACTANTS. CLEANSING AT ITS BEST AND MILDEST.  Parfuem
Kosmet 1995;76(1):28-32. 

The application of Mg contg. surfactants in cosmetics and toiletries for mild
formulations is described.  Comparison of Mg fatty alc. sulfates and ether
sulfates with Na analogous for skin compatibility and application properties
showed a low irritation potential for Mg types of surfactants in several
in-vitro and in-vivo tests. Investigations of cleansing, foaming, and
dermatol. properties showed favorable results for Mg surfactants, which
fulfill all requirements to become a major surfactant of cosmetics and
toiletries. 

164
Turowski A, Skrypzak W, Reng A, Juerges P. [CHARACTERIZATION OF MILD
SURFACTANTS FOR HAIR AND SKIN-CLEANSING PRODUCTS.]  Parfuem Kosmet
1995;76(1):16-27. (Ger)

A reveiw describing environmental friendly mild surfactants in cosmetic
formulations, esp. for hair and skin-cleansing prepns. with less irritation
potential, including alkyl ether sulfates, amide ether sulfates, alkyl
amidopropyl-betaines, alkyl ether carboxylic acids, sulfosuccinic acid ester,
ampho(di)acetate, fatty acid condensation products, and alkyl polyglycosides.
The synthesis route of some of these surfactants are described. The in vitro
methods for evaluation of irritation potentials to skin including
zein/isopropyl myristate protein solubilization testing, red blood cell test,
hemolysis test, Hb denaturing, and hen's egg chloroallantoic membrane test are
described.

165
European Centre for Ecotoxicology and Toxicology of Chemicals. SKIN IRRITATION
AND CORROSION: REFERENCE CHEMICALS DATA BANK. Tech Rep - ECETOC 1995;(66):247
pp.

Earlier ECETOC (Europen Center for Ecotoxicol. and Toxicol. of Chems.) has
published comprehensive listing of in vivo rabbit eye irritation data for 55
readily-available chems. of high purity. The establishment of such a data bank
allows investigators of in vitro or alternative methods to evaluate their own
techniques without the need to carry out in vivo testing of the ref. chems. A
companion data bank has now been developed for 176 chems. for which
comprehensive rabbit skin irritation/corrosion data are available.  No new in
vivo testing has been carried out to qualify a chem. for inclusion in this
list. The 176 chems. selected are readily available at high and consistent
purity and are expected to be stable on storage. They have been tested
undiluted in in vivo studies, excepting those chems. where high concns. of the
substance could be expected to cause severe effects.  The in vivo data have
been generated since 1981 in studies carried out according to OECD Test
Guideline 404 and following the principles of Good Lab. Practice. The data
presented were obtained from tests normally using at least three rabbits,
involving application of 0.5mL (or 0.5g) to the flank under semi-occlusive
patches and in which observations were made at least 24, 48, and 72 h after
application. The chems. represent a range of chem. classes (acids,
acrylates/methacrylates, alcs., aldehydes, alkalis, amides, amines, brominated
derivs., chlorinated solvents, esters, ethers, fatty acids and mixts.
fragrance oils, halogenated aroms., hydrocarbons (unsatd.), inorgs., ketones,
nitriles, phenolic derivs., S-contg. compds., soaps/surfactants,
triglycerides) and different degrees of irritancy. The chems. are ranked for
skin irritation potential on the basis of a 'primary irritation index'. They
should be of use in validation tests of promising alternatives to the in vivo
rabbit skin irritation/corrosion test. This is an essential step in the
progression to regulatory  acceptance. Classification schemes for chems. on
the basis of their skin irritation/corrosion properties are appended to the
report for the convenience of readers. 

166
Lalor CB, Flynn GL, Weiner AN.  FORMULATION FACTORS AFFECTING RELEASE OF DRUG
FROM TOPICAL VEHICLES. II. EFFECT OF SOLUBILITY ON IN VITRO DELIVERY OF A
SERIES OF N-ALKYL P-AMINOBENZOATES.  J Pharm Sci 1995; 84(6):673-6.

The major influence on the rate of drug transfer out of its vehicle and into
the skin is the thermodn. activity of the drug within its formulation. This
study addresses certain thermodn. dependencies of topical delivery in a model
system. Prototypical water-in-oil (W/O) and oil-in-water (O/W) emulsions and
their component phases are used as the test vehicles, polydimethylsiloxane is
the membrane, and three homologous n-alkyl p-aminobenzenzoate esters are the
test permeants. In an emulsion, the interaction of the compd. between the
water and oil phase can det. the extent of lowering of the thermodn. activity
in the external phase in contact with the membrane. The emulsifiers
(surfactants) impact strongly on partitioning and permeation as a result of
the extra solubilizing capacity contributed by the surfactant micelles. The
lower flux in the aq. phase of the O/W emulsion is the result of micellar
solubilization, and this solubilization increased with increasing ester chain
length. Solubilization is also an influence in  nonaq. phases, but permeant
hydrophobicity is without specific influence; therefore, transport becomes
less dependent upon the structure of the compd. 

167
Amdidouche D, Montassier P, Poelman MC, Duchene D.  EVALUATION BY  LASER
DOPPLER VELOCIMETRY OF THE ATTENUATION OF TRETINOIN INDUCED SKIN IRRITATION BY
BETA-CYCLODEXTRIN COMPLEXATION.  
Int J Pharm 1994;111(Oct 20 1994):111-6. 

The release of free tretinoin and its inclusion complex with beta-cyclodextrin
from a dermic gel was studied in vitro using Franz diffusion cells, and skin
irritation induced by the preparations was studied in 9 healthy subjects (mean
age 27 yr) using laser Doppler velocimetry. The in vitro study showed the
release of free drug and its inclusion complex from the gels. Eight h after
beginning the diffusion test, release rates were 10% for included drug and 20%
for free drug. Skin irritation was significantly reduced on complexation. The
attenuation of the irritation induced by including the drug in
beta-cyclodextrin was greater than 85%. It was concluded that laser Doppler
velocimetry may be used to assess skin irritation induced by a complex of
tretinoin and beta-cyclodextrin.

168
Schrader K.  [PHYSIOLOGICAL EVALUATION OF SURFACTANTS IN CONTACT WITH THE
SKIN.] Parfuem Kosmet 1994;75(Feb):68-85. (Ger)

A series of in vivo and in vitro test procedures to guarantee the
physiological compatibility of surfactants and their blends as raw materials
of detergents and cleaning agents with the skin surface are presented; the
results obtained by these methods are also discussed. 

169
Collier SW, Sardon S, Ruiz-Cabello J,  Johnson WA, Schwartz SL, et al. 
MEASUREMENT OF PHARMACODYNAMIC EFFECTS OF DEXAMETHASONE ON EPIDERMIS BY
PHOSPHORUS NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY IN VITRO.  J Pharm Sci
1994;83(Sep):1339-44. 

To assess topical corticosteroid bioequivalence, pharmacodynamic effects of
varying doses of dexamethasone acetate (dexamethasone-21-acetate) on perfused,
intact, viable swine epidermis were evaluated by measuring intracellular
phosphorus metabolism with nuclear magnetic resonance spectrometry.
Dexamethasone perfusion resulted in a biphasic effect on phosphomonoester
metabolism and a dose-dependent decrease in phosphocreatinine and nucleotide
triphosphate levels. In addition, log-linear relationship was observed between
dexamethasone dose and a decrease in phosphocreatinine. It was concluded that
these techniques may be useful for elucidating mechanisms of corticosteroid
action on the skin and may serve as a basis for developing a relevant
bioequivalence technique. 

170
Reifenrath WG, Lee B, Wilson DR, Spencer TS.  COMPARISON OF IN VITRO
SKIN-PENETRATION CELLS.  J Pharm Sci 1994;83(Sep):1229-33. 

A low volume flow-through diffusion cell that determines penetrant flux across
skin while minimizing the dilution of penetrant in receptor fluid and
eliminating the need for magnetic stirring was compared to a high volume flow
cell and  a magnetically stirred, manually sampled static cell with
hydrophilic and lipophilic penetrants. Results indicated that percutaneous
absorption and residue of tested drug did not differ between the low volume
and high volume diffusion cells. In addition, low volume diffusion flux
profiles accurately represented those of the static cell.

171
Su MH,  Srinivasan V,  Ghanem AH,  Higuchi WI.  QUANTITATIVE IN VIVO
IONTOPHORETIC STUDIES.  J Pharm Sci 1994;83(Jan):12-7. 

An experimental method was developed to evaluate in vitro-in vivo correlations
in iontophoretic drug delivery and the method was used to examine the release
of       tetraethylammonium bromide (tetrylammonium bromide) from an
iontophoretic device in vitro and its iontophoretic delivery in mice with and
without permeation enhancers. Good in vitro-in vivo correlation was found when
the dominant barrier to transport was the same in both situations. Blood
levels could be predicted using permeability coefficients and elimination
parameters, provided that iontophoretic flux was the same at the same current
density, skin metabolism and adsorption effects were insignificant, and
elimination kinetics were not significantly affected. It was concluded that
the method may be used to evaluate the efficacy of a permeation enhancer used
with iontophoresis. 

172
Barthes D, Bosset M, Rochard E, Courtois P.  EFFECT OF MICROFLORA ON THE IN
VITRO ALTERATION OF HYDROCORTISONE 17-BUTYRATE ON HEALTHY OR PSORIATIC SKIN:
HPLC DETERMINATION.  J Pharm Clin 1994;13(1):50-5. 

An HPLC method was used to determine the effect of microflora on the in vitro
alteration of hydrocortisone butyrate (hydrocortisone 17-butyrate; Locoid) on
healthy and on psoriatic skin. The skin induced transformation of
hydrocortisone butyrate into hydrocortisone at 37DGC, which was probably due
to biological alteration from bacteria associated with skin microflora. In
comparison with healthy skin, the increase in the number of bacteria such as
Klebsiella and Streptococcus A has been observed with psoriatic skin.
Therefore, it was concluded that a skin damaged by psoriasis may alter the
hydrocortisone butyrate faster and in a larger amount than healthy skin. 

173
Conti B,  Puglisi G, Ventura CA,  Giunchedi P,  Cutuli V,  et al.   TOLMETIN
POLY-D,L-LACTIDE MICROSPHERES: IN VITRO/IN VIVO EVALUATION.   STP Pharma Sci
1994;4(4):269-74. 

Poly-D,L-lactide microspheres containing tolmetin were prepared by
emulsification solvent evaporation or by spray drying and the microspheres
were evaluated in vitro and in rats. Spherical shaped particles with a size of
3.5-7.5 mum and drug content of 4.5-9.5% were obtained. The dissolution of
microspheres was dependent on the method of preparation, with drug release
being slower from spray-dried microspheres. Biological evaluation showed the
suitability of a microparticulate syste for chronic administration of the
drug. The anti-inflammatory effect of tolmetin-loaded microspheres in rat
models of acute and chronic inflammation was comparable to that of free drug,
but significantly sustained. It was concluded that poly-D,L-lactide
microspheres can be used for the sustained release of tolmetin.

174
Kobayashi D,  Matsuzawa T,  Sugibayashi K, Morimoto Y,  Kimura M.  ANALYSIS OF
THE COMBINED EFFECT OF l-MENTHOL AND ETHANOL AS SKIN PERMEATION ENHANCERS
BASED ON A TWO-LAYER SKIN MODEL.  Pharm Res 1994;11(Jan ):96-103. 

The effects of l-menthol ((-)-menthol) combined with ethyl alcohol (ethanol)
on the percutaneous absorption of model drugs were studied using 2 equations
and a 2-layer in vitro skin model. A nonlinear least-squares method was
employed to determine 6 coefficients using the 2 equations and experimentally
obtained permeability coefficient through full-thickness skin and
full-thickness skin/vehicle concentration ratio. Adding menthol to water and
40% ethyl alcohol increased  the diffusion coefficient of drugs in lipid and
pore pathways of stratum corneum. Adding ethyl alcohol to water and 5% menthol
increased drug solubility in the vehicle, decreased skin polarity, and
increased the role of the pore pathway to whole-skin permeation. It was
concluded that the effects of l-menthol and ethyl alcohol on percutaneous drug
absorption in vitro are synergistic.


GENOTOXICITY/MUTAGENESIS
175
Ronis MJ, Badger TM. TOXIC INTERACTIONS BETWEEN FUNGICIDES THAT INHIBIT
ERGOSTEROL BIOSYNTHESIS AND PHYSPHOROTHIOATE INSECTICIDES IN THE MALE RAT AND
BOBWHITE QUAIL (COLINUS VIRGINIANUS). Toxicol Appl Pharmacol
1995;130(2):221-8.

The potential for toxic interactions between ergosterol
biosynthesis-inhibiting      fungicides (EBIFs), used in U.S. agriculture or
clinically, and phosphorothioate insecticides was assessed in adult male rats
and adult male bobwhite quail (Colinus virginianus) by measuring inhibition of
plasma butyryl cholinesterase (BChE) following fungicide and insecticide
treatment. Male Sprague-Dawley rats (300 g) were administered corn oil or the
following EBIFs: propiconazole (400 mg/kg/day), vinclozolin  (400 mg/kg/day),
clotrimazole (100 mg/kg/day), or ketoconazole (100 mg/kg/day) for 3 days by
oral gavage. Forty-eight hours following the final dose, a single bolus of
parathion (0.4 mg/kg in corn oil) or malathion (150 mg/kg in corn oil) or corn
oil alone was given po. The rats were terminated 12 hr following parathion or
4 hr following malathion dosing. Significant (p < 0.05) inhibition of  BChE
was observed with parathion and malathion only following clotrimazole
treatment. In contrast, when  a similar experiment was performed in bobwhite
quail dosed with 12 mg/kg malathion following EBIF treatment, significant BChE
inhibition was observed following treatment with vinclozolin or ketoconazole,
but not with propiconazole or clotrimazole. Induction of cytochrome P450 in
rat and quail liver by EBIFs was accompanied by enhanced oxidative
desulfuration of malathion, parathion, and diazinon to toxic oxon products.
Increased detoxication via oxidative dearylation/esterolytic cleavage also 
occurred. However, while enhanced acute in vivo insecticide toxicity was
observed in both species with a number of EBIF-phosphorothioate combinations,
EBIF-induced oxidative activation of phosphorothioates by liver  microsomes in
vitro was not a good predictor of this effect.

176
Uuskula M, Jarventaus H, Hirvonen A, Sorsa M, Norppa H.  INFLUENCE OF GSTM1
GENOTYPE ON SISTER CHROMATID EXCHANGE INDUCTION BY STYRENE-7,8-OXIDE AND
1,2-EPOXY-3-BUTENE IN CULTURED HUMAN LYMPHOCYTES. Carcinogenesis
1995;16(4):947-50.

Glutathione S-transferase M1 (GSTM1), catalyzing the conjugation of various
reactive molecules with glutathione (GSH), shows genetic polymorphism in
humans. Almost half of all Caucasians lack the GSTM1 gene, being theoretically
at a higher risk from the toxic effects of substrates for GSTM1. The purpose
of the present study was to investigate whether the GSTM1 genotype of
lymphocyte donors influences the in vitro induction of sister chromatid
exchanges (SCEs) by styrene-7,8-oxide (SO) and  1,2-epoxy-3-butene (MEB), the
epoxide metabolites of styrene and butadiene respectively and potential
substrates for GSTM1. SCEs induced after a 48 h treatment (started 24 h after
culture initiation) by two different concentrations of SO (50 and 150 muM) and
MEB (50 and 250 muM) were analyzed in cultured (72 h) lymphocytes of six GSTM1
null (gene deleted) and six GSTM1-positive (gene present) donors. Both SO and
MEB were found to clearly increase SCEs. The GSTM1 genotype had no influence
on SCE  induction by SO. In contrast, MEB produced a higher level of SCEs
among the GSTM1 null than GSTM1-positive samples. At 250 muM MEB, the GSTM1
null donors showed 31 % more induced SCEs (on average seven more SCEs per
cell) than the GSTM1-positive donors (P = 0.02, acetone treatment as the
reference). Furthermore, the GSTM1 null genotype was associated with a slight
decrease in mitotic index and replication index, regardless of the treatment.
The results suggest that GSTM1-mediated GSH conjugation is an important
detoxification pathway for MEB, but not for SO, in cultured human lymphocytes.

177
Zeisig M, Moller L. 32P-HPLC SUITABLE FOR CHARACTERIZATION OF DNA ADDUCTS
FORMED IN VITRO BY POLYCYCLIC AROMATIC HYDROCARBONS AND DERIVATIVES.
Carcinogenesis 1995;16(1):1-9.

Analysis of DNA adducts demands both high sensitivity and good resolution. A
high-performance liquid chromatography method for 32P-postlabeled DNA adducts
(32P-HPLC) was used to investigate DNA adduct formation from 38 polycyclic
hydrocarbons and biphenyls in vitro. The 32P-HPLC method proved to be useful
for separation, detection and characterization of DNA adducts from most of the
substances. The in vitro method used to form the DNA adducts, with calf thymus
DNA, nucleotide 3'phosphates  and metabolic activation through S-9 liver
homogenate, gave poor quantitative reproducibility. However, the results
showed that the 32P-HPLC method was suitable for characterizing DNA adducts
from many substances. From 35 of the tested substances 365 DNA and nucleotide
3'-phospate adducts were detected and characterized concerning retention
times. Of the adducts, 171 were detected in DNA and 39 of them from five
substances were characterized concerning target nucleotides. The retention
time library built can be used in future analyses of DNA with complex patterns
of DNA adducts.

178
Naji-Ali F, Hasspieler BM, Haffner D, Adeli K. HUMAN BIOASSAYS TO ASSESS
ENVIRONMENTAL GENOTOXICITY: DEVELOPMENT OF A DNA REPAIR ASSAY IN HepG2 CELLS.
Clin Biochem 1994;27(6):441-8.

A direct assessment of the effects of environmental chemicals on human health
has been hampered by the lack of suitable experimental systems. We have
recently employed a human liver cell line (HepG2) to assess the biological
effects of pollutants at both cellular and DNA levels. A Neutral Red dye
uptake assay was used to assess potential cytotoxic effects of xenobiotics.
DNA damage was quantified using an unscheduled DNA synthesis assay that
measures repair that is induced following exposure to genotoxic compounds.
HepG2 cells responded to the known mutagens, 4-nitroquinoline N-oxide and
methylmethane sulfonate, both in the Neutral Red assay for cytotoxicity and
two DNA repair assays for genotoxicity (monitored autoradiographically or by
liquid scintillation counting). The HepG2 DNA repair and cytotoxicity assays
also responded to an extract (containing polycyclic aromatic hydrocarbons) of
sediment obtained from a polluted site in the Great Lakes. Results indicate
that this system can be deployed further to assess potential cyto- and
genotoxicity of pollutants. The development of human cell culture assays is a
critical step towards a full assessment of the risk that such pollutants pose
to human health.

179
Mackay JM, Fox V, Griffiths K, Fox DA, Howard CA, Coutts C, Wyatt I, Styles
JA.  TRICHLOROACETIC ACID: INVESTIGATION INTO THE MECHANISM OF CHROMOSOMAL
DAMAGE IN THE IN VITRO HUMAN LYMPHOCYTE CYTOGENETIC ASSAY AND THE MOUSE BONE
MARROW MICRONUCLEUS TEST. Carcinogenesis 1995;16(5):1127-33.

Trichloroacetic acid (TCA) was tested for its ability to induce chromosomal
damage in cultured human peripheral blood lymphocytes and in bone marrow cells
of male and female C57BL/6JfBL10/Alpk mice. Two in vitro cytogenetic assays
were conducted with TCA. In the first TCA, as free acid, was added to whole
blood cultures at final concentrations of 500, 2000 and 3500 micrograms/ml in
the presence and absence of an auxiliary metabolic activation system (rat
liver S9-mix). Statistically significant increases in the percentage of
aberrant cells compared with solvent control values were observed in cultures
treated with TCA at 2000 and 5000 mu/ml. Investigation into the effects of TCA
on the pH of the culture medium revealed significant reductions in pH at both
these TCA concentrations. Neutralized TCA was then tested at concentrations of
500, 2,000 and 5000 micrograms/ml, also in the presence and absence of S9-mix.
No statistically or biologically significant increases in the percentage of
aberrant cells were observed in any of these cultures. In the mouse
micronucleus test, neutralized TCA was administered in two equal
intraperitoneal doses 24 h apart to C57BL/6JfBL10/Alpk mice (337, 675 and 1080
mg/kg in males; 405, 810 and 1300mg/kg in females). These dose levels
represent 25%, 50% and 80% of the median lethal dose (MLD) in this strain of
mouse. Bone marrow samples were taken 6 and 24 h after the second dose and the
chromosomal damage assessed by analysis of the bone marrow for micronuclei. No
statistically or biologically significant increases in the incidence of
micronucleated polychromatic erythrocytes compared with the solvent control
dosed animals were observed in either sex at the 6 h sampling time or in the
females at the 24 h sampling time. A small but statistically significant
increase in micronucleated polychromatic erythrocytes was observed in male
mice 24 h after a dose of 675 mg/kg (50% MLD). Since no increases were noted
at the 25 or 80% MLD, and the levels recorded are within the range of the
concurrent solvent control values, the small increase observed in the males at
the 50%  LD is considered not to be biologically significant. Flow cytometric
studies on suspensions of isolated liver cell nuclei revealed that changes in
FITC binding (indicating altered chromatin conformation) were induced by pH
changes alone and were not caused by neutralized TCA.(ABSTRACT TRUNCATED AT
400 WORDS)

180
Burgeot T, His E, Galgani F. THE MICRONUCLEUS ASSAY IN CRASSOSTREA GIGAS FOR
THE DETECTION OF SEAWATER GENOTOXICITY. Mutat Res 1995;342(3-4):125-40.

The micronucleus (MN) test was performed in vivo and in vitro on the oyster
Crassostrea gigas to evaluate the genotoxic effect of the marine environment.
In vitro tests were carried out on adult and young (spat) specimens exposed to
benzo[a]pyrene (BaP: 0.5, 5, 500 and 1000 micrograms.l-1) and an effluent (5,
50, 75 and 100%) of Seine Bay, one of the most highly contaminated sites in
France. MN frequency observed after 48 h exposure to the two pollutants was
much greater in adults than spats. A preliminary test of the genotoxic effect
of BaP (0.05, 0.5, 1 and 500 micrograms.l-1), cupric sulfate (10, 25, 50 and
100 micrograms.l-1) and a paper mill effluent (1, 3, 10 and 30 mg.l-1) was
performed in C. gigas heart cells cultured for 6 days. Comparison of the MN
assay with the C. gigas larva test showed the clastogenic action of BaP and
the toxic effect of cupric sulfate on culture cells as well as the slighter
toxic effect of paper mill effluent on spats. An in vivo study was conducted
in an oyster-farming area contaminated by cadmium and copper. MN frequency was
not very sensitive to a pollution gradient but showed high interindividual
variability. The absence of precise criteria for MN identification in mollusks
and the identification of highly basophilic spherical inclusions in the
cytoplasm of gill tissue hemocytes in oysters during viral infection are
handicap for application of the micronuclei assay in the marine environment.
Another limitation of the assay is the particularly onerous requirement for
manual observation. Optimization of the assay by automated analysis is
necessary but can only be achieved if cytologic preparations are of good
quality.

181
Gollapudi BB, Linscombe VA, Wilmer JW. CLASTOGENICITY OF ISOAMYLENE OXIDE TO
RAT LYMPHOCYTES IN CULTURE. Mutat Res 1995;347(1):9-12.

The mutagenic activity of the aliphatic epoxide isoamylene oxide
(2-methyl-2,3-epoxybutane) is not readily detectable in the standard Ames
test. In this study, the clastogenic potential of isoamylene oxide was
evaluated using an in vitro mammalian cell culture system. Approximately 48 h
after establishing primary cultures of rat lymphocyte cultures, the cells were
treated for 4 h with various concentrations of isoamylene oxide (50, 166.7,
500,, 1666.7, and 5000 micrograms/ml in the initial assay and 500, 1000, 2000,
3000, 4000, and 5000 micrograms/ml in the confirmatory assay). The cultures
were harvested 24 h after termination of the treatment. Based upon the mitotic
indices, cultures treated with the three highest concentrations in both the
initial and confirmatory assays were evaluated to estimate the chromosomal
aberration frequencies. Isoamylene oxide demonstrated a strong clastogenic
activity in this assay: up to 29% aberrant cells (without gaps) were observed
at the highest concentration analyzed. The presence of an external metabolic
activation system (S9) did not seem to influence the magnitude of the response
at the dose levels analyzed.

182
Foellmann W, Hillebrand IE, Creppy EE, Bolt HM. SISTER CHROMATID EXCHANGE
FREQUENCY IN CULTURED ISOLATED PORCINE URINARY BLADDER EPITHELIAL CELLS
(PUBEC) TREATED WITH OCHRATOXIN A AND ALPHA. Arch Toxicol 1995;69(4):280-6.

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha
(OT-alpha) were investigated, to examine their potency to induce sister
chromatid exchanges (SCE) in cultured porcine urinary bladder epithelial cells
(PUBEC) (primary culture). Serum-free cultured PUBEC were incubated for 5 h
with either OTA or OT-alpha, respectively, and subsequently cultured in the
presence of 5-bromo-2-deoxyuridine (BrdU). After two cell cycles, mitosis was
inhibited by the colchicine derivative Colcemid,  cells were fixed and
chromosomes were prepared for SCE analysis. For OTA, a dose-dependent increase
in SCE frequency was measured in concentrations between 100 pM and 100 nM OTA.
At 100 nM OTA, SCE frequency increased by about 41%, compared to the base SCE
level (7.27 SCEs per chromosome set, solvent control). Higher concentrations
of OTA were cytotoxic. The metabolite OT-alpha also increased SCE  frequency,
but at higher concentrations. At a concentration of 10 muM OT-alpha, an
increase of  about 55% was detected. OT-alpha showed no cytotoxic effect.
These results indicate that OTA is genotoxic in this in vitro system, which
represents the urinary bladder epithelium, a target organ of OTA in vivo. It
could also be shown that OT-alpha, which is said to be non-toxic, is genotoxic
in this assay at higher concentrations.

183
Mao B, Xu J, Li B, Margulis LA, Smirnov S, Ya NQ, Courtney SH, Geacintov NE.
SYNTHESIS AND CHARACTERIZATION OF COVALENT ADDUCTS DERIVED FROM THE BINDING OF
BENZO(A)PYRENE DIOL EPOXIDE TO A-GGG- SEQUENCE IN A DEOXYOLIGONUCLEOTIDE.
Carcinogenesis 1995;16(2):357-65.

Direct synthesis and purification procedures are described for the preparation
of adducts derived from the covalent binding of 
7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene ((+)-anti-BPDE
or (+)-BPDE 2) to each of the three guanine residues (trans-N2-dG lesions) in
the oligodeoxyribonucleotide    d(CTATG1G2G3TATC). The positions of the
modified Gs are defined by Maxam-Gilbert sequencing techniques. Six different
oligonucleotides with one or two precisely positioned (+)-anti-BPDE residues
are identified. The absorbance, circular dichroism and fluorescence
characteristics are changed upon formation of duplexes with the complementary
strands d(GATACCCATAG). In the doubly-modified oligonucleotides, a  broad,
excimer-like long wavelength fluorescence emission band is observed with a
maximum near 455 nm only if the two (+)-anti-BPDE-modified Gs are adjacent to
one another. The covalently attached (+)-anti-BPDE residues decrease the
thermodynamic stabilities of  the duplexes; their melting points are markedly
dependent on the position of the lesions, being highest with the (+)-anti-
BPDE residue at G1 (Tm = 40~C, only 2~C lower than in the case of the
unmodified oligonucleotide) and lowest when it is situated at G3 (Tm = 29~C).
The implications of these and other physical characteristics are discussed.
The facile synthesis of these or similar site-specific and stereochemically
defined (+)-trans-anti-BPDE-N2-dG lesions in runs of contiguous guanines in 
oligodeoxyribo-nucleotides of specified base sequence should be useful for the
design of site-directed mutagenesis studies in vitro and in vivo. 

184
Morrison V, Ashby J. HIGH RESOLUTION RODENT BONE MARROW MICRONUCLEUS ASSAYS OF
1,2-DIMETHYLHYDRAZINE: IMPLICATION OF SYSTEMIC TOXICITY AND INDIVIDUAL
RESPONDERS.  Mutagenesis 1995; 10(2):129-35.

1,2-Dimethylhydrazine (DMH) is confirmed as active in male CBA mouse bone
marrow micronucleus (MN) assays conducted at non-toxic dose-levels between 25
and 45 mg/kg. Previous reports of the activity of DMH in rodent bone marrow MN
assays are reviewed. By the design of the present experiments it has been
possible to confirm the advice offered by the United Kingdom Environmental
Mutagen Society (UKEMS) that once 2000 polychromatic erythrocytes have been
assessed per animal, the use of larger  animal group sizes becomes the
critical means to increase assay sensitivity. Also, the use of historical
control data is shown to enhance assay sensitivity at low dose-levels by
enabling recognition of individual animals responding to the test chemical.
These outliers are seen before the group mean MPE value has been increased
enough to attain group statistical significance. It is concluded that careful
consideration of test data, within the context of concurrent and historical
control data, is sufficient to discern activity in the assay.

185
Bolcsfoldi G. THE DNA ALKALINE UNWINDING GENOTOXICITY TEST.  Methods Mol Biol
1995;43:257-66.

186
Zhu AX, Zhao Y, Moller DE, Flier JS.  CLONING AND CHARACTERIZATION OF p97MAPK,
A NOVEL HUMAN HOMOLOG OF RAT ERK-3.  Mol Cell Biol 1994; 14(12):8208-11.

Mitogen-activated protein kinases, or extracellular signal-regulated kinases
(ERKs), are serine/threonine protein kinases that are activated in response to
a wide variety of extracellular stimuli and are encoded by a multi-gene
family.  Little is known about the function of the ERK-3 subfamily.  To
explore the mol. diversity of the ERK-3 subfamily, the authors isolated a
novel human cDNA, designated Hu-ERK-3, from a fetal skeletal muscle library. 
Anal. of the complete 3,920-bp nucleotide sequence revealed that this clone
encodes a predicted protein of 720 amino acids.  In vitro
transcription-translation generates a 97-kDa protein referred to as p97MAPK.
Of all of the sequences compared, p97MARK is the most homologous to rat ERK-3.
Interestingly, although p97MAPK is highly (98%) homologous to ERK-3 at the
amino acid level within the N-terminal two-thirds of the coding region, it
diverges at the carboxyl terminus as a result of a unique extension of 178
amino acids.  Although expression of p97MAPK was detected in all of the
tissues tested by Northern (RNA) anal., the most abundant expression was seen
in skeletal muscle.  An antibody in an immune complex protein kinase assay,
the authors have shown that treatment of human fibroblasts with serum or
phorbol esters activates a myelin basic protein and histone H1 kinase activity
in immunoppts. P97MAPK appears to be the human homolog of rat ERK-3, and a
member of this family is an active protein kinase. 

187
Hasspieler BM, Ali FN, Alipour M, Haffner GD, Adeli K. HUMAN BIOASSAYS TO
ASSESS ENVIRONMENTAL GENOTOXICITY: DEVELOPMENT OF A DNA BREAK BIOASSAY IN
HepG2 CELLS. Clin Biochem 1995;28(2):113-6.

An in vitro assay was developed for the quantification of genotoxicity,
monitored as DNA single-strand breaks (SSB), in the HepG2 human hepatoma cell
line.  This assay procedure, which is based upon alk. unwinding and
hydroxylapatite DNA chromatog., is both rapid and simple to perform.  HepG2
cells responded to the std. mutagen, 4-nitroquinoline N-oxide, demonstrating
SSB formation at concns. >0.1 mumol/L.  Phenanthrene-9,10-quinone, a component
of diesel exhaust, mediated SSB formation at concns. >250 nmol/L.  Finally, an
ext. of contaminated sediment from the Great Lakes Basin mediated SSB
formation in a dose-dependent manner.  These results illustrate the utility of
this human genotoxicity assay for future use in screening of environmental
pollutants.

188
Gu Z. [IN VITRO MICRONUCLEUS ASSAY IN BALB/C-3T3 CELLS]. Gongye Weisheng Yu
Zhiyebing 1995; 20(4):204-6. (Chi)

The genotoxicity of benzo(a)pyrene, cyclophosphamide, 2-aminoanthracene,
2-nitrofluorene, nitrosated coal-dust exts., and cigaret-smoke condensate were
tested with the in vitro micronucleus assay using an established BALB/c-3T3
mammalian cell line.  The results showed that all chems. and complex mixts.
studied induced micronuclei in cells tested. These results indicate that
BALB/c-3T3 cells are capable of activating certain promutagens and
procarcinogens. It seems, therefore, that the micronucleus assay in BALB/c-3T3
cells without an exogenous activation system may be useful as an in vitro
assay for detecting genotoxicity.

189
Gollapudi BB, Mendrala AL, Linscombe VA. EVALUATION OF THE GENETIC TOXICITY OF
THE ORGANOPHOSPHATE INSECTICIDE CHLORPYRIFOS.  Mutat Res 1995;342(1-2):25-36.

The genetic toxicity of chlorpyrifos
(O,O,-diethyl-O-(3,5,6-trichloro-2-pyridinyl)phosphorothioate, C.A.S. Number:
2921-88-2)), an organophosphate insecticide, was examined by employing several
end points such as gene mutations in bacteria (Ames test) and mammalian cell
cultures (CHO/HGPRT assay), cytogenetic abnormalities in mammalian cells both
in vitro (rat lymphocyte chromosomal aberration test, RLCAT) and in vivo
(mouse bone marrow micronucleus test) and induction of DNA damage and repair
in rat hepatocytes in vitro. There was no indication of genotoxic activity for
chlorpyrifos in any of these assays. These results are consistent with the
reported lack of carcinogenic potential for chlorpyrifos in both mice and
rats. 

190
Bond JA, Recio L, Andjelkovish D. EPIDEMIOLOGICAL AND MECHANISTIC DATA SUGGEST
THAT 1,3-BUTADIENE WILL NOT BE CARCINOGENIC TO HUMANS AT EXPOSURES LIKELY TO
BE ENCOUNTERED IN THE ENVIRONMENT OR WORKPLACE. Carcinogenesis
1995;16(2):165-71.

1,3-Butadiene (BD) is a carcinogen in both rats and mice with mice being
substantially more sensitive than rats. It is not known if BD poses a
carcinogenic risk for humans. Findings from exposure assessment studies
indicate that potential industrial exposure to BD in monomer, polymer, and
end-user industries is typically <2 p.p.m. Epidemiologic studies of persons
occupationally exposed to BD are inconclusive. In vitro metabolism of BD in
rats, mice and human tissues indicate that there are  significant quantitative
species differences in the metabolic activation of BD to butadiene monoepoxide
(BMO) and butadiene diepoxide (BDE) and the detoxication of BMO.
Activation/detoxication ratios calculated using in vitro kinetic constants
reveal that ratios in mice were 12-fold greater than rats and humans. In rats
and mice exposed to BD, concentrations of BMO in blood and tissues of mice
were up to 14-fold higher than in rats and BDE was only detected in mice
thereby providing a strong  argument for why mice are highly sensitive to BD
carcinogenicity. The fact that human tissues do not appear to metabolize BMO
to BDE to any significant extent suggest that humans may not be sensitive to
BD carcinogenicity.  In mice, BDE is a more potent carcinogen than BMO. BDE is
mutagenic in vitro at the hprt locus in human TK6 lymphoblasts at
concentrations that were 100-fold less than the concentration of BMO required
to yield a similar mutation frequency. Importantly, the concentrations of  BDE
that were genotoxic in vitro are nearly identical to the concentrations of BDE
measured in blood and tissues of mice exposed to BD by inhalation. BD is
genotoxic in mice, but not rats, following inhalation exposure and this is
paralleled by species differences in observed tumor susceptibility. BD is not
genotoxic in occupationally-exposed workers. The genetic basis for BD
carcinogenicity appears to be primarily through induction of point mutations
and deletion events mediated via the potent  genotoxic metabolite, BDE. The
genotoxic endpoints induced by BDE (e.g., deletion and point mutations) rather
than BMO (e.g., point mutations) likely represent the underlying mechanism
responsible for the striking species differences observed in the genotoxicity
and carcinogenicity of BD in mice versus rats. In summary, the preponderance
of evidence which includes both epidemiological and mechanistic data in mice,
rats, and humans strongly suggests that BD will not be carcinogenic to humans
at  occupational or environmental exposures. Any cancer risk assessment for BD
should use in vitro human tissue metabolic data and in vitro and in vivo rat
data for estimation of human cancer risks.

191
Yamasaki H.  NON-GENOTOXIC MECHANISMS OF CARCINOGENESIS: STUDIES OF CELL
TRANSFORMATION AND GAP JUNCTIONAL INTERCELLULAR COMMUNICATION. Toxicol Lett
1995;77(1-3):55-61. 

It is widely accepted that a series of genetic changes accumulate during
carcinogenesis. In addition, it is likely that various non-genotoxic
mechanisms also operate at different stages of carcinogenesis. It is even
possible that non-genotoxic mechanisms indirectly generate genetic changes,
e.g., through induction of cell proliferation, active oxygen species or
cytosine methylation. This may partially explain why many carcinogens are
devoid of activity when tested in the usual genetic toxicology assays. In
vitro cell transformation mimics certain stages of in vivo carcinogenesis. It
has therefore been proposed that both genotoxic and non-genotoxic aspects of
carcinogenesis can be studied in cell transformation systems, with tumor
formation by transformed cells in syngenic animals or nude mice as the
endpoint. Many genotoxic as well as non-genotoxic carcinogens induce
transformation of Syrian hamster embryo, murine Balb/c 3T3 and murine
C3H10T1/2 cells; interaction of genotoxic and non-genotoxic mechanisms can be
clearly seen in 2-stage cell transformation studies in which a genotoxic
initiating agent and a non-genotoxic promoting agent act synergistically to
induce transformation of rodent cells. Aberrant control of gap junctional
intercellular communication (GJIC) in cell transformation and carcinogenesis
is well documented. Possible genotoxic as well as non-genotoxic mechanisms
involved in abnormal gap junction communication control in multistage
carcinogenesis are discussed. 

192
Shaw G, Connell D, Barron W. THE USE OF IN VITRO DNA ADDUCT FORMATION TO
ESTIMATE THE GENOTOXICITY OF RESIDUES AT CONTAMINATED SITES. Chemosphere
1995;30(10):1957-68.

