This
HuGE Review was published in the American Journal
of Epidemiology 2001; 154(1):1-13 |
d-Aminolevulinic
Acid Dehydratase (ALAD) Genotype and Lead Toxicity
Samir N. Kelada1,4, Erin Shelton2, Rachel B. Kaufmann3,
and Muin J. Khoury1
1 Office
of Genomics and Disease Prevention, Centers for Disease
Control and Prevention, Atlanta, Georgia 30341; e-mail: muk1@cdc.gov (MJK)
2 Department
of Environmental Health Sciences, School of Public Health,
University of Michigan, Ann Arbor, Michigan 48109; e-mail: erin.shelton@chmcc.org
3 Lead
Poisoning Prevention Branch, Division of Environmental
Hazards and Health Effects, National Center for Environmental
Health, Centers for Disease Control and Prevention,
Atlanta, Georgia; email: RKaufmann@cdc.gov
4 Address Correspondence to: Samir N. Kelada,
Department of Environmental Health, School of Public Health and Community Medicine, University of Washington, Box
357234, Seattle, WA 98195; email: skelada@u.washington.edu
AT-A-GLANCE
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The ALAD gene (chromosome 9q34) codes for d-aminolevulinic
acid dehydratase (ALAD) (E.C. 4.2.1.24). ALAD
catalyzes the second step of heme synthesis and
is polymorphic. The ALAD G177C polymorphism yields
two codominant alleles, ALAD-1 and ALAD-2, and
has been implicated in susceptibility to lead
toxicity. Genotype frequencies vary by geography
and race. The rarer ALAD-2 allele has been associated
with high blood lead levels and was thought to
increase the risk of lead toxicity by generating
a protein that binds lead more tightly than the
ALAD-1 protein. Other evidence suggests that ALAD-2
may confer resistance to the harmful effects of
lead by sequestering lead, making it unavailable
to participate pathophysiologically. Recent studies
have shown that individuals homozygous for the
ALAD-1 allele have higher cortical bone lead levels,
implying that they may have a greater body burden
and may be at higher risk of the long-term effects
of lead. Individuals exposed to lead in occupational
settings have been the most frequent subjects
of study. Genotype selection bias may limit inferences
from these studies. No firm evidence exists for
an association between ALAD genotype and lead
toxicity susceptibility at background exposure
levels; population testing for the ALAD polymorphism
is therefore not justified.
Keywords: lead, d-Aminolevulinic Acid Dehydratase
(ALAD), genetic susceptibility, epidemiology
Abbreviations: BLL = Blood lead level (mg/dl), ALAD = Aminolevulinic acid dehydratase, activity, ALA = Aminolevulinic acid.
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GENE |
The ALAD gene is located on chromosome 9q34
and is approximately 16 kilobases long (1). This
gene codes for the d-aminolevulinic acid dehydratase
(ALAD) enzyme (E.C. 4.2.1.24), also known as porphobilinogen
synthase, a 280 kilodalton protein that is composed
of eight identical subunits and requires eight
zinc ions as cofactors for full activity (2).
The ALAD enzyme catalyzes the second step in heme
synthesis, the asymmetric addition of two molecules
of aminolevulinic acid (ALA) to form the monopyrrole
porphobilinogen (PBG) (Figure 1), which is the
precursor of heme, as well as cytochromes and
cobalamins. ALAD is expressed in all tissues,
but the highest levels of expression are found
in erythrocytes and the liver (3). |
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GENE VARIANTS |
We searched Medline for relevant
publications using the MeSH headings “ALAD”
and “d-aminolevulinic acid dehydratase.”
Eight ALAD gene variants have been described in
the literature. This review will focus on one
polymorphism that yields two alleles, designated
ALAD-1 and ALAD-2, which exhibit a codominant
pattern of inheritance (4). The ALAD-2 allele
contains a G ® C transversion at position
177 of the coding region, resulting in the substitution
of asparagine for lysine at amino acid 59 (5).
