EXECUTIVE SUMMARY

ORGAN-SPECIFIC RESEARCH

Cardiovascular/Cardiomyopathy
Program Portfolio
Endocrinology
Program Portfolio
Bone Disorders
Program Portfolio
Immunology and AIDS
Program Portfolio
Pancreatitis
Program Portfolio
Liver Injury
Program Portfolio

EMERGING CROSS-CUTTING ISSUES

Metabolism
Program Portfolio
Epithelial Cells
Cell-Cell Communication and Intracellular Signaling
Mechanisms of Disease and Disease Monitoring
Model Systems

REFERENCES

APPENDICES

A: Subcommittee for Review of Biomedical Research Portfolio
B: Experts in Biomedical Research
C: NIAAA Program Staff
D: NIAAA Staff and Guests

The National Institute on Alcohol Abuse and Alcoholism’s (NIAAA) Subcommittee for the Review of the Extramural Research Portfolio for Biomedical Research met on May 2-3, 2000. The charge to the Subcommittee was to examine the appropriateness of the breadth, coverage, and balance of the biomedical research portfolio, identifying research areas that are well covered and others which are either under-investigated or which otherwise warrant significantly increased attention. The Subcommittee was asked also to provide specific advice and guidance on the scope and direction of the Institute’s extramural research activities in the biomedical research area.

The Subcommittee for the Review of the Extramural Research Portfolio for Biomedical Research consisted of a chair, NIAAA Advisory Council member, and an advisory group of seven individuals. Five of these individuals have demonstrated expertise in alcohol-related areas, and four individuals have demonstrated expertise in non-alcohol-related areas (see Appendix A).

The review process was initiated by having experts (see Appendix B) in biomedical research prepare written assessments of the state of knowledge, gaps in knowledge, and research opportunities. NIAAA program staff (see Appendix C) presented the current extramural portfolio, categorized into the areas of cardiovascular, endocrinology, bone disorders, immunology, AIDS, pancreatitis, liver, metabolism, and training and career development. All information was shared with experts, selected NIAAA staff, and the chair and advisory group before the meeting.

A summary of FY 99 biomedical research awards is detailed below.

¹ includes SBIR awards and reimbursable funds.

On May 2-3, 2000, experts and NIAAA program staff made abbreviated presentations of their material followed by discussion among all of the participants, including representatives from other NIH Institutes and guests (see Appendix D). After completing this process, the chair and advisory group, with input from the experts, delineated the following list of research priorities.

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(1)   Determine the basis for tissue-specific differences in ethanol metabolism. This can best be accomplished by using transgenics (knockouts of various cytokines, ADH, etc.); examining the role of innate immune systems; examining metabolite patterns and actions; and differentiating between direct and downstream effects.

(2)   New models of ethanol injury need to be developed. Examination of existing transgenics and emphasis on cofactors (priming, sensitization) can facilitate this process.

(3) Biomedical effects of moderate ethanol consumption, including pattern of consumption, should be emphasized and requires the development of new models.

(4) Resources for recording and organizing results of microarray analyses should be made available.

(5) Additional epidemiological studies are required to determine the biomedical consequences of alcohol consumption, especially in the areas of alcohol metabolism and genetics. Utilization of trans-NIH initiatives will be helpful.

Additional gaps in knowledge and research opportunities were determined by experts in each of the areas covered and are listed in the text of the report.

ALCOHOL ACTIONS ON THE CARDIOVASCULAR SYSTEM
AND CARDIOMYOPATHY

State of Knowledge (Andrew P. Thomas, Ph.D.)

Alcoholism is now recognized as the most frequent identifiable cause of heart muscle disease, and it has been estimated that alcohol abuse may underlie as much as 50% of all cases of congestive cardiomyopathy (Regan, 1990). Moreover, subclinical depression of heart function is apparent in a large proportion of alcoholics (Rubin and Urbano-Marquez, 1994). Alcoholic cardiomyopathy reflects a low-output congestive heart failure, the clinical symptoms and pathology of which are indistinguishable from other forms of dilated cardiomyopathy (Regan, 1990). The disease progresses from a preclinical (or asymptomatic) stage, in which the contractile function of the heart is compromised, initially manifest as a decreased diastolic function. These initial deficiencies are compensated by a variety of mechanisms, including ventricular dilation and cardiac hypertrophy. Eventually the compensatory mechanisms breakdown and the disease progressed through cardiac decompensation to cardiac failure. Alcoholic cardiomyopathy usually takes ten years or more of excessive drinking to develop. Women are as sensitive as men to the cardiac effects of alcohol (Fernandez-Sola et al., 1997). Subclinical depression of function is also widespread in alcoholics (Regan, 1990). The depression of cardiac contractile function and the increase in heart mass is correlated with lifetime alcohol consumption, indicating that the toxic effects develop in a dose-dependent fashion (Fernandez-Sola et al., 1997). Reversal of alcoholic cardiomyopathy can occur with abstention from alcohol, even after the development of end-stage congestive heart failure (Guillo et al., 1997).

During acute exposure to alcohol in vivo, there are two opposing effects. Indirect stimulation of the heart can result from alcohol-induced elevation of catecholamines, whereas the direct effect of alcohol on the heart muscle is to depress contractility (Thomas et al., 1994). It has been shown that alcohol interferes with the cardiac action potential and depresses the calcium transients that underlie excitation-contraction coupling (Kojima et al., 1993). Since a direct depression of contractile function is an early hallmark of alcoholic heart disease, and compensatory mechanisms probably play a role in the further development of the disease, the acute effects of alcohol to interfere with cardiac excitation-contraction coupling may contribute to the initiation of the processes leading to cardiomyopathy. Elevated catecholamine levels may play a role in the hypertrophic response to alcohol (Adams and Hirst, 1990). Another potential site of alcohol action is in the generation of energy, with disruption of mitochondrial function (Cunningham and Spach, 1994).