Genotoxic carcinogens such as polycyclic aromatic hydrocarbons (PAHs)
covalently bind to the bases in DNA to form adducts. The formation of DNA
adducts is significant with respect to chemical carcinogenesis. Many
contaminated sites contain quantities of carcinogens such as PAHs, and the
evaluation of the genotoxicity of these soils has important implications for
human risk  assessment. DNA adducts can be formed using an in vitro system
incorporating extracts from contaminated soils. The 32P-postlabelling assay is
a sensitive technique for the detection of DNA adducts from complex mixtures
of environmental carcinogens. These techniques have been used to form and
detect DNA adducts using soils from a number of coal gasworks sites. The
results show that the extent of adduct formation depends partially on the
petroleum hydrocarbon content of samples, but also on other undetermined
factors related to composition. While environmental weathering has been shown
to effect the PAH composition of samples, this is not an important factor in
controlling the genotoxicity of samples as estimated by DNA adduct formation. 

193
Santambien P, Sdiqui S, Hubert E, Girot P, Roche AC, Monsigny M, Boschetti E.
IN VITRO TOXICITY ASSAYS FOR DYE LIGANDS USED IN AFFINITY CHROMATOGRAPHY. J
Chromatogr B Biomed Appl 1995;664(1):241-6.

Some reactive textile dyes have been used for years as biomimetic ligands in
protein purification. There has been reluctance, however, to use these dyes on
a large scale for therapeutically applicable proteins for fear of possible dye
leakage and consequent contamination. Therefore, toxicological data are
necessary to quantify the level of this hazard. This study deals with a series
of in vitro toxicity investigations with eukaryotic cells (growth, polyploidy,
etc.) and with prokaryotic cells (Escherichia coli) for genotoxic studies.
Both approaches demonstrated a lack of or slight toxicity for Reactive Blue 2
and Reactive Red 120 and their derivatives over the range 10-62.5
micrograms/ml in several assays.

194
Parry EM, Henderson L, Mackay JM. PROCEDURES FOR THE DETECTION OF CHEMICALLY
INDUCED ANEUPLOIDY: RECOMMENDIATIONS OF A UK ENVIRONMENTAL MUTAGEN SOCIETY
WORKING GROUP.  Mutagenesis 1995;10(1):1-14.

The development of assays to detect numerical chromosome aberrations has not
kept pace with that for assays used to detect other genotoxicity endpoints
such as gene mutations and structural chromosome aberrations, even though the
importance of aneuploidy in relation to heritable defects in germ cells and to
carcinogenesis in somatic cells is acknowledged. Regulatory bodies at present
have no formal requirements concerning aneuploidy detection and decisions are
made on a case-by-case basis. The aim of this review is to indicate which
assays are available for the detection of chemically induced aneuploidy and
what aspects should be taken into account when testing for chemically induced
aneuploidy using in vitro, in vivo somatic and in vivo germ cell assays
without dictating exact protocols. Our recommendations concentrate on systems
that, to date, have been most extensively used and we indicate where future
developments may lie. It is important that the currently available and future
tests for chemically induced aneuploidy should be adequately validated before
being implemented into screening strategies or regulatory guidelines. This
requirement has not yet been met and is confounded by the lack of a well
defined reference database of animal and human chemical aneugens.

195
Tateno H, Kamiguchi Y. APPLICATION OF CRYOPRESERVED GOLDEN HAMSTER OOCYTES TO
IN VITRO GENOTOXICITY ASSAYS FOR HUMAN SPERM CHROMOSOMES. Environ Mol Mutagen
1995;25(3):263-5.

196
Smith ML, Chen IT, Zhan Q, O'Connor PM, Fornace AJ Jr. INVOLVEMENT OF THE p53
TUMOR SUPPRESSOR IN REPAIR OF U.V.-TYPE DNA DAMAGE. Oncogene
1995;10(6):1053-9.

The tumor suppressor p53 plays a central role in the cellular responses to
genotoxic stress. Besides its well known role in activation of the G1
checkpoint after exposure to agents like ionizing radiation and its role in
apoptosis, the possibility exists that p53 may have additional roles, such as
in DNA repair. For example, p53, is known to bind to single strand DNA such as
would occur during repair events, and the proteins encoded by two
p53-regulated genes have previously been found to bind to at least one protein
involved in DNA damage processing including nucleotide excision repair (NER).
NER is an important and versatile DNA repair mechanism, which is the major
pathway for repair of u.v.-type lesions and damage by a variety of important
carcinogens and mutagens. If components of the p53 pathway are involved in
NER, then disruption of p53 function by mutations or expression of certain
viral proteins could have important implications in carcinogenesis and cancer
treatment. In the present study we show that disruption of normal p53 function
in human colon carcinoma RKO cells with either the human papillomavirus E6
oncoprotein or a dominant-negative mutant p53 transgene results in reduced
repair of u.v.-induced DNA damage. The E6 and mutant p53-containing cell lines
demonstrated reduced repair of u.v.-induced DNA lesions in host cell
reactivation experiments with reporter plasmids, and reduced repair in in
vitro DNA repair assays. With this in vitro assay, extracts from the E6- and
mutant p53-containing lines also showed loss of induced repair following
cellular u.v.-irradiation. The reduced DNA repair activity of the transfected
cell lines also correlated with reduced clonogenic survival following
u.v.-irradiation.  These results indicate that p53 and/or p53-regulated gene
products function in the NER pathway and that this process is inducible by DNA
damage.

197
Sdiqui N, Santambien P, Roche AC, Hebert E, Girot P, Cochet S, Boschetti E,
Monsigny M, Bertrand O.  TOXICITY STUDIES ON NATIVE PROCION RED HE-3B AND
RELEASED DYE FROM AFFINITY MATERIAL EXPOSED TO DEGRADATIVE CHEMICAL
CONDITIONS. J Biochem Biophys Methods 1994;29(3-4):269-82.

Leached ligands from chromatographic packing material submitted to drastic
regeneration conditions can contaminate pure biological preparations. These
contaminants could have adverse effects from a toxicology point of view that
are very poorly documented in liquid chromatography for protein separation.
Investigations on toxicity level have been made on released material from
immobilized Procion Red HE-3B, after formal identification of the nature of
the leached chemical material.  Toxicity investigations in vitro involved a
number of tests on living cells (eucaryotic and procaryotic) covering
different aspects. Behaviour of cells in regular cultures, polyplo:idia
induction, genotoxicity as well as mechanisms of endocytosis have been
studied. Results showed no toxic effects within the range of concentration of
dye and dye derivatives studied. Genotoxicity studies in particular did not
show any toxic effect over a range of concentration much higher than the
regular level of dye leakage from the sorbent.

198
Marshall R. MEASUREMENT OF CHROMOSOME ABERRATIONS IN VITRO USING HUMAN
PERIPHERAL BLOOD LYMPHOCYTES. Methods Mol Biol 1995, 43:287-96.

199
Shaw G. AN EVALUATION OF MOLECULAR BIOLOGICAL AND CYTOGENETIC TESTING FOR
DETERMINATION OF HUMAN RISK FROM EXPOSURE TO CHEMICALS PRESENT IN CONTAMINATED
SITES.  J Environ Sci Health C  1995;13(1):75-103.

200
Mark H FL, Naram R, Singer JT, Rice RW, Bastan B, Beauregard LJ, Lamarche PH.
CYTOTOXICITY AND GENOTOXICITY OF WOOD DRYING CONDENSATE FROM SOUTHERN YELLOW
PINE: AN IN VITRO STUDY. Mutat Res 1995;342
(3-4):191-6.

We tested condensates from Southern Yellow Pine for potential cytotoxicity and
genotoxicity in CHO-WBL and human peripheral blood lymphocytes (PBL) in the   
absence of S-9 activation. Cytotoxicity was evaluated by the Trypan blue
exclusion assay, mitotic index (MI) and proliferative rate index (PRI).
Genotoxicity was measured by the chromosome aberration (CA) assay and sister
chromatid exchange (SCE) analysis. Both cytotoxic and genotoxic effects were
observed. Laboratory-generated Southern  Yellow Pine condensate reduced the
viability of CHO-WBL cells. The number of viable cells was roughly inversely
proportional to dosage over a range of 100% to 31% in treated groups, in both
experiments, as compared to 2.60%) in the control. The MI data in both CHO
cells and PBL also showed an inverse correlation. The highest scorable dose
limited by toxicity was determined to be 1 ml of Southern Yellow Pine
condensate in 10 ml total of medium. Lastly, a dose response curve was 
observed in CHO cells, as well as in PBL, using the CA assay and also with the
SCE analysis. The present findings corroborate the results from Ames testing
and represent the only information currently available on the genotoxic
potential of these chemicals. 

201
Shi CY, Hew YC, Ong CN. INHIBITION OF AFLATOXIN B1-INDUCED CELL INJURY BY
SELENIUM: AN IN VITRO STUDY. Hum Exp Toxicol 1995;14(1):55-60. 

Dietary selenium is an essential trace element in human nutrition. Selenium
has been shown in animal studies to inhibit aflatoxin hepatocarcinogenesis.
However, the cellular mechanism responsible for the inhibition has not been
thoroughly studied. This study examines the effect of two selenium compounds,
namely, sodium selenite and selenium-enriched yeast extract (SeY), on the
cytotoxicity, DNA-binding and mutagenicity of aflatoxin B1 (AFB1) in cultured
Chinese hamster ovary (CHO) cells. CHO  cells, after treatment with 2 mug ml-1
selenite or 80 mug ml-1 SeY, exhibited increased resistance to AFB1-induced
cell killing. At a concentration of 50 mug ml-1 AFB1, cell survival, measured
by the clonogenicity assay, was increased by 21- and 10-fold in selenite- and
SeY-treated cells, respectively. However, selenium treatment did not appear to
affect AFB1-DNA binding. Similarly, no effect was observed on AFB1
mutagenicity, as determined by the hypoxanthineguanine phosphoribosyl 
transferase (HPRT) gene mutation assay. The results showed that selenium could
effectively protect cells from AFB1 cytotoxicity in cultured cells but had no
effect on AFB1-DNA adduct formation or mutagenesis. It is suggested that there
are multiple pathways of  AFB1 toxicity and that selenium can modulate
AFB1-induced cell killing independent of its genotoxicity. 

202
Venkat JA, Shami S, Davis K, Nayak M, Plimmer JR, Pfeil R, Nair PP.  RELATIVE
GENOTOXIC ACTIVITIES OF PESTICIDES EVALUATED BY A MODIFIED SOS MICROPLATE
ASSAY.  Environ Mol Mutagen 1995;25(1):67-76.

The genotoxic activities of 47 pesticides were determined using a modified SOS
microplate assay in which the induction of beta-galactosidase in E. coli PQ37
was used as a quantitative measure of genotoxic activity. The results were
compared with those obtained with anethole, curcumin, and capsaicin, a few
examples of naturally occurring compounds present in foods. The assays were
conducted with pesticides dissolved either in a suitable solvent, such as 10%
DMSO in physiological saline or dispersed in sodium taurocholate micelles, to
simulate conditions in the small intestine from where these substances are
normally absorbed from the diet.  4-Nitroquinoline oxide (4-NQO) served as the
reference standard of a direct acting mutagen. In micellar form, 4-NQO and 25
of the 47 pesticides tested showed significantly higher genotoxic activities
than when they were tested in an organic solvent. In micellar form the SOS
inducing potency of 4-NQO was almost twice as high as in 10% DMSO in 
physiological saline. In taurocholate micelles, the five most active compounds
had activities in the range of 1,234-3,765 units/ mumol and in the order of
decreasing activities they were ranked as follows: malathion > dichlorvos >
lindane > chlordane > endrin. They were significantly less active than 4-NQO
(less than 40%). In micellar solution the naturally occurring compounds,
anethole, curcumin, and capsaicin gave activities of 4,594, 928, and 809
units/mumol, respectively. These studies  show that genotoxicity may depend
upon the environment in which cells are exposed to these potential genotoxins.
It appears that testing of the more hydrophobic compounds, both synthetic and
naturally occurring, are needed. 

203
Coates PJ, Save V, Ansari B, Hall PA. DEMONSTRATION OF DNA DAMAGE/REPAIR IN
INDIVIDUAL CELLS USING IN SITU END LABELLING: ASSOCIATION OF p53 WITH SITES OF
DNA DAMAGE. J Pathol 1995; 176(1):
19-26.

We describe the development and application of in situ end labelling (ISEL) to
identity sites of damaged DNA in the nuclei of individual cells. In cell
culture, exposure to a variety of genotoxic agents induced a dose and
time-dependent increase in nuclear labelling. In addition, examination of
histological sections of human skin exposed to solar-stimulated UV light
showed ISEL in both keratinocytes and superficial dermal cells, with the same
spatial and temporal distribution as that of a marker of DNA repair, PCNA
(proliferating cell nuclear antigen). Using co-localization techniques and
confocal microscopy, we found increased levels of p53 in many ISEL-positive
cells in vitro, with a similar distribution of labelling in the nucleus. This
observation provides further evidence for a direct role of p53 in the
recognition of damaged DNA. Thus, ISEL should prove a convenient method for
demonstrating genotoxic insult in individual cells and in histological
material, and may have value in toxicological screening. This high-resolution
microscopy technique can also be used to compare the spatial distribution of
various proteins implicated in the response to DNA damage with the sites of
the lesion. 

204
Benigni R.  MOUSE BONE MARROW MICRONUCLEUS ASSAY: RELATIONSHIPS WITH IN VITRO
MUTAGENICITY AND RODENT CARCINOGENICITY.  J Toxicol Environ Health
1995;45(3):337-47. 

In this article, the relationship was studied between the in vivo mutagenicity
assay of mouse bone marrow micronucleus (MIC), and four in vitro assays:
Salmonella typhimurium, chromosomal aberrations in Chinese hamster ovary (CHO)
cells,  sister chromatid exchanges (SCE) in CHO cells, and mutation in mouse
lymphoma L5178Y cells. A comparison with the rodent carcinogenicity data was
also undertaken. The MIC data on 49 chemicals were generated by Shelby et al.
(1993). The MIC assay system employed three daily exposures by intraperitoneal
injection; bone marrow samples were obtained 24 h following the final
exposure. A preliminary analysis indicated that the 49 chemicals selected by
Shelby et al. (1993) are a representative subset of the National Toxicology
Program database. This study showed that MIC has a number of particular
characteristics that are not shared by other biological systems. MIC is
basically different from rodent carcinogenicity, despite being an in vivo
system. At the same time, it responds to the chemicals in a different way from
that of the in vitro genotoxicity assays. These in vitro assays mainly differ
from each other in their different sensitivities to the genotoxins: MIC gives
just a few positives (limited sensitivity), but, at the same time, some of
these positives are detected only by the most sensitive assays, like the mouse
lymphoma or SCE assays. In terms of risk assessment, MIC does not complement
Salmonella for predicting chemical carcinogenicity, and would be better used
to verify if the in vitro positive chemicals are able to exert their genotoxic
potential in vivo.

205
James NH, Ashby J, Roberts RA. ENHANCED HEPATOCYTE COLONY GROWTH IN SOFT AGAR
AFTER IN VIVO TREATMENT WITH A GENOTOXIC CARCINOGEN: A POTENTIAL ASSAY FOR
HEPATOCARCINOGENS? Cancer Lett 1995;93(1):121-8.

We have shown previously that approximately 1 in 10,000 primary hepatocytes
isolated from untreated rats undergo clonal growth in soft agar in vitro in
response to the synergistic action of nafenopin, a peroxisome proliferator
(PP) and epidermal growth factor (EGF), a naturally occurring liver growth
regulator. Here, we demonstrate that prior treatment of the animals with the
genotoxic hepatocarcinogen diethylnitrosamine (DEN) caused a dose-dependent
increase in soft agar colony numbers formed in vitro. These data suggest that
the colony assay may offer a method of detecting in vitro hepatocytes
transformed in vivo by DEN. It is known that rats treated with DEN develop
enzyme altered foci prior to the development of tumours. The majority of these
foci express high levels of gamma-glutamyl transpeptidase (GGT). However, foci
promoted by PPs do not show this increased enzyme activity. In the present
study, the colonies we have       generated in vitro mimicked this pattern
since the majority (approximately 80%) of the spontaneous colonies expressed
GGT whereas colonies promoted by the synergistic action of nafenopin and EGF
were mainly (75%) GGT negative. The proportion of colonies positive for GGT
were similar using either hepatocytes isolated from control or from
DEN-initiated rats. Further studies are required to assess if the hepatocytes
selected for clonal expansion by this EGF/nafenopin regime reflect the
presumed pre-neoplastic cells induced by genotoxin in vivo and associated with
an increased propensity to cancer.

206
Hill CM, Lunec J, Griffiths HR, Herbert KE. CHARACTERIZATION OF TUMOR NECROSIS
FACTOR ALPHA RELEASE BY HUMAN GRANULOCYTES IN RESPONSE TO PROCAINAMIDE
CHALLENGE.  Biochem Pharmacol 1995; 49(12):1837-49.

The role of human granulocytes in the promotion of procainamide (PA) toxicity
in vitro has been studied and one of the agents responsible for DNA strand
scission and cell death in human target cells has been characterized. Crude
peripheral blood mononuclear cells (cPBMNs) isolated by density
centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were
exposed to PA, and DNA strand breaks were quantified by fluorescent analysis
of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused
dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in
cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM
PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with
significant induction of strand breaks also being observed for 10 and 25
microM concentrations. Toxicity was much reduced in lymphocyte cell lines
(maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A
similar decrease in toxicity was observed where N-acetyl procainamide (NAPA)
was substituted for PA (less than 50% of strand breaks at all concentrations).
Further investigations showed that the presence of a contaminating granulocyte
population in the cPBMN fraction was responsible for the induction of PA
toxicity. Incubation of a highly enriched       granulocyte population with PA
for 1 hr prior to exposure to purified peripheral blood mononuclear cells
(pPBMNs) led to the complete restoration of the toxic effects. The resulting
cyto- and genotoxicity were not significantly different to levels observed in
cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce
toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to
the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal
studies showed that the toxicity associated with the < 3000 Da fraction
appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da
fraction did not display significant toxicity until the 40-60 min period.
Further assessment of the nature of these agents indicated that the 30,000 Da
fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da
species appearing in granulocyte supernatants fter 40 min incubation with PA.
Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was
present in the > 30,000 Da fraction. Evidence that TNF alpha was the
high-molecular weight species responsible for PA-induced toxicity was obtained
from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT
TRUNCATED AT 400 WORDS) 

207
Caria H, Chaveca T, Laires A, Rueff J. GENOTOXICITY OF QUERCETIN IN THE
MICRONUCLEUS ASSAY IN MOUSE BONE MARROW ERTYHROCYTES, HUMAN LYMPHOCYTES, V79
CELL LINE AND IDENTIFICATION OF KINETOCHORE-CONTAINING (CREST STAINING)
MICRONUCLEI IN HUMAN LYMPHOCYTES. Mutat Res 1995;343(2-3):85-94.

Quercetin, a mutagenic flavonoid widely distributed in edible plants, was
studied for the induction of micronuclei (MN). We have carried out the MN
assay in bone marrow polychromatic erythrocytes in mice, in
cytokinesis-blocked human lymphocytes and in cytokinesis-blocked V79 cells. MN
assay in vitro was performed in the presence and in the absence of S9. To
further extend the study, an antikinetochore antibody (CREST staining) was
used to distinguish MN containing whole chromosomes (kinetochore positive)
from those containing acentric fragments (kinetochore negative). When tested
in vivo quercetin failed to induce micronuclei, a result which is in agreement
with other published reports. When tested in vitro in V79 cells quercetin
clearly induces micronuclei in the absence of S9 and also in the presence of
S9 for the highest dose used. When tested in vitro in human lymphocytes
quercetin shows a significant induction of micronuclei in the absence and in
the presence of S9. The presence of S9 compared to its absence is not
significant for any of the systems used. Both in the presence and absence of
S9, quercetin appears to behave as a clastogenic agent in human lymphocytes
inducing a significant majority of kinetochore-negative MN. 

208
Heidemann A, Tries S, Laufer S, Augustin J.  STUDIES ON THE IN VITRO AND IN
VIVO GENOTOXICITY OF [2,2-DIMETHYL-6-
(4-CHLOROPHENYL)-7-PHENYL-2,3-DIHYDRO-1H-PYRROLIZINE-5-YL]-ACETIC ACID. 
Arzneimittelforschung 1995;45(4):486-90.

[2,2-Dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrroliz- ine-5-
yl]-acetic acid (CAS 156897-06-2, ML 3000) was examined for genotoxic activity
in bacteria and mammalian cells in vitro as well as in vivo. The substance did
not increase gene mutation frequencies either in a bacterial system or in a
cultured V79 cell line of the Chinese hamster. Both in vitro tests were
conducted in the presence and absence of S9-mix. In the unscheduled DNA
synthesis assay in vitro with primary rat hepatocytes, negative results were
also obtained. A cytogenetic analysis of the bone marrow of male and female
Wistar rats was performed. After oral application ML 3000 did not increase the
number of cells with structural chromosomal aberrations. The results suggest
that ML 3000 has no genotoxic potential in vitro and in vivo.

209
Anderson D, Basaran N, Blowers SD, Edwards AJ.  THE EFFECT OF ANTIOXIDANTS ON
BLEOMYCIN TREATMENT IN IN VITRO AND IN VIVO GENOTOXICITY ASSAYS. Mutat Res
1995;329(1):37-47.

Antioxidants are thought to be important in protecting against damage from
active oxygen species. The effects of the antioxidant nutrients vitamins C and
E have been investigated after bleomycin treatment in the Salmonella
typhimurium bacterial mutation assay, in the human peripheral lymphocyte
chromosome aberration assay, and in the mouse micronucleus assay in peripheral
blood and bone marrow cells. There were no protective effects from vitamins C
and E in the bacterial mutation assay, but vitamin C and not vitamin E
abolished chromosome damaging responses in human peripheral lymphocytes, and
both vitamins reduced responses in micronuclei from peripheral blood cells in
mice. This would suggest that in human cells in vitro and mouse cells in vivo
these vitamins could have a protective role.

210
Fryxell D, Li BY, Mohanraj D, Johnson B, Ramakrishnan S. GENETIC CONSTRUCTION
OF A PHOSPHORYLATION SITE IN RICIN A CHAIN:
SPECIFIC RADIOLABELING OF RECOMBINANT PROTEINS FOR LOCALIZATION AND
DEGRADATION STUDIES. Biochem Biophys Res Commun 1995;210(2):253-9.

Ricin A chain was modified by the addition of the heptapeptide LRRASLG
(Kemptide) and a histidine tag for bacterial expression. The mutagenized toxin
was purified by nickel column and could be phosphorylated in vitro by protein
kinase A as demonstrated by labeling with [gamma-32P] ATP. Kemptide-A chain
could be labeled even after reassociation with ricin B chain or disulfide
linkage to antibody to form an immunotoxin. The 32P label in all cases was
associated only with the A chain; ricin B chain and antibody were not kinase
substrates alone or after conjugation. Kemptide-immunotoxin was tested in
cytotoxicity assays and used to monitor internalization of the toxin moiety
after [32P] phosphorylation.

211
Leroy T, Lison D, Lauwerys R. PRELIMINARY IN VITRO INVESTIGATION INTO THE USE
OF ALKALINE ELUTION ASSAY FOR THE BIOMONITORING OF HUMANS EXPOSED TO GENOTOXIC
AGENTS. Hum Exp Toxicol 1995;14(1):61-8. 

1. This in vitro study was undertaken as a preliminary approach before
assessing whether the alkaline elution assay can be applied to peripheral
blood lymphocytes (PBL) for the monitoring of humans exposed to genotoxic
agents such as polycyclic aromatic hydrocarbons (PAH). We have compared in
vitro, with the aid of the alkaline elution assay, the formation and the
repair of DNA single-strand breaks (ssb) induced by different genotoxic agents
[gamma-irradiation, ethyl methanesulfonate (EMS), benzo<a>pyrene diol epoxide
(BPDE)] on quiescent and PHA-stimulated human lymphocytes and on a fibroblast
cell line. 2. Gamma-irradiation (4 Gy) induced an equivalent amount of DNA ssb
in the three cell types. On the other hand, after treatment with EMS (10 mM)
and BPDE (50 microM), a higher production of DNA ssb was observed in
replicating cells (PHA-stimulated lymphocytes and fibroblasts) when compared
with quiescent lymphocytes. 3. After gamma-irradiation, all cell types
repaired more than 65% of ssb within 1 h. After treatment with EMS, we noted a
deficient DNA repair capacity in quiescent lymphocytes in comparison with
replicating cells. In all cell types treated with BPDE, more breaks were
observed after a 2 h repair period than immediately after treatment,
demonstrating the involvement of a slow repair mechanism after BPDE
treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

212
Douglas GR, Gingerich JD, Soper LM. EVIDENCE FOR IN VIVO NON-MUTAGENICITY OF
THE CARCINOGEN HYDRAZINE SULFATE IN TARGET TISSUES OF lacZ TRANSGENIC MICE.
Carcinogenesis 1995;16(4):801-4.

Transgenic mouse models permit the confirmation of in vitro mutagenicity in
vivo without the constraints in the selection of tissues imposed by other in
vivo assays. This feature is of particular importance in the determination of
mutagenicity in the target tissues of carcinogens, especially those that are
in vitro mutagens. Such information is critical in the determination of
whether a chemical is carcinogenic via a genotoxic or non-genotoxic mechanism.
Hydrazine sulfate is an in vitro mutagen that induces lung and liver tumours
in mice. Transgenic mice from strain 40.6 (Mutamouse) were administered single
oral doses up to a toxic concentration (400 mg/kg). No dose induced any lacZ
mutations in lung, liver or bone marrow. Since the highest single dose used is
higher than the cumulative dose that induced tumours in previous studies, it
may be that either hydrazine sulfate is genotoxic in target tissues in vivo
only when given in multiple doses or that it is a non-genotoxic carcinogen.  

213
Dhillon VS, Singh J, Singh H, Kler RS. IN VITRO AND IN VIVO GENOTOXICITY OF
HORMONAL DRUGS. VI. FLUOXYMESTERONE.  Mutat Res 1995;342
(3-4):103-11.

Genotoxic evaluation of a commonly used synthetic steroidal androgen,
fluoxymesterone, was undertaken using a combination of in vitro and in vivo
assays. The clastogenic potential of fluoxymesterone was evident from the
chromosome aberrations and sister chromatid exchanges induced by it in the
cultured human lymphocytes and also from the increased frequencies of 
micronuclei and sister chromatid exchanges in bone marrow cells of mice.
However, in Ames Salmonella assay both with and without S9 mix and in
host-mediated assay using bacterial strains of S. typhimurium as indicator
organism, fluoxymesterone did not cause any significant increase/decrease in
His+ revertants.

214
Fekedu K, Parzefall W, Kronberg L, Franzen R, Schulte-Hermann R, Knasmueller
S.  INDUCTION OF GENOTOXIC EFFECTS BY CHLOROHYDROXYFURANONES, BYPRODUCTS OF
WATER DISINFECTION, IN E. COLI K-12 CELLS RECOVERED FROM VARIOUS ORGANS OF
MICE. Environ Mol Mutag 1994;24(4):317-24.

The genotoxic effects of three chlorohydroxyfuranones (CHFs),
3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX),
3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)furanone (CMCF) and
3,4,-dichloro-5-hydroxy-2(5H)furanone (MCA), which are formed as byproducts of
water disinfection with chlorine, were investigated in bacterial differential
DNA repair assays in vitro and in animal-mediated assays in vivo. As
indicators of DNA damage, E. coli K-12 strains were used that differ in their 
repair capacity (uvrB/recA vs. uvr+/rec+). Liquid incubation of the compounds
without metabolic activation caused a pronounced reduction of the viability of
the repair-deficient strain relative to the repair-proficient wild-type
strain. The order of potency of genotoxic activity in vitro (dose range
0.004-10 mug/ml) was MX > CMCF > MCA. Addition of mouse S-9 mix or bovine
serum albumin to the incubation mixtures resulted in an almost complete loss
of the activity of all three test compounds. In the animal-mediated assays,
mixtures of the indicator bacteria were injected intravenously into mice which
were subsequently treated with the test compounds (200 mg/kg b.w.). Two hours
later, the cells were recovered from various organs and the relative survival
frequencies determined. Under these conditions, all three compounds caused
pronounced genotoxic effects, MX and CMCF being stronger genotoxins than MCA.
The strongest effects were consistently found in the gastrointestinal tract,
but statistically significant DNA damage was also observed in indicator cells
recovered from lungs, liver, spleen and kidneys. In a further experiment, the
effects of lower doses of MX (4.3, 13 and 40 mg/kg) were investigated. In
these experiments dose-dependent effects were measured in all organs. CMCF and
MA caused only marginal effects at 40 mg/kg except in the stomach where
approximately a 50% reduction of relative survival frequency was observed with
CMCF. The results of these animal-mediated  assays indicate that (i) all three
CHFs cause genotoxic effects in the living animal, and (ii) the potencies of
the three compounds observed under in vivo conditions are not commensurate
with their extremely high activities measured in vitro. One possible
explanation for the weaker responses observed in the animal-mediated assays
might be that CHFs are inactivated by  nonspecific protein binding. 

215
Daugel'-Dauge NO, Durnev AD, Kulakova AV, Seredenin SB, Velichkovskii BT. 
[CORPUSCULAR MUTAGENESIS AND ITS PREVENTION.] Vestn Ross Akad Med Nauk
1995;1:29-38. (Rus)

The carcinogenic and mutagenic activity of dust containing chrysotile-asbestos
and zeolites, as well as the role of active oxygen species in their cytotoxic
and mutagenic actions are discussed. Superoxide dismutase (50 mg/ml) was
demonstrated to prevent the mutagenic effects of chrysotile-asbestos and
latex, catalase (20 mg/ml) to prevent the same of zeolites in experiments on
cultured human whole blood. The intraperitoneal administration of dusts of
chrysotile-asbestos and zeolites in a dose of 50 mg/kg to C57B1/6 mice was
found to elevate the count of cells with chromosomal aberrations in the
peritoneal liquid and bone marrow cells of mice, which was dependent on dust
exposure time. It was revealed that ascorbic acid, rutin, chemically modified
flavonoid of Scutellaria Baicalensis Georgy, drugs such as bemitil and
thomersol in the broad range of       concentrations (10(-7)-10(-3) M)
decreased or completely reduced the clustogenic action of zeolites and
chrysotile-asbestos on cultured human whole blood. The ability of bemitil
(1.8-19 mg/kg) rather than the others to prevent the mutagenic effect of
chrysotile-asbestos was confirmed by the method of recording chromosomal
aberrations in the cells of peritoneal liquid and bone marrow in mice. The
findings suggest that the mutagenic effects of the corpuscular xenobiotics
under study are mediated by active oxygen species and that the use of the
models in vitro and in vivo is adequate for investigations into corpuscular
mutagenesis. Based on their own data and literature data, the authors have
defined possible lines of further research of corpuscular mutagenesis.

216
Donnelly KC, Safe SH, Randerath K, Randerath E.  BIOSASSY-BASED RISK
ASSESSMENT OF COMPLEX MIXTURES. J Hazard Mater 1995; 41(2-3):341-50. 

In order to compare a std. chem.-based risk assessment with in vitro
genotoxicity assays, two complex environmental mixts. from a wood-preserving
site were analyzed in the Salmonella/microsome and E. coli prophage induction
assays. Using GC/MS, sample 003 was found to contain relatively low levels of
polycyclic arom. hydrocarbons (PAHs) and elevated levels of polychlorinated
dibenzo-p-dioxins (PCDDs), while sample 005 had higher levels of PAHs and
relatively low levels of PCDDs.  The complex mixts. were sequentially extd.
with methylene chloride and methanol for anal. in Salmonella, or extd. with a
1:1 hexane:acetone mixt. for anal. in the E. coli prophage induction assay. 
At a dose of 1.0 mg/plate in Salmonella strain TA98 with metabolic activation,
the methanol ext. of sample 003 induced 197 net revertants, while sample 005
induced 436 net revertants.  In the prophage induction assay, with activation,
the hexane:acetone ext. of sample 003 induced a genotoxic response that was
slightly lower than that obsd. with sample 005.  The estd. incremental
carcinogenic risk for ingestion of PAHs  was 1.5E-3 for sample 003, while for
sample 005 the estd. risk was 1.5E-2.  Thus, the sample which induced the max.
response in both bioassays also had the highest estd. cancer risk.  However,
the frequency of PAH-DNA adducts in both skin and liver tissues was
appreciably higher with sample 003 than with sample 005.  A combined testing
protocol, using both biol. and chem. anal., therefore provides more accurate
information from which to assess risk than the use of either method alone. 

217
De Deyne PG, Kirsch-Volders M.  IN VITRO EFFECTS OF THERAPEUTIC ULTRASOUND ON
THE NUCLEUS OF HUMAN FIBROBLASTS. Phys Ther 1995;75(7):629-34.

The purpose of this investigation was to examine the cytological effects of
therapeutic ultrasound on human fibroblasts. MATERIAL AND METHODS. Using an in
vitro approach, the number of cells recovered, the morphology of chromosomes,
and the presence of mitotic spindles were studied after sonication of human
fibroblasts in culture. The ultrasound output had a frequency of 1 MHz and was
delivered in a pulse mode of 2 milliseconds "on" and 8 milliseconds "off."
Sonication was given for 0, 30, 60, and 90 seconds. RESULTS. There was a
time-dependent decrease in number of cells recovered, a fourfold increase in
mitotic index in the cells that survived the treatment, and a nearly eightfold
increase of chromosomal aberrations with loss of mitotic spindles. CONCLUSION
AND DISCUSSION. The dose-dependent lytic effect of nonthermal ultrasound could
result in fractionation of cells, which might facilitate phagocytosis during
chronic inflammation. In addition, we observed an increase in chromosomal
aberrations and a loss of mitotic spindles as a result of an extremely short
ultrasound treatment (30 seconds). Whether these alterations are mutagenic or
not warrants further study. It is possible that in our in vitro experiments,
the ultrasound received was greater than would occur in a clinical situation. 

218
Wilmer JL, Luster MI.  CHEMICAL INDUCTION OF INTERLEUKIN-8, A PROINFLAMMATORY
CHEMOKINE, IN HUMAN EPIDERMAL KERATINOCYTE CULTURES AND ITS RELATION TO
CYTOGENETIC TOXICITY.  Cell Biol Toxicol 1995;11(1):37-50. 

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis
inhibitors can modulate cell cycle kinetics of various cell types, stimulate
production of reactive oxygen species, and induce keratinocytes to produce
interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils
and T lymphocytes. The aim of this study was to determine whether
perturbations of cytogenetic responses correlated with the induction of IL-8
expression. Cultures of primary human keratinocytes were grown in serum-free
medium with 5 mumol/L bromodeoxyuridine to label DNA and exposed either to
phorbol-13-myristate-12-acetate (PMA) (0.0001-100 ng/ml), cycloheximide (CHX)
(0.01-50 mug), lipopolysaccharide (0.1-100 mug/ml), tumor necrosis
factor-alpha (TNFalpha) (3.13-50 ng/ml), or interleukin-1alpha (IL-1alpha)
(1-182 pg/ml). Metaphase chromosome preparations were stained by a
fluorescence-plus-Giemsa technique to differentiate sister chromatids. For
IL-8 production, keratin grown to 70% confluency and then exposed to chemicals
for 24 h. Immunoreactive IL-8 was       quantitated from the supernatants by
ELISA. With the exception of benzo(a)pyrene used as a positive control, none
of the agents induced sister chromatid exchanges. However, PMA and TNFalpha
induced IL-8 production that coincided with significant cell cycle inhibition.
IL-1alpha had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold
increase in IL-8. CHX inhibited cell cycle progression and mitotic  activity
at concentrations that were 200 times lower than required for IL-8 induction;
however, puromycin (0.31-10 mug/ml), another protein synthesis inhibitor, did
not induce IL-8. At all concentrations tested, TNFalpha reduced the mitotic
index byuced a flat, albeit large, IL-8 response at concentrations : 12.5
ng/ml. These agent-specific response patterns suggest that induction of IL-8
production is not always the inevitable result  of cell cycle perturbations or
genetic damage.

219
Cravino V, Kropko ML, Rothwell CE, Hovey CA, Theiss JC.  THE GENOTOXICITY
PROFILE OF ATORVASTATIN, A NEW DRUG IN THE TREATMENT OF HYPERCHOLESTEROLEMIA. 
Mutat Res 1995;343(2,3):95-107.

While HMG-CoA reductase inhibitors such as fluvastatin, lovastatin,
pravastatin and simvastatin demonstrate lack of in vitro and in vivo
mutagenicity and clastogenicity in bacterial and mammalian cells, long term
rodent carcinogenicity studies resulted in an increased incidence in neoplasms
at high doses.  These effects may be attributable to an exaggeration of the
desired biochem. effect of the drug and/or a tumor promoting effect.  The
genotoxicity of atorvastatin, a newly developed HMG-CoA reductase inhibitor,
was evaluated in a variety of test systems.  In bacterial mutagenicity tests,
the E. coli tester strain WP2(uvrA) and S. typhimurium strains TA98, TA100,
TA1535, TA1537, and TA1538 were exposed to concns. of atorvastatin as high as
5000 mug/plate both in the absence (S9-) and presence (S9+) of metabolic
activation.  Atorvastatin was not mutagenic in either E. coli or S.
typhimurium.  Chinese hamster lung V79 cell cultures were exposed to
atorvastatin at concns. of 50-300 mug/mL (S9-) and 100-300 mug/mL (S9+) and
structural chromosome aberrations were assessed.  Mutation at the hgprt locus
was assessed at concns. of 100-300 mug/mL (S9-) and 150-275 mug/mL (S9+). 
Atorvastatin was neither mutagenic nor clastogenic in the absence or presence
of S9.  The lack of in vitro genotoxicity was corroborated in vivo in a mouse
micronucleus study in which single oral doses of atorvastatin were
administered to male and female CD-1 mice at 1, 2500, or 5000 mg/kg.  No biol.
significant increases in the frequency of     micronucleated polychromatic
erythrocytes in bone marrow at 24, 48, or 72 h postdosing were obsd.  Thus,
atorvastatin, as with the other tested HMG-CoA reductase inhibitors, is not
genotoxic. 

220
Takahashi N, Shibahara T, Shiragiku T, Kanbe T, Kanbe K, Sugawara M, Yamashita
S. REDUCTION OF IN VITRO CLASTOGENICITY INDUCED BY THE MIXTURE OF OPTICAL
ISOMERS OF NADIFLOXACIN DURING STORAGE. Arzneimittelforschung
1995;45(2):195-7.
 