These two alleles determine three isozymes, designated
1-1, 1-2, and 2-2, all of which display similar
activities but have different charges (4). Asparagine
is a neutral amino acid, whereas lysine is positively
charged. Therefore, ALAD 1-2 heterozygotes produce
an enzyme that is more electronegative than that
of ALAD-1 homozygotes, and ALAD-2 homozygotes
produce an enzyme that is more electronegative
than 1-2 heterozygotes. This forms the basis of
the electrophoretic technique originally used
to identify the polymorphism and phenotype individuals
(4).
The prevalence of the ALAD-2 allele ranges from
0 to 20 percent depending on the population. Generally,
Caucasians have the highest frequency of ALAD-2
allele, with approximately 18 percent of the population
being ALAD 1-2 heterozygotes and 1 percent being
2-2 homozygotes. In comparison, African and Asian
populations have low ALAD-2 allele frequencies,
with few or no ALAD-2 homozygotes found in such
populations. Table 1 lists genotype frequencies
from around the world (6-26). All of these frequencies
are in Hardy-Weinberg equilibrium. The listed
genotype frequencies were determined in the early
1980s by phenotyping. In 1991, Wetmur et al. (5)
devised a PCR-based genotyping technique, which
correctly identified all 93 ALAD-2 heterozygotes
and homozygotes tested, i.e., there was a 100%
genotype-phenotype correspondence. Most investigators
have subsequently used this technique.
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Table 1: ALAD genotype frequencies
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Most of the studies presented in Table 1 that
document genotype or phenotype frequencies gave
little detail about the study population (e.g.,
age or source of donors), making it hard to rule
out any potential biases due to subject selection.
These populations are referred to as “general”
in the table. Alternatively, some studies used
hospital-based study samples. Other studies (e.g.,
6, 7, 26) used samples comprised of individuals
with relatively high levels of lead exposure from
occupational studies. This may also promote bias
in the results, as persons with “at risk”
genotypes may have been selected against during
the course of employment and therefore may not
have been represented in the study sample. Background
and Epidemiology of Lead Poisoning
Lead has been a known toxin for thousands of
years and remains a persistent environmental health
threat. Exposure to lead can result in significant
adverse health effects to multiple organ systems.
Toxic effects to the nervous, hematological, renal,
and reproductive systems have been studied extensively
and are well documented (27, 28). Since lead was
phased out as a gasoline additive (tetra-ethyl
lead) in the 1970s and its use in paint and food
containers (e.g., ceramicware and tin cans) was
curtailed, blood lead concentrations have decreased
significantly; however, other sources of lead
and its unknown threshold of subclinical toxicity
continue to make lead an issue of public health
concern.
There are many risk factors for lead poisoning.
Generally, living in a home built before 1950
is considered a risk factor because of the presence
of multiple avenues of exposure to lead. Old pipes
with lead solder can contaminate the water supply,
and lead-based paint is still a notorious source
of lead in these houses (29). Additionally, living
in close proximity to lead-emitting industrial
facilities can present a significant source of
cumulative exposure to lead via air, water, and
soil. Occupational exposure to lead is most often
encountered at lead smelters and battery manufacturing
facilities, as well as in housing renovation projects
in which workers inhale and ingest lead-contaminated
fumes and dust from lead-based paint.
Children’s hand-to-mouth activity, increased
respiratory rates, and increased intestinal absorption
of lead make them more susceptible than adults
to lead exposure (30, 31). Lead-based paint remains
the predominant source of high-dose lead poisoning
in children. Poor nutrition, particularly inadequate
calcium and iron intake, is likely an important
risk factor for children as well (32).
Blood lead level (BLL, mg/dl) is the biological
index most often used by health care providers
as an indicator of recent lead exposure (33).
Two analytical techniques, anodic stripping voltammetry
(ASV) and atomic absorption spectroscopy (AAS),
are used to measure BLL and have detection limits
< 1 mg/dl (34). In addition to BLL, other lead
exposure indices include free erythrocyte protoporphyrin
(FEP) and zinc protoporphyrin (ZPP); both are
precursors of heme whose levels elevate upon moderate
to high exposure to lead. However, FEP and ZPP
are neither sensitive enough nor specific enough
to be used as primary indicators of lead exposure
(27, 35). Lead levels in plasma, urine, bone,
and teeth (dentin lead) are less commonly used
measures of exposure and body burden.
At steady state, 90 percent of lead is in the
skeleton (27). The association of lead and bone
is due to lead’s similar valence to calcium.
Measurements of lead in trabecular or spongy bone
(e.g., patella), in which lead has a relatively
short half-life, and lead in cortical bone (e.g.
tibia), which represents a site of long-term lead
storage, have been used to estimate the distribution
of lead in bone and total body burden (24, 27).
Reliable, non-invasive techniques such as X-ray
fluorescence (XRF) have been developed to measure
bone lead levels. Lead in bone can leach out and
constitutes a significant long-term source of
lead to the blood (27). Chelating agents such
as dimercaptosuccinic acid (DMSA) have been used
therapeutically to extract lead from tissues (28).
It has been shown that chelatable lead correlates
well with lead in trabecular bone (36). Administration
of chelators has also been used in research studies
to estimate body burden.
Subclinical lead toxicity remains a problem for
both adults and children (37-39). Blood lead concentrations
of 10 mg/dl in children have been associated with
cognitive deficits, aggressive behavior, and hearing
dysfunction (40-45). Alarmingly, evidence is indicating
that no detectable threshold exists for the adverse
effects of lead exposure on neurodevelopment (45,
46). Using National Health and Nutrition Examination
Survey (NHANES) III survey data, the Centers for
Disease Control and Prevention (CDC) estimated
that 890,000, or 4.4 percent, of U.S. children
aged 1 through 5, have blood lead concentrations
of 10 mg/dl, the current level of concern, or
higher (47). The current mean blood lead concentration
for children 1-5 years old is 2.7 mg/dl (48).
In the adult population, BLLs measured in NHANES
II and Phase I of NHANES III showed a decrease
from 13.1 mg/dl to 3.0 mg/dl, and currently more
than 90 percent of adults have BLLs < 10 mg/dl
(49). With respect to the occupational arena,
the current goal of the U.S. Department of Health
and Human Services (DHHS) is to eliminate all
occupational exposures resulting in BLLs >
25 mg/dl (33). The National Institute for Occupational
Safety and Health (NIOSH) used to maintain the
Adult Blood Lead Epidemiology and Surveillance
(ABLES) program, which reported the prevalence
of elevated BLLs among adults in 28 US states.
At last report, in the third quarter of 1998,
3322 (16%) of the 20,511 adults for whom BLLs
were reported had BLLs ³ 25 mg/dl; of these,
182 (6%) had BLLs ³ 50 mg/dl (50). Both of
these prevalence statistics represent declines
from previous quarters of ABLES reporting.
Lead and ALAD
One of lead’s primary effects is hematotoxicity,
specifically heme synthesis inhibition. Lead inhibits
three enzymes in the heme biosynthesis pathway
(Figure 1) -- ALAD, coporphyrinogen oxidase, and
ferrochelatase -- but its effects on ALAD are
most profound (51). Lead inhibits ALAD stoichiometrically
(52-54), and the degree of erythrocyte ALAD inhibition
has been used clinically to gauge the degree of
lead poisoning. At the molecular level, lead displaces
a zinc ion at the metal binding site, not the
active site (55), producing inhibition through
a change in the enzyme’s quaternary structure.
ALAD inhibition results in the build up of ALA,
detectable in the plasma and urine at BLLs < 10 mg/dl. ALA resembles g-aminobutyric acid (GABA)
and can stimulate GABA receptors in the nervous
system; this is thought to be one of primary mechanisms
of lead-induced neurotoxicity (55-57).