The fluoroquinolone antibacterial agent, nadifloxacin (NDFX, CAS 124858-35-1),
is a racemic compound. The storage effect on the in vitro clastogenicity of a
solution of the racemic compound and a mixture solution of the optical isomers
of NDFX, prepared by mixing equal amounts of S- and R-enantiomers, was
investigated. The potential of NDFX and the enantiomer mixture, prepared from
equal amounts of each S- and R-enantiomer, to induce chromosomal aberrations
in vitro was investigated in cultured fibroblasts derived from Chinese hamster
lung cells immediately, 2 and 4 weeks after preparation of the test solutions
(stored at 20 degrees C, protected from light) using 24 h of continuous
treatment method. In the results, NDFX did not significantly increase the
incidence of chromosomal aberrations at 200 micrograms/ml regardless of the
storage period. On the other hand, the mixture significantly increased the
incidence of chromosomal aberrations at 200 micrograms/ml immediately after
preparation to an extent similar to that of S-enantiomer alone, but the
mixture did not do so after 2 and 4 weeks of storage.       Neither S- nor
R-enantiomer changed the chromosomal aberration inducibility during storage.
The content and optical purity of the test substances in each test solution
also did not change during storage. These facts suggested that the molecular
condition of each optical isomer in the mixture solution became equivalent to
that in the racemic solution during storage periods.

221
Suzuki O, Ishimura K, Takahashi T, Miyauchi S. [IN VITRO CHROMOSOME ABERRATION
TEST OF SODIUM HYALURONATE IN CULTURED MAMMALIAN CELLS.] Oyo Yakuri
1995;50(1):73-7. (Jpn)

As part of a mutagenicity testing program, sodium hyaluronate (Na-HA) with a
mol. wt. of 276.times.104 was examd. for its clastogenicity by using cultured
Chinese hamster lung fibroblast cells.  Concns. of 1,250, 2,500 and 5,000
mug/mL were tested with and without metabolic activation.  Examn. of 200
mitosis per concn. for chromosome aberrations showed that Na-HA did not differ
significantly from the neg. control.  It is concluded that Na-HA with a mol.
wt. of 276.times.104 is not a clastogen under the conditions of the expt.

222
Wening JV, Marquardt H, Katzer A, Jungbluth KH, Marquardt H.  CYTOTOXICITY AND
MUTAGENICITY OF KEVLAR: AN IN VITRO EVALUATION.  Biomaterials
1995;16(4):337-40.

Toxicity and mutagenicity of Kevlar 49 (PPPT;
poly-para-phenylene-terephthalamide) was tested in six strains of Salmonella
typhimurium (Ames test; TA97, TA98, TA100, TA102, TA1535, TA1537) with and
without an external metabolic activation system (S9), as well as in a
mammalian cell mutagenesis assay using V79 Chinese hamster cells. For the Ames
test, liquid preincubation, which is considered particularly sensitive, was
used. The cells were incubated for 24 h at a temperature of 37 degrees C
either directly with Kevlar49 or with ethanol- or chloroform-extracted
Kevlar49. The experiments were performed at least twice. The Ames test with
six different Salmonella typhimurium strains featuring either base pair
substitution or frameshift mutations revealed no cytotoxic or mutagenic
activity of Kevlar49. In the mammalian cell mutagenesis assay, using
8-azaguanine (AG) as a selective agent, Kevlar49 was also devoid of cytotoxic
or mutagenic activity. Both tests have to be regarded as an initial
exploratory screening due to the chosen testing conditions and should be
supplemented by tests at different temperatures. 

223
Jacobson-Kram D, Rosenthal SL.  MOLECULAR AND GENETIC TOXICOLOGYOF
1,3-BUTADIENE. Mutat Res 1995;339(2):121-30. 

During the last 9 years, there have been many studies published concerning the
mutagenic potential of butadiene in mammalian systems, including alterations
at the molecular level. Butadiene has tested positive in several mouse in vivo
and in vitro assays, but has generally tested negative in rat studies. Most of
these studies are cytogenetic and include positive data in mice for
chromosomal aberrations, micronucleus formation, and sister chromatid
exchanges. Butadiene also induces mutations in lung, spleen, and bone marrow
of transgenic mice. The positive bone marrow cytogenetic and transgenic data
may be significant in view of the increased lymphohematopoietic malignancies
observed in mice and probably in humans. In addition, butadiene causes
mutations in the K-ras protooncogene and in the p53 tumor suppressor gene in
mouse studies. Mutations in these genes are associated with oncogenesis in
humans as well as in rodents. Also, positive mutagenicity data have been
obtained in a pilot study of workers exposed to butadiene. Positive dominant
lethal studies in rodents suggest that exposure to butadiene can result in
germ cell mutation and heritable risk. These mutagenicity and molecular data
suggest that butadiene is both a somatic and germ cell mutagen in mammals,
possibly including humans.

224
Guo YJ, Lu SX, Liang YY.  [ALTERATIONS OF ONCOGENES IN HUMAN FETAL ESOPHAGEAL
EPITHELIUM INDUCED BY N-METHYLBENZYLNITROSAMINE (NMBzA).] Chunghua Chung Liu
Tsa Chih 1994;16(6):407-10. (Chi)

Epidemiological investigation showed that N-methylbenzylnitrosamine (NMBzA)
has been associated with increased incidence of esophageal cancer (EC) in
Linxian county, a high incidence area. In present study, our results indicate
that NMBzA can induce amplification and over-expression of EGFr gene in human
fetal esophageal epithelium (HFE) treated with NMBzA for 24 hours as shown by
southern blot assay and immunohistochemistry. The papillary hyperplasia was
induced in HFEs that cultured with NMBzA for 1 to 3 weeks. Amplification of
c-myc and int-2 gene in HFEs treated by NMBzA for 1 week and 3 weeks was
found, respectively. Deletions of p53 and Rb gene were found in human fetal
esophageal carcinomas induced by NMBzA. Overexpression of p53 protein in human
fetal esophageal carcinomas detected by immunohistochemical methods indicates
that p53 gene mutation(s) may be occured. The HFE explants treated in vitro
with NMBzA for 3 weeks were inoculated subcutanously into balb/c nude mice. No
tumor was found in 5 months after inoculation, suggesting that only changes of
oncogene(s) are insufficient to induce full transformation. Other genetic
alterations (such as functional inactivation of Rb or/and p53 tumor suppressor
genes) may be necessary in the further progression of malignant lesions.

225
Tateno H, Kamiguchi Y.  APPLICATION OF CRYOPRESERVED GOLDEN HAMSTER OOCYTES TO
IN VITRO GENOTOXICITY ASSAYS FOR HUMAN SPERM CHROMOSOMES. Environ Mol Mutagen
1995;25(3):263-5.

226
Turk PW, Laayoun A,  Smith SS, Weitzman SA.  DNA ADDUCT
8-HYDROXYL-2'-DEOXYGUANOSINE (8-HYDRONYGUANINE) AFFECTS FUNCTION OF HUMAN DNA
METHYLTRANSFERASE. Carcinogenesis 1995;16(5):1253-5 .

8-Hydroxyl-2'-deoxyguanosine (also referred to as  8-hydroxyguanine [8-OH-dG]
or 7,8-dihydro-8-oxoguanine), a common DNA adduct resulting from injury to DNA
via reactive oxygen  species, affects the in vitro methylation of nearby
cytosine moieties by the human DNA methyltransferase. The exact position of
8-OH-deoxyguanosine relative to a CpG dinucleotide appears important to this
effect. Our data indicate that 8-OH-deoxyguanosine diminishes the ability of
the methyltransferase to methylate a target cytosine when the
8-OH-deoxyguanosine is one or two nucleotides 3' from the cytosine, on the
same strand. On the other hand 8-OH-deoxyguanosine does not diminish the
ability of the enzyme to respond to a methyl director (5-methylcytosine) when
the 8-OH-deoxyguanosine is on the same strand but one or two nucleotides 3'
from the methyl director. Differences in methylation rates as great as 13-fold
have been detected using various 8-OH-deoxyguanosine-containing
oligonucleotides as substrates in methylation assays. Our findings suggest
that oxidative damage of parental strand guanines would permit normal copying
of methylation patterns through maintenance methylation, while oxidative
damage of guanines in the nascent strand DNA would inhibit such methylation.

227
Erexson GL, Bryant MF, Kwanyuen P, Kligerman AD. BLEOMYCIN SULFATE-INDUCED
MICRONUCLEI IN HUMAN, RAT, AND MOUSE PERIPHERAL BLOOD LYMPHOCYTES. Environ Mol
Mutagenesis 1995;5(1):31-6.

The sensitivity to micronucleus (MN) induction of human, mouse, and rat
peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in
vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the
PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 mug/ml BLM was
added to 5 ml aliquots of whole blood for 4 hr at 37~C in a 5% CO2 atmosphere.
Leukocytes were isolated on a density gradient and cultured in the presence of
phytohemagglutinin to stimulate  blastogenesis, and cytochalasin B was added
to each culture at 21 hr postinitiation to prevent cytokinesis. A total of
4,000 BNs/concentration/species was analyzed for MN in two independent
experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation
index was determined. Significant increases both in total MN-BNs and multiple
MN-BNs were observed at all concentrations in all species. All three species'
concentration-response curves gave good fits (r2 values from 0.87 to  0.95) to
either a linear or a square root model (y = mx + b or y = m(x)0.5 + b,
respectively; where y = the percentage of MN-BN, m is the slope, and b is the
y-intercept). The MN induction in the human and rat PBLs was not statistically
different, but both were significantly less sensitive than the response shown
by the BLM-exposed mouse PBLs. This difference in MN susceptibility was
observed only at BLM test concentrations : 20 mug/ml. The nucleation index was
significantly decreased  in all species at either 80 or 160 mug/ml. 

228
Hinton A Jr, Hume ME.  SYNERGISM OF LACTATE AND SUCCINATE AS METABOLITES
UTILIZED BY VEILLONELLA TO INHIBIT THE GROWTH OF SALMONELLA TYPHIMURIUM AND
SALMONELLA ENTERITIDIS IN VITRO.  Avian Dis 1995;39(2):309-16.  

The inhibition of salmonellae growth by a Veillonella bacterium isolated from
the cecal contents of adult chickens was examined. The Veillonella isolate was
grown on an agar medium supplemented with 175 mumol of lactate or
succinate/ml. Either 0, 100, 125, 150, or 175 mumol of succinate/ml was added
to the lactate medium; either 0, 100, 125, 150, or 175 mumol of lactate/ml was
added to the succinate medium; and the pH of all media was adjusted to 6.0.
Agar overlays of Veillonella cultures  grown on the media were inoculated with
Salmonella typhimurium or S. enteritidis. The largest zones of inhibition of
salmonellae growth were produced by Veillonella cultures grown on medium
supplemented with 175 mumol/ml of both lactate and succinate. The widths of
the zones of inhibition decreased as the concentration of lactate was reduced
in the succinate medium and as the concentration of succinate was reduced in
the lactate medium. Analyses of lactate broth and succinate broth  inoculated
with Veillonella indicated       that inhibition of salmonellae growth on the
agar media was related to the production of volatile fatty acids by
Veillonella, the presence of residual succinate in the media, and the final pH
of the media.

229
Mothersill C.  HUMAN ESOPHAGEAL CULTURE. Methods Mol Biol 1995;43:75-9. 

230
Jorritsma U, Cornet M, Van Hummelen P, Bolt HM, Vercruysse A, Kirsch-Volders
M, Rogiers V. COMPARATIVE MUTAGENICITY OF 2-METHYLPROPENE (ISOBUTENE), ITS
EPOXIDE 2-METHYL-1,2-EPOXYPROPANE AND PROPYLENE OXIDE IN THE IN VITRO
MICRONUCLEUS TEST USING HUMAN LYMPHOCYTES. Mutagenesis 1995;10(2):101-4.

2-Methylpropene (isobutene), a gaseous compound widely used in chemical
industries, is metabolized to the epoxide 2-methyl-1,2-epoxypropane. The
parent compound has previously been shown to be non-mutagenic in a modified
Ames test, whereas the epoxide metabolite gave a positive result. In this
study, both compounds have been tested in the in vitro micronucleus test using
human lymphocytes. Propylene oxide, a well known mutagenic compound, served as
a positive control. It was found that 2-methylpropene had no mutagenic effect,
whereas its epoxide induced a statistically significant dose-dependent
increase in the number of micronuclei. The effect observed was comparable with
that obtained for propylene oxide.


IMMUNOTOXICITY
231
Honda T, Miwatani T, Yabushita Y, Koike N, Okada K. A NOVEL METHOD TO
CHEMICALLY IMMOBILIZE ANTIBODY ON NYLON AND ITS APPLICATION TO THE RAPID AND
DIFFERENTIAL DETECTION OF TWO VIBRIO       PARAHAEMOLYTICUS TOXINS IN A
MODIFIED ENZYME-LINKED IMMUNOSORBENT ASSAY. Clin Diag Lab Immunol
1995;2(2):177-81.

A new method of chemically immobilizing antibody on nylon was developed. The
method consists of serial treatments with HCl, polyethylene imine, and maleic
anhydride methylvinyl ether copolymer, which resulted in the stable
immobilization of sufficient amounts of antibodies on nylon. This principle
was used to differentially detect two immunologically related but nonidentical
hemolysins (thermostable direct hemolysin (TDH) and TDH-related hemolysin
(TRH)) of Vibrio parahaemolyticus in a  modified enzyme-linked immunosorbent
assay with antibodies immobilized on nylon slips (NSIT). The results (dark
purple color on nylon slips) were easily evaluated by the naked eye. The
results with NSIT were compatible with those obtained by using DNA probes or a
conventional bacterial culture test, not only with cultured specimens but also
with clinical specimens (diarrheal stool samples). Furthermore, the NSIT
differentially detected TDH and TRH in a single test. The antibody
immobilization  method developed here is applicable to various immunological
detection methods and may improve their sensitivity and specificity.

232
Nicklin S. IMMUNE FUNCTION ASSAYS. Methods Mol Biol, 1995;43:245-56.

233
House RV, Thomas PT, Bhargava HN.  SELECTIVE MODULATION OF IMMUNE FUNCTION
RESULTING FROM IN VITRO EXPOSURE TO METHYLENEDIOXYMETHAMPHETAMINE (ECSTASY). 
Toxicology 1995; 96(1):59-69.

Abuse of illicit analogs of methamphetamine (i.e., 'designer drugs')
represents a growing problem. One of the most popular methamphetamine analogs
is (:)-3,4- ethylenedioxymethamphetamine (MDMA), commonly known as Ecstasy.
The authors demonstrated previously that in vitro exposure to methamphetamine
results in modulation of immune functional parameters necessary for host
defense. The current study was performed to assess the potential direct (in
vitro) immunomodulatory effect of exposure to a  modified methamphetamine.
Splenocytes or peritoneal macrophages from B6C3F1 mice were cultured in vitro
at MDMA concentrations of 0.0001-100 muM. T-cell regulatory function was
assessed by anti-CD3-mediated production of IL-2 and IL-4, B-cell function    
was assessed by quantitating cellular proliferation, natural immunity was
assessed by quantitating natural killer (NK) cell activity, T-cell effector
function was evaluated as a  unction of cytotoxic T-lymphocyte (CTL) activity,
and macrophage  function was assessed by IL-6 tumor necrosis factor (TNF)
production. In vitro exposure to MDMA had no effect on B-cell proliferation at
any concentration tested. In comparison, in the absence of direct cellular
toxicity, production of IL-2 was enhanced at concentrations as low as 0.0001
muM. IL-4 production was not affected by exposure to any concentration of MDMA
examined, suggesting a differential alteration in T-helper cell function by
this compound. Basal and augmented NK cell function  were enhanced at MDMA 
concentrations between 0.0001 and 1.0 muM when examined at an effector:target
ratio of 100:1. CTL induction was significantly suppressed at a concentration
of 100 muM. Finally,  macrophage production of TNF was slightly suppressed at
10 and 100 muM MDMA, although this inhibition was not statistically
significant. 

234
Jeong TC, Matulka RA, Jordan D, Yang KH, Holsapple MP.  ROLE OF METABOLISM IN
COCAINE-INDUCED IN IMMUNOSUPPRESSION IN SPLENOCTYE CULTURES FROM B6C3F1 FEMALE
MICE.  Immunopharmacology 1995;29(1): 37-46.

Cocaine has been reported to directly suppress the in vitro immune responses
at very high concentrations. In the present study, the possible role of 
metabolism in cocaine-induced immunosuppression was investigated in splenocyte
cultures isolated from B6C3F1 female mice. Since cocaine can be metabolized by
both esterase and P-450 monooxygenase, we studied the direct effects of
cocaine, benzoylecgonine and norcocaine on the in vitro T-dependent antibody
response to SRBC. Direct exposure to  cocaine only produced a modest (30%) but
nonsignificant suppression of the antibody response, while benzoylecgonine, a
primary product of metabolism by the esterase pathway, was devoid of activity.
In contrast, direct exposure to norcocaine, the initial product of
N-demethylation by the P-450 pathway, produced significant suppression at
concentrations greater than or equal to 10 muM. Similar results were observed
in studies measuring LPS and Con A mitogenicity. Furthermore, a significant 
suppression was observed when splenocytes were preincubated for 1 h with 1 mM
cocaine in the presence of liver S-9 fractions isolated from
phenobarbital-induced mice. Meanwhile, no suppression was obtained when
splenocytes were preincubated in the presence of untreated S-9 fractions. To
characterize the mechanism of our results, the capacity of both untreated and
phenobarbital-induced microsomes to produce formaldehyde from cocaine was
compared. The Ar-demethylation of cocaine was  NADPH-dependent and
phenobarbital-induced microsomes produced approx. 6-times higher amounts of
formaldehyde, indicating a greater portion of cocaine could be metabolized
through the P-450 pathway to its toxic metabolites. Finally, because
benzoylecgonine shares with cocaine the presence of a methyl group on the
tropane nitrogen, we also compared the ability of N-demethylation from cocaine
and benzoylecgonine in mouse liver microsomes. Our results indicated that
benzoylecgonine could not be  demethylated as determined by a failure to
generate any formaldehyde. These results offer further support that the
N-demethylation pathway is a critical step to cause its immunotoxicity. 

235
Kuehn U, Lempertz U, Knop J, Becker D.  A NEW METHOD FOR PHENOTYPING
PROLIFERATING CELL NUCLEAR ANTIGEN POSITIVE CELLS USING FLOW CYTOMETRY:
IMPLICATIONS FOR ANALYSIS OF THE IMMUNE RESPONSE IN VIVO. J Immunol Methods
1995;179(2):215-22.

The incorporation of radioactive nucleotides into newly synthesized DNA has
been established as a standard method for the detection of proliferation in
eucaryotic cells. Unfortunately the use of this method makes it harder to
obtain information on the phenotype of proliferating cells in mixed cell
populations. For this reason we established a flow-cytometric approach
employing a monoclonal antibody specific for murine as well as human
proliferating cell nuclear antigen (PCNA) and a double  labeling technique for
detection of cell membrane-expressed phenotypic markers. The efficiency of
this immunostaining procedure was confirmed by simultaneous and highly
specific detection of PCNA in nuclear structures as well as cell
membrane-expressed antigens using cytological techniques. In vitro experiments
with mitogen- and alloantigen-stimulated murine lymph node cells (LNC) and
human peripheral blood mononuclear eukocytes (PBML) revealed a good
correlation of total (3H)thymidine  incorporation into DNA and expression of
PCNA. For the analysis of proliferating cells activated in vivo the method was
employed to evaluate the local lymph node assay which assesses the
allergenicity of small chemicals. LNC prepared from the cervical lymph nodes
of mice treated on 4 consecutive days with sensitizing concentrations of the
contact allergens oxazolone, TNCB and DNFB as well as the irritants benzoic
acid and SLS in comparison to the solvent control showed a dramatic increase
in  the total amount of proliferating cells for contact allergen-treated
animals in comparison to the solvent control and irritant-treated mice. In
addition a detailed phenotyping of the proliferating cell populations was
possible. This approach offers  an easy to perform, non-radioactive method for
the assessment of proliferation of murine as well as human leukocytes in vitro
and especially in vivo and will be of great advantage for situations where the
phenotype of proliferating cellular subsets  in heterogeneous populations is
of interest.

236
Garssen J, Van der Vliet H, De Klerk A, Goettsch W, Dormans JA, Bruggeman CA,
Osterhaus AD, Van Loveren H.  A RAT CYTOMEGALOVIRUS INFECTION MODEL AS A TOOL
FOR IMMUNOTOXICITY TESTING. Eur J Pharmacol 1995;292
(3-4):223-31.

A rat cytomegalovirus infection model for use in immunotoxicity testing has
been developed. In resistance against viruses, natural killer cells and
cytotoxic T-cells play an important role. Therefore, this model complements
other rat host resistance models for immunotoxicity testing, i.e. existing
bacterial and parasitic infection models in which cytotoxic T-cells and
natural killer cells play a minor role. Host resistance against
cytomegalovirus infections in the rat was determined by titrating infectious
virus levels in organs after cytomegalovirus infection in an in vitro
infectivity test denoted as the Plaque Forming Unit (PFU) Test. In this test,
homogenates of different organs were investigated for infectious virus titers
on rat embryonic cell monolayers. We demonstrated that in the salivary gland,
the major target organ for rat cytomegalovirus, virus was detectable from 8
days onward after intraperitoneal infection. To show that this model is
suitable for the detection of immunotoxicity four different methods for
immunosuppression were investigated: 1. gamma-irradiation, 2. congenitally
athymic rats, 3. chemically induced immunosuppression, 4. ultraviolet-B (UVB)
irradiation. Rat cytomegalovirus titers in the salivary glands of irradiated
(500 rad 1 day prior to infection) or congenitally athymic rats were
significantly increased as compared to non-irradiated rats and euthymic
control rats respectively. In TOX-Wistar rats, given 20 or 80 mg
bis(tri-n-butyltin)oxide (TBTO) per kg food beginning 6 weeks before
cytomegalovirus infection, a regimen known to have immunotoxic effects,
cytomegalovirus titers in the salivary glands were significantly increased as
compared to non-TBTO-treated cytomegalovirus infected rats.(ABSTRACT TRUNCATED
AT 250 WORDS) 

237
Yokozeki H, Katayama I, Nishioka K. EXPERIMENTAL STUDY FOR THE DEVELOPMENT OF
AN IN VITRO TEST FOR CONTACT ALLERGENS: 1. PRIMARY ACTIVATION OF
HAPTEN-CONJUGATED EPIDERMAL CELLS. Int Arch Allergy Immunol
1995;106(4):394-400.

We conducted a study on the primary in vitro activation of T cells from
nonsensitized mice by using hapten-conjugated Pam 212 cells (keratinocyte cell
line). Furthermore, we attempted to develop a simple, quantitative in vitro
test to assess the sensitizing potency of contact allergens and applied it to
determine the stimulation index (SI) of various chemicals with known degrees
of sensitizing potency. Monolayered Pam 212 cells were incubated with a
variety of chemicals exhibiting allergenic potential. Washed and fixed T cells
depleted of autoreactive T cells and macrophages from spleens of nonsensitized
Balb/c mice were cocultured for 5 days with those monolayered Pam cells
conjugated with chemicals. They were then harvested and restimulated with
mitomycin-C-treated spleen cells conjugated with chemicals in 96-well culture
plates to inhibit the proliferation of stimulator cells. We evaluated the
sensitizing potency of the following chemicals: oxazolone, TNBS, DNFB and FITC
(strong  sensitizers); p-phenylendiamine (p-PD), nickel chloride and potassium
dichromate (potent sensitizers); betamethasone and budesonide
(corticosteroids), and methyl salicylate (MS) as an irritant. T cells
sensitized in vitro with TNP-Pam cells and macrophages demonstrated
antigen-specific proliferation when restimulated in vitro with
mitomycin-C-treated TNP-spleen cells. Subcutaneous injection of these T cells
induced contact sensitivity in vivo in an antigen-specific fashion. While T'
cells cocultured with TNP-3T3 could not be activated even in the presence of
macrophages. The SI of strong sensitizers was about 4.00 and that of p-PD was
2.36. The SIs of budesonide, betamethasone and MS were 1.62, 0.98 and 1.21,
respectively. These results suggest that this method of producing primary
hapten-specific T cell activation demonstrated its potential to become an in
vitro test for contact allergens in the future. 


238
Koh WS, Yang KH, Jeong TC, Delany B, Kaminski NE.  2-ACETYLAMINO-FLUORENE
INHIBITS THE ACTIVATION OF IMMUNE RESPONSES BY BLOCKING CELL CYCLE PROGRESSION
AT G1 PHASE. Arch Toxicol 1995;69(5): 350-6.

2-Acetylaminofluorene (AAF) inhibited in a dose dependent manner mouse spleen
cell blastogenesis in response to phorbol 12-myristate 13-acetate
(PMA)/Ionomycin (Io) activation, the T-cell lectin, concanavalin A (Con A),
and following stimulation by alloantigens as measured by the mixed lymphocyte
response (MLR). AAF also markedly suppressed the T-cell dependent antibody
forming cell (AFC) response to sRBC. AAF was most inhibitory on both the sRBC
IgM AFC response and Con A stimulated  proliferation when added during the
first 24 h following initiation of culture. Direct addition of high
concentrations of AAF (100 muM) to spleen cell cultures at 48 h following Con
A stimulation produced a very modest inhibition (<20%) of T-cell proliferation
as compared to 90% when added at the time cultures were initiated. Similarly,
AAF (75 and 100 muM) produced a greater than 80% inhibition of the in vitro
AFC response when spleen cells were sensitized with antigen in presence of
AAF. In  contrast, no inhibition of the IgM AFC response was produced when AAF
(75 muM) was added to spleen cell cultures 48 or 72 h after antigen
sensitization. Con A-triggered cell-cycle progression was attenuated at the G1
stage by the addition of AAF (50 and 100 muM) with no inhibition of S to G2/M
phase transition. These results suggest that the mechanism of AAF-mediated
immune suppression is through a blockade of cell cycle progression from G1 to
S phase. 

239
Post J, Vooys WC, de Gast GC, Bast BJ. COMPARISON OF VARIOUS IN VITRO ASSAYS
FOR EFFICACY SCREENING OF IMMUNOTOXINS.  Leuk Res 1995;19(4):241-7.

Before using immunotoxins in vivo, their efficacy is evaluated in in vitro
assays.  In this study we compare six different assays for the evaluation of
immunotoxins: protein and DNA synthesis inhibition assay, chromium release
assay, cell line colony assay, limiting diln. assay and clonogenic assay.  All
assays except the chromium release assay show specificity of the immunotoxins
in appropriate concns. The protein and DNA synthesis inhibition assays are
easy to perform and, therefore, suitable for initial screening, while the
clonogenic assay seems to be the best one for immunotoxin efficacy detn.

240
House RV, Thomas PT, Bhargava HN. IN VITRO EVALUATION OF FENTANYL AND
MEPERIDINE FOR IMMUNOMODULATORY ACTIVITY. Immunol Lett 1995; 46(1,2):117-24.

Exposure to drugs, either ethical pharmaceuticals or illicit street drugs,
often results in medical complications, including alterations in the immune
system.  Among the drugs assocd. with immunomodulatory potential are the
analgesics fentanyl and meperidine.  The purpose of this study was to det. the
potential of these drugs to alter immunol. parameters subsequent to in vitro
exposure at a range of concns. This potential immunotoxicity was assessed
using a series of in vitro assays measuring B-lymphocyte proliferation,
cytokine prodn. by T-helper lymphocytes, T-lymphocyte cytolytic function,
natural killer (NK) cell function, and macrophage function. Exposure to these
analgesics was assocd. with a differential suppression of interleukin-4 prodn.
by T-cells, as well as a more generalized suppression of cytokine prodn. by
macrophages.  In addn., T-cell cytolytic activity was suppressed at high drug
concns.  B-cell proliferation and NK cell activity were also inhibited, but to
a lesser degree than noted with T-cell function.  Addn. of naltrexone to the
cultures did not reverse these alterations in immune function, suggesting that
these changes are not mediated via opioid receptors. 


NEUROTOXICITY
241
Ennis MD, Stjernloef P, Hoffman RL, Ghazal NB, Smith MW, Svensson K, Wikstroem
H, Haadsma-Svensson SR, Lin C.  STRUCTURE-ACTIVITY RELATIONSHIPS IN THE
8-AMINO-6,7,8,9-TETRAHYDRO-3H-BENZ[E]INDOLE RING SYSTEM. Part 2: EFFECT OF
8-AMINO NITROGEN SUBSTITUTION ON SEROTONIN RECEPTOR BINDING AND PHARMACOLOGY.
J Med Chem 1995;38(12):2217-30.

A series of analogs of the potent and selective 5-HT1A agonist
8-(di-n-propylamino)-6,7,8,9-tetrahydro-3H-benz[e]indole-1-carbal- dehyde
(OSU191) was prepd. in which the dipropylamino group was modified to bear a
variety of substituents. These compds. were evaluated for both in vitro and in
vivo effects, including the establishment of a receptor binding profile for
these analogs at the 5-HT1A, dopamine D-2, dopamine D-3, 5-HT1Dalpha, and
5-HT1Dbeta sites.  Several of the analogs were evaluated for their biochem.
effects in reserpinized rats, specifically with regard to in vivo changes in
brain levels of 5-HTP and DOPA. Nearly all of the compds. prepd. for this
study were exceedingly potent at the 5-HT1A receptor, although most also
displayed significant affinity for the dopamine D-2 receptor. A strong
preference for the 5-HT1Dalpha over the 5-HT1Dbeta receptor was also apparent.
An analog bearing a butylglutarimide side chain, S-7k, was extremely selective
for the 5-HT1A receptor. Although this compd. possessed a Ki of 0.6 nM, it
elicited only modest changes in 5-HTP brain levels. However, this compd. did
not appear as an antagonist when tested in a cyclic-AMP-based intrinsic
activity assay. 

242
Van Muiswinkel FL, Jongenelen CA M, Schepens HT W, Stoof JC, Drukarch B. 
EFFECTS OF CHRONIC ACTIVATION OF DOPAMINE D-2 RECEPTORS IN CULTURES OF RAT
FETAL DOPAMINERGIC NEURONS: INDICATIONS FOR ALTERATIONS IN FUNCTIONAL
ACTIVITY.  Dev Brain Res 1995;85(1):128-36.

In Parkinsonian patients, previously subjected to neuronal grafting therapy,
the survival and functional status of dopaminergic grafts might be impaired by
the concurrent pharmacotherapy with L-DOPA and/or dopamine (DA) D-2 receptor
agonists. To test this hypothesis in vitro, we studied the effects of chronic
DA D-2 receptor activation on the functional capacity of cultured fetal rat
mesencephalic DA neurons, using the activity of tyrosine hydroxylase (TH) and
the intracellular dopamine content as neurochem. parameters. In cellular exts.
prepd. from our cultures, TH activity (as detd. by the release of 3H2O from
3H-[3,5] tyrosine) appeared to be tetrahydrobiopterin-, Fe2+, and temp.
sensitive, while in intact cells, the catalytic activity of TH could be
induced by K+-evoked       depolarization in a Ca2+-dependent way. In
contrast, no acute DA D-2 receptor mediated inhibitory effects could be
demonstrated in intact cells, either when tested under basal or depolarizing
conditions. Nevertheless, after chronic exposure to DA D-2 receptor agonists
for 14 days clear differences were obsd. in the functional status of cultured
fetal dopaminergic neurons. Thus, whereas the overall survival and basal TH
activity of cultured fetal dopaminergic neurons remained virtually unaltered,
the depolarization induced activation of TH was enhanced in agonist-treated
cultures. Moreover, after long-term treatment for 14 or 21 consecutive days,
the intracellular DA content of agonist treated cultures appeared to be
higher, as compared to untreated controls. It is concluded that chronic
activation of  DA D-2 receptors may induce adaptive alterations in the      
functional activity of cultured fetal dopaminergic neurons. The possible
consequences of these changes for the functional activity of (grafted) fetal
dopaminergic neurons are discussed. 

243
Zhang Z, Wu D. [EFFECT OF METHYLMERCURY CHLORIDE ON BRAIN MUSCARINIC RECEPTOR
IN RAT.] Hua Hsi I Ko Ta Hsueh Hsueh Pao 1994;25(4):388-92. (Chi)

In this study, we found that methylmercury chloride inhibited the binding of
3H-QNB to muscarinic receptor of rat brain tissue in vitro with IC50 values of
0.0137 +/- 0.0037mol/L. It also decreased the densities of muscarinic receptor
and affinity to 3H-QNB. In vivo test system, the rats were exposed to
methylmercury chloride (ip) 0, 1.5, 2.5, 3.5 mg/kg.d on the 7-12th day of
gestation. On the 7, 14 and 21st day after birth, the offsprings were killed,
and the cerebrum and cerebellum were immediately dissected on ice and
homogenizated. The ligand assays were carried out. The result showed the
effect of inhibition on the binding to cerebrum and cerebellum with
significant correlation.

244
Heaton MB, Paiva M, Swanson DJ, Walker DW.   ALTERATIONS IN  RESPONSIVENESS TO
ETHANOL AND NEUROTROPHIC SUBSTANCES IN FETAL SEPTOHIPPOCAMPAL NEURONS
FOLLOWING CHRONIC PRENATAL ETHANOL EXPOSURE. Devel Brain Research
1995;85(1):1-13. 

Pregnant Long-Evans rats were maintained on three diets: a liquid diet in
which ethanol accounted for 35-39% of the total calories, a similar diet with
the isocaloric substitution of sucrose for ethanol, and a lab chow control
diet. At gestation day 18, the fetuses were taken and cultures of septal and
hippocampal neurons prepared. Neuronal survival and neurite outgrowth were
compared in cultures from the three diet groups, using the following media
supplements: ethanol (1.2, 1.8 or 2.4 g/dl),  neurotrophic factors (nerve
growth factor (NGF) with the septal cultures, basic fibroblast growth factor
(bFGF) with the hippocampal cultures), or ethanol plus neurotrophic factors.
Both the septal and hippocampal neurons responded to ethanol in a
dose-dependent manner. The neurons from both populations from fetuses which
had been exposed prenatally to ethanol, however, tolerated considerably higher
ethanol concentrations before decreases in survival or outgrowth were seen.
These  ethanol-exposed neuronal populations were also less responsive to
neurotrophic factors: in hippocampal cultures, process outgrowth was
significantly enhanced by bFGF in control but not ethanol-derived cultures,
and in septal and hippocampal cultures, the neurotrophic factors significantly
ameliorated ethanol neurotoxicity in control cultures, but not in those from
the ethanol-exposed fetuses. The possible relevance of these observations to
the fetal alcohol syndrome is discussed. 

245
Abdulla EM, Calaminici M, Campbell IC.   COMPARISON OF NEURITE OUTGROWTH WITH
NEUROFILAMENT PROTEIN SUBUNIT LEVELS IN NEUROBLASTOMA CELLS FOLLOWING MERCURIC
OXIDE EXPOSURE. 
Clin Exper Pharmacol Physiol 1995;22(5):362-3.

1. The objectives of the study were to establish that inhibition of neuronal
differentiation in culture (assessed by neurite outgrowth) can be used as a
broad spectrum in vitro measure of neurotoxicity. 2. To establish whether a
rapid measure of neurite outgrowth could be used. Thus the study examined the
relationship between the degree of neurite outgrowth assessed directly by
image analysis and neurofilament protein subunit levels measured by an ELISA.
3. SKNSH neuroblastoma cells, exposed  for up to 6 days to mercuric chloride
during initiation and continuation of differentiation, had lower levels of
neurofilament proteins than unexposed cells. 4. Preliminary data from parallel
examinations of neurite outgrowth assessed by image analysis and neurofilament
protein subunit levels assessed by ELISA support a correlation when
neurofilament protein levels are decreased by sub-cytotoxic doses of mercuric
chloride in SKNSH cells.

246
Monnet-Tschudi F, Zurich MG, Riederer BM, Honegger P.  EFFECTS OF TRIMETHYLTIN
(TMT) ON GLIAL AND NEURONAL CELLS IN AGGREGATE CULTURES: DEPENDENCE ON THE
DEVELOPMENTAL STAGE.   Neurotoxicology 1995;16(1):97-104.

Long term effects of trimethyltin (TMT) applied at concentrations below the
cytotoxic level were examined in three-dimensional cell cultures of fetal rat
telencephalon using biochemical, immunochemical and morphological criteria. It
was found that in immature cultures low concentrations of TMT (10-8 M)
specifically induced a gliotic response in astrocytes, with increased
immunoreactivity for glial fibrillary acidic protein, and a greater number of
astrocytic processes. Significant changes in  oligodendrocytic and neuronal   
parameters were found only at 10-6 of TMT. In differentiated cultures,
distinct changes in cell type-specific parameters occurred at 10-6 of TMT (the
lowest effective concentration). In addition, different patterns of responses
were found for astrocytes and oligodendrocytes, as compared to immature
cultures. These results suggest that among neural cells, astroblasts are most
sensitive to TMT, and that the glial responses to this neurotoxicant are 
development-dependent.

247
Chute SK, Flint OP, Durham SK.  ANALYSIS OF THE STEADY-STATE DYNAMICS OF
ORGANELLE MOTION IN CULTURED NEURITES: PUTATIVE INDICATOR OF NEUROTOXIC
EFFECT.  Clin Exp Pharmacol Physiol 1995;22(5):360-1.

The objective of this study was to develop a physiol. based method to evaluate
the neurotoxic potential of drug candidates in vitro. Rat embryo midbrain
cells were grown in micromass culture, and the movement of mitochondria
labeled with the fluorescent dye rhodamine 123 was quantified in fasciculated
neurites by using a laser cytometer. The rhodamine 123 signal in a defined
region of the fascicle was quantified and photobleached with the laser. A
series of post-photobleach scans revealed the movement of fluorescent-labeled
mitochondria into the bleached region from adjacent unbleached regions.
Recovery of fluorescence is a measure of the size of the mobile pool of
mitochondria relative to the total (moving plus stationary) pool. The
steady-state levels of fluorescence recovery were dependent on intracellular
Ca2+ and Mg2+ concns., energy status (ATP), and microtubule integrity
(post-taxol or post-vinblastine treatment). This technique may be a useful
indicator of neurotoxic effect.

248
Mueller U, Krieglstein J. PROLONGED PRETREATMENT WITH ALPHA-LIPOIC ACID
PROTECTS CULTURED NEURONS AGAINST HYPOXIC, GLUTAMATE-, OR IRON-INDUCED INJURY.
J Cereb Blood Flow Metab 1995;15(4):624-30.