The ALAD-1/2 Polymorphism as a Modifier of Lead’s
Effects Initial Studies
Early studies conducted on the ALAD polymorphism
and lead poisoning focused on differences in BLLs
by genotype in populations with relatively high
levels of lead exposure, either from home or occupation
(Table 2). Ziemsen et al. (58) were the first
to describe differences in BLLs by genotype. They
found that lead-exposed workers (n = 202) with
the ALAD 1-2 genotype* had higher BLLs than ALAD
1-1 homozygotes (44 vs. 38 mg/dl) and that ALAD
2-2 homozygotes had higher BLLs at 56 mg/dl. Astrin
et al. (25) subsequently found a higher than expected
proportion of individuals with the ALAD 1-2 or
2-2 genotype among individuals with lead poisoning
screened by BLLs > 50 mg/dl or FEP > 30
mg/dl (n = 1074). The ascertainment bias in the
sampling technique was noted in the paper. Astrin
et al. also reported that the ALAD-2 allele was
associated with a four-fold increase in the ability
to retain BLLs above 30 mg/dl. Further, Wetmur
et al. (26) found significant differences in BLLs
in a group of lead-exposed workers (n = 202) and
New York City children (n = 1278) screened by
elevated FEP. They found median BLLs 9 mg/dl and
11 mg/dl higher among ALAD-2 carriers in these
two populations, respectively. All three of these
studies examined populations with exposure levels
higher than normal whose BLLs were often > 30 mg/dl, a previously designated cutoff used
as evidence of lead poisoning.
Hypotheses generated to support these results
were based on the charge of the ALAD-2 isozyme
(3, 25, 26). Since the ALAD-2 allele codes for
a more electronegative enzyme, the ALAD-2 protein
is thought to be able to bind positively charged
lead ion more tightly than the ALAD-1 protein.
Carriers of the ALAD-2 allele who are exposed
to lead might then retain it in their blood and
tissues longer, increasing the chance of an adverse
effect due to inhibition of ALAD and consequent
build up of ALA and perhaps due to lead itself,
which can initiate oxidative damage and change
the structure of cellular components (27). From
these initial studies, it is safe to conclude
that the kinetics of lead in blood are modified
by ALAD genotype, although perhaps only at relatively
high levels of exposure. These studies also imply
that the ALAD 1-2 and 2-2 genotypes are the “at
risk genotypes” at high exposure levels.
Further Studies
Subsequent studies (Table 2) were again primarily
occupational epidemiologic studies but often used
new sets of measures for lead exposure and body
burden. Bone lead measurements, in particular,
began to be used as measures of outcome. In 1995,
Schwartz et al. (59) used an occupational cohort
of employees from three lead storage battery factories
(n = 307) and found that the ALAD-2 allele was
not clearly associated with higher blood levels
(i.e., no difference in BLL by genotype) but that
individuals with the 1-2 genotype (there were
no 2-2 subjects) were 2.3 times more likely to
have BLL3 40 mg/dl, although the 95 percent
confidence interval (CI) contained 1.0. No relationship
was found between genotype and ZPP. They did find,
however, that the 1-2 genotype was associated
with occupational exposures of more than 6 years
(Odds Ratio (OR) = 2.6, 95 percent CI: 1.2, 5.8),
suggesting that the ALAD-2 allele conferred a
protective effect. In support, ALAD 1-2 heterozygote
workers with high exposure histories had lower
ZPP levels than ALAD-1 homozygotes with equivalent
exposure histories. The authors cited this as
a possible genotype-selection factor and proposed
that the ALAD-2 subunit of the protein keeps lead
in a non-bioavailable form so that these individuals
(ni = 4) were protected from lead’s effects
and could tolerate longer exposures to lead than
ALAD 1-1 subjects.