The antioxidant dihydrolipoic acid has been shown to reduce hypoxic and
excitotoxic neuronal damage in vitro. The present study tested whether
pretreatment with alpha-lipoic acid, which presumably allows endogenous
formation of dihydrolipoic acid, can protect cultured neurons against injury
caused by CN-, glutamate, or Fe ions, using the trypan blue exclusion method
to det. neuronal damage. One hour of preincubation with dihydrolipoic acid (1
muM), but not with alpha-lipoic acid, reduced damage to neurons from chick
embryo telencephalon caused by 1 mM NaCN or Fe ions. Alpha-Lipoic acid (1 muM)
reduced CN--induced neuronal damage when added 24 h before hypoxia, and
pretreatment with alpha-lipoic acid for >24 h enhanced this neuroprotective
effect. Both the R- and the S-enantiomers of alpha-lipoic acid exerted a
similar neuroprotective effect.  Pretreatment with alpha-lipoic acid (1 muM)
from the day of plating onward prevented the degeneration of chick embryo
telencephalic neurons that had been exposed to Fe2+/Fe3+. Alpha-Lipoic acid (1
muM) added to the culture medium the day of plating also reduced neuronal
injury induced by 1 mM L-glutamate in rat hippocampal cultures, whereas 30 min
of preincubation with alpha-lipoic acid failed to attenuate glutamate-induced
neuronal damage. The results indicate that neuroprotection by prolonged
pretreatment with alpha-lipoic acid is probably due to the radical-scavenger
properties of endogenously formed dihydrolipoic acid.

249
Sawyer T. PRACTICAL APPLICATIONS OF NEURONAL TISSUE CULTURE IN IN VITRO
TOXICOLOGY.  Clin Exp Pharmacol Physiol 1995;22(4):295-6.

1. Primary chick embryo forebrain neurones are relatively easy to culture and
are quite resilient to treatment manipulation. These characteristics have
allowed application in a surprisingly diverse number of areas. 2. These
cultures have been used to investigate the anticholinesterase potencies of
many organophosphate (OP) nerve agents and insecticides. 3. These cultures
have been used to quantitate levels of OP in 'spiked' unknowns and in
OP-contaminated soil samples. 4. These cells have also  been used to test a
variety of compounds using the MTT and neutral red cytotoxicity assays. 

250
Freeman JK, Goldberg MP. CONFOCAL MICROSCOPIC VISUALIZATION OF MK-801-INDUCED
CYTOPLASMIC VACUOLES IN VITRO. Psychopharmacol Bull 1994;  30(4):541-7.

We examined neuroprotective and neurotoxic effects of the NMDA antagonist,
MK-801, in primary cell cultures derived from embryonic mouse neocortex. Brief
deprivation of oxygen and glucose, or direct application of
N-methyl-D-aspartate (NMDA), resulted in acute neuronal swelling followed by
neuronal death during the next day. This excitotoxic neuronal injury could be
blocked by inclusion of a wide variety of NMDA antagonists in the cell culture
medium. MK-801 attenuated neuronal death in the low micromolar range; 1 to 10
microM concentrations were sufficient to maximally reduce injury from NMDA
toxicity, oxygen deprivation, or combined deprivation of oxygen and glucose.
MK-801 alone caused no apparent toxicity at these concentrations in exposures
of 24 to 48 hours. However, 24-hour exposure to 100 microM MK-801 resulted in
appearance of cytoplasmic vacuoles, which could be visualized with
immunofluorescence against the microtubule associated protein, MAP2, together
with laser scanning confocal microscopy. Thus, at concentrations sufficient to
block NMDA receptors, MK-801 is neuroprotective rather than neurotoxic for
cortical neurons in vitro. This model system may provide a method to examine
cellular mechanisms underlying the neurotoxicity of MK-801 at very high
concentrations.

251
Abdulla EM, Atterwill C, Campbell IC. WORKSHOP ON IN VITRO NEUROTOXICITY
TESTING THE OBSTACLES. THE WAY FORWARD.
VAL MORIN CANADA JULY 1994. Clin Exp Pharmacol Physiol 1995;22(4):277-80.

252
Vaalavirta L, Tahti H. EFFECTS OF SELECTED ORGANIC SOLVENTS ON THE ASTROCYTE
MEMBRANE  ATPase IN VITRO. Clin Exp Pharmacol Physiol 1995;22(4):293-4.

1. The present study deals with astrocyte cultures as a model for studying the
membrane-mediated central nervous system-depressing effect of organic
solvents. 2. The primary astrocyte cultures were prepared from neonatal rat
cerebella. The cells were cultured in modified essential medium. The astrocyte
membranes isolated from the cultures were exposed to solvents in incubation
mixture at different dose levels (3, 6 and 9 mmol/L) for 1 h. The
physiologically important integral proteins Na+,  K+-ATPase and Mg2+-ATPase
were studied. 3. The aromatic hydrocarbons (benzene, toluene, styrene, xylene
and ethylbenzene) inhibited the ATPase activities according to their lipid
solubilities. n-Hexane and cyclohexane clearly had less effect than aromatic
hydrocarbons, despite their greater lipid solubilities. 4. Astrocytes were
shown to be sensitive targets to the effects of organic solvents, measured as
the inhibition of the integral enzymes Na+, K+-ATPase and Mg2+-ATPase.

253
Martenson CH, Sheetz MP, Graham DG. IN VITRO ACRYLAMIDE EXPOSURE ALTERS GROWTH
CONE MORPHOLOGY. Toxicol Appl Pharmacol 1995;31(1):119-29. 

Acrylamide intoxication leads to degeneration of the longest axons of the
central and peripheral nervous systems in humans and laboratory animals.
Axonal derangements resulting from in vivo acrylamide exposure are first noted
within synapses of the longest axons before involving more proximally located
axonal segments or shorter axons, thus illustrating the specificity of
acrylamide for the terminal axonal regions. As a possible model system for
investigating the mechanism of toxicity of  acrylamide on the distal axon, we
exposed neurite-extending chick dorsal root ganglion (DRG) cells to acrylamide
in vitro and then examined growth cones for alterations in morphology and
function. Exposing DRG explants to media containing from 0.125 to 1.0 mM
acrylamide for 16 hr leads to specific and dose-responsive alterations of
growth cone morphology including: a nearly total loss of filopodial elements,
the preservation of highly active but two-dimensional lamellar structures, an
inappropriate extension of the axonal cytoskeleton into the forward region of
most growth cones, and a frequent breakdown of the central and peripheral
growth cone domains. The sulfhydryl alkylating agents ethacrynic acid,
iodoacetamide, and iodoacetic acid were tested and none produced
acrylamide-like morphological alterations at any dose. DRG cultures were also
exposed to the neurotoxic acrylamide analogs glycidamide, N-hydroxy
methacrylamide (HM-ACR), and methacrylamide (M-ACR). At  concentrations of
0.25 to 1.0 mM, glycidamide exposure resulted in acrylamide-like growth cone
alterations. HM-ACR exposure also resulted in growth cones that were
acrylamide-like but only at concentrations > 1.5 mM. M-ACR did not produce
acrylamide-like growth cones at doses of up to 16.6 mM. Thus, in vitro
exposure of DRG explants to acrylamide and two neurotoxic acrylamide analogs
leads to reproducible and specific morphological alterations that are
dose-dependent and       separable from the effects of sulfhydryl alkylation.

254
Ehrich M. USING NEUROBLASTOMA CELL LINES TO ADDRESS DIFFERENTIAL      
SPECIFICITY TO ORGANOPHOSPHATES. Clin Exp Pharmacol Physiol 1995; 22(4):291-2.

1. Organophosphates can cause acute toxicity, which follows inhibition of
acetylcholinesterase (AChE), or delayed neuropathy, which follows inhibition
of       neuropathy target esterase (NTE). 2. Human neuroblastoma SH-SY5Y
cells contain AChE and NTE. 3. Organophosphates actively able to inhibit AChE
in animal models inhibited AChE in neuroblastoma cells. 4. Inhibition of NTE
in neuroblastoma cells could identify active organophosphates capable of
causing delayed neuropathy in animal models and  distinguish these
organophosphates from those that do not cause delayed neuropathy in animal
models.

255
Durham HD, O'Brien C, Nalbantogul J, Figlewicz DA. USE OF TISSUE CULTURE
MODELS TO STUDY ENVIRONMENTAL-GENETIC INTERACTIONS RELEVANT TO
NEURODEGENERATIVE DISEASES. Clin Exp Pharmacol Physiol 1995;22(5): 366-7.

1. Clonal cell lines, primary cultured neurones and transgenic animals
expressing mutant genes linked to familial forms of neurodegenerative diseases
provide models in which to examine the interaction between expression of a
predisposing gene and exposure to neurotoxic chemicals. Methods of
establishing these models are reviewed. 2. Mutations in the gene encoding
Cu/Zn-superoxide dismutase (SOD-1) have been identified in cases of familial
amyotrophic lateral sclerosis linked to chromosome 21. We report that in
clonal lines of PC12 cells, the cytotoxicity of a glutathione-depleting
epoxide, styrene oxide, varied with SOD activity in a manner similar to that
previously demonstrated for redox cycling chemicals. These preliminary data
suggest that either low or high SOD-1 activities       may be associated with
greater toxicity of a variety of neurotoxic chemicals and their metabolites.

256
Carbonnelle P, Lison D, Leroy JY, Laurerys R. EFFECT OF THE BENZENE METABOLITE
HYDROQUINONE, ON INTERLEUKIN-1 SECRETION BY HUMAN MONOCYTES IN VITRO. Toxicol
Appl Pharmacol 1995;132(2):220-6.

Decreased interleukin-1 (IL-1) production by mononuclear phagocytes has been
shown to contribute to benzene myelotoxicity in animals. The study presented
here was designed to examine the relevance of this mechanism in humans. Fresh
human blood monocytes were exposed to 0.001-10 muM hydroquinone (HQ) and
assessed for their ability to release IL-1alpha and IL-1beta in response to a
stimulation with endotoxin. Both cytokines were measured by specific ELISA.
Exposure of human monocytes to micromolar concentrations of HQ for 2 hr
resulted in a dose-dependent reduction of IL-1 secretion. For both IL-1alpha
and IL-1beta, the decreases were statistically significant at concentrations
of 5 muM and above. HQ also inhibited RNA and protein synthesis in a
dose-dependent manner, with 50% inhibitory concentrations of 21 : 11 and 10 :
9 muM, respectively. Furthermore, monocytes treated with 5 muM HQ also
displayed a reduced total protein content when compared with control cells.
These data suggest tha the reduction of IL-1 production caused by HQ results
from a global impairment of       monocyte essential functions such as
transcription or translation. Taken as a whole, our results support a
mechanism whereby HQ may contribute to the myelotoxicity of benzene in humans
by inhibiting the production by mononuclear phagocytes of cytokines involved
in the regulation of hematopoiesis.

257
Kohila T, Hypponen S, Tahti H. THE NEUROTOXICITY OF LEAD STUDIED WITH RAT
SYNAPTOSOMAL INTEGRAL PROTEINS ATPase AND ACETYL-CHOLINESTERASE. Neurosci Res
Commun 1995;16(3):173-80.

The effects of inorganic lead on synaptosomal cell membrane integral proteins
were studied both in vivo and in vitro. In in vivo studies, outbred WI:Han
rats from Laboratory Animal Centre, Helsinki University were exposed to
inorganic lead (PbCO3), 29.0 mg/kg in the diet ad libitum. In the control diet
the amount of lead was 0.58 mg/kg. The experimental animals were exposed to
lead as follows: the mothers were eating the test diet during the whole
pregnancy and the suckling. After weaning the pups were fed with the test diet
for two months. This exposure caused a slight increase of blood lead level
(B-Pb 0.35 muM/l) compared to controls (B-Pb 0.20 muM/l). In the in vitro
study, incubation mixtures had known lead concentrations of 3, 6, 9, 15 and 30
muM/l. Synaptosomal membranes were isolated from rat cerebrum by using a
nontoxic isoosmotic Percoll gradient system. The integral proteins studied
were ATPase and acetylcholinesterase (AChE), both important in the normal
functioning of synaptosomal membranes. Inorganic lead in vitro caused a marked
dose-dependent decrease in Na+,K+-ATPase activity, but not in Mg2+-ATPase and
in AChE activity. After in vivo exposure, there was a slight but statistically
not significant decrease in the enzyme activities studied.

258
Kerr JE, Allore RJ, Beck SG, Handa RJ. DISTRIIBUTION AND HORMONAL REGULATION
OF ANDROGEN RECEPTOR (AR) AND AR MESSENGER RIBONUCLEIC ACID IN THE RAT
HIPPOCAMPUS.  Endocrinology 1995; 136(8):3213-21.

The actions of androgens in both peripheral and central tissues are linked in
part to their ability to specifically bind and activate androgen receptors
(ARs). ARs have been well studied in the rat hypothalamus and peripheral
reproductive tissues, where they are directly involved in endocrine feedback
mechanisms and reprodn. Previous studies revealed relatively high levels of AR
and AR mRNA in the rat hippocampus; however, the action of androgen in this
brain region remains unclear. To begin to address this issue, we used a
multidisciplinary approach to quantitate hippocampal AR and AR mRNA levels and
investigate their regulation after various hormonal manipulations. In vitro
binding assays revealed a single, saturable, high affinity binding site for
androgen in hippocampal cytosols. The expression of AR mRNA in the intact
adult male rat hypothalamus and hippocampus was demonstrated using reverse
transcription-polymerase chain reaction and quantified using a RNase
protection assay. Comparable levels of  AR mRNA were found in the hippocampus
and hypothalamus. In addn., in situ hybridization anal. revealed a unique
distribution of AR mRNA in the hippocampus. AR mRNA was found predominately in
the CA1 pyramidal cells, which form the major signal output of the hippocampal
trisynaptic circuit. Reverse transcription-polymerase chain reaction of total
RNA from microdissected hippocampal regions confirmed this distribution. RNase
protection assay demonstrated a significant decrease in the AR mRNA content of
the hippocampus in animals killed 4 days after castration or in intact rats
after four daily injections of the AR antagonist, flutamide (15 mg/animal),
compared to that in intact controls.  In contrast, a 35% increase in the
hippocampal AR mRNA content was found in old (22-mo-old) compared to young
(5-mo-old) male rats. In both cases, [3H]dihydrotestosterone binding to the
cytosolic prepn. did not parallel the changes obsd. in the AR mRNA content.
Taken together, these data demonstrate that hippocampal cells contg. AR can
respond to circulating androgen to alter AR gene expression. Furthermore, AR
mRNA autoregulation appears to be both age and tissue specific and does not
directly follow the regulatory patterns described for other steroid hormone
receptors found in the hippocampus. 

259
Skofitsch G. LIGHT MICROSCOPIC IN VITRO RECEPTOR AUTORADIOGRAPHY: X-RAY FILM
VISUALIZATION OF NEUROPEPTIDE RECEPTOR BINDING SITES IN RAT BRAIN.  Mod
Methods Anal Morphol [Proc. Int. Workshop Mod. Anal. Methods Histochem.]
1994;361-79. 

A general and simple autoradiog. procedure to visualize neuropeptide receptor
binding sites in rat brain tissue sections is described in detail. The method
utilizes unfixed slide-mounted cryostat tissue sections of the rat brain which
are incubated with 125I labeled neuropeptides. Visualization of binding sites
is achieved by direct exposure of radiolabeled tissue sections to x-ray film
in autoradiog. cassettes. For example, binding of 125I-porcine galanin to
coronal sections of the rat brain is shown. Details of the autoradiog.
procedure are discussed. 

260
Zhou A, Guo J, Wang H, Gu B, Du Y.  ENHANCEMENT OF NGF GENE EXPRESSION IN RAT
BRAIN BY THE MEMORY-ENHANCING PEPTIDE
AVP(4-8).  Peptides 1995;16(4):581-6.

Northern blot anal. of nerve growth factor (NGF) was used to evaluate the
effect of exogenous AVP(4-8) on the transcription of NGF gene in rat brain.
NGF expression was significantly enhanced by exogenous AVP(4-8) in the
hippocampus as well as in the cerebral cortex in a time period of 12 h. This
effect was inhibited by an antagonist to AVP(4-8). In addn., gel mobility
shift assay was also used to observe the in vitro expression of c-fos gene in
rat hippocampal slices. The results suggest that NGF gene is one of the target
genes responsible for memory-enhancing responses induced by AVP(4-8) and that
the enhancement of NGF gene expression may share the signaling pathway
mediated by AVP(4-8) receptor and c-fos gene expression.

261
Li J, Bigge CF, Williamson RM, Borosky SA, Vartanian MG, Ortwine DF. POTENT,
ORALLY ACTIVE, COMPETITIVE N-METHYL-D-ASPARTATE (NMDA) RECEPTOR ANTAGONISTS
ARE SUBSTRATES FOR A NEUTRAL AMINO ACID UPTAKE SYSTEM IN CHINESE HAMSTER OVARY
CELLS.  J Med Chem 1995; 38(11):955-65.

A series of enantiomerically pure (phosphonomethyl)-substituted phenylalanine
derivs. related to SDZ EAB 515 (I) were prepd. as competitive
N-methyl-D-aspartate (NMDA) receptor antagonists. Unlike most known
competitive NMDA antagonists, analogs in this series with the S-configuration
are potent NMDA antagonists whereas analogs with the unnatural R-configuration
are weak NMDA antagonists, as detd. by receptor binding expts. and their
anticonvulsant action in mice. Examn. in a previously reported competitive
NMDA pharmacophore model revealed that receptor affinity can be explained
partially by a cavity that accommodates the biphenyl ring of I, while the
biphenyl ring of the R-enantiomer extends into a disallowed steric region. We
proposed that analogs with the natural S-configuration and a large hydrophobic
moiety would have an advantage in vivo over analogs with an R-configuration by
being able to use a neutral amino acid uptake system to enhance both
peripheral adsorption and transport into the brain.  Examn. in a system L
neutral amino acid transport carrier assay shows that 1 competes with L-Phe
for transport in an apparent competitive and stereospecific manner (estd. Ki =
50 .mu.M).  The 1- and 2-naphthyl derivs. (II and III) were among the most
potent, competitive NMDA antagonists yet discovered, being ca. 15-fold more
potent than I in vitro and in vivo, with a long duration of action. The title
compd. II had potent oral activity in MES (ED50 = 5.0 mg/kg). II also retains
its ability to compete, albeit more weakly than I (estd. Ki = 200 muM), for
L-Phe uptake to CHO cells. In this series, analogs with the R-configuration
are not substrates for the system L neutral amino acid transport carrier. 
These results provide evidence that central nervous system active agents can
be designed as substrates of a neutral amino acid transporter as a means to
enhance penetration of the blood-brain barrier. 

262
Dicicco-Bloom E, Black IB. BLOOD-BRAIN BARRIER TRANSPORTERS OF NEUROLOGICAL
AGENTS DERIVED FROM GROWTH FACTORS.  PCT Int. Appl. PATENT NO. 95 07092
03/16/95 (University of Medicine and Dentistry of New Jersey).

A method for inducing neuronal precursor cells (NPCs) in vitro and in vivo
using proliferation factors to promote mitosis, survival or differentiation of
at least one type of NPC in the treatment of neuronal cell disorders caused by
disease, injury, and other neural disorders is described. Methods for
transporting mols. across the blood brain barrier by linking them to fragments
of growth factors to effectively introduce neurol. agents capable or incapable
of independently crossing the blood brain barrier into the central nervous
system is a major component of the treatment. Such substances include peptides
having the amino acid sequence Val Phe Phe. The in vivo and in vitro
regulation of cerebellar granule cell neurogenesis by basic fibroblast growth
factor is demonstrated.

263
Prasad JA,  Shukla VK, Lemaire S. SYNTHESIS AND BIOLOGICAL ACTIVITY OF
HISTOGRANIN AND RELATED PEPTIDES. Can J Physiol Pharmacol 1995; 73(2):209-14.

Histogranin (HN) was first isolated from bovine adrenal medulla and shown to
be a pentadecapeptide displaying N-methyl-D-aspartate (NMDA) receptor
antagonist       activity. To det. the active pharmacophore of HN, fragments
of the peptide were synthesized and their structure-activity relationships
studied by measuring their ability to displace the binding of [125I][Ser1]HN
to rat brain membrane prepns. and to block NMDA-induced convulsions in mice.
In the binding assay, only the full length peptide HN(1-10) displayed a high
affinity (Ki of 72 and 162 nM, resp.). All other tested fragments with
deletions at the N- and(or) C-terminals of the mol. showed large
(16-2500-fold) decreases in potency. The least active peptide fragment tested
was HN(6-10) (Ki of 164 muM). In vivo, HN and HN(2-15) (100 nmol; i.c.v.)
produced 94 and 40% protection against NMDA-induced convulsions in mice, resp.
None of the other peptide fragments displayed significant anticonvulsant
activity. The protective activity of HN (60 and 100 nmol) was markedly
antagonized by coadministration of HN(1-10) (100 nmol). The results indicate
that the in vivo anti-NMDA and in vitro binding activities of HN and related
peptides, with the exception of HN(1-10), depend upon the integrity of the
mol.  The high affinity of HN(1-10) for HN binding sites correlates well with
its antagonist effects towards the activity of the parent peptide.

264
Kiso K, Watanabe Y, Shibuya T.  CENTRAL MECHANISM OF A NOVEL NEUROTOXIC,
PARAQUAT, AND ITS RELATIONSHIP TO INCREASED AMOUNTS OF EXCITATORY AMINO ACIDS.
Neurosciences 1994;20(4):169-79.

The neurotoxic mechanism of paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridylium) in
the central nervous system due to the excessive release of brain excitatory
amino acids was studied using glia-rich and -poor cell cultures derived from
neonatal rat cerebellar granule cells. In glia-poor cell cultures, a 15-min
exposure to PQ (2 mM, 37.degree.) significantly enhanced the 50 mM KCl-evoked
glutamate (Glu) and taurine (Tau) release, but not glutamine (Gln) and glycine
(Gly) release. In glia-rich cell cultures, PQ tended to amplify the high
potassium-evoked release of the 4 amino acids, but these increases were not
significant. The enhancement of Glu release by PQ in the glia-poor cell
cultures was much greater than that in the glia-rich cell cultures. These
differences might be explained by the uptake of much of the released Glu by
the glial cells, because the potentiation of Gln synthetase activity was
higher in glia-rich cell cultures than in glia-poor cell cultures after the
treatment with PQ. Neuronal cell function in glia-poor cell cultures was
markedly damaged after a 30-min exposure to PQ (2 mM, 37.degree.) using the
lactate dehydrogenase (LHD) assay. Furthermore, in previously reported in
vitro studies, PQ increased the sustained rise of Ca2+ levels in the rat brain
synaptosomal fraction, and such Ca2+ increase was remarkably potentiated by 50
mM KCl (Pharmacol. and Toxicol., 1993). The above results suggest that part of
the PQ-induced neurotoxicity is assocd. with the increased release of Glu
caused by the unstable depolarizing condition of brain neuronal cells. 

265
Woodward RM,  Huettner JE, Guastella J, Keana JF, Weber E.  IN VITRO
PHARMACOLOGY OF ACEA-1021 AND ACEA-1031: SYSTEMICALLY ACTIVE QUINOXALINEDIONES
WITH HIGH AFFINITY AND SELECTIVITY FOR N-METHYL-D-ASPARTATE RECEPTOR GLYCINE
SITES.  Mol Pharmacol 1995; 47(3):568-81.

N-methyl-D-aspartate (NMDA) receptor antagonists show therapeutic potential as
neuroprotectants, analgesics, and anticonvulsants. In this context, we used
elec.       recording techniques to study the in vitro pharmacol. of two novel
quinoxalinediones, i.e., ACEA-1021 and ACEA-1031 (5-nitro-6,7-dichloro- and
5-nitro-6,7-dibromo-1,4-dihydro-2,3-quinoxalinedione, resp.). Assays with NMDA
receptors expressed by rat brain poly(A)+ RNA in Xenopus oocytes and with NMDA
receptors in cultured rat cortical neurons indicated that ACEA-1021 and
ACEA-1031 are potent competitive antagonists at NMDA receptor glycine sites.
Apparent dissocn. consts. (Kb values) for ACEA-1021 and ACEA-1031 ranged
between 6 and 8 nm for oocyte assays and between 5 and 7 nM for neuronal
assays. Cloned NMDA receptors expressed in oocytes showed up to 50-fold
variation in sensitivity, depending upon subunit compn. For example, using
fixed agonist concns. (10 muM glycine and 100 muM glutamate) IC50 values for
ACEA-1021 with four binary combinations were as follows: NMDA receptor
(NR)1A/2A, 29 nM; NR1A/2B, 300 nM; NR1A/2C, 120 nM; NR1A/2D, 1500 nM.
Measurement of EC50 for glycine and calcn. of Kb for the inhibitors indicated
that differences in IC50 values are due to subunit-dependent variations in
glycine affinity (EC50 ranged between .apprx.0.1 and 1 muM) combined with
variations in affinity of the antagonists themselves (Kb of .apprx.2-13 nM).
In addn. to the strong antagonism of NMDA receptors, ACEA-1021 and ACEA-1031
were also moderately potent competitive inhibitors of non-NMDA receptors
activated either by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
or by kainate.  Antagonist affinities were similar whether measured with
receptors expressed by rat brain poly(A)+ RNA in oocytes (Kb of 1-2 muM) or
with cultured neurons (Kb of 1.5-3.3 .mu.M). Our results suggest that the in
vivo neuroprotective actions of  ACEA-1021 and ACEA-1031 are predominantly due
to inhibition at NMDA receptor glycine sites, although addnl. inhibition at
non-NMDA receptors may play an ancillary role. 

266
Bilsky EJ, Calderon SN, Wang T, Bernstein RN, Davis P, Hruby VJ, McNutt RW,
Rothman RB, Rice KC, Porreca F. SNC 80, A SELECTIVE, NONPEPTIDIC AND
SYSTEMICALLY ACTIVE OPIOID DELTA AGONIST.  J Pharmacol Exp Ther
1995;273(1):359-66.

The present study has investigated the pharmacol. of SNC 80, a nonpeptidic
ligand proposed to be a selective delta agonist in vitro and in vivo.  SNC 80
was potent in producing inhibition of elec. induced contractions of mouse vas
deferens, but not in inhibiting contractions of the guinea pig isolated ileum
(IC50 values of 2.73 nM and 5457 nM, resp.). The delta selective antagonist
ICl 174,864 (1 muM) and the mu selective antagonist CTAP (1 muM) produced 236-
and 1.9-fold increases, resp., in the SNC 80 IC50 value in the mouse vas
deferens.  SNC 80 preferentially competed against sites labeled by
[3H]naltrindole (delta receptors) rather than against those labeled by
[3H]DAMGO (mu receptors) or [3H]U69, 593 (kappa receptors) in mouse
whole-brain assays.  The ratios of the calcd. Ki values for SNC 80 at mu/delta
and kappa/delta sites were 495- and 248-fold, resp., which indicates a
significant degree of delta selectivity for this compd. in radioligand binding
assays. SNC 80 produced dose- and time-related antinociception in the mouse
warm-water tail-flick test after i.c.v., i.th. and i.p. administration.  The
calcd. A50 values (and 95% C.I.) for SNC 80 administered i.c.v., i.th. and
i.p. were 104.9 (63.7-172.7) nmol, 69 (51.8-92.1) nmol and 57 (44.5-73.1)
mg/kg, resp.  The i.c.v. administration of SNC 80 also produced dose- and
time-related antinociception in the hot-plate test, with a calcd. A50 value
(and 95% C.I.) of 91.9 (60.3-140.0) nmol.  I.p. SNC 80 antinociception was
antagonized by pretreatment with i.c.v.       naloxone (3 nmol), with i.c.v.
or i.th. N,N-diallyl-Tyr-(Aib)2-Phe-Leu-OH(Aib=alpha-amino isobutyric acid)
(4.4 nmol) or with i.p. naltrindole (20 mg/kg), but not i.c.v. or i.th.
beta-FNA (18.8 nmol at -24 h).  Furthermore, the antinociceptive effects of
i.c.v. SNC 80 were antagonized by i.c.v. pretreatment with either
[D-Ala2,Leu5,Cys6]enkephalin (a putative delta1 antagonist) or [D-Ala2,
Cys4]deltorphin (a putative delta2 antagonist), but not by beta-funaltrexamine
(a  mu antagonist).  This suggests that the antinociceptive actions of SNC 80
are produced via both opioid delta1 and delta2, but not mu, receptors.  On the
basis of its profile in vivo and in vitro, SNC 80 is perhaps the first highly
selective, nonpeptidic and systemically active opioid delta agonist.  SNC 80
promises to be a useful compd. for the exploration of opioid delta-receptor
pharmacol. and provides a basis for the further identification of selective
nonpeptidic delta ligands.

267
Greer JJ, Carter JE.  EFFECTS OF CYANIDE ON THE NEURAL MECHANISMS CONTROLLING
BREATHING IN THE NEONATAL RAT IN VITRO.   Neurotoxicology 1995;16(2):211-5.

The effects of hydrogen cyanide (HCN) on the neutral mechanisms controlling
breathing were studied. Two in vitro experimental models were utilized: the
brain stem-spinal cord and the medullary slice preparations isolated from
neonatal rats. Cyanide, at concentrations deemed lethal in vivo (50 muM),
caused a modest (< 15%) depression of the frequency and amplitude of
inspiratory rhythmic discharge when added to the bathing media. Moreover, the
neuronal network underlying respiratory rhythmogenesis continued to function
for hours in the presence of very high concentrations of cyanide (600 muM). We
hypothesize that the rapid suppression of breathing caused by cyanide in vivo
is due to changes in neuronal excitability in respiratory modulating
populations in the CNS rather than due to perturbations of cellular oxidative
metabolism of neurons within respiratory rhythm generating centres.

268
Deshpande SS, Smith CD, Filbert MG. ASSESSMENT OF PRIMARY NEURONAL CULTURE AS
A MODEL FOR SOMAN-INDUCED NEUROTOXICITY AND EFFECTIVENESS OF MEMANTINE AS A
NEUROPROTECTIVE DRUG. Arch Toxicol 1995;69(6):384-90.

An in vitro mammalian model neuronal system to evaluate the intrinsic toxicity
of soman and other neurotoxicants as well as the efficacy of potential
countermeasures was investigated. The link between soman toxicity, glutamate
hyperactivity and neuronal death in the central nervous system was
investigated in primary dissociated cell cultures from rat hippocampus and
cerebral neocortex. Exposure of cortical or hippocampal neurons to glutamate
for 30 min produced neuronal death in almost 80% of  the cells examined at 24
h. Hippocampal neurons exposed to soman for 15-120 min at 0.1 mum
concentration caused almost complete inhibition (> 90%) of
acetylcholinesterase but failed to show any evidence of effects on cell
viability, indicating a lack of direct cytotoxicity by this agent.
Acetylcholine (ACh, 0.1 mM), alone or in combination with soman, did not
potentiate glutamate toxicity in hippocampal neurons. Memantine, a drug used
for the therapy of Parkinson's disease, spasticity and  other brain disorders,
significantly protected hippocampal and cortical neurons in culture against
glutamate and N-methyl-D-aspartate (NMDA) excitotoxicity. In rats a single
dose of memantine (18 mg/kg) administered 1 h prior to a s.c. injection of a
0.9 LD50 dose of soman reduced the severity of convulsions and increased
survival. Survival, however, was accompanied by neuronal loss in the frontal
cortex, piriform cortex and hippocampus. 

269
Struzynska L, Dabrowska-Bouta B, Lenart J, Zborowska J, Rafalowska U.  SOME
METABOLIC EFFECTS IN RAT BRAIN SYNAPTOSOMES AFTER EXPOSURE TO LEAD IN VIVO AND
IN VITRO.  Bull Pol Acad Sci Biol Sci 1994;42(1):55-62.

The mechanisms of the toxicity effect of lead on the central nervous system
are not clear. Our earlier investigation on isolated synaptosomes confirmed a
hypothesis, that Pb2+/Ca2+ interactions may play an important role in release
of neurotransmitter from synaptosomes. However, we have been also shown, that
Pb-toxicity effect on uptake of neurotransmission is not necessarily connected
with Pb2+/Ca2+ interactions. Thus, we look for other mechanisms, which can be
responsible for Pb-toxicity effects on neurotransmitters uptake. In the
present work we demonstrated that administration of Pb(CH3COO)2 in vivo and in
vitro did not affect phospholipid composition and content in synaptosomes and
the lipid peroxidation, but inhibit Na+-K+-ATPase activity. The decrease of
Na+-K+-ATPase activity can change the gradients of sodium and potassium across
the cell membrane and can be the cause of the disturbances in
neurotransmitters uptake.

270
Matsuda M,  Tsukada N,  Miyagi K, Yanagisa WA.  ADHESION OF LYMPHOCYTES TO
ENDOTHELIAL CELLS IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS BEFORE AND AFTER
TREATMENT WITH ENDOTOXIN LIPOPOLYSACCHARIDE. Int Arch Allergy Immunol
1995;106(4):335-44.

We investigated the in vitro adhesion of 51Cr-labeled lymphocytes to cultured
brain endothelial cells and the in vivo expression of intercellular adhesion
molecule-1 (ICAM-1) on cerebral endothelial cells in a rat model of
experimental allergic encephalomyelitis (EAE) before and after treatment with
lipopolysaccharide (LPS). Adhesion of lymphocytes to cerebral endothelial
cells was significantly increased in EAE compared with controls (p <0.01), and
was significantly correlated with the  percentage of major histocompatibility
complex class II antigen-positive cells in lymph node cells (p<0.001). LPS
enhanced ICAM-1 expression on endothelial cells and lymphocyte adhesion to
those cells, and caused a significant increase in the in vivo expression of
ICAM-1 compared with controls (p<0.001). Lymphocyte adhesion to endothelial
cells was significantly blocked by monoclonal antibodies against ICAM-1,
lymphocyte function-associated antigen-1, or very late activation antigen-4.
Our  findings suggest that lymphocyte adhesion to brain endothelial cells may
contribute to lymphocyte migration across the blood-brain barrier in EAE and
that LPS may cause progression of EAE lesions.

271
Mundy WR, Freudenrich T, Shafer TJ, Nostrandt AC. IN VITRO ALUMINUM INHIBITION
OF BRAIN PHOSPHOINOSITIDE METABOLISM: COMPARISON OF NEONATAL AND ADULT RATS. 
Neurotoxicology 1995;16(1):35-44.

Recent evidence indicates that the neurotoxic metal aluminum interferes with
the phosphoinositide second messenger system in adult rats both in vitro and
in vivo. We have examined the age-related effects of aluminum chloride (AlCl3)
on receptor-stimulated inositol phosphate (IP) accumulation in brain slices
from neonatal and adult rats in vitro. Carbachol-stimulated (1 mM) IP
accumulation was greatest in frontal cortex slices from 7 day old rats,
decreased in 14 day old and 21 day old rats,  and was lowest in adults (120
days old). AlCl3 (500 muM) inhibited both basal and carbachol-stimulated IP
accumulation in neonatal and adult rats. The effects of AlCl3 were
concentration-related and produced significant decreases (15-25%) in IP
accumulation at 500 and 1000 muM. The concentration-response curve for AlCl3
was similar in 7 day old and adult rats. AlCl3 reduced carbachol-,
norepinephrine- and quisqualate-stimulated IP accumulation in both 7 day old
and adult rats. The effects of 500 muM AlCl3 were examined on
carbachol-stimulated IP accumulation in slices prepared from frontal cortex,
hippocampus, striatum, and cerebellum. Although IP accumulation was greater in
slices from the 7 day old rats        compared to adults in each tissue, AlCl3
(500 muM) decreased IP accumulation by approximately 20% in all regions at
both ages. Aluminum produced concentration-dependent inhibition of
phospholipase C in cortical homogenates which was similar in 7 day old and
adult rats. These results show that in vitro exposure to aluminum decreases IP
accumulation through a mechanism which is not age-dependent. 

272
Jett DA, Guilarte TR. DEVELOPMENTAL LEAD EXPOSURE ALTERS N-MEYTHL-D-ASPARTATE
AND MUSCARINIC CHOLINERGIC RECEPTORS IN THE RAT HIPPOCAMPUS: AN
AUTORADIOGRAPHIC STUDY. Neurotoxicology 1995; 16(1):7-18.

We have used quantitative autoradiography of the N-Methyl-D-Aspartate (NMDA)
receptor non-competitive antagonist (+)-5-methyl-10,
11-dihydro-5H-dibenzo(a,d)-cyclohepten-5, 10-imine maleate in the tritiated
form ((3H)-MK-801) and the muscarinic cholinergic receptor antagonist
(3H)-N-Methylscopolamine ((3H)-NMS) to determine the effects of developmental
exposure to lead (Pb) on NMOA and muscarinic receptors in the rat hippocampus.
Exposure to Pb during development resulted in age-specific changes in the
level of binding of both ligands to their respective receptors.  Pb exposure
caused significant increases (19-49%) in (3H)-MK-801 binding throughout the
hippocampus and entorhinal cortex (ECTX) of postnatal day (PN) 14 rats
relative to controls. Small but significant region-specific reductions
(14-18%) and increases (12-15%) in (3H)-MK-801 binding were measured in PN28
rats. No significant differences in (3H)-MK-801 binding were observed among
any of the groups at PN56. Unlike (3H)-MK-801 binding, the       most
significant effects of Pb on (3H)-NMS binding were reductions in binding of
10-20% observed in the hippocampus and ECTX of Pb-exposed rats at PN14.
Additionally, Pb in vitro had no effect on the binding of (3H)-NMS to brain
sections (10 muM Pb acetate) or neuronal membrane preparations (0.1 nM to 0.1
mM Pb acetate) from normal neonatal or adult rats. These findings indicate
that there may be a critical developmental window during which Pb effects are
most pronounced, and the magnitude and direction of these changes is dependent
upon the age of the animal and the neurotransmitter system being examined. It
is suggested that NMDA and muscarinic receptors play an important role in the
developmental toxicity of Pb. 

273
Kisby GE, Ross SM, Spencer PS, Gold BG, Nunn PB, Roy DN.  CYCASIN AND BMAA:
CANDIDATE NEUROTOXINS FOR WESTERN PACIFIC AMYOTROPHIC LATERAL
SCLEROSIS/PARKINSONISM-DEMENTIA COMPLEX. Neurodegeneration 1992;1(1):73-82.