Using a group of 122 carpenters with relatively
low blood lead levels for study (average BLL =
7.8 mg/dl), Smith et al. (24) avoided the bias
of previous studies that used individuals with
high blood lead levels. They found no association
between ALAD genotype and BLL, implying that ALAD
genotype may be a modifier of BLL only at high
blood lead concentrations. They also found no
association between genotype and tibial or patellar
bone lead levels, which was measured using XRF
methods. However, using the difference of lead
levels in patellar versus tibial bone as an indicator
of effect of the genotype on the distribution
of lead in bone, a difference of borderline significance
was found between 1-1 and 1-2/2-2 genotypes (p
= 0.06). ALAD-1 homozygotes had a smaller difference
in patella – tibia bone lead levels than
1-2/2-2 individuals (3.4 ± 12.0 vs. 8.6
± 9.5 mg Pb/g bone mineral). This indicates
that 1-1 individuals have increased uptake of
lead into cortical bone, the long-term storage
depot, relative to 1-2/2-2 individuals. It was
hypothesized that 1-2/2-2 individuals partition
less lead into cortical bone because of the increased
affinity of the ALAD-2 subunit for lead. Hence,
ALAD-1 homozygotes would be at increased long-term
risk as they build up higher cortical bone lead
levels that can leech out at times of bone lead
redistribution (e.g., during pregnancy). These
investigators also observed a relationship between
ALAD-2 and subclinical renal toxicity, as evidenced
by elevated blood, urea, and nitrogen (BUN), uric
acid, and creatinine levels in ALAD-2 subjects.
In contrast, Bergdahl et al. (60) found lower
levels of urinary creatinine and calcium in 1-2/2-2
genotype subjects in a study of 89 lead-exposed
and 34 unexposed workers in Sweden. No association
between genotype and lead in blood, bone, or urine
among the exposed group was observed in this study.
The frequency of the ALAD-2 allele was less than
expected among lead workers (p value from c2 test
= 0.0025), and the authors cited this finding
as potential evidence of a genetic-healthy worker
effect, in which ALAD-2 individuals who reach
high blood lead levels would be removed from the
workplace (by Swedish occupational health standards)
and therefore would not be represented in the
study sample.
Several studies have yielded supporting evidence
for the hypothesis that ALAD genotype also modifies
the kinetics of lead in bone. In a study of 381
lead smelter workers, Fleming et al. (6) observed
increased uptake of lead from blood into bone
in ALAD-1 homozygotes, which was seen by the increased
slope of the line relating bone lead levels to
a cumulative blood level index (CBLI, mg/dl) in
1-1s compared to 1-2/2-2s. This effect was most
pronounced in trabecular bone (the calcaneus)
of workers hired after the implementation of a
lead safety initiative at the plant in 1977 (p
< 0.001, vs. p < 0.04 in cortical bone).
This study also provided more evidence for the
influence of ALAD genotype on the kinetics of
lead in blood at moderate to high exposure levels
(mean BLL = 23.3 mg/dl), as 1-2/2-2 genotype individuals
had 10 percent greater blood lead levels (p <
0.04).
Schwartz et al. (61) used the chelator DMSA to
test for differences in bioavailable lead by genotype
in a group of 57 lead battery manufacturing workers.
The data showed that 1-1 individuals yielded more
lead in 4-hour urine samples in response to chelation
therapy (5 mg/kg orally) versus 1-2/2-2 individuals
with the same exposure history (92.9 ±
45.1 vs. 70.3 ± 42.1 mg, respectively,
p = 0.07). Another study by Schwartz et al. (62)
corroborated these findings (Table 2). Given that
DMSA chelatable lead is used as a measure of bioavailable
lead, these data indicate that ALAD 1-2 subjects
have lower levels of bioavailable lead and therefore
may be at decreased risk compared to ALAD-1 homozygotes.
Schwartz and colleagues (61) also saw higher levels
of ALA in plasma (ALAP) of 1-1 individuals (17.3
vs. 11.8 ng/ml, p = 0.03), a finding that was
replicated by Sithisarankul et al. (p = 0.012)
(63). Similar differences in ALAP were seen in
a recent study of 192 Japanese male lead workers
by Sakai et al (17). These findings suggest that
ALAD-1 homozygotes may be at greater neurological
risk due to the build up of ALA in the plasma.