Medicinal and food use of seed of the neurotoxic cycad plant (Cycas spp.) is a
possible etiological factor for a model, age-linked neurodegenerative disease
frequenting certain Chamorro (Guam and Rota, Mariana Islands), Japanese
(Honshu island) and Papuan (west New Guinea) populations. The cycad plant
toxins beta-N-methylamino-L-alanine (BMAA) and cycasin (methylazoxymethanol
beta-Dglucoside) enter rodent brain tissue in vitro by a sodium-dependent
mechanism (BMAA) or via a mechanism compatible  with glucose transport
(cycasin). The uptake of radiolabel from (3H)-BMAA into mouse brain
synaptosomes is dependent on protein concentration and optimal at pH 7.4-9.0.
Cycasin uptake into mouse cortical explants is concentration- and
time-dependent with significant levels of the ultimate toxic metabolite
methylazoxymethanol (MAM) produced 24 h after glucoside treatment. Cycasin
(100 muM) significantly inhibited the uptake of (3H)-2-deoxyglucose (2-DG), a
marker for glucose transport,  into mouse cortical explants. Treatment of
mouse cortical explants with cycasin (1.0 muM-1000 muM) induced selective
neuronal degeneration with extensive vacuolation of neuronal perikaraya,
neuropil and post-synaptic elements. The uptake and subsequent intracellular
actions of these two cycad chemicals are potential mechanisms by which these
agents trigger their neurotoxic effects. Further research is underway to
characterize the intracellular actions of BMAA and cycasin, and to determine
their potential role in the prototypical western Pacific neurodegenerative
disorder. 

274
Gerasimyak GR, Rozanov VA, Shafran LM. [STUDY OF BIS-(N-TRIBUTYLTIN)-OXIDE
EFFECT ON THE BRAIN GABA-ERGIC SYSTEM IN VITRO.] Ukr Biokhim Zh
1994;66(2):71-9. (Ukr)

Wide concentration range (10-14-10-4 M) of bis-(n-tributyltin)-oxide effect on
Na+-dependent uptake, spontaneous and K+-stimulated release, specific receptor
binding and GABA metabolism were studied in vitro experiments using brain
slices, synaptic membrane fraction and brain tissue homogenates. It is shown
that the dependence concentration-effect is of non-linear character in all
cases. Prevailing supression of Na+-dependent uptake and specific receptor
binding during  K+-stimulated release and metabolism (production and
utilization) of GABA activation were marked as a general tendency. Mechanisms
of TBTO effect on the studied processes and the involvement of GABA-ergic
system in realization of TBTO neurotoxic effects are discussed.

275
Kurek A, Ledwozyw A. [NEUROTOXIC ESTERASE ACTIVITY OF HEN'S BRAIN HOMOGENATES
IN VITRO.] Bromatol I Chemia Toksykol 1994;27(2):175-9. (Pol)

The usefulness of phenyl butyrate, phenyl valerate, and phenyl caproate as
substrates for the determination of neurotoxic esterase activity, was
ascertained. The activity of hen's brain homogenate neurotoxic esterase was
measured, and I50 values of potent acetylcholinesterase inhibitors: mipafox
and DFP (diisopropyl fluorophosphate), were determined.

276
Walker I, Coleman MD.  THE BLOOD-BRAIN BARRIER: IN VITRO METHODS AND
TOXICOLOGICAL APPLICATIONS. Toxicol In Vitro 1995;9(2):191-204.

The blood-brain barrier (BBB) is reviewed with reference to in vitro cell
culture models and their use and potential use in toxicological studies. The
structure, function and in vitro study of brain microvessel endothelial cells
(BMEC) is briefly described, as well as the effects of a number of
xenobiotics, such as solvents, metals, polycations and herbicides, on the
viability and barrier function of the BBB model. The biotransformation of
xenobiotics is increasingly thought to be responsible for many toxic reactions
seen in living systems. Few studies have addressed the effects of the products
of biotransformation on the integrity of the barrier model. Many of the
specific human bioactivating enzymes, such as       cytochrome P-450s, can now
be conveniently studied in eukaryotic in vitro gene expression systems. The
combination of such systems with a well characterized porcine BMEC culture
model might be useful in the study of reactive metabolites on the BBB, in
terms of changes in indices of functional and structural BMEC viability. The
potential applications and the value of such an experimental approach are
discussed. 

277
Samynathan YM, Bondy SC.  INHIBITION OF PLASMA MEMBRANE AND MITOCHONDRIAL
TRANSMEMBRANE POTENTIALS BY ETHANOL.  Neurochem Res 1995;20(2):171-6.

The actions of ethanol and its primary oxidative metabolite, acetaldehyde, on
plasma membrane and mitochondrial transmembrane potentials were examined in
rat brain using fluorescence techniques. Subchronic treatment of adult rats
with ethanol resulted in a significant depolarization of both the plasma and
mitochondrial membranes when the mean blood ethanol level of the rats was 59 :
11 mM (mean : SEM, n = 6). Acute dosing of animals (4.5 g/kg, i.p.) failed to
show any significant alterations. Various concentrations of ethanol, added in
vitro to a crude synaptosomal preparation isolated from the rat cerebrocortex
(P2) from untreated animals, depolarized both the plasma and mitochondrial
transmembrane potentials in a dose-related manner. Addition of acetaldehyde in
vitro did not reveal any significant effects on plasma or mitochondrial
transmembrane potential.

278
Mokrzan EM, Kerper LE, Ballatori N, Clarkson TW.  METHYLMERCURY-THIOL UPTAKE
INTO CULTURED BRAIN CAPILLARY ENDOTHELIAL CELLS ON AMINO ACID SYSTEM L.  J
Pharmacol Exp Ther 1995;272(3):1277-84.

Recent in vivo studies suggest that the neurotoxin methylmercury (MeHg) is
transported into brain as an L-cysteine complex by amino acid transport system
L. To test this hypothesis, the mechanism of MeHg uptake into cultured calf 
brain capillary endothelial cells, an in vitro model of the blood-brain
barrier, was examined. Uptake of Me203Hg-L-cysteine followed Michaelis-Menten
kinetics, with a Km of 234 : 58 muM (mean : S.E.) and a Vmax of 57 : 25
pmolsec-1. Uptake of 10 muM MeHg-L-cysteine was stereoselective and Na+
independent and it was inhibited by the system L substrates L-leucine,
2-amino-2-norbornanecarboxylic acid and L-methionine (5 mM), consistent with
transport of MeHg-L-cysteine by the L amino acid carrier. L-Glutamate and
methylaminoisobutyric acid, which are transported by the acidic and A amino
acid carriers, respectively, had no effect. Moreover, uptake of 3H-L-leucine
(5 muM) was inhibited by 1 mM MeHg-L-cysteine. These observations provide
direct evidence that MeHg-L-cysteine is transported into brain capillary
endothelial cells by the L carrier. Uptake of other MeHg-thiols was also
measured. MeHg-D, L-homocysteine uptake was 82 : 11% of MeHg-L-cysteine
uptake, whereas uptakes of MeHg complexes of L-penicillamine,
dimercaptosuccinic acid, N-acetyl-L-cysteine and glutathione were 57 t 16%, 19
: 7%, 10 : 4% and 8 : 5% of MeHg-L-cysteine uptake, respectively. These
results illustrate the potential to  minimize transport of MeHg across brain
capillary endothelium by the careful choice of a thiol complexing agent.

279
Kowalski C, Crest M, Vuillet J, Pin T, Gola M, Nieoullon A. EMERGENCE OF A
SYNAPTIC NEURONAL NETWORK WITHIN PRIMARY STRIATAL CULTURES SEEDED IN
SERUM-FREE MEDIUM. Neuroscience 1995;64(4):979-93.

In order to investigate the basic cellular mechanisms involved in neuronal
interactions within the striatum, we prepared a primary striatal cell culture
from rat fetal brain in chemically defined medium. Using morphological and
whole-cell recording methods, we observed that an intensive neuritic
elongation with a progressive build up of a sodium-dependent electrogenesis
occurred during the first week of culture. Morphologically mature synapses
began to develop after 10 days in vitro. By this  time, most of the neurons
(82 + 9%) received spontaneously synaptic potentials, which led them to fire
(71 + 11%). The spontaneous firing was prevented by cadmium (200 muM) and
tetrodotoxin (5 muM), which suggested that a Ca2+-dependent release of
neurotransmitters was involved in the synaptic activation. We further obtained
evidence that GABA, and to a lesser extent acetylcholine, contributed to these
spontaneous synaptic potentials. At 15 days in vitro, it was possible to
observe up to four  synaptic contacts on a given dendrite. By this time,
whole-cell recordings performed on pairs of neurons showed that the mature
neurons were interconnected by excitatory synapses. As the number of synapses
increased, the striatal neurons gradually formed a large network in which
spontaneous activity developed, which tended to be organized into synchronized
bursting patterns. 

280
Green S. VALIDATION AND IN VITRO NEUROTOXICITY. Clin Exp Pharmacol Physiol
1995;22(5):383-4.

1. Validation of in vitro systems for studying neurotoxicants generally has
not been accomplished, although in vitro tests have been used as screens to
identify potential neurotoxic hazards and to study mechanisms. 2. A number of
factors need to be  taken into account when a test is validated: (i) a
rationale for developing the test; (ii) clear biological or pathophysiological
relevance of the endpoint to the effect detected in vivo; (iii) a standardized
protocol and evidence of intra- and  interlaboratory reproducibility; (iv)
testing of chemicals representative of the categories of interest, including
very toxic, moderately toxic and relatively non-toxic substances; and (v) a
method to statistically evaluate the data. 3. Proper validation should lead to
methods which can be used by regulatory agencies to make decisions regarding
hazard/risk.

281
Capo MA, Alonso CE, Sevil MB, Frejo MT.  "IN VITRO" EFFECTS OF METHYL-MERCURY
ON THE NERVOUS SYSTEM: A NEUROTOXICOLOGIC STUDY. 
J Environ Pathol Toxicol Oncol 1994;13(2):117-23.

Many of the currently prevailing toxicologic problems are due to the use of
organic mercurial compounds in pesticides and fungicides. During recent years,
environmental pollution has originated from the incorrect use of these
organometals. Methyl-mercury (Me-Hg) is absorbed quickly from the
gastrointestinal tract and is distributed to most tissues. The most important
effect of Me-Hg is on the nervous tissue and is more relevant in the fetal
brain. We were interested in assessing the neurotoxic effects of Me-Hg on the
central and peripheral nervous system. Neuronal cells cultures from 14-day-old
fetal Wistar rats and ciliary ganglion cells cultures from 8-day-old chick
embryos were used. Various Me-Hg concentrations (10-3 M to 10-8 M) were added
to these cultures after 36 hr to study the morphologic changes. At 10-3 M and
10-4 M concentrations, cellular degeneration and death in the central nervous
system (CNS) were noted. At 10-5 M concentrations, axonal and nerve fibers
degeneration, loss of synapsis, and inhibition in the cellular development in
CNS were seen; regroupment and destruction in the peripheral nervous system
(PNS) was noted. Finally, at 10-6 M and 10-7 M concentrations, there were
hardly any modifications in the CNS, whereas only the nervous processes were
affected in the PNS.

282
Audesirk T, Shugarts D, Cabell-Kluch L, Wardle K.  THE EFFECTS OF TRIETHYL
LEAD ON THE DEVELOPMENT OF HIPPOCAMPAL NEURONS IN CULTURE. Cell Biol Toxicol
1995;11(1):1-10.

Triethyl lead is the major metabolite of tetraethyl lead, which is used in
industrial      processes and as an antiknock additive to gasoline. We tested
the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5 mumol/L)
interfere with the normal development of cultured E18 rat hippocampal neurons,
possibly through increases in intracellular free calcium ion concentration,
(Ca2+)in. The study assessed survival and differentiation using morphometric
analysis of individual neurons. We also looked at short-term (up to 3.75-h)
changes in intracellular calcium using the calcium-sensitive dye fura-2.
Survival of neurons was significantly reduced at 5 mumol/L, and overall
production of neurites was reduced at : 2 mumol/L. The length of axons and the
number of axons and dendrites were reduced at : 1 mumol/L. Neurite branching
was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases
in intracellular calcium were observed during a 3.75-h exposure of newly
plated neurons to 5 mumol/L triethyl lead. These increases were prevented by
BAPTA-AM; which clamps (Ca2+)in at about 100 nmol/L. Culturing neurons with
BAPTA-AM and 5 mumol/L triethyl lead did not reverse the effects of triethyl
lead, suggesting that elevation of (Ca2+)im is not responsible for decreases
in survival and neurite production. Triethyl lead has been shown to disrupt
cytoskeletal elements, particularly neurofilaments, at very low levels,
suggesting a possible mechanism  for its inhibition of neurite branching at
nanomolar concentrations. 

283
Walum E, Forsby A. MEASUREMENT OF CELL MEMBRANE TOXICITY BY MEANS OF
2-DEOXY-D-GLUCOSE. Methods Mol Biol 1995; 43:129-35.

284
Spoerri PE, Srivastava N, Vernadakis A. GABA ATTENUATES THE NEUROTOXIC EFFECTS
OF ETHANOL IN NEURON-ENRICHED CULTURES FROM 8-DAY-OLD CHICK EMBRYO CEREBRAL
HEMISPHERES. Devel Brain Res 1995;86(1-2):94-100.

Neuron-enriched cultures were prepared from 8-day-old chick embryo cerebral
hemispheres and exposed to ethanol (50 mM), GABA (10-5 M) and ethanol (50 mM)
+ GABA (10-5 M) from day 4 to 8 in culture. At day 8, control, ethanol, GABA
and ethanol + GABA-treated cultures were examined morphologically and
biochemically. Choline acetyltransferase (ChAT) and glutamic acid
decarboxylase (GAD) activities were used as markers for cholinergic and
GABAergic neuronal phenotypic expression, respectively. Control cultures
showed more numerous and large neuronal aggregates as well as prominent
neuritic bundles. Moreover, cultures treated with GA-BA depicted even more
numerous neuronal aggregates with interconnecting neurites as compared to
control. In contrast, ethanol-treated cultures exhibited smaller neuronal
aggregates with less prominent neuritic bundles than control. However,
cultures treated concomitantly with ethanol + GABA exhibited numerous and
larger aggregates than cultures treated  with ethanol alone. Neuritic bundles
which were highly reduced in ethanol-treated cultures became prominent in the
presence of GABA. As previously reported,  ethanol alone enhanced ChAT and
reduced GAD activities. GABA given alone enhanced the expression of both
neuronal phenotypes. When GABA was given concomitantly with ethanol the
decline in GAD and the rise in ChAT observed in ethanol-treated cultures was
restored by GABA to almost control levels. Thus, ethanol-induced alterations
in morphology and neuronal phenotypes were counteracted by the neurontrophic
effect of GABA. 

285
Uto A, Dux E, Kusumoto M, Hossmann KA. DELAYED NEURONAL DEATH AFTER BRIEF
HISTOTOXIC HYPOXIA IN VITRO. J Neurochem 1995;64(5):2185-92.

The effect of three metabolic inhibitors - iodoacetate, potassium cyanide, and
potassium arsenate - on neuronal viability was studied in primary rat cortical
and hippocampal CA1 neuronal cultures. iodoacetate (0.1 mM) applied for 5 min
to 8-day-old cultures resulted in delayed neuronal death within 3-24 h in
cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by
transient inhibition of energy metabolism to synthesis tohe neuronal death
were prevented by the free radical scavenger vitamin E but not by the
glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure
could not protect against toxicity, and there was no increase of intracellular
calcium concentration during and shortly after iodoacetate treatment. Cyanide
and arsenate produced only partial neuronal degeneration, even at a dose of 10
mM. These observations demonstrate that brief exposure of  neurons to low
concentrations of iodoacetate produces a delayed type of neuronal death that
is not mediated by either calcium or glutamate. The therapeutic effect of
vitamin E points to a free-radical mediated injury and suggests that this type
of pathology may also be involved in delayed neuronal death after transient
energy depletion in vivo.

286
Wagner M, Toews AD, Morell P.  TELLURITE SPECIFICALLY AFFECTS SQUALENE
EPOXIDASE: INVESTIGATIONS EXAMINING THE MECHANISM OF TELLURIUM-INDUCED
NEUROPATHY.  J Neurochem 1995;64(5):2169-76.

A peripheral neuropathy characterized by a transient
demyelinating/remyelinating sequence results when young rats are fed a
tellurium-containing diet. The neuropathy occurs secondary to a systemic block
in cholesterol synthesis. Squalene accumulation suggested the lesion was at
the level of squalene epoxidase, a microsomal monooxygenase that uses NADPH
cytochrome P450 reductase to receive its necessary reducing equivalents from
NADPH. We have now demonstrated directly specificity for squalene epoxidase;
our in vitro studies show that squalene epoxidase is inhibited 50% in the
presence of 5 muM       tellurite, the presumptive in vivo active metabolite.
Under these conditions, the activities of other monooxygenases, aniline
hydroxylase and benzo(a)pyrene hydroxylase, were inhibited less than 5%. We
also present data suggesting that tellurite inhibits squalene epoxidation by
interacting with highly susceptible -SH groups present on this monooxygenase.
In vivo studies of specificity were based on the compensatory response to
feeding of tellurium. Following tellurium intoxication, there was
up-regulation of squalene epoxidase activity both in liver (11-fold) and
sciatic nerve (fivefold). This induction was a specific response, as
demonstrated in liver by the lack of up-regulation following exposure to the
nonspecific microsomal enzyme inducer, phenobarbital. As a control, we also
measured the microsomal monooxygenase activities of aniline hydroxylase and
benzo(a)pyrene  hydroxylase. Although they were induced following  
phenobarbital exposure, activities of these monooxygenases were not affected
following tellurium intoxication, providing further evidence of specificity of
tellurium intoxication for squalene epoxidase.

287
Soderstrom S, Ebendal T.  IN VITRO TOXICITY OF METHYL MERCURY: EFFECTS ON
NERVE GROWTH FACTOR (NGF)-RESPONSIVE NEURONS AND ON NGF SYNTHESIS IN
FIBROBLASTS. Toxicol Lett 1995;75(1-3):133-44.

The effect of methyl mercury chloride (MeHgCl) on the chick sympathetic and
sensory dorsal root ganglia was studied in a biological in vitro assay. These
cultures were not affected by the addition of MeHg up to a concentration of 2
muM. However, after an addition of 4-5 muM MeHg the capability of the neurons
to respond to added nerve growth factor (NGF) was completely inhibited. The
effect of MeHg was also examined in a fibroblast cell line, mouse 3T3 cells.
After the addition of mercury to the culture medium at concentrations as low
as 0.1 muM, an elevated production of the NGF protein was observed. However,
NGF mRNA measured in the individual fibroblast cells by in situ hybridization
was found to be reduced to about 80% of the control in the low level at day 2
of exposure. These results suggest that the release of NGF is actively
enhanced from the 3T3 cells by addition of low levels of mercury. The results
thus show that MeHg at low to moderate concentrations has adverse effects on
NGF responses in cultured neurons and moreover alter levels of NGF production
in cells, suggestive of mechanisms for mercury toxicity in the developing
nervous system.

288
Stehrer-Schmid P, Wolf HU. EFFECTS OF BENZOFURAN AND SEVEN BENZOFURAN
DERIVATIVES INCLUDING FOUR CARBAMATE INSECTICIDES IN THE IN VITRO PORCINE
BRAIN TUBULIN ASSEMBLY ASSAY AND DESCRIPTION OF A NEW APPROACH FOR THE
EVALUATION OF THE TEST DATA. Mutat Res 1995;339(1):61-72.

The influence of benzofuran and 7 benzofuran derivatives, including the
carbamate insecticides benfuracarb, carbofuran, carbosulfan, and furathiocarb,
on the in vitro assembly kinetics of porcine brain tubulin was investigated. A
new approach to the evaluation of the raw data was made based on polynomial
regression and the calculation of a polynomial function of the 11th degree
fitting the raw data. By this procedure it is possible to calculate the
parameters defining the shape of the  absorbance curves and more parameters
than those used so far can be included in the analysis of substance effects.
In detail, the following curve parameters of the dependence of optical
absorption on time were included in the evaluation of the substances of
interest: the difference between maximum and minimum absorbance as a measure
for the polymerization degree, the coordinates of the turning point of the
curve, the slope of the tangent at the turning point which represents the
maximum  reaction velocity, the mean slope between the points with 10%
absorbance increase and 90% absorbance increase and the duration of the lag
phase. Out of the eight compounds tested, only the carbamate insecticides had
distinct effects on the in vitro polymerization of tubulin, whereas benzofuran
and the three 2,3-dihydro-2,2-dimethylbenzofuran derivatives without a
carbamate function were inactive. Benfuracarb, carbofuran, carbosulfan, and
furathiocarb led to a dose-dependent reduction of the polymerization degree of
tubulin as well as to reduction of the maximum and mean reaction velocities.
The strongest effects were obtained with furathiocarb and benfuracarb.


OCULAR TOXICITY
289
Earl LK, Jones PA, Dixit MB, O'Brien KA.  COMPARISON OF FIVE POTENTIAL METHODS
FOR ASSESSING OCULAR IRRITATION IN VITRO. Toxicol In Vitro 1995;9(3):245-50.

Thirty-three test substances comprising household and personal products and a
pure surfactant were used in an independent evaluation of the ability of five
selected in vitro assays to predict eye irritation potential. The data were
compared with historical archived data from rabbit eye irritation tests
conducted on the same subsamples. The data were assessed in three ways. The
linear correlation for all 33 test substances with in vivo data was poor for
each assay. However, the correlation improved greatly for some assays when a
group of similar test substances was considered. Anal. of the data by Cooper's
criteria suggested that fluorescein diacetate uptake by Chinese hamster V79
cells (V79) and the Microtox test kit were the best of the assays evaluated in
discriminating correctly between the irritants and non-irritants that were
identified by rabbit eye irritation testing. Some of the data were selected
for further anal. because it was possible to predict differences, based on
experience learned from animal data on similar substances, between the test
substances without ref. to exptl. data. Near or more concd. test substances
were predicted to more irritant than dilns. and an exptl. laundry powder
formulation contg. 10% sodium metasilicate (MTS) was predicted to be more
irritant than two analogous non-MTS contg. formulations. The exptl. data were
then compared with the prediction. The in vivo rabbit eye irritation tests and
Microtox distinguished correctly between all the test substances in this anal.
Eytex was unable to distinguish correctly between any of the test substances
in this anal. The other assays were able to distinguish between some of the
test substances. No single assay performed well in every type of anal. and it
is concluded that a battery of assays is required to obtain reliable
predictive data. Overall, Microtox, V79 fluorescein diacetate accumulation and
3T3 neutral red uptake were the most promising assays. Extreme caution should
be used in comparing in vitro results with rabbit eye irritation data to make
definitive conclusions. 

290
Pasternak AS, Miller WM.  FIRST-ORDER TOXICITY ASSAYS FOR EYE IRRITATION USING
CELL LINES: PARAMETERS THAT AFFECT IN VITRO EVALUATION. Fundam Appl Toxicol
1995;25(2):253-63.

First-order toxicity assays can be used to rapidly screen test agents.
Investigators in many labs. have used cultured cell lines to obtain
correlations between first-order assay end-points and in vivo eye irritation
(Draize test) for a wide variety of compds. Since validation is a key step in
assay acceptance, it is important to understand which factors alter the
responses of cell-line-based assays. In this study the authors examine: (1)
the presence and configuration of a type I collagen gel; (2) the responses of
epithelial (Sf-I-Ep) and fibroblast (Sirc and 3T3) cell lines; (3) the total
glutathione content, ATP content, methionine incorporation, and neutral red
absorption endpoint assays; (4) alc. (C2-C8), surfactant (Tween 20), and heavy
metal (NiCl) test agents; and (5) test agent exposure time (1 to 24 h). The
presence of a collagen gel and the cell type did not significantly affect
endpoint assay R50 (test agent concn. that decreases assay response by 50%)
values for a 1-h exposure to hexanol. The ATP and glutathione endpoints (after
1-h exposure) are able to distinguish between the relative in vivo toxicities
of C2-C8 normal alcs. All four endpoint assays detected sublethal damage, with
the ATP and methionine endpoints being the most sensitive. The type of test
agent affects the endpoint response, as shown by the lack of a glutathione R50
value for a 1-h exposure to Tween 20 or NiCl. Even for a single test agent,
endpoint assay R50 values may decrease continuously (ATP), decrease and then
stabilize (glutathione), or remain unchanged (methionine incorporation) during
a 24-h exposure. 

291
Nishi C, Nakajima N, Ikada Y.  IN VITRO EVALUATION OF CYTOTOXICITY OF DIEPOXY
COMPOUNDS USED FOR BIOMATERIAL MODIFICATION.  J Biomed Mater Res
1995;29(7):829-34.

The toxicity of various diepoxy compds. used for biomaterials crosslinking was
investigated with a cell culture method and compared with an in vivo method.
The       neutral red uptake by cells was used to count the no. of cells still
alive after contact with the diepoxy compds., because this method was more
sensitive in cell counting than the other four methods studied in this work.
The amt. of neutral red taken up by cells depended strongly on the activity of
cells in comparison with other methods; only small amts. of neutral red were
taken up when cells were in a low activity state even if they were still
alive. The in vitro toxicity of diepoxy compds. evaluated by the neutral red
method revealed a good correlation with that found by the in vivo Draize test.
The in vitro cytotoxicity to a cell line of L929 was closely related to that
of primary culture cells of the normal rabbit cornea epidermal cell. The
toxicity of diepoxy compds. was lower as their chain was longer, probably
because of the lower chem. reactivity. All the diepoxy compds. investigated in
this study exhibited lower cytotoxicity than formaldehyde, glutaraldehyde, and
a water-sol. carbodiimide. 

292
Rasmussen ES. PROSPECTS FOR USE OF IN VITRO METHODS FOR ASSESSMENT OF HUMAN
SAFETY. Levnedsmiddelstyr (National Food Agency of Denmark); 1995. Ministry of
Health Publication No. 229. 126 p. 

The prospects for use of in vitro methods as replacements for or supplements
to animal expts. currently used for nonmedical assessment of human safety are
discussed. The present report concs. on the potential introduction of
scientifically validated in vitro alternatives to the acute toxicol. animal
tests such as the ocular and dermal Draize tests for corrosivity and irritancy
and the LD50 expts. for acute systemic effects. 

293
Durrani AM, Farr SJ, Kellaway IW.  INFLUENCE OF MOLECULAR WEIGHT AND
FORMULATION pH ON THE PRECORNEAL CLEARANCE RATE OF HYALURONIC ACID IN THE
RABBIT EYE. Int J Pharm 1995;118(2):243-50.

Hyaluronic acid is a natural polymer which, due to its water retaining
capability, binds to cell membranes and can therefore be considered as a
putative vehicle for controlled ocular delivery. In an in vitro mucoadhesion
test, the force of detachment was significantly greater for Healon (HA-Na)
compared to low mol. wt. hyaluronic acid. Also, this bioadhesion was stronger
for Healon at pH 5 than at pH 7.4. The precorneal clearance of sodium
hyaluronate (0.2%) was investigated at pH 5.0 and 7.4 by employing gamma
scintigraphic imaging of the 111In-labeled biopolymer. Protonation of the
macromol. did not result in any increase in ocular mucoadhesion as the mean
residence time at pH 5.0 was not significantly longer than at pH 7.4. The
effect of mol. wt. of hyaluronic acid on the corneal retention was also
investigated. There was a statistically significant difference (p<0.05) in the
clearance half-life (t0.5) and AUC of % activity remaining vs time plot for   
Healon (mol. wt 2.2.times.106) compared to those obsd. for the two lower
mol. wt. hyaluronic acid samples (mol. st 134 000 and 620 000). 

294
Northover AM.  THE USE OF THE BOVINE ISOLATED CORNEA AS A POSSIBLE IN VITRO
TEST FOR OCULAR IRRITANCY.  Methods Mol Biol 1995;43:205-10.

295
Spielmann H.  HET-CAM TEST. Methods Mol Biol 1995;43:199-204.

296
Vian L, Vincent J, Maurin J, Fabre I, Giroux J, Cano JP.  COMPARISON OF THREE
IN VITRO CYTOTOXICITY ASSAYS FOR ESTINATING SURFACTANT OCULAR IRRITATION.
Toxicol In Vitro 1995;9(2):185-90.

Three in vitro cytotoxicity assays (neutral red uptake assay (NRU), MTT test
and total protein content determination (TPC)) were analysed to assess their
value for predicting the ocular irritancy potential of 20 surfactants. For
each test, three established cell lines (SIRC rabbit corneal cells, Balb/c 3T3
and L929 mouse fibroblasts) were used. The concentration that induced 50%
inhibition relative to controls (IC50) was calculated for each test, cell line
and chemical. In vivo ocular  irritancy data were compared with in vitro
results. None of these assays provided a marked correlation of surfactant
ocular irritation (Spearman or Pearson correlation coefficient lower than
0.65, P < 0.01 and moderate agreement with Kappa test). An IC50 threshold of
700 mug/ml was set to discriminate between surfactant irritation and
non-irritation. Three compounds were detected as false negatives (CHAPS,
CHAPSO and sodium taurocholate), and two as false positives (Triton X155 and
Brij 35).

297
Joller PW, Coquette A, Noben J, Pirovano R, Southee JA, Logemann PK.  EUROPEAN
INTERLABORATORY EVALUATION OF AN IN VITRO OCULAR       IRRITATION MODEL SKIN-2
MODEL ZK1100 USING 18 CHEMICALS AND       FORMULATED PRODUCTS. In: Reinhardt
CA, editor. Alternatives to Animal Testing: New Ways in the Biomedical
Sciences, Trends and Progress; Symposium; 1992 Nov 30; Zurich, Switzerland.
New York: VCH Publishers, Inc: 1994. p. 159-64.


PULMONARY TOXICITY
298
Herman EH,  Hasinoff BB,  Zhang J, Raley LG, Zhang TM, Fukuda Y, Ferrans VJ. 
MORPHOLOGIC AND MORPHOMETRIC EVALUATION OF THE EFFECT OF ICRF-187 ON
BLEOMYCIN-INDUCED PULMONARY TOXICITY. Toxicology 1995;98(1-3):163-75.

Morphologic and morphometric studies were made of the protective effects of
ICRF-187 against the pulmonary damage induced by bleomycin in male and female
C57/BL6 mice. Sixty minutes prior to the subcutaneous administration of 15
mg/kg of bleomycin, animals received either saline or ICRF-187 (300 or 150
mg/kg) intraperitoneally, twice a week for 4 weeks. The lungs of animals
treated with bleomycin alone showed inflammation, hyperplasia of type II
epithelial cells, squamous cell metaplasia and fibrosis. The extent of
fibrosis was quantified by means of a color videometric system and histologic
sections of lung stained according to a modified Masson trichrome method. The
severity of  these alterations, particularly of fibrosis, was reduced in all
groups of animals pretreated with ICRF-187. The fibrosis was reduced to a
similar extent in female mice treated with the 300 mg/kg and the 150 mg/kg
doses of ICRF-187, from 39.3% to 17.6% and 13.3%, respectively. ICRF-187
induced significantly different degrees of reduction in fibrosis in the 2
groups of male mice treated with the 150 mg/kg and the 300 mg/kg doses, from
30% to 19.7% and 12.2%, respectively. In vitro studies indicated that both
ICRF-187 and its open-ring hydrolysis product (ADR-925) remove iron slowly
from the bleomycin-iron complex. This observation provides a basis for the
concept that ICRF-187 protects by chelating iron involved in the formation of
the bleomycin-Fe3+ complex that generates reactive oxygen radicals capable of
causing pulmonary damage. 


RENAL AND HEPATIC TOXICITY
299
Lave T, Schmitt-Hoffmann AH, Coassolo P, Valles B, Ubeaud G, Ba B, Brandt R,
Chou RC. A NEW EXTRAPOLATION METHOD FROM ANIMALS TO MAN: APPLICATION TO A
METABOLIZED COMPOUND, MOFAROTENE. Life Sci 1995; 56(26):PL473-PL478.

Allometric scaling (a technique which uses data obtained in lab. animals to
predict human pharmacokinetics) works well for drugs that are cleared intact,
but is less successful with extensively metabolized compds. This paper
describes a new method to improve the accuracy of such projections, by
integrating metabolic data obtained in vitro (e.g., with liver microsomes or
hepatocytes) into these calcns. The approach was used prospectively, to
predict the clearance of mofarotene (Ro 40-8757) in humans from in vivo
kinetic data obtained in mice, rats and dogs. This compd. was selected to
illustrate this approach because it is exclusively eliminated through metab.
Without the metabolic correction and by using brain wt. as an empirical
correcting factor, the oral clearance values predicted for man were 2.7 and
0.6 mL/min/kg, resp. This fell outside the range subsequently obtained in
healthy volunteers treated orally with 300 mg mofarotene (7.5 .+-. 4.0
mL/min/kg).  However, inclusion of the microsomal or hepatocyte data gave
values of 5.1 and 4.2 mL/min/kg, resp., illustrating that the integration of
in vitro metabolic data improves the accuracy of kinetic extrapolations. In
contrast to the existing empirical techniques, this approach offers a rational
basis for predicting clearance of metabolized compds. in humans.

300
Khan SA, Ghosh S, Wickstrom M, Miller LA, Hess R, Haschek WM, Beasley VR.  
COMPARATIVE PATHOLOGY OF MICROCYSTIN-LR IN CULTURED HEPATOCYTES, FIBROBLASTS,
AND RENAL EPITHELIAL CELLS. Nat Toxins 1995;3(3):119-28.

The cyanobacterial toxin microcystin-LR (MCLR) is a potent inhibitor of
protein       phosphatases 1 and 2A, and is selectively toxic to the liver in
vivo and to isolated hepatocytes in vitro. This selectivity is believed to be
due to toxin uptake via bile acid carriers. We investigated at the light and
ultrastructural levels the effects of high concentrations of MCLR and long
incubation times to determine in vitro whether fibroblasts and kidney cells
(non-target cells) respond in the same manner as  do hepatocytes (target
cells) at low concentrations and short incubation times. Cultured rat skin
fibroblasts (ATCC 1213) and rat kidney epithelial cells (ATCC 1571) were
incubated with MCLR at 133 muM for 1-24 hr. Lesions in these cells were
compared with those in cultured hepatocytes incubated with MCLR at 13.3 muM
from 1 to 32 min. Lesions in hepatocytes, kidney cells, and fibroblasts were
noted at 4 min, 1 hr, and 8 hr, respectively, after initial exposure to MCLR.
Lesions in all three cell  types progressed and included plasma membrane
blebbing, loss of cell-to-cell contact, clumping and rounding of cells,
cytoplasmic vacuolization, and redistribution of cytoplasmic organelles. Loss
of microvilli, whorling of rough endoplasmic reticulum, dense staining and
dilated cristae in mitochondria, and pinching off of membrane blebs were noted
only in hepatocytes. Nuclear changes typical of apoptosis were observed only
in fibroblasts and kidney cells.      Similarities in responses of different 
cell types to MCLR exposure probably reflect a common biochemical mechanism of
action, i.e., inhibition of protein phosphatases 1 and 2A as described by
others. The observed differences in the responses of the cell types examined
in this study may reflect differences in the proteins phosphorylated and the
severity of hyperphosphorylation. 

301
Ferro M.  HEPATOMA CELL CULTURES AS IN VITRO MODELS FOR HEPATOTOXICITY.
Methods Mol Biol 1995;43:51-7.

302
James NH, Roberts RA.  SPECIES DIFFERENCES IN THE CLONAL EXPANSION OF
HEPATOCYTES IN RESPONSE TO THE COACTION OF EPIDERMAL GROWTH FACTOR AND
NAFENOPIN, A RODENT HEPATOCARCINOGENIC PEROXISOME PROLIFERAR. Fundam Appl
Toxicol 1995;26(1):143-9.

Peroxisome proliferators are members of the nongenotoxic family of rodent
hepatocarcinogens. There exist substantial species differences in response to 
peroxisome proliferators among mammalian species. We have reported
previously that peroxisome proliferators can synergize with epidermal growth
factor (EGF) to promote the clonal expansion of rat hepatocytes associated
with the early stages of hepatocarcinogenesis. The aim of the present study
was to determine whether responsiveness in this in vitro assay reflected the
known species differences in response to peroxisome proliferators. The process
of tumorigenicity was modeled in the soft agar cloning assay since growth in
soft agar is thought to reflect the early stages of tumorigenesis. This is
because clonal expansion under these conditions requires the cells to survive,
to undergo mitosis, and to escape from the contact-dependent growth associated
with normal cell behavior. The data presented here show that mouse hepatocytes
are able to undergo clonal expansion in soft agar in response to nafenopin and
EGF giving a three- to fourfold increase in colony numbers over control. This
result is comparable to the fivefold increase in rat hepatocyte colony numbers
that we have reported previously. In contrast, hamster, guinea pig, and human
hepatocytes did not respond to the concerted action of EGF and nafenopin
despite their ability to respond to EGF as a mitogen in monolayer culture.
These data demonstrate that the clonal  expansion of rodent hepatocytes in
soft agar in response to peroxisome proliferators and EGF displays the same
species differences as other pleiotropic responses to these compounds and is
likely therefore to be relevant to the process of hepatocarcinogenesis. 

303
Shen HM, Ong CN, Shi CY.   INVOLVEMEMT OF REACTIVE OXYGEN SPECIES IN AFLATOXIN
B1-INDUCED CELL INJURY IN CULTURED RAT HEPATOCYTES. Toxicology
1995;99(1-2):115-23.

The role of reactive oxygen species (ROS) in AFB1-induced cell injury was
investigated using cultured rat hepatocytes. Malonaldehyde (MDA) generation
and lactate dehydrogenase (LDH) release were determined as indices of lipid
peroxidation and cell injury, respectively. Exposure to AFB1 for up to 72 h
resulted in significantly elevated levels of LDH being released into the
medium as well as the MDA generation in cultured hepatocytes. These effects
were dose-dependent,  indicating that AFB1 was  capable of inducing oxidative
damages in the cell. Further, MDA generation and LDH release were effectively
inhibited by the addition of the following: (1) superoxide dismutase (500
units/ml), (2) catalase (1500 units/ml), (3) 10 mM desferrioxamine (a specific
iron chelator), or (4) 260 mM dimethyl sulfoxide (a hydroxyl radical
scavenger). These evidences therefore suggest that ROS, such as superoxide
radicals, hydroxyl radicals and hydrogen peroxides, are involved in
AFB1-induced cell injury in cultured rat hepatocytes. 

304
Steinmassl D, Pfaller W, Gstraunthaler G, Hoffmann W.  LLC-PK1 EPITHELIA AS A
MODEL FOR IN VITRO ASSESSMENT OF PROXIMAL TUBULAR  NEPHROTOXICITY.  In Vitro
Cell Dev Biol Anim 1995;31(2):94-106.