Finally, Schwartz et al. (62) noted an elevation
in hemoglobin A1 (HbA1) levels in 1-1 subjects
(n = 38) versus 1-2 subjects (n = 19) (6.3 ±
1.0 vs. 5.9 ± 1.0 %, p = 0.08), which led
them to conclude that both ALAD and HbA1 are important
lead binding sites that influence the excretion
of chelated lead.
It should be noted that the studies by Schwartz
et al. (59, 61, 62) and Sithisarankul et al. (63)
contained overlapping study samples. The degree
of overlap is, however, unknown. The studies are
presented separately for the purposes of this
review, but the results should be interpreted
with caution since they are based on study samples
that may have contained substantial redundancy.
Most recently, Schwartz et al. (64) reported
on a study of 798 Korean lead workers and 135
unexposed controls. ALAD 1-1 workers yielded substantially
more chelated lead after 10 mg/kg DMSA (see Table
2). Logistic regression modeling of chelated lead
showed that creatinine clearance was an important
predictor (b = 0.006, p < 0.001) and ALAD genotype
modified this relationship (p value for ALAD-creatinine
interaction = 0.04). ALAD-2 subjects had larger
increases in chelated lead with increasing creatinine
clearance. (The effect of a polymorphism in the
Vitamin D Receptor gene was also investigated
and is discussed below under gene-gene interactions.)
In a study by Bergdahl et al. (65), the ALAD
enzyme was found to be the principal lead- binding
site in erythrocytes. The investigators found
a higher percentage of lead bound to erythrocyte
ALAD in lead-exposed ALAD 1-2 subjects versus
1-1 subjects (84 percent vs. 81 percent, p = 0.03).
One study of a group of 134 lead smelter workers
in British Columbia by Alexander et al. (7) examined
differences in sperm count and sperm concentration
by genotype, in addition to BLL, ZPP, heme, and
coporphyrin (CPU). In this group, blood lead levels
were higher in ALAD 1-2 subjects, 28.4 versus
23.1 mg/dl for 1-1s (p = 0.08); ALAD 1-2 subjects
had higher sperm counts but the difference between
the two groups was not significant. Focusing on
the relationships of ALAD genotype, ZPP, and CPU
at blood lead levels ³ 40 mg/dl, Alexander
et al. observed that ALAD-1 homozygotes had significantly
higher ZPP levels (86.0 ± 41.1 vs. 50.0
± 18.4 mg/dl, p = 0.03) and higher CPU
levels (46.5 ± 31.6 vs. 13.6 ± 7.8
mg/l, p = 0.01). Markers other than BLL, in other
words, indicated that ALAD-1 homozygotes in this
study exhibited more inhibition of heme synthesis
after exposure to lead. The authors noted that
the non-random method of study subject ascertainment
(solicitation by postal questionnaire) and the
lack of women in the sample were limitations of
the study.
The sole population-based study was conducted
by Hsieh et al. (23) in a Taiwanese population
(n = 660). They measured BLL and found 20 percent
higher levels in the 1-2/2-2 group (7.83 ±
5.95 vs. 6.51 ± 5.03 mg/dl), but this result
was not statistically significant (p = 0.17).
The authors suggested that the difference in BLL
by genotype was not significant possibly because
of the small number of individuals in the 1-2/2-2
group (n = 30). They also postulated that blood
lead levels in Taiwan may be relatively low due
to the rarity of the ALAD-2 allele in that population.
ALAD Genotype and Neurological Outcomes
In 1994, Bellinger et al. (66) published a report
in which adolescents (n = 72) with high (>
24 mg/g) and low (< 8.7 mg/g) dentin lead levels
were studied, and the results suggested that the
body burden and the effects of lead were worse
in ALAD-1 homozygotes. Although the study had
only five subjects with the 1-2 genotype, these
five had lower dentin lead levels than subjects
with the 1-1 genotype and consistently scored
better on neuropsychological tests (no p values
given because of small n).> This is the only
study on the ALAD polymorphism to date that has
used a neurological outcome measure. The 1-2 genotype
subjects were also less likely to have tibial
lead concentrations > 6 mg/g and more likely
to have patellar lead concentrations > 6 mg/g.
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INTERACTIONS |
In addition to ALAD genotype, other factors to
consider in determining overall susceptibility to
lead toxicity include substances that inhibit the
ALAD enzyme and nutritional status, primarily calcium
and iron intake. The interaction between these factors
and ALAD may be important when considering the health
effects of lead. Additionally, the genes encoding
the vitamin D receptor (VDR) and the hemochromotosis-MHC
Class I protein (HFE), are both polymorphic and
have been recently implicated in lead poisoning
susceptibility. Thus, gene-environment and gene-gene
interactions may produce enhanced effects and deserve
further exploration Gene-Environment Interactions
The ALAD enzyme is inhibited by alcohol and smoking
(67). Three of the studies examined in this review
measured smoking (24, 61, 66), and one of them
controlled for smoking in regression models of
outcome (61). Three studies measured alcohol use
(24, 61, 66), and one controlled for alcohol use
in a model (24). No studies explicitly examined
ALAD-alcohol or ALAD-smoking interactions.
Calcium status has been shown to influence the
intake and effects of lead. Lead binds to calcium
binding proteins and may also compete directly
for absorption in the intestine. Mahaffey et al.
(68) showed that blood lead levels are lower in
children with higher calcium intakes. In addition,
several studies in experimental animals have clearly
demonstrated that prior intake of calcium reduces
the absorption of lead and that absorption of
lead is higher in calcium-deficient animals compared
to normal animals. Studies also show that persons
subjected to fasting absorb more lead than when
not fasting (69); dietary intake in general is
therefore an important factor as well. No studies
have explored interactions between ALAD and any
of these factors.
Gene-Gene Interactions
The effects of calcium on lead intake and absorption
are mediated through calcium binding proteins
that are, in turn, mediated through the bloodborne
form of vitamin D, calcitriol. Calcitriol binds
to the Vitamin D receptor, and thus genetic variations
in the vitamin D receptor are also important in
this pathway. A common polymorphism in the VDR
gene, a restriction fragment length polymorphism
detected by digestion with BsmI that results in
the B and b alleles, has already been shown to
affect bone mineral density (BMD). The BB genotype,
which signals no BsmI restriction site, exists
in about 7-32 percent of the population (70) and
has been shown by meta-analysis to be associated
with lower BMD (70). Two recent studies by Schwartz
et al. (65, 71) explored the role of this polymorphism
in tibial bone lead levels in lead workers. Schwartz
et al. (71) first reported small differences in
bone lead levels by VDR genotype among a group
of former lead workers (13.9 ± 7.9, 14.3
± 9.5, and 15.5 ± 11.1 mg Pb/g bone
mineral in the bb, Bb, and BB genotypes, respectively;
adjusted p value for linear trend = 0.16). The
relationship between years since last exposure
and tibial bone lead concentration was also modified
by VDR genotype. In their second report (65),
Schwartz and colleagues noted larger differences
in BLL by VDR genotype than by ALAD genotype.
On average, the VDR B allele gave a 4.2 mg/dl
increase in BLL, while the ALAD-2 allele yielded
an increase of 3.6 mg/dl in BLL. They also explored
the role of a possible gene-gene interaction between
VDR and ALAD and found no evidence of an interaction.
Interestingly, they did find an association between
ALAD and VDR genotypes which varied by exposure
status. Lead workers with ALAD 1-1 genotype were
less likely to have the VDR bb genotype (OR =
0.29, 95 percent CI: 0.06 – 0.91) while
unexposed controls with the ALAD 1-1 genotype
were more likely to have the VDR bb genotype (OR=2.5,
95 percent CI: 0.23-14.84). This may be indicative
of genotype selection in the occupational environment.