LLC-PK1 cells, an established epithelial cell line derived from pig kidney,
were used as a model system for assessment of nephrotoxic side effects of
three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime.
Toxic effects of these xenobiotics were monitored on confluent monolayers by
light and electron microscopy and by the release of cellular marker enzyme
activities into the culture medium. In addition, LLC-PK1 cells were grown on
microporous supports, and cephalosporin-induced alteration of epithelial
functional integrity was monitored by a novel electrophysiologic approach. For
this purpose, an Ussing chamberlike experimental setup was used. The
dose-dependent effects on transepithelial ionic permselectivity were monitored
under conditions in which defined fractions of the apical culture medium NaCl
contents were replaced iso-osmotically by mannitol. This method of determining
the functional intactness of the epithelial barrier by measuring dilution
potentials was found to be far more sensitive than monitoring cell injury by
means of morphology or measurement of enzyme release. As expected from animal
experimental data, a dose-dependent disruption of monolayer integrity was
detected with all three methodologies applied. Cephaloridine was found the
most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime,
where a 10-fold dose of that of cephaloridine was needed to produce cell
injury. Measurement of transepithelial       dilution potentials was more
sensitive as compared to the release of the apical plasma membrane marker
enzyme activities alkaline phosphatase and gamma-glutamyltranspeptidase, the
cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase.
The data were compared to the effects of the       aminoglycoside antibiotic
gentamicin, which at least with respect to its effects on LLC-PK1 morphology
and enzyme release, but not transepithelial electrical properties, was already
investigated. 

305
Nath KA, Balla J, Croatt AJ, Vercellotti GM.  HEME PROTEIN-MEDIATED RENAL
INJURY: A PROTECTIVE ROLE FOR 21-AMINOSTEROIDS IN VITRO AND IN VIVO.  Kidney
Int 1995;47(2):592-602.

21-aminosteroids ("lazaroids") have recently excited much interest by virtue
of their ability to inhibit lipid peroxidation in vitro and to protect against
neural injury in vivo. We tested the effect of these compounds in models of
heme protein-mediated renal injury in vitro and in vivo. We devised an in
vitro model of heme protein-induced toxicity in which renal epithelial cells
were exposed to heme proteins for one hour, after which they were subjected to
glutathione depletion by 1-chloro-2,4-dinitrobenzene (CDNB). This model was
associated with more than a threefold increase in lipid peroxidation (as
measured by thiobarbituric acid reactive substances, TBARS) and a marked
reduction in cellular glutathione content. In this model, 21-aminosteroids
virtually prevented cytotoxicity as measured by the 51-chromium release assay,
and significantly reduced TBARS in a       dose-dependent manner. Catalase was
partially protective in this model, thereby indicating hydrogen
peroxide-dependent toxicity. While pursuing mechanisms accounting for enhanced
cellular generation of hydrogen peroxide, we uncovered the first direct
evidence that  the heme prosthetic group per se directly stimulates cellular
generation of hydrogen peroxide; complementing these findings is the
remarkable efficacy of 21-aminosteroids in protecting against cytotoxicity
induced by hydrogen peroxide. We also tested the capacity of 21-aminosteroids
to protect against heme protein-mediated renal injury in vivo. Prior
administration of 21-aminosteroids attenuated reductions in GFR and renal
blood flow rates following the systemic infusion of methemoglobin in normal
rats. 21-aminosteroids also attenuated renal injury observed over three
successive days in the glycerol model of heme protein-mediated injury when
this model was induced at a higher dose of glycerol (8 ml/kg body wt) but not
at a lower dose (5 ml/kg body wt). We       conclude that 21-aminosteroids
protect against heme protein-mediated renal injury in vitro and in vivo. We
suggest that these compounds are potentially useful in such clinical
conditions as rhabdomyolysis, intravascular hemolysis and renal injury
associated with hemoglobin-based red blood cell substitutes.

306
Vickers AE.  USE OF HUMAN ORGAN SLICES TO EVALUATE THE BIOTRANSFORMATION AND
DRUG-INDUCED SIDE-EFFECTS OF PHARMACEUTICALS.  Cell Biol Toxicol
1994;10(5-6):407-14.

Human liver and kidney organ slices were used to investigate the
biotransformation competence of the slices in combination with several markers
of cell viability and function. The immunosuppressant cyclosporin A (CSA) is
extensively metabolized in liver slices to the three known primary metabolites
and many secondary metabolites. In kidney cortex slices the biotransformation
of CSA is far more pronounced in humans than in rats. In human liver slices,
levels of CYP3A, the proteins metabolizing CSA, are depressed about 25% by 1
and 10 mumol/L CSA within 24 h, indicating that high blood or tissue
concentrations will inhibit CSA clearance. A clinical marker for liver damage
is the release of cellular alpha-glutathione-S-transferases (alpha GST). In
this study the alpha GST levels were used to assess donor organ quality, organ
slice incubation conditions, and       compound exposure. A marker for cell
death in human cells is the solubilization and release of nuclear matrix
proteins (Numa). Increases were apparent only after 48 h of culture. A
side-effect of CSA is that it induces hypertension and perturbs the lipid
profile of transplant recipients. A potential marker for lipid disturbances is
levels of serum lipoprotein (a) (Lp(a)), which is synthesized in the liver and
found only in humans, apes, and nonhuman primates. CSA increases Lp(a) levels
in the human liver slice cultures about 2-fold. This study has demonstrated
that the biotransformation capability of the organ slices contributes to the
optimization of the in vitro system and to the evaluation of markers for drug
induced side-effects or toxicity.(ABSTRACT TRUNCATED AT 250 WORDS) 

307
White DJ, Seaman C.  LLC-RK1 CELL SCREENING TEST FOR NEPHROTOXICITY.   Methods
Mol Biol 1995;43:11-6.

308
Knapek DF, Serrano M, Beach D, Trono D, Walker CL. ASSOCIATION OF RAT
P15INK4B/p16INK4 DELETIONS WITH MONOSOMY 5 IN KIDNEY EPITHELIAL CELL LINES BUT
NOT PRIMARY RENAL TUMORS. Cancer Res1995;55(8):
1607-12.

Recently the putative tumor suppressor gene p16INK4 was mapped to human
chromosome 9p21, which is homologous to rat chromosome 5. Monosomy of rat     
chromosome 5 occurs with high frequency in rat kidney tumor-derived cell lines
(ERC lines). Thus, we studied these lines in order to investigate the
involvement of p15INK4B in the genesis of this tumor type. p15INK4B and
p16INK4 were found by Southern blot analysis to be codeleted in five of seven
of these lines. This was confirmed by Northern blot analysis with a probe for
the rat p15INK4B gene. In normal rat tissues, expression of p15INK4B was
abundant in lung (2.5 and 2.0 kilobases), less abundant in testis (2.5, 2.0,
1.1, and 0.9 kilobases), barely detectable in liver (2.0 kilobases), and not
detectable in neonatal kidney, adult kidney, brain, heart, or spleen. In the
ERC lines, p15INK4B was expressed as a single 2.0-kilobase transcript observed
only in those cell lines in which the gene was detected by  Southern blot
analysis. However, neither p15INK4B nor p16INK4 were deleted in 12 of 12
primary kidney tumors examined, suggesting that deletion of these genes is not
directly involved in the process of renal tumor development but may be related
to tumor progression or autonomous growth in vitro. A panel of rat kidney
epithelial cell lines chemically transformed in vitro (TRKE lines) that had
high-frequency monosomy 5 were also examined, but deletion of p15INK4B and
p16INK4 was  observed in only one of six of the TRKE lines. To our knowledge,
this is the first reported investigation of these genes in rodent tumors and
cell lines, and its data support the theory that alterations of genes located
in the INF region of rat chromosome 5 may play a role in rodent cell
transformation. 

309
Rankin GO, Valentovic MA, Nicoll DW, Ball JG, Anestis DK, Brown PI, Hubbard
JL.  ACUTE RENAL AND HEPATIC EFFECTS INDUCED BY 3-HALOANILINES IN THE FISCHER
344 RAT. J Appl Toxicol 1995;15(2):139-46.

Haloanilines are commonly used as chemical intermediates in the manufacture of
a wide range of products. The purpose of this study was to examine the in vivo
nephrotoxic and hepatotoxic potentials of the 3-haloanilines. The in vitro
effects of the 3-haloanilines on renal function were also examined. In the in
vivo experiments, male Fischer 344 rats (four rats/group) were administered a
single intraperitoneal (i.p.) injection of an aniline hydrochloride (1.0 or
1.25 mmol kg-1) or vehicle.  Renal and hepatic function were monitored at 24
and/or 48 h post-treatment. None of the 3-haloanilines were potent
nephrotoxicants at either dose level. The greatest effects on renal function
were observed following administration of 3-chloroaniline at a dose of 1.25
mmol kg-1 (oliguria,       glucosuria, hematuria, decreased p-aminohippurate
accumulation by renal cortical slices and increased blood urea nitrogen
concentration). 3-Chloroaniline also was the only aniline compound to increase
plasma  ALT/GPT activity at 48 h. In the in vitro experiments, the ability of
ananiline (10-5-10-3 M) to decrease organic ion accumulation in renal cortical
slices from untreated rats was examined. The decreasing order of in vitro
nephrotoxic potential was 3-iodoaniline > 3-bromoaniline > 3-chloroaniline >
aniline > 3-fluoroaniline. These results indicate that the 3-haloanilines are
not potent nephrotoxicants or hepatotoxicants at sublethal doses. In addition,
the reasons why the 3-haloanilines  have different orders of nephrotoxic
potential in vivo and in vitro are not clear at this time.

310
Valentovic MA, Ball JG, Anestis DK, Rankin GO.  COMPARISON OF THE IN VITRO
TOXICITY OF DICHLOROANILINE STRUCTURAL ISOMERS. Toxicol In Vitro
1995;9(1):75-81.

Acute exposure to certain dichloroaniline (DCA) isomers results in renal and
hepatic toxicity in vivo. In the present study we examined whether
dichloroaniline structural isomers were cytotoxic to liver and kidney slices
in vitro and compared the toxicities of the different structural isomers.
These studies were necessary in order to validate the use of an in vitro slice
system for examination of the cellular mechanisms for toxicity. Renal cortical
and hepatic slices were incubated for 90 min  with 2,3-DCA, 2,4-DCA, 2,5-DCA,
2,6-DCA, 3,4-DCA or 3,5-DCA at a final concentration of 0-1 mM.
Pyruvate-directed gluconeogenesis was measured following an additional 30-min
incubation with 10 mM pyruvate. Cytotoxicity was also determined by
measurement of lactate dehydrogenase (LDH) release 120 min after the addition
of dichloroaniline isomers at a final concentration of 0, 0.5, 1 or 2 mm.
Gluconeogenesis in renal cortical slices was inhibited by all of the isomers
beginning at a concentration  of 0.5 mM. Renal slice LDH leakage was elevated
above control levels by 1-2 mm 3,4-DCA or 3,5-DCA. A final concentration of 2
mM was needed for 2,3-DCA, 2,4-DCA, 2,5-DCA or 2,6-DCA in order to detect a   
significant (P < 0.05)  increase in renal slice LDH leakage. Hepatic slices
incubated with 0.5-2 mM 2,3-DCA or 2 mm 2,5-DCA exhibited diminished
pyruvate-directed gluconeogenesis. After exposure to 2,4-DCA, 2,6-DCA, 3,4-DCA
or 3,5-DCA,       pyruvate-directed gluconeogenesis was similar to that in the
controls. LDH leakage was increased significantly (P < 0.05) above control
values by exposure to 2 mm 3,4-DCA or 3,5-DCA. In conclusion, DCA structural
isomers were toxic in vitro to liver and kidney slices. These results
indicated that the kidney was more sensitive than the liver to DCA isomers,
and that the most toxic isomer was 3,5-DCA. These results are similar to those
previously observed in vivo. 

311
Willinger CC,  Thamaree S, Schramek H, Gstraunthaler G, Pfaller W.  IN VITRO
NEPHROTOXICITY OF RUSSELL'S VIPER VENOM. Kidney Int 1995;47(2):518-28.

To assess direct nephrotoxicity of Russell's viper venom (RVV; Daboia russelii
siamensis), isolated rat kidneys were perfused in single pass for 120 min. Ten
mug/ml and 100 mug/ml RVV were administered 60 minutes and 80 minutes,
respectively, after starting the perfusion. Furthermore, cultured mesangial
cells and renal epithelial LLC-PK1 and MDCK cells were exposed to RVV (100 to
1000 mug/ml) for 5 minutes up to 48 hours. The IPRK dosedependently exhibited
reductions of renal perfusate flow  (RPF, 7.7 : 2.4 vs. 16.5 : 0.7 ml/min g
kidney wt in controls, experimental values given are those determined 10
minutes after termination of 100 mug/ml RVV admixture), glomerular filtration
rate (GFR 141 : 23 vs. 626 : 72 mul/min g kidney wt) and absolute reabsorption
of sodium (TNa 8 : 1.7 vs. 79 : 9 mumol/min g kidney wt), and an increased
fractional excretion of sodium (FENa 60 : 7 vs. 8 : 0.8%) and water (FEH2O 68
: 3.2 vs. 13 : 1.2%). Urinary flow rate (UFR) showed both oliguric and
polyuric phases. Functional alterations of this type are consistent with ARF.
Light and electron microscopy of perfusion fixed IPRK revealed an extensive
destruction of the glomerular filter and lysis of vascular walls. Various
degrees of epithelial injury occurred in all tubular segments. In cell culture
studies RVV induced a complete disintegration of confluent mesangial cell
layers, beginning at concentrations of 200 mug/ml. In epithelial LLCPK, and
MDCK cell cultures only extremely high doses of RVV (>600 and 800 mug/ml,
respectively) led to microscopically discernible damage. These results clearly
demonstrate a direct dose dependent toxic effect of RVV on the IPRK, directed
primarily against glomerular and vascular structures, and on cultured
mesangial cells. 

312
Toyokuni S, Sagripanti JL, Hitchins VM.  CYTOTOXIC AND MUTAGENIC EFFECTS OF
FERRIC NITROLOTRIACETATE ON L5178Y MOUSE LYMPHOMA CELLS. Cancer Lett
1995;88(2):157-62.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal
tubular       necrosis that leads to a high incidence of renal adenocarcinoma
in rodents. Others have shown that Fe-NTA induces modified DNA base products
both in vitro and in vivo. However, Fe-NTA is negative in the Ames Salmonella
test with or without S9      activation. The goal of this project was to
determine if Fe-NTA  is cytotoxic and mutagenic using the L5178Y (TK +/-)
mouse lymphoma assay. Our experiments showed a relationship between the
concentration of Fe-NTA (0 to 1 mM) and the decrease in relative survival. An
exposure-dependent increase in the number of mutations was observed with
increasing concentrations of Fe-NTA. At 14% relative survival, there was about
a 4-fold increase in mutations (trifluorothymidine resistance) over unexposed,
control cells. Ferric nitrate or nitrilotriacetic acid alone induced a
relatively low 1.5- or 1.1-fold increase in mutation, respectively. Our
results establish that  Fe-NTA is mutagenic in the L5178Y mouse lymphoma assay
system. 

313
James PE, Jackson SK, Grinberg OY, Swartz HM.   THE EFFECTS OF ENDOTOXIN ON
OXYGEN CONSUMPTION OF VARIOUS CELL TYPES IN VITRO: AN EPR OXIMETRY STUDY. 
Free Radic Biol Med 1995;18(4):641-7.

We have studied the effects of bacterial endotoxin on the oxygen consumption
of a variety of target cells, and found that the rate of utilization of oxygen
by treated cells was decreased in a time- and dose-dependent manner. Precise
EPR measurement of oxygen concentrations enabled us to demonstrate that this
effect was linked to mitochondrial dysfunction and was particular to each cell
type. Such detailed knowledge on oxygen utilization by viable whole cells and
the varied effects of endotoxin are as yet undocumented. Oxygen consumption
was shown to decrease quite markedly in CHO cells and kidney cells from the
cortex region. Cells from the kidney medulla region had lower baseline
consumption and were stimulated to increased levels of oxygen consumption on
addition of similar doses of endotoxin. Macrophages exhibited a dual response
in that in addition to inhibiting mitochondrial oxygen consumption, endotoxin
pretreatment primed these cells to exhibit an enhanced oxidative burst on 
stimulation with Zymosan. These results show that endotoxin has a direct
effect on normal cellular oxygen     consumption and is an important parameter
that must be considered when following the early effects on cells and tissues
during the septic syndrome. 

314
Mattana J,  Singhal PC.  HEPARIN ATTENUATES THE EFFECT OF MITOGENIC
VASOCONSTRICTORS ON MESANGIAL CELL PROLIFERATION AND HANDLING OF
IMMUNOGLOBULIN G COMPLEXES.  J Pharmacol Exp Ther 1995;273(1):80-7.

Angiotensin II (ANG II) and endothelin-1 (ET-1) may both play significant
roles in causing mesangial expansion and glomerulosclerosis after renal injury
by enhancing mesangial cell (MC) proliferation and by increasing MC uptake of
macromols. Heparin has been demonstrated to significantly ameliorate the
development of glomerulosclerosis in animal models of progressive renal
injury, although the mechanism is unknown. We undertook the present study of
examine in an in vitro system the mechanism by which heparin might modulate
the effects of ANG II and ET-1 on MC proliferation and MC uptake of IgG
complexes. Heparin was found to suppress MC uptake of IgG complexes in a
concn.-related manner. Whereas ANG II significantly enhanced uptake of IgG
complexes, this increase was significantly antagonized by coincubation of MC
with heparin at therapeutic concn. (1 U/mL). ET-1 also was found to
significantly increase MC uptake of IgG complexes, and this increase also was
significantly antagonized by coincubation with heparin.  Heparin was found to
inhibit surface binding of IgG to MC in a concn.-related manner (55% redn. at
1 U/mL). MC [3H]thymidine incorporation was significantly enhanced by both ANG
II and ET-1 and these mitogenic effects were significantly attenuated by
coincubation of cells with heparin (and, resp.). The antiproliferative effect
of heparin was attenuated by the nitric oxide synthase inhibitor
Nomega-nitro-L-arginine methylester and by the guanylate cyclase inhibitor
methylene blue suggesting that a nitric oxide-cGMP-dependent mechanism may
account for the antiproliferative effect of heparin. By using the
diazotization assay, heparin significantly increased MC nitric oxide prodn. in
a concn.-related manner. These data demonstrate that heparin can antagonize
the mitogenic effects of  ANG II and ET-1 on MC as well as the ability of
these agents to enhance MC uptake of IgG complexes. These effects may play a  
role in the obsd. ability of heparin to afford protection against
progressive glomerulosclerosis of renal injury.


REPRODUCTIVE TOXICITY
315
Davies WJ, Freeman SJ.  THE HYDRA ATTENUATA ASSAY.  Methods Mol Biol
1995;43:321-6.

316
Davies WJ, Freeman SJ.  THE DROSOPHILA MELANOGASTER ASSAY. Methods Mol Biol
1995;43:317-20.

317
Davies WJ, Freeman SJ.  FROG EMBRYO TERATOGENESIS ASSAY.  Methods Mol Biol
1995;43:311-6.

318
Davies WJ, Freeman SJ.  CHICK EMBRYOTOXICITY SCREENING TEST CHEST I AND II.
Methods Mol Biol 1995;43:307-10.

319
Cosenza ME, Bidanset J. EFFECTS OF CHLORPYRIFOS ON NEURONAL DEVELOPMENT IN RAT
EMBRYO MIDBRAIN MICROMASS CULTURES.  Vet Hum Toxicol 1995;37(2):118-21.

320
Brunstrom B, Engwall M, Hjelm K, Lindquist L, Zebuehr Y. EROD INDUCTION IN
CULTURED CHICK EMBRYO LIVER: A SENSITIVE BIOASSAY FOR DIOXIN-LIKE
ENVIRONMENTAL POLLUTANTS. Environ Toxcol Chem 1995;14(5):837-42.

A technique for studying 7-ethoxyresorufin O-deethylase (EROD) induction in
chick embryo liver in vitro was developed. Livers from 8-d-old embryos were
cultured in rotating vials at 37~ C for 48 h in a medium to which
DMSO-dissolved test compounds had been added. This bioassay proved to be
highly sensitive to dioxin-like compounds, and its usefulness for assessing
the toxic potency of such compounds in environmental samples was demonstrated.
Concentration-response curves were determined for 
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3'4,4',5-pentachlorobiphenyl
(PCB IUPAC no. 126), 3,3',4,4'-tetrachlorobiphenyl (PCB 77),
2,3,3',4,4'-pentachlorobiphenyl (PCB 105), and benzo(k)fluoranthene (BkF).
TCDD induced EROD in a concentration-dependent manner, having an EC50 of
5.0-12 M. The cultured embryo livers were extremely sensitive to TCDD, and
about 30 fg of this compound per liver (2as enough to significantly induce
EROD. The EC50 values obtained for PCBs 126, 77, 105, and BkF were 4.4 M,
1.6nduction levels obtained for three different preparations of
polychlorinated naphthalenes (PCNs) were less than those of the other
compounds tested. When the technical PCN mixture Halowax 1014 was
coadministered with TCDD, the induction was lower than that caused by TCDD
alone. An organic extract of fly ash from a municipal waste combustion plant
was very potent. Considering its contents of polychlorinated
dibenzo-p-dioxins/furans, expressed as TCDD equivalents, the EC50 obtained was
close to that for TCDD. 

321
Mothersill C. HUMAN ESOPHAGEAL CULTURE. Methods Mol Biol 1995;43:75-9.

322
Mclean M, Watt MP, Berjak P, Dutton MF.  AFLATOXIN B1: ITS EFFECTS ON AN IN
VITRO PLANT SYSTEM. Food Addit Contam 1995;12(3):435-43.

The phytotoxic effects of aflatoxin B1 (AFB1) on in vitro cultures of
differentiating calli and regenerating plantlets of Nicotiana tabacum were
assessed. Callus appeared more sensitive to the effects of AFB1, with fresh
mass accumulation and callus chlorophyll levels affected at low (approximately
0.5 mug/ml) aflatoxin concentrations. Transmission electron microscopy
revealed early deteriorative alterations in chloroplast morphology. Inhibitory
effects of the toxin (up to and including 10  mug/ml) on callus fresh mass
accumulation were reversed following a 3 week toxin-free recovery period. In
tobacco plantlets, root and leaf development, and root and leaf mass were
significantly inhibited in a dose-dependent fashion with increasing AFB1
concentration above 0.5 mug/ml. Inhibitory effects on plantlet root
development were more pronounced that on leaf development.

323
Peters JM, Duncan JR, Wiley LM, Keen CL. INFLUENCE OF ANTIOXIDANTS ON CADMIUM
TOXICITY OF MOUSE PREIMPLANTATION EMBRYOS IN VITRO.  Toxicology
1995;99(1-2):11-8.

To test the hypothesis that the developmental toxicity of cadmium (Cd) is due
in part to oxidative damage, embryos were cultured in medium containing 0.0,
1.0, 3.0, or 6.0 muM Cd with or without various antioxidants for 72 h.
Ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and
glutathione (GSH) were all effective at ameliorating 1.0 muM Cd-induced
embryotoxicity. For embryos cultured in medium containing either 3.0 or 6.0
muM Cd, GSH was effective at ameliorating Cd toxicity while the other
antioxidants tested were ineffective. Pretreating embryos with antioxidants
for 24 h prior to exposing them to Cd and antioxidants did not significantly
alter the previously observed improvement with the exception that pretreatment
with GSH virtually eliminated Cd-induced embryotoxicity between 1.0 and 6.0
muM Cd. A 4-h exposure to GSH prior to culture in Cd markedly improved embryo
development suggesting that GSH taken up during pretreatment can provide
protection  against Cd-induced embryotoxicity. This work supports the
hypothesis that the developmental toxicity of Cd is in part due to oxidative
damage that can be modulated by select antioxidants. 

324
Kishimoto T, Oguri T, Tada M.  METHYLMERCURY-INJURY EFFECT ON TUBE FORMATION
BY CULTURED HUMAN VASCULAR ENTOTHELIAL CELLS. Cell Biol Toxicol
1995;11(1):29-36.

The effect of methylmercury chloride (MeHg) on growth and tube formation by
cultured human umbilical vein endothelial cells (HUVECs) was investigated.
HUVECs were collected by enzymatic digestion with collagenase. Precultivation
of HUVECs with MeHg at concentrations of 1.0-50.0 mumol/L exerted negligible
effects on the viable cell number, while the viable cell number was slightly
reduced at 100 mumol/L and fell to zero at concentrations exceeding 500.0
mumol/L MeHg. The viable cell number was  depressed in a
concentration-dependent manner. Tube formation was studied by culturing the
cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with
0.15.0 mumol/L MeHg for 24 h inhibited tube formation dose-dependently. Fetal
bovine serum       (FBS) increased tube formation in a dose-dependent manner,
with half-maximum stimulation of tube formation at approximately 3.4% FBS. The
length of tube formation decreased time-dependently at concentrations of 0.1
and 1.0 mumol/L MeHg.  Pretreatment of Matrigel with 1 mumol/L MeHg before the
cell seeding reduced the tube formation by HUVECs. These results suggest that
the growth and tube formation by HUVECs is susceptible to MeHg cytotoxicity,
and that MeHg could be injurious to endothelial cell function. 

325
Renault JY, Caillaud JM, Chevalier J. ULTRASTRUCTURAL CHARACTERIZATION OF
NORMAL AND ABNORMAL CHONDROGENESIS IN MICROMASS RAT EMBRYO LIMB BUD CELL
CULTURES. Toxicol Appl Pharmacol 1995;130(2):
177-87.

Inhibition of chondrogenesis in limb bud cell micromass cultures has been
proposed as a short-term teratogen detection test. Validation studies were
performed by testing large series of reference compounds and comparing their
teratogenic potential with their ability to inhibit chondrogenesis; however,
there are few reports describing the histological and ultrastructural changes
associated with inhibition of chondrogenesis in vitro. The objective of this
study was to provide a qualitative  description of the histological and
ultrastructural alterations induced by three chondrogenesis inhibitors:
retinoic acid (RA) and 6-aminonicotinamide (6AN), two teratogens, and
doxylamine succinate (DS), a nonteratogen compound. In addition, in order to
have a basis for the interpretation of the morphological alterations induced
by the test compounds, the histological and ultrastructural changes which
occur during the time course of chondrogenesis in control cultures were
described and  compared with those in rat embryo limb buds. We found that RA
at 0.5 mug/ml led to a marked decrease in the number and size of cartilaginous
foci; most cells lacked morphological signs of differentiation but their
ability to proliferate was unaffected. At concentrations of 2 mug/ml and more,
6AN delayed cell proliferation, reduced staining of the extracellular matrix,
and induced the formation of endoplasmic cisternae. DS at 50 mug/ml affected
both differentiation and proliferation; pigment deposits were observed in
chondrocytes, suggesting phospholipid metabolism disorders. In conclusion,
this study showed that inhibition of chondrogenesis in this simple cell
culture system can be associated with different types of histological and
ultrastructural alterations. Examination of these alterations can provide
useful information about the teratogenic potential of tested compounds and
their mechanism of action. 

326
Hooghe RJ, Ooms D. USE OF THE FLUORESCENCE-ACTIVITED CELL SORTER (FACS) FOR IN
VITRO ASSAYS OF DEVELOPMENTAL TOXICITY. Toxicol In Vitro 1995;9(3):349-54.

Our objective is to predict embryotoxicity with reliable in vitro techniques.
In several exptl. systems, differentiation is accompanied by changes in the
glycosylation pattern of cell-surface glycoconjugates. This is also the case
with embryonal carcinoma cells. We have monitored the expression of receptors
for wheat germ agglutinin (WGA). Murine embryonal carcinoma cells (P19 and F9)
were exposed in vitro to xenobiotics for 1-3 days, then incubated successively
with WGA-biotin (15 mug/mL) and streptavidin-phycoerythrin (SA-PE) (20
mug/mL), each for 30 min at room temp. Cell-surface fluorescence was then
analyzed using a fluorescence-activated cell sorter (FACS).  Exposure to 1 muM
retinoic acid, a known inducer of differentiation, altered glycosylation as
indicated by changes in WGA binding. Clear-cut effects were also obsd. after
exposure to salts of arsenic (20 muM), or nickel (50 muM), and to methotrexate
(1 mug/mL), fluorouracil (1.3 mug/mL) or actinomycin D (0.04 mug/mL). These
compds. affected the percentage of pos. cells, the intensity of labeling, or
both. Two non-teratogenic compds. (metronidazole and sulfonilamide) have also
been tested and had no effect.  Lectin histochem. of embryonal carcinoma cells
exposed to potentially toxic agents holds promise as a method for predicting
embryotoxicity.  FACS anal. allows rapid quantification.

327
Andrews JE, Ebron-McCoy M, Nau H, Kavlock RJ.  VALDIATION OF AN IN VITRO
TERATOLOGY SYSTEM USING CHIRAL SUBSTANCES: STEREOSELECTIVE TERATOGENICITY OF
4-YN-VALPROIC ACID IN CULTURED  MOUSE EMBRYOS. Toxicol Appl Pharmacol
1995;132(2):310-6.

In vitro systems are important for toxicity testing as well as for
investigating the mechanism of action of xenobiotics. The validation of such
in vitro systems is often incomplete and extrapolation to the in vivo
situation is equivocal.  In the present study, the authors studied the effects
of enantiomers of an analog of the antiepileptic drug valproic acid
(VPA):R(+)- and S(-)-4-yn-VPA (R- and S-2-n-propyl-4-pentynoic acid), which
have previously been shown to induce selective teratogenicity in mice after in
vivo administration, in mouse whole-embryo culture (WEC).  Aq. solns. of the
sodium salts of the pure R- and S-enantiomers as well as R,S-4-yn-VPA (racemic
mixt.) or VPA itself were added to the culture medium at 0, 0.075, 0.15, 0.3,
0.6, or 1.2 mmol/L and embryos were evaluated 24 h later. The S-4-yn-VPA
enantiomer induced clear concn.-dependent dysmorphogenesis that was evident
even at the lowest concn. The primary anomalies were neural tube defects,
erratic neural seams, blisters, and rotational defects. Embryolethality was
obsd. at 1.2 mmol/L. The R-4-yn-VPA enantiomer was neither embryotoxic nor
dysmorphogenic at any tested concn. The lack of biol. activity over 24 h in
WEC with the R-enantiomer suggests also that, as previously shown in vivo,
there was no racemization of this isomer to the more active S-enantiomer. The
racemic mixt. of R and S isomers appeared to be slightly more embryolethal and
dysmorphogenic than VPA. Overall, the potency of the S-enantiomer was approx.
four times that of VPA. Therefore, the rank order of the four chems. tested
was S(-) >> S(-), R(+) > VPA >>> R(+), which is in agreement with the effects
obsd. in in vivo exposed mice. These data demonstrate a direct stereoselective
effect of these compds. on the embryo. This is the first illustration of the
stereoselectivity of a xenobiotic in the WEC in vitro test system. Pure and
stable enantiomers, which induce stereoselective toxicity in vivo, are
demonstrated to be valuable for validation of this in vitro system.

328
Kishi J, Noda Y, Goto Y, Nakayama T, Nonogaki T, Mori T.  ANALYSIS OF IN VITRO
DEVELOPMENTAL BLOCK OF RAT EMBRYOS: ASSESSMENT FROM THE VIEW POINT OF OXYGEN
TOXICITY.  J Reprod Dev 1994;40(4):285-91.

The authors have already reported that the block to development in cultured
1-cell rat embryos was overcome using HECM-1, a protein-free chem. defined
medium without glucose or phosphate. In addn., the authors showed the
beneficial effect of low oxygen tension in rat embryos. In this study, in
order to clarify the reason why in vitro developmental block of rat embryos is
overcome in HECM-1, the authors examd. the effects of phosphate, glucose, and
L-cysteine on the in vitro development of rat embryos.  Furthermore, the
authors examd. their effects on the prodn. of H2O2 in embryos by the
fluorometric method. The addn. of KH2PO4 even at only 1 muM completely
inhibited the development of rat (Wistar) embryos either from the 1-cell stage
or the 2-cell stage. The addn. of glucose at 1000 mug/mL significantly
decreased the blastulation rate. The prodn. of H2O2 in embryos cultured under
5% O2 was significantly (P<0.01) lower than that of embryos cultured under 20%
O2 in HECM-1. H2O2 prodn. in embryos cultured in mKRB was significantly
(P<0.01) higher than that in embryos cultured in HECM-1, irresp. of the oxygen
tension. The addn. of neither phosphate nor glucose to HECM-1 affected the
H2O2 prodn. at any concn. examd. L-Cysteine significantly decreased the H2O2
prodn. in embryos in mKRB, but developmental block could not be overcome by
the addn. of L-cysteine to mKRB. These results revealed that glucose and
phosphate independently inhibited the development of  rat embryos in vitro,
and suggest that the mechanism of in vitro developmental block of rat embryos
could not be explained simply in terms of oxygen toxicity. 

329
Lane M, Gardner DK.  REMOVAL OF EMBRYO-TOXIC AMMONIUM FROM THE CULTURE MEDIUM
BY IN SITU ENZYMATIC CONVERSION TO GLUTAMATE. J Exp Zool 1995;271(5):356-63.

An enzymatic method for removing embryo-toxic ammonium from culture medium has
been developed. Ammonium, produced by both embryo metabolism and spontaneous
breakdown of amino acids at 37~C, is transaminated by glutamate dehydrogenase
to nontoxic glutamate. Initially, the individual components of the
transamination reaction were titrated against mouse embryo development in
vitro to determine embryo-safe levels. ADP, an allosteric activator of
glutamate dehydrogenase, was found to inhibit embryo development and was
therefore omitted from the final formulation (alpha-ketoglutarate, 0.44 mM;
glutamate dehydrogenase, 0.375 U; NADH, 0.12 mM). It was found that 0.30 mM
ammonium could be removed from the culture medium in situ in 3 h. In situ
removal of ammonium significantly increases both blastocyst cell number,
implantation, fetal development, and fetal weight after transfer. Removal of
ammonium by the conventional method of renewing the culture medium also
increased blastocyst cell number but did not affect postimplantation
development. In conclusion, it is possible to alleviate the toxic effects of
ammonium in vitro on pre- and postimplantation mouse embryo development by its
transamination in situ, thereby facilitating the continual exposure to
embryo-derived factor(s) which stimulates both pre- and postimplantation
development. 

330
Pinski J,  Schally AV, Yano T,  Groot K, Srkalovic G, Serfozo P, Reissmann T, 
Bernd M, Deger W, et al.  EVALUATION OF THE IN VITRO AND IN VIVO ACTIVITY OF
THE L-, D,L-  AND D-Cit6 FORMS OF THE LH-RH ANTAGONIST CETRORELIX (SB-75). 
Int J Pept Protein Res 1995;45(5):410-7.

The objective of this study was to examine the in vivo and in vitro
gonadotropin-inhibiting potencies, edematogenic activities and the receptor
binding affinities of the D-Cit6, and L-Cit6 forms of the LH-RH antagonist
Cetrorelix.  To demonstrate the suppressive effects of two different
diastereomers of SB-75 and their racemic mixt. on LH and FSH release, [D-Cit6]
SB-75 was injected s.c. in doses of 2.5 and 10 mug/rat, [DL-Cit6]-SB-75 in
doses of 5 and 20 mug/rat and [L-Cit6]-SB-75 in doses of 12.5 and 50 mug/rat
to castrated male rats.  Two hours after administration, there was no
difference in LH levels between rats injected with the L-form and control
animals, indicating a low activity and(or) a rapid enzymic degrdn. of this
peptide.  The (1:1) diastereomeric mixt. was only about half as potent in
suppression in LH release compared to [D-Cit6]-SB-75.  Serum FSH levels were
suppressed for more than 48 h after the administration of 10 mug
[D-Cit6]-SB-75 and 20 mug of [DL-Cit6]-SB-75, resp.  [D-Cit6]-SB-75
administered at a dose of 2 mug/rat induced 100% inhibition of ovulation,
while 4 mug/rat of the DL-Cit6 peptide were necessary to produce the same
effect.  [L-Cit6]-SB-75 given at a high dose of 40 mug/rat produced only 14%
inhibition of ovulation.  The D-Cit6 form of SB-75 produced skin lesions with
a much smaller diam. than the L-isomer, and was about 34 times less
edematogenic. [D-Cit6]-SB-75 was bound more powerfully to high-affinity
pituitary LH-RH receptors than either DL-Cit6 or L-Cit6 analogs.  In vitro
assays based on the superfusion of dispersed rat pituitary cells on a column,
followed by RIA for LH, also demonstrated a lower inhibitory activity for the
L-Cit6 analog, but the differences between D-, DL- and L-citrulline analogs
were smaller than in vivo.  The results indicate that the LH-RH antagonist
[D-Cit6]-SB-75 is more effective in suppression of gonadotropin release in
vivo and in vitro, less edematogenic and possesses higher binding affinity to
pituitary LH-RH receptors than the DL- and L-citrulline decapeptide analogs.

331
Hansen DK, Grafton TF.  COMPARISON OF DEXAMETHASONE-INDUCED EMBRYOTOXICITY IN
VITRO IN MOUSE AND RAT EMBRYOS. Teratogenesis Carcinog Mutagen
1994;14(6):281-9. 

Previous work demonstrated that rat embryos were more susceptible to the
growth retardation effect of the synthetic glucocorticoid dexamethasone (DEX)
in vivo than were mouse embryos. The purpose of this study was to examine this
species difference using an in vitro system. Embryos of CD rats and CD-1 mice
were cultured in a whole embryo culture system with concentrations of DEX from
5 to 250 mug/ml. Rat embryos were explanted on day 9 of gestation (GD 9: plug
day = GD 0), while mouse embryos  were removed on GD 8. After 48 h in culture,
each viable embryo was evaluated for morphological score, and the number of
somite pairs, crown-rump, and head lengths, as well as DNA and protein
concentrations were determined. A reduced morphological score was observed for
mouse embryos at 5 mug DEX/ml, but a significant decrease in this parameter
was only observed at DEX concentrations of : 100 mug/ml in rat embryos.
Significant reductions in the number of somite pairs were observed at 25 
mug/ml for mouse embryos and 100 mug/ml for rat embryos. Crown-rump and head
lengths as well as DNA and protein concentrations were significantly decreased
at 100 mug/ml in mouse embryos and 150 mug/ml in rat embryos. Therefore, in
vitro mouse embryos were adversely affected by lower concentrations of DEX
than were rat embryos for each of the six end points examined  in this study.
This species sensitivity in vitro could be due to inherent genetic differences
or to the slightly different developmental stages evaluated using the culture
system. The species sensitivity observed in vivo may be due to a maternal
difference in the pharmacodynamics of the drug.

332
Shibata M,  Watanabe T.  IN VITRO ASSESSMENT OF TERATOGENIC POTENTIAL OF
CIS-1-[4-(P-MENTHANE-8-YLOXY) PHENYL] PIPERADINE.  J Toxicol Sci 1995;
20(1):9-14.