Like calcium status, iron deficiency also increases
the absorption and toxic effects of lead (72).
Ferritin, the iron transport protein, binds lead
and is increased in iron-deficient anemia. Counter-intuitively,
persons homozygous for the HFE mutation that induces
hemochromotosis, a disease of iron overload, have
been shown to have higher blood lead levels than
wild-type HFE persons (5.6 ± 0.6 vs. 3.6
± 0.5 mg/dl, respectively, [73]), and heterozygotes
had intermediate levels (4.1 ± 0.5 mg/dl),
suggesting that carriers of the mutant gene absorb
more lead. This finding was not, however, replicated
in a recent study by Åkesson et al. (74).
To date, no studies have evaluated an HFE-ALAD
interaction.
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LABORATORY
TESTS |
Early studies used the phenotyping
technique developed by Battistuzzi et al. (4)
to classify individuals as ALAD 1-1, 1-2, or 2-2.
In this procedure, whole blood samples are taken
and the red blood cells are isolated and lysed.
Isolation and electrophoresis of the ALAD protein
enables distinction of the phenotypes due the
charge differences of the isozymes. Wetmur et
al. (5) developed the PCR-based genotyping technique
that has been used by most investigators. A 916
base-pair sequence containing the ALAD-1/2 polymorphic
site is amplified and then cleaved with MspI.
The cleavage products are then analyzed on an
agarose gel. Studies using this technique should
include positive (e.g., a gene encoding an essential
enzyme) and negative controls.
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POPULATION TESTING
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There is inadequate evidence to support population-based
testing at this time. |
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SUMMARY |
The evidence surrounding the ALAD G177C polymorphism
and lead poisoning can be summarized as follows: at
high levels of exposure and in comparison to ALAD 1-1
genotype individuals, ALAD 1-2/2-2 genotype individuals
have increased blood lead levels, lower concentrations
of ALAP, lower ZPP levels, lower cortical bone lead
concentrations, higher concentrations of trabecular
(spongy) bone lead, and lower amounts of DMSA chelatable
lead. Thus, ALAD genotype modifies the kinetics of lead
in both blood and bone. Although ALAD-2 subjects may
achieve higher BLLs when exposed to lead, they may experience
less heme synthesis inhibition compared to ALAD-1 homozygotes.
When lead binds and inhibits the ALAD enzyme, ALAD expression
is increased in response (75, 76). Therefore individuals
with ALAD-2 may be better able to compensate than ALAD-1
homozygotes as more lead is bound to ALAD-2 enzyme (65).
This hypothesis might help explain the genotype selection
observed by Schwartz et al. (59) in which ALAD-2 subjects
seemed to tolerate longer exposures to lead in the occupational
setting. The data also suggest that ALAD-1 homozygotes
may be at greater risk of neurotoxicity than ALAD 1-2
individuals, as ALAD-1 homozygotes have higher levels
of ALAP (61, 63). Finally, a study by Bellinger et al.
(66) gave preliminary evidence that ALAD 1-2 individuals
may have better neuropsychological performance than
ALAD-1 homozygotes of similar lead exposure history.
It is difficult to make a decision as to which genotype
is in fact “at risk” because different measures
of outcome indicate that each genotype is more susceptible
to one or more adverse effects compared to the other.
This problem is complicated by the use of different
measures in studies. Indeed, the question of which measures
are most appropriate for estimating body burden and
health risk is one that remains to be answered and merits
discussion. The lack of available data on the effect
of this polymorphism on endpoints such as cognitive
deficits and/or neuropsychological performance, in particular,
is troubling. In addition, most studies used occupationally
exposed individuals with relatively high levels of lead
exposure. Bias in study subject selection is often encountered
in these studies. Very few studies used samples from
the general population, for whom exposure levels are
generally much less than in occupational settings; and
in both these and the occupational studies the percentage
of samples including women is unknown. Thus, it is difficult
to make inferences for the general population. Results
from current research projects investigating these issues
using community samples may help resolve these issues.
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