The teratogenic potential of cis-1-[4-(p-menthane-8-yloxy) phenyl] piperadine
(YM9429) was assessed by an in vitro culture method using fetal palatal
tissues of CD rats. YM9429 induced fetal cleft palate in vivo in CD rats when
dams were orally treated during days 11-14 of pregnancy at a dose of 500
mg/kg/day, but no abnormalities were detected when treated on days 15-16
(Shibata, 1993). After 4 successive days of oral treatment during days 11-14
of pregnancy, the maternal plasma levels of the drug were <5 mug/mL on day 15
and <1 mug/mL on day 16. After a single oral dosing on day 14, the fetal
levels were .apprx.0.3 mug/g-fetus at 2 h postdosing. When the fetal maxillary
regions removed from the normal fetuses on day 15 of pregnancy were cultured
with YM9429 at concns. of 5 and 1 mug/mL-medium for 48 or 72 h, normal and
full contact of the palatal shelves was obsd. These results supported the
previous findings that YM9429 demonstrated no teratogenic effects in vivo by
maternal treatment on days 15-16 of pregnancy, and       suggested other
mechanisms than the direct influences on the fetal palatal shelves in the
latter phases of morphogenesis including reorientation, horizontal extension,
and adhesion. 

333
Daston GP, Baines D, Elmore E, Fitzgerald MP, Sharma S. EVALUATION OF CHICK
EMBRYO NEURAL RETINA CELL CULTURE AS A SCREEN FOR DEVELOPMENTAL TOXICANTS.
Fundam Appl Toxicol 1995; 26(2):203-10.

This paper describes a study to evaluate the concordance with in vivo results
of an in vitro screen for developmental toxicants. The screen is a primary
culture of chick embryo neural retina cells (CERC) which undergo processes of
cell-cell recognition and interaction, growth, and differentiation over a
7-day culture period. Each of these developmentally significant events is
measured sep. as formation of multicellular aggregates, protein content, and
glutamine synthetase activity, resp.  A total of 45 chems., 24 of which have
been shown to be teratogenic at some dosage to mammalian embryos in utero, 7
of which are embryotoxic (but not teratogenic) in utero a high dosage, and 14
of which have not produced developmental toxicity in vivo, were evaluated in
this assay by investigators who were blinded to the identity of the chems.
Chems. were tested up to concns. that were frankly cytolethal, or up to a max.
of 4 mg/mL.  Chems. were present only during the first 24 h of culture.  The
chems. were selected to be    representative of a variety of chem. classes
(e.g., solvents, metals, food additives, anticonvulsants, antineoplastics). 
In several cases, pairs of structurally similar compds. with different
developmental toxic potencies (e.g., valproate and 2-en-valproate, formamide,
and N,N-dimethylformamide) were tested. Of the 31 developmental toxicants, 25
affected at least one endpoint in the assay at concns. which are achievable in
vivo (i.e., below the systemic concn. at a LD), yielding a false-neg. rate of
19%.  Two of the nondevelopmental toxicants, saccharin, and penicillin G, had
adverse effects at concns. below those that may be biol. achievable in vivo,
giving a false-position rate of 14%.  Overall concordance with in vivo results
by these criteria was 82%. Quant. comparisons were also made between the
lowest obsd. effect concn. (LOEC) in the assay and (i) lowest      
developmentally toxic dosage (mostly i.p.) reported in rats or mice in vivo
and (ii) LOEC in rodent whole embryo culture. In the first instance, 77% of
the LOECs (LOELs) were within an order of magnitude and 93% were within a
factor of 30. In the second instance 81% of the LOECs were within an order of
magnitude. Potency ranking of four alkoxy acids was comparable in CERC and in
vivo rodent embryo. These results indicate that the CERC assay is concordant
with developmental toxic potential and potency for the diverse group compds.
selected, and that it could serve as a preliminary screen for developmental
toxicity. 

334
Murakami M, Hosokawa S, Yamada T, Harakawa M, Ito M, Koyama Y, Kimura J, 
Yoshitake A, Yamada H.  SPECIES-SPECIFIC MECHANISM IN RAT LEYDIG CELL
TUMORIGENESIS BY PROCYMIDONE.  Toxicol Appl Pharmacol 1995;131(2): 
244-52.

To clarify the mechanism of species difference in the induction of testicular
interstitial cell tumor (ICT, Leydig cell tumor) between rats and mice, male
Sprague-Dawley rats and ICR mice were fed procymidone at dietary
concentrations of 700, 2000 or 6000 ppm and 1000, 5000, or 10,000 ppm,
respectively, for 3 months. The Leydig cell functions were evaluated by serum
testosterone and luteinizing hormone (LH) levels, testosterone levels in the
testis, LH levels in the pituitary, the capacity of  the testis to respond to
gonadotropin stimulation, i.e., the production of testosterone in vitro, and
by the testicular binding of labeled human chorionic gonadotropin (hCG).
Measurement of testosterone and LH levels in rat serum, the testis, or the
pituitary showed that both hormones were enhanced throughout the 3-month
treatment period. The hypergonadotropism was associated with the increase of
interstitial cell response to hCG in vitro for up to 3 months. As with rats,
both serum and  pituitary LH were increased in mice at 4 weeks but not at 13
weeks. However, in contrast to rats, no significant increase in testosterone
was observed in mice either in vivo or ex vivo during the course of the study.
This suggests a difference between the rat and mouse in the response of the
Leydig cell to the LH stimulation associated with procymidone administration.
These differences in the response of interstitial  cells to procymidone may be
the basis for the distinct species responses to  procymidone-induced Leydig
cell tumorigenesis. The sustained response of the Leydig cells to stimulation
in the rat results in chronic hyperplasia and subsequent benign tumor
formation, while the attenuated response of Leydig cells in the mouse is
associated with neither hyperplasia nor neoplasia. 

335
McMaster ME, Munkittrick KR, Jardine JJ, Robinson RD, Van Der Kraak GJ.
PROTOCOL FOR MEASURING IN VITRO STEROID PRODUCTION BY FISH GONADAL TISSUE. 
Can Tech Rep Fish Aquatic Sci 1995;1961:I-VIII,1-78.

336
Lazo JS, Kondo Y, Dellapiazza D, Michalska AE, Choo K HA, Pitt BR.  ENHANCED
SENSITIVITY TO OXIDATIVE STRESS IN CULTURED EMBRYONIC CELLS FROM TRANSGENIC
MICE DEFICIENT IN METALLOTHIONEIN I AND II GENES.  J Biol Chem
1995;270(10):5506-10.

Embryonic cells from transgenic mice with targeted disruption of
metallothionein I and II genes expressed no detectable metallothionein either
constitutively or after treatment with cadmium, in contrast to cultured cells
that were wild type or heterozygous for the loss of the metallothionein genes.
Metallothionein null cells were most sensitive to the cytotoxic effects of
cadmium, the membrane permeant oxidant tert-butylhydroperoxide, and the redox
cycling toxin paraquat. No marked differences  were seen among the wild type,
heterozygous, or metallothionein null cells in glutathione levels or in the
activity of CuZn-superoxide dismutase, glutathione peroxidase, or catalase.
Nevertheless, metallothionein null cells were more sensitive to
tertbutylhydroperoxide-induced oxidation as ascertained by confocal
microscopic imaging of dichlorofluoroscein fluorescence. These results
indicate basal metallothionein levels can function  to regulate intracellular
redox status in mammalian cells. 

337
Seeley MR, Faustman EM.  TOXICITY OF FOUR ALKYLATING AGENTS ON IN VITRO RAT
EMBRYO DIFFERENTIATION AND DEVELOPMENT.  Fundam Appl Toxicol
1995;26(1):136-42.

The relative developmental toxicity of four direct acting, alkylating agents
was determined in primary cultures of differentiating rat embryo midbrain
(CNS) and limb bud (LB) cells and compared with that observed in the rat whole
embryo postimplantation culture system. The alkylating agents tested include
methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate
(MMS), and ethyl methanesulfonate (EMS). These alkylating agents have been
shown to produce developmental toxicity  following either in vitro or in vivo
exposure. Viability for both CNS and LB was assessed by a neutral red dye
assay. Differentiation of CNS cells was assessed by hematoxylin staining of
neurons; differentiation of LB cells was assessed by Alcian blue staining of
extracellular proteoglycans. Relative potencies of these compounds in the cell
culture system were not the same as those observed in the embryo culture
system. Whereas rank order of potency in the cell culture system, for
viability and  differentiation, was MMS > MNU > ENU > EMS, rank order in the
embryo culture system, for embryo lethality and malformations, was MNU > ENU >
MMS > EMS. Effective concentrations for cell culture viability and
differentiation by MNU and ENU in cell culture were about three to nine times
higher than comparable values previously reported for embryos, while effective
concentrations for MMS and EMS were two to seven times lower than those
observed in the embryos. Differences in potency between  the two culture
systems may be related to differences in formation and repair of DNA adducts,
as well as differences in culture conditions.

338
Sandberg JA, Murphey LJ, Olsen GD.  PHARMACOKINETICS AND METABOLISM OF COCAINE
IN MATERNAL AND FETAL GUINEA PIGS.  Neurotoxicology 1995; 16(1):169-78.

The pharmacokinetics and metabolism of cocaine (COC) were determined in late
gestation maternal and fetal guinea pigs. After a single i.v. dose of 2-12
mg/kg, the  average : SD total body clearance of COC was 59 : 16 ml/min/kg and
was not dose dependent. However, volume of distribution was 2.1 and 3.9 l/kg,
mean resident time (MRT) was 42 and 57 min, and elimination half-life was 34
and 49 min at the 2 and 4 mg/kg dose of COC, respectively. With the exception
of an increased MRT, the pharmacokinetics were similar after s.c. COC
administration. Benzoylecgonine (BE) and benzoylnorecgonine (BN) were major
and persistent metabolites. Norcocaine (NOR) concentrations were low and
transient. After chronic maternal administration of 6 mg/kg COC s.c., there
was no difference between maternal and fetal plasma COC concentrations one
hour after the last injection, but COC and BN accumulated in amniotic fluid.
Examination of in vitro metabolism of COC in fetal and maternal guinea pig 
hepatic microsomes demonstrated minimal fetal N-demethylation and induction of
maternal N-demethylation by chronic COC exposure. The minimal fetal
N-demethylation suggests BN seen previously in vivo after chronic maternal COC
administration resulted from maternal formation of NOR and subsequent maternal
and/or fetal hydrolysis to BN.

339
Srivastava SC, Kumar R, Prasad AK, Srivastava SP.  EFFECT OF
HEXACHLORO-CYCLOHEXANE (HCH) ON TESTICULAR PLASMA MEMBRANE OF RAT. Toxicol
Lett 1995;75(1-3):153-7. 

The study deals with the analysis of residue of hexachlorocyclohexane (HCH)
and its possible damaging potential on testicular plasma membrane of rats. In
vitro studies were conducted by exposing plasma membrane of testis with 1.46
1.46ibition in the activity of the Ca2+-ATPase, Na+ + K+ + Mg2+-ATPase and 5'
Nucleotidase. In vivo studies were carried out following  repeated dermal
exposure to HCH at a dose level of 50 or 100 mg/kg/day for 60 days to male
rats. The results show significant decrease in the activities of
5'-Nucleotidase, Ca2+-ATPase, Na+ + K+-ATPase and Mg2+-ATPase in the plasma
membrane of testis following exposure to HCH. The analysis of the residues of
HCH reveals the presence of significant quantities of its different isomers
viz., alpha, beta, gamma and delta in the testicular plasma membrane of rats
given in vivo dermal exposure of this pesticide. These results suggest that
the presence of HCH residue may be a factor in inhibiting the marker enzymes
of the plasma membrane of testis. 

340
Singh PB, Kime DE. IMPACT OF GAMMA-HEXACHLOROCYCLOHEXANE ON THE IN VITRO
PRODUCTION OF STEROIDS FROM ENDOGENOUS AND EXOGENOUS PRECURSORS IN THE
SPERMIATING ROACH, RUTILUS RUTILUS. Aquatic Toxicol 1995;31(3): 231-40. 

Testicular fragments from spermiating roach were incubated with 0, 1, 10 and
20 mg/L of gamma-hexachlorocyclohexane (gamma-HCH), and either carp
hypophyseal homogenate (chh) or 3H-17-hydroxyprogesterone (3H-17P). The
endogenous production of testosterone (T), 17-hydroxyprogesterone (17P),
17,20beta-dihydroxy-4-pregnen-3-one (17,20,betaP), 11-ketotestosterone (KT),
testosterone glucuronide (TG), and 17,20,beta-dihydroxy-4-pregnen-3-one
glucuronide (1 7,20betaPG) was measured by radioimmunoassay.  of gamma-HCH on
the testicular metabolism of 3H-17P was examined by thin layer and high
performance liquid chromatography. Endogenous production of T was stimulated
by 1 mg/L gamma-HCH but inhibited at higher doses, while its glucuronide was
higher in all incubations containing gamma-HCH compared with controls. 17P and
KT production was lower at all concentrations of gamma-HCH   compared with
controls, but the low endogenous 17,20betaP production was unaffected.
gamma-HCH had a much lower impact o metabolism of exogenous 17P than on
endogenous precursors. The major metabolite of 17P in the testis, 17,20betaP,
showed a significant increase in yield in response to gamma-HCH together with
a decrease in production of its glucuronide. There was also a significant
increase in yield of testosterone at some concentrations, but none of the
other metabolites were affected by gamma-HCH exposure. The results show that
the balance of steroid production by testes of spermiating roach is
significantly perturbed by gamma-HCH and suggests that the pesticide affects 
predominantly the stages of steroidogenesis leading to 17-hydroxyprogesterone
production.

341
Kemp RB.  CYTOTOXICITY IN AN ANCHORAGE-INDEPENDENT FIBROBLAST CELL LINE
MEASURED BY A COMBINATION OF FLUORESCENT DYES.   Methods Mol Biol
1995;43:211-8.

342
Kristen U, Kappler R.  THE POLLEN TUBE GROWTH TEST. Methods Mol Biol 1995;
43:189-98.

343
Knoll M, Shaoulian R, Magers T, Talbot P.  CILIARY BEAT FREQUENCY OF HAMSTER
OVIDUCTS IS DECREASED IN VITRO BY EXPOSURE TO SOLUTIONS OF MAINSTREAM AND
SIDESTREAM CIGARETTE SMOKE.  Biol Reprod 1995;53(1):29-37. 

Epidemiological data support a correlation between smoking and increased
incidence of ectopic pregnancy, yet the causal mechanism responsible for this
relationship is unknown. The purpose of this study was to examine the effect
of solutions containing dissolved mainstream (MS) or sidestream (SS) cigarette
smoke on ciliary beat frequencies (CBF) in explants of hamster oviducts. MS
smoke is the puff inhaled by an active smoker, while SS smoke leaves the
burning end of cigarette. SS smoke is inhaled by both active and passive
smokers. Experiments were performed in handmade perfusion chambers using
infundibula from hamster oviducts. After a short incubation in Earle's
balanced salt solution containing HEPES buffer (EBSS-H), chambers were flushed
with one of six types of smoke solution prepared in EBSS-H, and incubation
continued 19 min. A second perfusion (washout) was then done using EBSS-H
alone to determine whether effects induced by the smoke solutions could be
reversed. CBF were determined at three times in both the smoke and washout
solutions, and means were compared to values obtained in the initial EBSS-H
incubation. All smoke solutions except the SS particulate solution inhibited
CBF in a dose-dependent manner. Whole MS and whole SS smoke solution at the
highest strength tested caused the greatest inhibition and in some cases
completely stopped ciliary beating. Both single-strength and 0.1-strength MS
gas phase solutions, which contained concentrations of  nicotine in the range
found in typical human smokers, produced about 50% inhibition of ciliary
beating. Inhibition was generally seen within 2-12 min of adding smoke
solutions. In all cases, washout of smoke solution caused an increase in CBF.
In some washouts, CBF returned to initial values found in EBSS-H within 2 min
of incubation in the recovery medium. However, in both MS and SS whole smoke
solutions, CBF did not return to the initial EBSS-H values during the washout
interval. In SS gas  phase solutions, inhibition was not observed during
exposure to the smoke solution but did occur during washout, suggesting that
the mechanism of action of SS gas phase solutions differs from the other types
of smoke solution tested. These results show that components in both MS and SS
cigarette smoke can inhibit beating of oviductal cilia in vitro and that
inhibition can be at least partially reversed when the smoke solution is
replaced by EBSS-H. 

344
Hoyes KP,  Johnson C, Johnston RE, Lendon RG, Hendry JH, Sharma HL, Morris ID.
TESTICULAR TOXICITY OF THE TRANSFERRIN BINDING RADIONUCLIDE 114mIn IN ADULT
AND NEONATAL RATS.  Reprod Toxicol 1995;9(3):297-305.

Adult (70 d) and neonatal (7 d) male rats were dosed (i.p.) with 37 MBq/kg (1
mCi/kg; approximately 1 mug elemental indium/kg) 114mIn, a transferrin-binding
radionuclide. In adults, approximately 0.25%  of the injected activity
localised within the testis by 48 h postinjection and remained constant for up
to 63 d. In neonates,  0.06% of the activity was in the testis by 48 h, and
this declined such that by 63 d only 0.03% remained. At 63 d, treated rats had
reduced sperm head counts and  abnormal testicular histology that was more
marked in animals dosed as adults than as neonates. In vitro, uptake of 114mIn
into seminiferous tubules isolated from 7-, 20-, or 70-d-old rats was compared
with that of 125I. Both radionuclides were readily accumulated by the tubules.
Whilst 114mIn uptake into 20- and 70-d tubules was inhibited by excess
transferrin, uptake into 7-d tubules was unchanged. 125I uptake was not
affected by excess transferrin. These data support the  contention that some
radionuclides may cross the blood-testis barrier by utilisation of the
physiologic irontransferrin pathway, which may lead to greater testicular
damage in adult compared to neonatal animals. 

345
Genbacev O,  Bass KE, Joslin RJ, Fisher SJ.  MATERNAL SMOKING INHIBITS EARLY
HUMAN CYTOTROPHOBLAST DIFFERENTIATION. Reprod Toxicol 1995;9(3):245-55. 

Differentiation of the specialized epithelial cells of the placenta, termed
cytotrophoblasts, is a particularly important aspect of placental development
during the first trimester of pregnancy. During this process cytotrophoblast
stem cells either fuse to form the syncytium or aggregate to form cell columns
that adhere to, then invade the uterus. We found that chorionic villi from
early gestation placentas of mothers who smoke showed a marked reduction in
cell columns, a defect that could not  be corrected by placing them in
culture. We used two different in vitro models to determine if nicotine plays
a role in the etiology of this defect. Exposing early gestation chorionic
villi from nonsmoking women to nicotine inhibited subsequent cell column
formation in vitro. Nicotine also inhibited normal first trimester
cytotrophoblast invasion, apparently by reducing the ability of treated cells
to synthesize and activate the 92 kDa type IV collagenase, an important
mediator of invasion in  vitro. These results suggest that maternal cigarette
smoking inhibits the trophoblast differentiation pathway that leads to column
formation and uterine invasion. This effect, which is due at least in part to
the effects of nicotine, may contribute to the growth retardation observed in
fetuses of mothers who smoke during pregnancy. 

346
Jahan I,  Bai L, Iijima M,  Kondo T, Namba M.   KARYOTYPIC ANALYSIS IN THE
PROCESS OF IMMORTALIZATION OF HUMAN CELLS TREATED WITH 4-NITROQUINOLINE
1-OXIDE. Acta Med Okayama 1995;49(1):25-8. 
Holladay SD, Smith BJ, Luster MI. B LYMPHOCYTE PRECURSOR CELLS REPRESENT
SENSITIVE TARGETS OF T2 MYCOTOXIN EXPOSURE. Toxicol Appl Pharmacol
1995;131(2):309-15.

Exposure of experimental animals and humans to the Fusarium trichothecene
metabolite, T2 toxin, has been associated with a variety of immunosuppressive
effects, including altered parameters of humoral-mediated immunity. Although
T2 toxin is cytotoxic in vitro to lymphocytic cells, limited information is
presently available regarding the contribution of such a mechanism to
immunosuppression in vivo, or to potential immune cell targets. In the present
report, subchronic T2 toxin treatment of timed-pregnant B6C3F1 mice resulted
in significant and selective depletion of fetal liver cells expressing low
levels of surface CD44 and CD45 antigens, suggestive of possible lymphoid
progenitor cell sensitivity to this agent. Evaluation of CD45R antigen
expression in fetal liver supported such a hypothesis, demonstrating a
significant reduction in fetal liver B lymphocytic cells in animals exposed to
T2 toxin. Subsequent in vitro T2 toxin exposure of fetal liver cells enriched
for prolymphocytes by differential density gradient centrifugation
demonstrated the presence of a highly sensitive subpopulation of cells that
was eliminated in a selective, and near-complete, manner by T2 toxin exposure.
This       sensitive cell population was observed to have light-scatter
characteristics of CD45R+ B-lineage lymphocytes. Additional studies in adult
mice demonstrated a reduction in CD44lo and CD45R+ bone marrow cells similar
to that seen in fetal liver, indicating that T2 toxin may  also target
immature B lymphocytes in this hematopoietic compartment. Taken together,
these data suggest that the precursors of B cells may represent, for unknown
reasons, highly sensitive targets of T2 toxin exposure.

347
Fukuoka M, Kobayashi T, Hayakawa T.  MECHANISM OF TESTICULAR ATROPHY INDUCED
BY DI-N-BUTYL PHTHALATE IN RATS: VI. A POSSIBLE ORIGIN OF TESTICULAR IRON
DEPLETION. Biol Pharm Bull 1994;17(12):1609-12.

In previous studies we have described mechanisms of testicular atrophy whereby
di-n-butyl phthalate (DBP) caused a sloughing of the germ cells, prior to the
testicular atrophy; this sloughing might be attributed to iron depletion in
the blood and the testicular interstitial cells. To determine whether the iron
depletion is mediated by iron-release from hemoglobin (Hb), the effects of DBP
upon erythrocytes have been studied. In the in vivo studies, it was observed
that DBP induced glutathione  (GSH) depletion, a decrease in GSH reductase
activity and Heinz body formation in the red blood cells, and iron release
from Hb. In the in vitro studies, in which mono-n-butyl phthalate (MBP), a
metabolite of DBP, was incubated with erythrocytes, Heinz bodies and iron
release from Hb were observed. The present study proposes that a mechanism for
the testicular atrophy induced by DBP might involve Heinz body formation,
accompanied by iron release from Hb followed by depletion of iron in the 
blood and testes. 

348
Gray LE, Klinefelter G, Kelce W, Laskey J, Ostby J,  Ewing L.  HAMSTER LEYDIG
CELLS ARE LESS SENSITIVE TO ETHANE DIMETHANESULFONATE WHEN COMPARED TO RAT
LEYDIG CELLS BOTH IN VIVO AND IN VITRO.   Toxicol Appl Pharmacol
1995;130(2):248-56.

It has been reported that ethane dimethanesulfonate (EDS) is a Leydig cell
toxicant that affects rats and hamsters (Kerr et al., 1987), while, in
contrast, the Leydig cells of mice are relatively insensitive to the toxicant.
In the rat, there is a rapid decline in levels of testosterone (T) within
hours after EDS administration. However, T production, spermiogenesis, and
fertility are restored within a few weeks as new Leydig cells are formed from
undifferentiated cells in the interstitium of  the testis. In an earlier
study, we found, as expected. that ejaculated sperm counts (ESCs) reached a
nadir 10 days after adult rats were dosed with EDS at 65 mg/kg ip along with
serum and testicular T, testis and seminal vesicle weights, and in vitro T
production, while, in contrast, EDS at 65 mg/kg had no effect on these
endpoints in the Syrian hamster (Gray et al., 1992). In the current study,
when EDS was administered to 6, 12, and 18 month old hamsters at 100 mg/kg, it
produced subtle effects on serum T and sex accessory gland weights, while
dramatic effects were seen in similarly exposed rats. In addition, when testes
were examined by light microscopy all treated rats displayed severely reduced
Leydig cell numbers, while, in contrast, only one-third of the EDS-treated
hamsters were affected, having moderately reduced Leydig cell numbers. In
support of the histological data, 3beta-HSD enzyme activity was reduced by 99%
of control in EDS-treated rats, but it was reduced by onl 35% of control in
EDS-treated hamsters. An in vitro analysis of the effects of EDS on
LH-stimulated T production by quartered testes demonstrated that the hamster
testis was less sensitive to the direct effects of EDS than the rat testis.
The IC50 after 3 hr in culture was greater than 1800 mug EDS/ml for the
hamster quarter testes, while the IC50 for the rat quarter testes was 320 mug
EDS/ml. In summary, these results demonstrate in vivo and in vitro that Leydig
cells of hamsters are less sensitive to EDS than those of the adult rat. 

349
Eisses KT.  DIFFERENCES IN TERATOGENIC AND TOXIC PROPERTIES OF ALCOHOL
DEHYDROGENASE INHIBITORS PYRAZOLE AND 4-METHYLPYRAZOLE IN DROSOPHILA
MELANOGASTER: II. ADH ALLOZYMES IN AN ISOGENIC BACKGROUND. Teratogenesis
Carcinog Mutagen 1994;14(6):
291-302.

Pyrazole and 4-methylpyrazole (4-MP) are in vivo and in vitro inhibitors of
alcohol       dehydrogenase activity in mammals. The fruitfly Drosophila
melanogaster has been used to demonstrate the influence of genetic variation
in alcohol dehydrogenase alleles on the results of larval treatment with
pyrazole and 4-MP. Genetic polymorphism of organisms involved in experiments
with teratogenic and toxic agents is not often considered. Administration of
pyrazole to larvae of isogenic D. melanogaster  strains, differing mainly in
their Adh alleles, caused large Notch-like teratogenic aberrations,
macrochaetae  multiplication, and pupal mortality. The level of teratogenicity
and developmental-toxicity of pyrazole was both concentration and
Adh-genotype-dependent. The strain with the highest ADH activity showed
smaller effects after the treatments with the two concentrations used. 4-MP
does not cause morphological aberrations, although treatment of larvae with an
isogenic background caused a  high pupal mortality due to non-differentiated
material in the pupal case. 

350
Heaton MB, Bradley DM.  ETHANOL INFLUENCES ON THE CHICK EMBRYO SPINAL CORD
MOTOR SYSTEM: ANALYSES OF MOTONEURON CELL DEATH, MOTILITY, AND TARGET TROPHIC
FACTOR ACTIVITY AND IN VITOR ANALYSES OF NEUROTOXICITY AND TROPHIC FACTOR
NEURO-PROTECTION.  J Neurobiol 1995;26(1):47-61.

A series of in vivo and in vitro experiments were conducted to determine the
influence of prenatally administered ethanol on several aspects of the
developing chick embryo spinal cord motor system. Specifically, we examined:
(1) the effect of chronic ethanol administration during the natural cell death
period on spinal cord motoneuron numbers; (2) the influence of ethanol on
ongoing embryonic motility; (3) the effect of ethanol exposure on neurotrophic
activity in motoneuron target tissue (limb  bud); and (4) the responsiveness
of cultured spinal cord neurons to ethanol, and the potential of
target-derived neurotrophic factors to ameliorate ethanol neurotoxicity. These
studies revealed the following: Chronic prenatal ethanol exposure reduces the
number of motoneurons present in the lateral motor column after the cell death
period (embryonic day 12 (E12)). Ethanol tends to inhibit embryonic motility,
particularly during the later stages viewed       (E9-E11). Chronic ethanol
exposure  reduces the neurotrophic activity contained in target muscle tissue.
Such diminished support could contribute to the observed motoneuron loss.
Direct exposure of spinal cord neurons to ethanol decreases neuronal survival
and process outgrowth in a dose-dependent manner, but the addition of target
muscle extract to ethanol-containing cultures can ameliorate this ethanol
neurotoxicity. These studies demonstrate ethanol toxicity in a population not
previously viewed in this regard and suggest a mechanism that may be related
to this cell loss (i.e., decreased neurotrophic support). 

351
Cosenza ME,  Bidanset J.  EFFECTS OF CHLORPYRIFOS ON NEURONAL DEVELOPMENT IN
RAT EMBRYO MIDBRAIN MICROMASS CULTURES.  Vet Hum Toxicol 1995;37(2):118-21. 

352
Ciapetti G,  Granchi D, Stea S, Cenni E, Schiavon P, Giuliani R,  Pizzaferrato
A.  ASSESSMENT OF VIABILITY AND PROLIFERATION OF IN VIVO SILICONE-PRIMED
LYMPHOCYTES AFTER IN VITRO RE-EXPOSURE TO SILICONE.  
J Biomed Mater Res 1995;29(5):583-90. 

The functional response of peripheral blood lymphocytes isolated from 22
patients with silicone gel-filled breast implants was assessed after in vitro
re-exposure to silicone. Using cell culture test methods to quantify
proliferation and viability and/or activation of lymphocyte microcultures,
i.e., the uptake of tritiated thymidine (3H-TdR uptake test) and the reduction
of formazan salts (MTT assay), interesting data were obtained. Peripheral
blood lymphocytes purified from patients wearing  silicone gel-filled breast
implants react in vitro to silicone showing a statistically significant
increase of both proliferation and viability, while healthy subjects do not
respond on in vitro exposure to silicone. Differences resulted even more
statistically significant when patients were divided into two      groups
depending on the type of surgery they underwent: patients with breast
augmentation for aesthetic reasons seem to have an increased responsiveness in
vitro to silicone compared to patients who experienced a reconstructive
surgery of the breast. Although they are still preliminary, being referred to
a limited population, these results suggest that the lymphocytes of patients
with silicone gel-filled breast implants could be sensitized in vivo toward
silicone; the re-exposure of these cells to silicone leads to a higher
functional response which could be looked for by using quantitative in vitro
test methods.

353
Canteros G,  Rettori V,  Franchi A, Genardo A, Cebral E , Faletti A, Gimeno M,
McCann SM.  ETHANOL INHIBITS LUTEINIZING HORMONE-RELEASING HORMONE (LHRH)
SECRETION BY BLOCKING THE RESPONSE OF LHRH NEURONAL TERMINALS TO NITRIC OXIDE.

Proc Natl Acad Sci USA 1995;92(8):3416-20. 

It has previously been shown that alcohol can suppress reproduction in humans,
monkeys, and small rodents by inhibiting release of luteinizing hormone (LH).
The principal action is via suppression of the release of LH-releasing hormone
(LHRH) both in vivo and in vitro. The present experiments were designed to
determine the mechanism by which alcohol inhibits LHRH release. Previous
research has indicated that the release of LHRH is controlled by nitric oxide
(NO). The proposed pathway is via  norepinephrine-induced  release of NO from
NO-ergic neurons, which then activates LHRH release. In the present
experiments, we further evaluated the details of this mechanism in male rats
by incubating medial basal hypothalamic (MBH) explants in vitro and examining
the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to
prostanoids, and production of cGMP. The results have provided further support
for our theory of LHRH control. Norepinephrine increased the release of NO  as
measured by conversion of (14C)arginine to (14C)citrulline, and this increase
was blocked by the a, receptor blocker prazosin. Furthermore, the release of
LHRH induced by nitroprusside (NP), a donor of NO, is related to the
activation of soluble guanylate cyclase by NO since NP increased cGMP release
from MBHs and cGMP also released LHRH. Ethanol had no effect on the production
of NO by MBH explants or the increased release of NO induced by
norepinephrine. Therefore, it does not act  at that step in the pathway.
Ethanol also failed to affect the increase in cGMP induced by NP. On the other
hand, as might be expected from previous experiments indicating that LHRH
release was brought about by PGE2, NP increased the conversion of
(14C)arachidonic acid to its metabolites, particularly PGE2. Ethanol
completely blocked the release of LHRH induced by NP and the increase in PGE2
induced by NP. Therefore, the results support the theory that norepinephrine
acts to stimulate NO  release from NO-ergic neurons. This NO diffuses to the
LHRH terminals where it activates guanylate cyclase, leading to an increase in
cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP
increases intracellular free calcium, activating phospholipase A2 to provide
arachidonic acid, the substrate for conversion by the activated cyclooxygenase
to PGE2, which then activates the release of LHRH. Since alcohol inhibits the
conversion of labeled arachidonic acid to PGE2,  it must act either directly
to inhibit cyclooxygenase or perhaps it may act by blocking the increase in
intracellular free calcium induced by cGMP, which is crucial for activation of
both phospholipase A2 and cyclooxygenase. 

354
Esterman AL, Rosenberg C, Brown T, Dancis J.  THE EFFECT OF ZIDOVUDINE AND
2'3'-DIDEOXYINOSINE ON HUMAN TROPHOBLAST IN CULTURE.  Pharmacol Toxicol
1995;76(1):89-92.

Trophoblast from term and first trimester placenta, maintained in culture,
were exposed to 20 mumoles/l zidovudine or 2'3'dideoxyinosine. Several indices
of function were measured and compared to control trophoblast in parallel
culture. The results from individual placentas were examined by Student's
t-test and cumulative results by ANOVA. Neither zidovudine or
2'3'-dideoxyinosine had statistically significant effects on the function of
term trophoblast, following a 48 hr exposure to the drug as indicated by hCG
secretion, protein synthesis and glucose consumption. In one of five placentas
exposed to zidovudine, progesterone secretion was reduced as compared to its
control but remained in the high range. Zidovudine had no significant effect
on cultured trophoblast isolated from first trimester placenta even after
prolonged exposure to the drug for eleven days. Both term and first trimester
trophoblast in culture tolerate prolonged exposure to high concentrations of
zidovudine or 2'3'-dideoxyinosine. Human trophoblast in culture provides a
safe in vitro model for the screening of drugs intended for use during
pregnancy.

355
Cain L, Chatterjee S, Collins TJ.  IN VITRO FOLLICULOGENESIS OF RAT PREANTRAL
FOLLICLES. Endocrinology 1995;136(8):3369-77.

The impact of various gonadotropic hormones on the growth and development of
secondary follicles from primordial and primary follicles obtained by enzymic
dissocn. of the ovaries of immature 14-day-old rats was studied in vitro.  The
substratum-adherent culture technique developed for studying folliculogenesis
in the current study permitted direct visualization of follicular growth on a
day-to-day basis by avoiding the cumbersome process of fixing and sectioning
follicles in culture.  The cultures were maintained in a serum-free modified
McCoy's medium is a humidified atm. contg. 5% CO2 at 37 C. Daily observation
of the culture dishes under the phase contrast microscope revealed that the
follicles grew and developed from primordial to primary and secondary
follicular stages in the presence of FSH. Large antral follicles were able to
secrete estradiol and progesterone into the medium, indicating that the
follicles are not merely formed by cellular reorganization, but are physiol.
functional competent units. The organized release of the oocyte with
accompanying corona radiata was made possible in some secondary follicles with
large antral structures by introducing LH into the culture medium. However,
introduction of hCG (which has the biol. properties of LH) into the cultures
on day 1 resulted in follicular degeneration within 3-4 days of culture.
Follicular organization was also disrupted when LH was introduced together
with FSH into the medium on day 1 of culture. Primordial or primary follicles
obtained from the ovaries could survive, but could not transform to secondary
follicles in the absence of FSH. The results of these in vitro studies
indicate, and therefore are in agreement with earlier in vivo studies, that
FSH alone is essential for the progression of folliculogenesis to the
preovulatory condition, and that LH is essential for the organized expulsion
of the oocyte from a mature follicle. This technique, described in the current
study, for producing physiol. functional secondary follicles in culture not
only allows progress in folliculogenesis to be monitored very closely, but
also serves as a model for studying the various intrinsic factors that may be
involved in the successful development of dominant mature Graafian follicles
that can finally ovulate. It also facilitates access to the growing follicle
along with its oocyte, which can, therefore, be used as a powerful model to
study the effects of various test substances in follicular development.
Addnl., the oocyte may be exptl. manipulated and subjected to in vitro
fertilization for producing animal species that could be used for research
purposes, clin. trials, and restoring species that are on the brink of
extinction.

356
Bourget P, Roulot C, Fernandez H. [TRANSPLACENTAL PASSAGE AND FETOPLACENTAL
METABOLISM OF DRUGS: STUDY DESIGN, THERAPEUTIC CONTRIBUTION AND IMPLICATIONS.]
Therapie 1994;49(6):481-97. (Fre) 

Pregnancy is a specific dynamic state and the potential usefulness of caring
for a fetal and/or adjacent disorder by treating the mother is now well
established.  Pregnant women being excluded from the investigational field of
clin. trials, only few studies exist concerning evaluation of the
pergestational metab. or transplacental transfer (TPT) of drugs. Questions are
extensive and complex. Does TPT occur at a given gestational age (GA), in the
context of a particular type of pathol., when a drug is administered by a
certain dosage regimen and if this is the case, what is the rapidity of
penetration of the products of conception by the drug (bearing in mind its
phys.-chem. characteristics); need harmful adverse effects on the child be
feared; is such penetration desirable, of no consequence or dangerous; does
the possibility exist of accumulation in the placenta, fetal tissue or
amniotic fluid; should such findings modify the therapeutic regimens of drugs
given to expectant mothers.  After dealing with the ethical and physiol.
context in which such research is undertaken, the authors review methods for
the study of TPT developed both in vitro and in vivo.  The current review
covers the period between 1972 and 1993.  Exchange mechanisms are complicated
and models developed in vitro only partially reflect the actual equil. which
develop.  These include: 1. the perfused cotyledon model, which while simple,
elegant and inexpensive, offers only a localized and fixed view of pregnancy;
2. the necessary study, using microsomes, of placental metabolic capacity
(enzyme cartog.).  In vivo study of TPT is based upon various
multicompartmental pharmacokinetic models, some of which have been relatively
validated in animals.  The simplest indicator for the in vivo evaluation of
TPT of a drug in the human species is detn. of a feto-maternal blood concns.
ratio (usually performed at the time of sepn.).  The usefulness and
limitations of this parameter are controversial, and it would seem preferable
to assoc. it with a kinetic profile of variations in blood concns. established
in the mother.  Any extrapolation of a single result to fetal and adjacent
tissues must be done with the greatest caution. 

357
Hoermann R, Poertl S, Liss I, Amir SM,  Mann K.  VARIATION IN THE THYROTROPIC
ACTIVITY OF HUMAN CHORIONIC GONADOTROPIN IN CHINSES HAMSTER OVARY CELLS ARISES
FROM DIFFERENTIAL EXPRESSION OF THE HUMAN THYROTROPIN RECEPTOR AND
MICROHETEROGENEITY OF THE HORMONE.  J Clin Endocrinol Metab
1995;80(5):1605-10.

The role of hCG as a stimulator of the human thyroid has been a subject of
controversy, because discrepant results have been obtained in different in
vitro assays. To explain the variation obsd. in the thyroid response to hCG,
the authors investigated the ability of hCG and that of its isoforms and
glycosylation variants to inhibit [125I]bovine (b) TSH binding and stimulate
adenylate cyclase in 2 clones HO09 and JP26, of Chinese hamster ovary cells
stably transfected with the human TSH receptor (hTSHr). The two clones
differed with respect to the no. of hTSHr expressed per cell (34,000 in JP09
and 2,000 in JP26 cells). Both responded extremely well to bTSH; the cAMP
response to 0.001 IU/L bTSH was distinguishable from basal values.
Interestingly, JP09 cells were readily stimulated       by hCG (20-100 mg/L;
0.52-2.6.times.10-6 mol/L) to release cAMP,  whereas JP26 cells showed little
if any response. Also, cAMP stimulation produced by asialo-hCG was 12-fold in
JP09 cells and only 4-fold in JP26 cells compared to 45- and 67-fold
stimulations by bTSH, resp. Stimulation by asialo-hCG was approx. 30% that of
bTSH in JP09 cells, but less than 6% in JP26 cells. When assessing the
thyrotropic activity of the micro-heterogeneous isoforms of hCG, more alk. pI
forms were more active than those of a more acidic pI regardless of whether
they were derived from normal or molar pregnancy urine. Further studies with
hCG, asialo-hCG, asialo-galacto-hCG, and deglycosylated hCG revealed that
removal of sialic acid caused a marked increase in both its affinity for hTSHr
and its cAMP-releasing potency, whereas removal of further carbohydrate,
although it slightly enhanced receptor binding, was detrimental to adenylate
cyclase activation.  In conclusion, differences in hTSHr expression may cause
a variation in the cAMP response to hCG or its glycosylation variants, as does
the microheterogeneity of the hormone itself. These mechanisms may be
responsible at least in part for the divergent responses of different cell
types to hCG and render interpretation of the physiol. meaning of the data
obtained in recombinant receptor systems difficult. 

358
Ealey PA, Yateman ME, Sandhu R,  Dattani MT, Hassan MK, Holt SJ, Marshall NJ. 
THE DEVELOPMENT OF AN ELUTED STAIN BIOASSAY (ESTA) FOR HUMAN GROWTH HORMONE.
Growth Regul 1995;5(1):36-44. 

The basic characteristics of MTT-formazan prodn. by both quiescent Nb2 cells
and those activated by fetal calf serum or human growth hormone (hGH) are
described. These characteristics are exploited for the development of an
MTT-ESTA bioassay for purified prepns. of lactogens such as growth hormone.
The resulting in vitro bioassay is sensitive and precise, with a detection
limit of about 0.05 mU hGH/L (19 ng/L) and a within-assay imprecision of 2.5%
in the presence of 0.3 mU hGH/L (114 ng/L).  When utilizing quiescent Nb2
cells for bioassays, large magnitudes of response are obsd. The major
component of the response is clearly derived from metabolic activation of the
cells, rather than increased cell proliferation. The response was abolished by
anti-human growth hormone. Delayed addn. of the latter demonstrated that the
presence of the hormone is required for the entire 96 h of the recommended
bioassay incubation period to obtain the max. response. At high doses, the
dose-response relation reaches a prolonged plateau which covers 4 orders of
magnitude of incremental hormone concns. A decline in response is obsd. at the
highest dose tested, 106 mU hGH/L (385 mg/L). This auto-inhibition is
consistent with recent reports of a redn. in response due to stoichiometric
blockade of sequential receptor dimerization which is crucial for activation
of both somatogenic and lactogenic receptors by hGH. 

359
Yuan YY, Gu ZP, Shi QX, Qin GW, Xu RS, Cao L.  [IN VITRO INHIBITION OF
SPERMATOZOA FERTILIZATION ABILITY OF GUINEA PIG BY CELASTROL.]   Yaoxue Xuebao
1995;30(5):331-5. (Chi)

The effects of celastrol (Cel), isolated from Tripterygium wilfordii, on
guinea pig sperm forward motility (FM), capacitation (Cap), the acrosome
reaction (AR) and sperm penetration assay (SPA) were assessed in vitro. Cel (5
mug.cntdot.mL-1) was found to inhibit these spermatozoal functions, and the
inhibitions were proportional to the concns. of Cel used. The potency in
inhibition of Cel on the fertilizing ability in guinea pig spermatozoa in
vitro seems to follow the order: Cap > FM > SPA > AR.  The inhibitory effect
appeared to be reversible after washing away Cel if the duration of exposure
of spermatozoa to Cel was shorter than 3 h. In a comparative study, the
inhibitory effects of Cel on guinea pig sperm FM and AR were significantly
stronger than those of gossypol acetic acid.

360
Mares A, DeBoever J, Stans G, Bosmans E, Kohen F.  SYNTHESIS OF A NOVEL
BIOTIN-ESTRADIOL CONJUGATE AND ITS USE FOR THE DEVELOPMENT OF A DIRECT, BROAD
RANGE ENZYME IMMUNOASSAY FOR PLASMA ESTRADIOL. J Immunol Methods
1995;183(2):211-9.

The synthesis and purifn. by high pressure liq. chromatog. of a novel
biotinoyl-diaminodioxaoctane-estradiol conjugate is described. The conjugate
was used for the development of an enzyme immunoassay (EIA) for the direct
detn. of estradiol in human plasma. This assay was characterized by a low
limit of detection (77 pM) and a broad working range. Estradiol concns.
ranging from 77 to 24300 pM, i.e. from the lower levels obsd. in the
follicular phase of the normal menstrual cycle to the very high levels
attained during hyperstimulation for in vitro fertilization, could be detd.
directly in undiluted plasma samples. Intra-assay variation ranged from 3.6 to
10.9% and interassay variation from 6.1 to 12%. The results obtained by the
present EIA correlate well with target values obtained by isotope-diln. gas
chromatog.-mass spectrometry (r = 0.96) and with results obtained by a direct
RIA (r = 0.97). The EIA requires no special app., is simple, fast and robust,
and could be applied in any clin. lab.

361
Liu C, Litscher ES, Wassarman PM.  TRANSGENIC MICE WITH REDUCED NUMBERS OF
FUNCTIONAL SPERM RECEPTORS ON THEIR EGGS REPRODUCE NORMALLY.  Mol Biol Cell
1995;6(5):577-85.

To initiate fertilization in mice, free-swimming sperm binds to mZP3, an
.apprx.83-kDa glycoprotein present in the ovulated egg zona pellucida (ZP).
MZP3 is located       periodically along the filaments that constitute the ZP.
Sperm recognize and bind to specific oligosaccharides linked to one or more of
five Ser residues clustered in the carboxy-terminal one-third of the mZP3
polypeptide. When all five Ser residues are converted to nonhydroxy amino
acids by site-directed mutagenesis of the mZP3 gene, an inactive form of mZP3,
called mZP3[ser], is secreted by embryonal carcinoma cells stably transfected
with the mutated gene. Here, seven independent transgenic mouse lines were
established that harbor the mutated mZP3 gene. In all lines, the mutant gene
is expressed by growing oocytes and mZP3[ser] is synthesized, secreted, and
incorporated into the ZP.  Purified mZP3[ser] prepd. from ovaries of
transgenic mice, like mZP3[ser] from transfected embryonal carcinoma cells, is
inactive in sperm binding assays in vitro. On the other hand, the presence of
mZP3[ser] in the ZP does not significantly affect either the binding of sperm
to ovulated eggs in vitro or the reprodn. of the mice, i.e., the transgenic
mice are fertile, breed at normal intervals, and produce litters of normal
sizes. These results indicate that the no. of functional sperm receptors in
the ZP can be reduced by more than 50% without adversely affecting
fertilization of eggs in vivo.


RESPIRATORY TOXICITY
362
Giri SN,  Hollinger MA.  EFFECT OF CADMIUM ON LUNG LYSOSOMAL ENZYMES IN VITRO.
Arch Toxicol 1995;69(5):341-5.

Labilization of lysosomal enzymes is often associated with the general process
of inflammation. The present study investigated the effect of the pneumotoxin
cadmium on the release and activity of two lung lysosomal enzymes. Incubation
of rat lung lysosomes with cadmium resulted in the release of
beta-glucuronidase but not acid phosphatase. The failure to "release" acid
phosphatase appears to be the result of a direct inhibitory effect of cadmium
on this enzyme. The K1 for cadmium was determined to be 26.3 muM. The
differential effect of cadmium on these two lysosomal enzymes suggests that
caution should be exercised in selecting the appropriate enzyme marker for
assessing lysosomal fragility in the presence of this toxicant. Furthermore,
the differential basal release rate of the two enzymes from lung lysosomes may
reflect the cellular heterogeneity of the lung.

363
Balls M, Fentem J. IN VITRO TEST SYSTEMS AND RESPIRATORY TOXICOLOGY. In:
Jenkins PG, et al., editors. International Programme on Chemical Safety IPCS
Joint Series, 18. Respiratory Toxicology and Risk Assessment; International
Symposium; 1992 Oct 6-9; Hanover, Germany. Stuttgart: Wissenschaftliche
Verlagsgesellschaft MBH: 1994. p. 291-314. 

364
Hirabayashi T, Ochiai H, Sakai S, Nakajima K, Terasawa K.  INHIBITORY EFFECT
OF FERULIC ACID AND ISOFERULIC ACID ON MURINE INTERLEUKIN-8 PRODUCTION IN
RESPONSE TO INFLUENZA VIRUS INFECTIONS IN VITRO AND IN VIVO.  Planta Med
1995;61(3):221-6.

We investigated the effect of ferulic acid (FA) and isoferulic acid (IFA),
which are active components of the rhizoma of Cimicifuga species used
frequently as anti-inflammatory drugs in Japanese Oriental medicines, on
murine interleukin-8 (IL-8) production in response to influenza virus
infections in vitro and in vivo by antibody-sandwich enzyme-linked
immunosorbent assay. In the in vitro study, the murine macrophage cell line
RAW 264.7 was infected with influenza virus at a dose of 10 plaque forming
units (PFU)/cell and cultured in the presence or absence of drugs. Both FA and
IFA reduced the IL-8 levels in the 20-h conditioned medium in comparison with
control in a dose-dependent manner. The effect of IFA was greater than that of
FA: IL-8 levels were reduced to 43% and 56% of the control in the presence of
100 micrograms/ml of IFA and FA, respectively. In the in vivo study, mice were
infected with 1,000 PFU of virus and received daily oral administrations of
Cimicifuga heracleifolia extract (5 mg/mouse/day), FA (0.5 mg/mouse/day), IFA
(0.125 mg/mouse/day), or phosphate buffered saline. The three drugs showed a
tendency to reduce IL-8 levels in bronchoalveolar lavage (BAL) obtained 2 days
after infection. Moreover, both FA and IFA also significantly reduced the
number of exuded neutrophils into BAL. However, the drug administrations did
not affect the virus yields in BAL. These data suggest that FA and IFA are
novel and potent inhibitors of murine IL-8 production and might act as one of
the main components of anti-inflammatory rhizoma of Cimicifuga species.

365
Kier LD, Wagner LM, Wilson TV, Li AP, Short RD, Kennedy GL Jr.  CYTOTOXICITY
OF ETHYLENE OXIDE/PROPYLENE OXIDE COPOLYMERS IN CULTURED MAMMALIAN CELLS. Drug
Chem Toxicol 1995;18(1):29-41.

Cytotoxicity was measured in vitro for 8 ethylene oxide/propylene oxide
copolymers (EO/PO copolymers) using lactate dehydrogenase release from
cultured mammalian cells as the endpoint. Three cell types were used in these
assays: Chinese hamster ovary cell line (AS52), rat lung epithelial cell line
(LEC), and freshly isolated rat alveolar macrophages (RAM). A range of
cytotoxicity was seen with toxic effects observed from 20 to > 20,000
micrograms/ml. The same relative order of toxicities were observed for all 3
cell lines although RAM cells appeared to be somewhat more sensitive. The in
vitro cytotoxicity, as measured by LDH release and microscopic observations of
the cells, correlated poorly with the in vivo inhalation toxicity. The most
lethal compounds following acute inhalation (UCON 50-HB-5100 and UCON
50-HB-2000) were among the least toxic in the in vitro cytotoxicity screen.
Conversely the 2 compounds which were the most toxic in vitro (Pluronic 17 R1
and Pluronic L64) did not produce any unusual degree of toxicity in inhalation
studies. The results of these experiments indicate that these in vitro
mammalian cell assays will not be useful, at least for these classes of
chemistry, in prediction of in vivo inhalation toxicity.

366
Fiala ES, Sodum RS, Hussain NS, Rivenson A, Dolan L.  SECONDARY NITROALKANES:
INDUCTION OF DNA REPAIR IN RAT HEPATOCYTES, ACTIVATION BY ARYL
SULFOTRANSFERASE AND HEPATOCARCINOGENICITY OF 2-NITROBUTANE AND 3-NITROPENTANE
IN MALE F344 RATS. Toxicology 1995;99(1-2):89-97.

The secondary nitroalkanes, 2-nitropropane, 2-nitrobutane, 3-nitropentane,
2-nitroheptane, nitrocyclopentane and nitrocyclohexane, as well as the primary
nitroalkanes, 1-nitropropane, 1-nitrobutane, 1-nitropentane and
1-nitroheptane, were examined for their ability to induce DNA repair in rat
hepatocytes and to serve as substrates for activation by partially purified
rat liver aryl sulfotransferase in vitro. All of the secondary, but none of
the primary nitroalkanes examined, induced significant DNA repair in rat
hepatocytes. Also, the nitronates of all of the secondary nitroalkanes, but
none of the primary nitroalkanes, served as substrates for the aryl
sulfotransferase-catalysed production of 8-aminoguanosine and 8-oxoguanosine
from guanosine in vitro. In a carcinogenicity assay using male F344 rats, the
secondary nitroalkanes, 2-nitrobutane and 3-nitropentane, produced a highly
significant incidence of hepatocarcinoma with metastases to the lungs,      
whereas the primary nitroalkane, 1-nitrobutane, was not carcinogenic. While a
low incidence of hepatocarcinoma was also produced by cyclopentanone oxime,
the results were not statistically significant. Since the secondary
nitroalkane, 2-nitropropane, in contrast to the primary nitroalkane,
1-nitropropane, was also previously shown to be hepatocarcinogenic in rats, it
is probable that secondary nitroalkanes constitute a hitherto unrecognized
class of chemical carcinogens.

367
Bianchi V.  V79 CYTOTOXICITY TEST FOR MEMBRANE DAMAGE. Methods Mol Biol
1995;43:151-60.

368
Price RJ, Renwick AB, Wield PT, Beamand JA, Lake BG. TOXICITY OF
3-METHYLINDOLE, 1-NITRONAPHTHALENE AND PARAQUAT IN PRECISION-CUT RAT LUNG
SLICES. Arch Toxicol 1995;69(6):405-9.

The toxicity of 3-methylindole, 1-nitronaphthalene and paraquat has been
studied in precision-cut rat lung slice cultures. Lung slices were prepared
from male Sprague-Dawley rats using an agarose gel instilling technique with a
Krumdieck tissue slicer and cultured for 24 h in a dynamic organ culture
system. Treatment of rat lung slices with 3-methylindole, 1-nitronaphthalene
or paraquat produced concentration dependent decreases in lung slice protein
synthesis and potassium content. EC50  values (concentration to produce a 50%
inhibition) for protein synthesis were 0.024, 0.27 and 0.57 mM for paraquat,
1-nitronaphthalene and 3-methylindole, respectively. These results demonstrate
that precision-cut lung slices are a useful in vitro model system for studying
the pulmonary toxicity of xenobiotics. Lung slices offer the potential as a
rapid in vitro screen for identifying pulmonary toxicants and to evaluate
species differences in response. 

369
Boor PJ, Gotlieb AI, Joseph EC, Kerns WD, Roth RA, Tomaszewski KE.
CHEMICAL-INDUCED VASCULATURE INJURY. Summary of the symposium presented at the
32nd annual meeting of the Society of Toxicology, New Orleans, Louisiana,
March 1993. Toxicol Appl Pharmacol 1995;132(2):177-95.

The cross-sectional structure of the vasculature is comparatively simple,
comprising three layers-the intima, adjacent to the lumen, the media, and the
adventitia. Notwithstanding this simplicity, the vessels are host to a variety
of reactions to injury. Two cell types, endothelial cells of the intima and
smooth muscle cells of the media, are principal targets of damage and repair.
The endothelial cells of the intimal layer of the vessel wall present a
macromolecular barrier and are important  in maintaining vessel integrity.
When the integrity is compromised by physical or chemical injury, endothelial
cells play a key role in the repair processes. The use of single-cell wound
models allows the mechanisms of damage and subsequent repair to be studied in
depth. Repair processes can be observed using time-lapse photography and
differences between cytoskeleton changes during repair and
reendothelialization of small and large wounds can be discriminated. In rats
treated with the plant  toxin monocrotaline, pulmonary vascular injury occurs
which manifests as thrombosis and remodeling with consequent progressive
pulmonary hypertension. In vivo and in vitro studies of the mechanism of
monocrotaline toxicity suggest that the endothelial cells are an important
target. In vitro studies show monocrotaline to be directly cytotoxic; in cells
that survive, there are functional changes to the endothelial cells, resulting
in a decreased repair capability which may lead to the complex, progressive
lung lesions that develop. The other target cells of the vasculature are the
smooth muscle cells of the media. Ingestion of primary amines allylamine and
beta-aminopropionitrile (beta-APN) results in chronic vasculotoxicity to the
aorta and medium-sized arteries. For allylamine, subtle changes in smooth
muscle result in medial hypertrophy and subintimal proliferation. The changes
are slow to occur, taking weeks or months of repeated treatment. For beta-APN,
which is the active ingred of the toxic sweet pea Lathyrus odoratous, vascular
toxicity is manifested by fatal rupture of aortic aneurysms. When these agents
are administered concomitantly, a synergistic acute smooth muscle necrosis
occurs in large elastic arteries and degenerative changes are seen in muscular
arteries. A change in the target for toxicity of allylamine toward the
vasculature may be responsible for this       synergistic toxic insult. Medial
smooth muscle necrosis is also noteworthy after administration of certain
pharmaceutical agents of diverse structure and pharmacological activity. These
agents induce arteriopathies in dogs and rats, although at different sites. In
dogs, the coronary arteries are susceptible, whereas in rats the mesenteric
arteries are the principal sites of injury. Since these agents are diverse in
structure and biochemical activity, the mechanism of toxicity of these agents
appears to be related to their pharmacodynamic activity rather than to a
direct effect. Studies on  the relationship between vasodilatory and
hypotensive properties of these agents and toxic response suggest the
mechanism may be related to profound and exaggerated hemodynamic properties. 

370
Nakamura Y, Romberger DJ, Tate L, Ertle RF, Kawamoto M, Adachi Y, Mio T,
Sisson JH,  Spurzem JR, Rennard SI. CIGARETTE SMOKE INHIBITS LUNG FIBROBLAST
PROLIFERATION AND CHEMOTAXIS.  Am J Respir Crit Care Med 1995;
151(5):1497-503.

Cigarette smoking is the most clearly recognized cause of pulmonary emphysema.
Since loss of lung tissue, which characterizes emphysema, represents a balance
between injury and repair, we hypothesized that cigarette smoke might
contribute to the development of emphysema by inhibiting fibroblast
proliferation and migration. To evaluate this, we examined the effect of
cigarette smoke extract (CSE) on the proliferation and migration of human lung
fibroblasts in vitro. CSE inhibited fibroblast  proliferation and migration at
noncytotoxic concentrations. When CSE was treated to remove volatile
components, it showed less inhibitory activity on fibroblast proliferation.
Therefore, we also examined acrolein and acetaldehyde, which are volatile
components of cigarette smoke. Micromolar concentrations of acrolein and
millimolar concentrations of acetaldehyde induced significant inhibition of
fibroblast proliferation. In contrast, removal of volatile       components
did not eliminate the  inhibitory activity of CSE for fibroblast migration,
although acetaldehyde and acrolein alone were also capable of inhibiting
chemotaxis. Cigarette smoke-induced inhibition of fibroblast proliferation and
migration may impair lung repair following lung injury, and may thus
contribute to the development of pulmonary emphysema.

371
Cherpillod P, Amstad PA.  BENZO(a)PYRENE-INDUCED MUTAGENESIS OF p53 HOT-SPOT
CODONS 248 AND 249 IN HUMAN HEPATOCYTES. Mol Carcinog 1995;13(1):15-20.

Human tobacco-related cancers show a high frequency of G-to-T transversions in
several mutation hot-spot regions of the p53 tumor suppressor gene, probably
the result of specific mutagens in tobacco smoke, most notably benzo(a)pyrene.
To gain insight into the mechanism of formation of these G-to-T transversions
in tobacco-associated carcinogenesis, we studied the mutagenesis of p53 codons
247-250 by benzo(a)pyrene in human hepatocellular carcinoma cells by
restriction fragment length  polymorphism-polymerase chain reaction genotypic
analysis. Benzo(a)pyrene preferentially induced G-to-T transversion in the
second and third positions of codon 248 and C-to-A transversion in the first
position of codon 248. However, benzo(a)pyrene did not induce base-pair
changes in codon 249, which is a mutational hot-spot in aflatoxin-related
hepatocarcinogenesis, in which predominantly G-to-T transversion in the third
position of codon 249 is observed. The benzo(a)pyrene-induced G-to-T
transversion in the middle position of codon 248, in  which arginine is
changed into leucine, is frequently observed in tumors of the lung. The other
two benzo(a)pyrene-induced base-pair changes in codon 248, namely the C-to-A
transversion in the first position and G-to-T transversion in the third
position, do not lead to a change in the amino-acid composition of the p53
protein. These mutations are silent and therefore are not selected in tumors.
It follows that benzo(a)pyrene-induced. mutability on the DNA level in p53
codons 247-250 correlates well with the type of mutation found in tumors of
the lung, Therefore, our results support the hypothesis that benzo(a)pyrene is
the etiological agent in tobacco-related cancers.

372
Lee RP, Forkert P-G. PULMONARY CYP2E1 BIOACTIVATES 1,1-DICHLOROETHYLENE IN
MALE AND FEMALE MICE.  J Pharmacol Exp Ther 1995;273(1):561-7.

Pulmonary cytotoxicity induced by 1,1-dichloroethylene (DCE) has been linked
to the generation of reactive intermediates through a cytochrome
P450-dependent pathway. In the present studies, our objectives were to
investigate and compare cytochrome P450 isozyme-selective bioactivation of DCE
in vitro in the lungs of male and female mice. Our results showed that
CYP2E1-dependent p-nitrophenol hydroxylation was significantly higher in
microsomes from female (0.45 : 0.01 nmol/mg protein/min) than  from male (0.38
: 0.02 nmol/mg protein/min) mice. Lung microsomes from male mice incubated in
the presence of an NADPH-generating system and increasing amounts of DCE (5-20
mM) exhibited corresponding decreases in p-nitrophenol hydroxylase activity
(19%-50%);       however, greater decreases (26%-70%) were observed in lung
microsomes from female mice incubated under the same conditions. In contrast,
alterations in CYP2B1-dependent 7-pentoxyresorufin O-dealkylation and
CYP1A1-dependent 7-ethoxyresorufin O-dealkylation were not detected in any
microsomal preparation incubated with DCE. Reaction with an anti-CYP2E1
antibody abolished the inhibition of p-nitrophenol ydroxylation by DCE.
Protein immunoblotting revealed significant decreases in the intensity of the
bands of microsomal samples incubated previously with DCE; in contrast,
alterations in heme content were not evoked by reaction with DCE. Our results
have demonstrated that CYP2E1, and not CYP2B1 or CYP1A1, mediated  the
bioactivation of DCE. Furthermore, this bioactivation occurred to a greater
extent in lung microsomes from female than from male mice, which suggests that
females may be at slightly greater risk for DCE-induced pneumotoxicity.

373
Miller RC, Marino SA, Brenner DJ, Martin SG, Richards M, Randers-Pehrson G,
Hall EJ.  THE BIOLOGICAL EFFECTIVENESS OF RADON-PROGENY ALPHA PARTICLES: II.
ONCOGENIC TRANSFORMATION AS A FUNCTION OF LINEAR ENERGY TRANSFER.  Radiat Res
1995;142(1):54-60.

Epidemiological studies have established an association between exposure to
radon and carcinoma of the lung. However, based on data for either lung cancer
in uranium miners exposed to radon or bronchial epithelial carcinomas in
Japanese A-bomb survivors, it has not been possible to assign estimates of
risk of. lung cancer for the general population exposed to radon in their
homes. Based on past success with the excellent quantitative properties of the
C3H 10T1/2 in vitro oncogenic transformation assay system, the relative
biological effectiveness (RBE) for radiation-induced transformation for
charged particles of defined LET has been determined. As the LET of the
radiation was increased, the rate of induction of oncogenic transformation
increased and the RBEm approached 20. At higher LETs, RBE dropped
precipitously. The rapid drop in effectiveness for et particles with LETs
between 120 and 265 keV/mum implies a lower quality factor than the 20-25     
currently considered appropriate when estimating lung cancer mortality.

374
Walles SA, Victorin K, Lundborg M.  DNA DAMAGE IN LUNG CELLS IN VIVO AND IN
VITRO BY 1,3-BUTADIENE AND NITROGEN DIOXIDE AND THEIR PHOTOCHEMICAL REACTION
PRODUCTS. Mut Res 1995;328(1):11-9.

A UV-irradiated mixture of 1,3-butadiene and nitrogen dioxide (NO2) was tested
for its potency to induce DNA damage measured as single-strand breaks (SSB) in
lungs of mice. Both gases were also tested separately. After 16 h exposure a
UV-irradiated mixture of 40 ppm butadiene + 20 ppm NO2, but not 20 ppm
butadiene + 10 ppm NO2 + UV, induced a significant increase in SSB as measured
by the alkaline unwinding technique. There was no increase in the level of SSB
using the alkaline elution  technique during the same testing conditions.
However, after 5 h exposure to 60 ppm butadiene + 30 ppm NO2 + UV both methods
demonstrated a significant increase in SSB. Mice were also exposed to
butadiene at 80 and 200 ppm for 16 h and at 500 ppm for 5 h. DNA damage was
demonstrated in both liver and lung after 5 and 16 h (only at 200 ppm) of
exposure using the unwinding technique. Using the alkaline elution assay, a
significant increase in the level of SSB in lung and liver was found only 
after 5 h of exposure. When mice were exposed to 30 ppm NO2 for 16 h or 50 ppm
for 5 h, a significant increase in SSB was found with the unwinding technique.
Alveolar macrophages from mice were also exposed in vitro to the gas mixture
and to butadiene and NO2 separately. In these experiments, the DNA damage was
studied with the unwinding technique. A significant effect was demonstrated
with 40 ppm butadiene + 20 ppm NO2 + UV. NO2 itself contributed to some extent
to the increase. Reasons for the discrepancies between the unwinding and the
alkaline elution techniques are discussed. 

375
Price RJ, Renwick AB, Beamand JA, Escalagon F, Wield PT, Walters DG, Lake BG. 
COMPARISON OF THE METABOLISM OF 7-ETHOXYCOUMARIN AND COUMARIN IN PRECISION-CUT
RAT LIVER AND LUNG SLICES.  Food Chem Toxicol 1995;33(3):233-7.

The metabolism of 7-ethoxycoumarin and (3-14C)coumarin was compared in
precision-cut rat liver and lung slices. The lung slices were prepared using
an agarose gel instilling technique enabling the production of tissue
cylinders followed by lung slices employing a Krumdieck tissue slicer. Both 50
muM 7-ethoxycoumarin and 50 muM (3-14C)coumarin were metabolized by rat liver
and lung slices. 7-Ethoxycoumarin was converted to 7-hydroxycoumarin (7-HC)
which was conjugated with both D-glucuronic acid and sulfate. 7-HC sulfate was
the major metabolite formed by both liver and lung slices. (3-14C)Coumarin was
metabolized by rat liver and lung slices to both polar products and to
metabolite(s) that bound covalently to tissue slice proteins. The polar
products included unidentified metabolites and 3-hydroxylation pathway
products, with only very small quantities of 7-HC being formed. These results
demonstrate that precision-cut lung slices are a useful model in vitro system
for studying the pulmonary metabolism of xenobiotics. Moreover, the
precision-cut tissue slice technique may be employed for comparisons of
hepatic and extrahepatic xenobiotic metabolism.

376
Stringer B, Imrich A, Kobzik L.  FLOW CYTOMETRIC ASSAY OF LUNG MACROPHAGE
UPTAKE OF ENVIRONMENTAL PARTICULATES. Cytometry 1995;20(1):23-32.

We sought to establish a quantitative method using flow cytometry to study
uptake of environmental particulates by alveolar macrophages (AMs). We used
right angle light scatter (RAS) to measure uptake of titanium dioxide, quartz,
and diesel particulates. After incubation with TiO2 in vitro, AMs showed
dose-dependent increases in both cell-associated particles visualized by
microscopy and RAS measured by flow cytometry (e.g., fold increase RAS at 4,
8, 16, 32, and 80 micrograms/ml, respectively, = 2 +/- 0.1, 4.0 +/- 0.5, 5.5
+/- 0.5, 9.1 +/- 2.5, 14.3 +/- 0.9; mean +/- SEM). Similar results were
obtained with quartz and diesel particles. A strong correlation was observed
between particle load per cell and AM RAS after uptake of fluorescent latex
beads or fluorescent TiO2 (coated with BODIPY-BSA) (R2 = 0.984, 0.997,
respectively). Using this technique, we found AM uptake of environmental
particulates to be substantially greater than that of a panel of
myelomonocytic and epithelial cell lines, consistent with their physiologic
role in pulmonary defenses. RAS measurements have also identified both      
calcium-dependent and calcium-independent components in AM interactions with
inert particles. Although this technique does not allow precise quantitation
of particle number or mass per cell, flow cytometric analysis of relative
increases in RAS is a useful tool to study AM interactions with a variety of
environmental particulates. 

377
Spaggiari L, Alfieri R, Cattelani L, Bobbio A, Rusca M, Urbani S, Foletti G,
Tecchio T, Carbognani P, Petronini P, et al.  HAEMMACCEL, A KEY FOR LUNG
PRESERVATION? Acta Biomed Ateneo Parmense 1995;65(3-4):147-55.

Several and different solutions have been used for lung preservation but, at
present, fluids and solutions are quite alike. In the last years extracellular
type solutions have been progressively tested in experimental researches and
have shown a better protection vs intracellular one. In this research we have
studied the effect of a low-potassium solution normally used as plasma
expander (Haemmaccel, HM) on isolated foetal human fibroblasts (WI-38). HM has
been compared with Belzer solution (UWS) after 16 hrs incubation at 10 degrees
C and low-potassium solutions with dextran 2% and 5% after 6 hrs and 16 hrs
incubation at 10 degrees C. Wi-38 cells have been seeded at the density of
9x10(4)/cm2 onto plastic well plates. Cellular viability was measuring using
the rate of protein synthesis through the incorporation of 3H leucine in
growth medium during a       1 hr incubation at 37 degrees C. The results were
expressed as nmol 3 H leu/mg of proteins/minute and presented as means +/- SD;
the comparison has been performed by the one way variance analysis test. After
16 hrs incubation at 10 degrees C HM preserves WI-38 cells significantly
better than UWS. Comparing HM with LPD 2% and 5% a significant difference both
at 6 hrs and at 16 hrs was observed. Our preliminary in vitro results confirm
that low-potassium solutions are less toxic on isolated lung cells than
intracellular one. Polygelin contained in HM could be considered a suitable
colloidal substance that determine a better preservation if compared with
Low-Potassium Dextran solutions.

378
Malkusch W, Rehn B, Bruch J.  IN VITRO METHOD FOR MEDICAL RISK ASSESSMENT OF
LASER FUMES. Opt Laser Technol 1995;27(1):39-43.

Laser processing of different materials may produce toxic fumes. In preventive
occupational medicine it is necessary to evaluate valid hygienic stds. for
work places. The basis for such hygienic stds. is the classification of laser
fumes by their fibrogenic, emphysematous, immunol. or other harmful potencies
in biol. assay systems. This paper is part of a European project on laser
safety. The part in this project is the development of a method for the
investigation of lung responses using in vitro cell assays.  The appropriate
laser fume samples will be supplied by other groups in this European project. 
In contrast to the cell assays usually used in risk assessment, the method is
based on isolated target cells in the lung, such as alveolar macrophages. The
test criteria are mediator release, surfactant reactions, release of reactive
oxygen species and cell proliferation. As demonstrated in the lung response to
other dusts (minerals, fibers etc.) these parameters are medically relevant
factors in the pathogenic alveolar dust response.  The paper gives basic
information about the method using lung cell assays and the results of known
substances, in comparison with a dust generated by laser processing. 


STRUCTURE ACTIVITY
379
Zhao F, Mayura K, Hutchinson RW, Lewis RP,  Burghardt RC, Phillips TD. 
DEVELOPMENTAL TOXICITY AND STRUCTURE-ACTIVITY RELATIONSHIPS OF CHLOROPHENOLS
USING HUMAN EMBRYONIC PALATAL MESENCHYMAL CELLS.  Toxicol Let
1995;78(1):35-42.

The chlorophenols (CPs) comprise a major class of widely distributed and
frequently occurring environmental contaminants. Previous studies have
demonstrated the adverse effects of CPs on embryonic and fetal development.
HEPM (human embryonic palatal mesenchymal) and MOT (mouse ovarian tumor) cell
lines have been utilized in complementary bioassays for the detection of
teratogens, but not the CPs. In this study, the objectives were 2-fold: (1) to
det. if the HEPM assay could be used to complement other bioassay systems of
nonhuman origin, i.e., Hydra attenuata (HA) and rat whole embryo culture
(WEC), in the evaluation of the developmental toxicity of CPs, and (2) to
delineate the ability of the HEPM assay to evaluate structure-activity
relationships of pentachlorophenol (C5P), 2,3,4,5- tetrachlorophenol (C4P),
2,3,5-trichlorophenol (C3P), 3,5-dichlorophenol (C2P), 4-monochlorophenol
(CP), phenol, and CP derivs. (i.e., acetates, sodium phenates and anisoles).
HEPM cells were seeded into each well of a 24-well plate and cultivated for 24
h. The medium was replaced with fresh medium contg. various concns. of test
chems. dissolved in DMSO (0.1%). After culturing for 72 h, the medium was
removed, cells were trypsinized, and cell no. detd. The HEPM cell growth
inhibition assay demonstrated a linear relation between the IC50 values of 
the CPs and degree of chlorine substitution. The IC50 values of C5P, C4P, C3P,
C2P, CP, and phenol were 18.8, 21.5, 27.5, 63.0, 150.0 and 470.0 muM, resp. A
clear structure-activity relation was obsd. between toxicity of CPs and the
degree of chlorine substitution.  The rank order of CP toxicity from the HEPM
assay (i.e., C5P > C4P > C3P > C2P > CP > phenol) is in excellent agreement
with previous in vitro and in vivo studies. However,       contrary to
published reports, the HEPM assay predicted that all CPs were teratogenic
(false positives). These findings suggest that the HEPM cell growth inhibition
bioassay may be useful to discriminate between subtle differences in
structure-activity and, in combination with other bioassays, might facilitate
the rapid detection and prioritization of diverse cytotoxins, including
various developmental toxicants. Importantly, conclusions about the
teratogenicity of a test chem. (via HEPM testing) should be approached with
caution and confirmed with other teratogen-sensitive systems. 

380
Stehmann C, De Waard MA. RELATIONSHIP BETWEEN CHEMICAL STRUCTURE AND
BIOLOGICAL ACTIVITY OF TRIAZOLE FUNGICIDES AGAINST BOTRYTIS CINEREA. Pestic
Sci 1995;44(2):183-95.

The inhibitory activity of commercial and experimental triazole fungicides on
the target enzyme, sterol 14alpha-demethylase (P45014DM), was studied in a
cell-free sterol synthesis assay of Botrytis cinerea Pers. ex Fr. In order to
assess structure-activity relationships, the inhibitory activities of the
compounds on radial growth of the fungus were tested as well. The EC50 values
(concentrations of fungicide inhibiting radial growth of B. cinerea on PDA by
50%) of all triazoles tested  ranged between 10-8 and 10-5 M. IC50 values
(concentrations of fungicides inhibiting incorporation of (2-14C)mevalonate
into C4-desmethyl sterols by 50%) generally ranged between 10-9 and 10-7 M and
correlated with inhibition of  radial mycelial growth. However, differences in
IC50 values did not reflect quantitatively the observed differences in EC50
values, since the ratio between EC50 and IC50 increased with decreasing
fungitoxicity. For a limited number of compounds the correlation between
intrinsic inhibitory activity and fungitoxicity was low. Both in-vitro tests
were used to investigate structure-activity relationships for stereoisomers of
cyproconazole, SSF-109 and tebuconazole. Fungitoxicity and the  potency to
inhibit cell-free C4-desmethyl sterol synthesis correlated for all
stereoisomers tested. Mixtures of isomers of tebuconazole or cyproconazole
were slightly less active than the most potent isomer. The high activity of
several commercial triazoles in both experiments implies that poor field
performance of triazole fungicides against B. cinerea is due neither to
insensitivity of the P45014DM nor to low in-vitro sensitivity of the fungus. 

381
Luker KE, Tyler AN, Marshall GR, Goldman WE. TRACHEAL CYTOTOXIN STRUCTURAL
REQUIREMENTS FOR RESPIRATORY EPITHELIAL DAMAGE IN PERTUSSIS.  Mol Microbiol
1995;16(4):733-43.

The respiratory epithelial pathol. of pertussis (whooping cough) can be
reproduced by tracheal cytotoxin (TCT), a disaccharide-tetrapeptide released
by Bordetella pertussis.  TCT is a muramyl peptide, a class of
peptidoglycan-derived compds. which have many biol. activities including
adjuvanticity, somnogenicity, pyrogenicity, and cytotoxicity.  The structural
requirements for muramyl peptides to produce some of these biol. effects have
been partially characterized. Using in vitro assays with respiratory
epithelial cells and tissue, we have previously detd. that the disaccharide
moiety of TCT is not involved in toxicity and that the side-chain functional
groups of diaminopimelic acid (A2pm) are crucial for toxicity. In this study,
we det. the importance of every amino acid, functional group and chiral center
in the peptide portion of TCT. Although lactyl tetrapeptides are the most
toxic of the TCT fragments, producing dose-response curves identical to TCT,  
the smallest analogs of TCT which are active in our assay are of the form
X-gamma-(D)-Glu-meso-A2pm, where X may be an amino acid or a blocking group.
Within this active substructure, main-chain chirality and all functional
groups are essential for toxicity. This definition of the core region of TCT
indicates that the TCT interaction site is unlike almost all other muramyl
peptide interaction sites for which structure-activity data are available.