Alcohol abuse is associated with increased risk for arrhythmia, and both acute alcohol intoxication and chronic alcohol consumption can lead to arrhythmias (Regan, 1990). Arrhythmias could result from effects of alcohol either at the level of the triggering mechanisms (altered action potential firing) or by creating the substrate through structural alterations in the cardiac tissue. The most common rhythm disturbances observed in connection with alcohol abuse are atrial fibrillation and ventricular dysrhythmia. In most cases, atrial fibrillation brought on by acute alcohol consumption reverses within 24 hours of cessation of alcohol intake (Zakhari, 1991). Several studies have reported prolongation of the QT interval as a result of alcohol, even in alcoholic patients without cardiac dysfunction (Rossinen et al., 1999), and this is a known risk factor for malignant ventricular arrhythmias. Such arrhythmias are one of the major factors precipitating sudden death in alcoholics (Regan, 1990). Electrocardiographic abnormalities in alcoholics are associated with an adverse prognosis, especially for sudden cardiac death (Day et al., 1993).

Potential mechanisms for alcohol-induced arrhythmias can be divided into disturbances in the initiation of the electrical depolarization, interference in the propagation of the electrical impulses, and alterations in the normal path and sequence of electrical excitation (Rosenqvist, 1998). Alcohol can also interfere with the regulation of heart rate and impulse conduction by neurohormones (Zakhari, 1991).

Effects of Alcohol on Coronary Artery Disease, Myocardial Infarction, and Atherosclerosis

A large number of epidemiology studies have suggested that moderate alcohol consumption is associated with reduced risk of coronary artery disease (CAD) (Goldberg et al., 1999). Nonfatal acute myocardial infarction has been reported to be more prevalent in abstainers than in drinkers consuming a broad range of alcohol, but when CAD mortality is considered, the protective effect of alcohol is restricted to light to moderate drinkers (Klatsky, 1994). This gives rise to a U- or J-shaped relationship between alcohol consumption and CAD mortality with a value of 1-2 drinks per day apparently affording optimal protection against CAD. It is clear from a wide variety of epidemiological studies that sustained consumption of high levels of alcohol (>3 drinks per day) is associated with increased risk of sudden death from other cardiovascular causes (Anderson et al., 1993).

It is likely that several mechanisms contribute to the apparent reduction in CAD risk in those consuming light to moderate alcohol amounts. Alcohol-induced increase in HDL can explain about half of the protective effect of alcohol against CAD (Langer et al., 1992). It has been noted that alcohol use is associated with elevated levels of the apolipoproteins associated with the formation of HDL, which are also closely correlated with reduced risk of CAD (Okamoto et al., 1988). Alcohol may also reduce CAD risk through an antithrombotic effect (Rubin, 1999). Important targets for alcohol inhibition in platelets include the thrombin-induced formation of the second messenger inositol trisphosphate and the release of arachidonic acid. Low levels of alcohol can enhance fibrinolysis, apparently by upregulating the expression of endothelial cell tissue plasminogen activator (t-PA) urokinase activity (Booyse et al., 1999).

In addition to the apparent protective effect of moderate alcohol consumption against CAD, moderate alcohol use may improve the recovery after acute myocardial infarction.

Chronic, excessive alcohol consumption is associated with hypertension, and this appears to be independent of other known risk factors for hypertension (Hillbom, 1998). In contrast to the effects of excessive consumption, a number of epidemiological studies have reported that moderate alcohol consumption is associated with a lower blood pressure than observed in abstainers (Hillbom, 1998).

Alcohol modifies the secretion of many hormones and neurotransmitters that regulate cardiac function and vasculature, which can result in changes in heart rate, force of contraction, vascular resistance, and the distribution of blood flow (Hillbom, 1998). Ethanol exposure increases sympathetic activity that can contribute to hypertension (Russ et al., 1991). It has also been reported that alcohol decreases the sensitivity of the baroreceptors, which provide feedback control over blood pressure by lowering heart rate and vascular resistance (el-Mas and Abdel-Rahman, 1993).

Alcohol can have direct effects on the contractile properties of vascular smooth muscle to affect blood pressure (Zakhari, 1991). Acute alcohol administration reduces intracellular calcium levels and can cause relaxation of aortic tissue (Zhang et al., 1992), whereas chronic alcohol exposure is associated with increased calcium fluxes into aortic smooth muscle (Vasdev et al., 1991). Of interest is the possible relationship between alterations in calcium fluxes and the hypomagnesmia observed in alcoholics (Altura et al., 1996).

Alcohol has been reported to have both positive (moderate drinking) and negative (heavier drinking) effects with respect to the risk of stroke. There is a clear association between heavy alcohol consumption and an increased incidence of stroke (Hillbom, 1999). It has been suggested that light alcohol consumption may have a protective effect against ischemic stroke (Gorelick and Kelly, 1992). A U- or J-shaped relationship was found between alcohol consumption and relative risk of stroke, with decreases in stroke incidence of up to 50% at alcohol consumption levels of 1-2 drinks per day. Nevertheless, higher levels of consumption are universally found to increase the risk of stroke (Anderson et al., 1993). The risk of stroke increased by 250-450% at consumption levels in the order of 5 or more drinks per day (Rodgers et al., 1993).

Several mechanisms are likely to contribute to the increased incidence of stroke in heavy drinkers. Other cardiovascular effects of alcohol described above are known risk factors for stroke. In particular, there is a well-characterized relationship between hypertension and enhanced risk of stroke. In addition, the effects of alcohol to interfere with blood clotting may contribute to the increased likelihood of hemorrhage. A further contributing factor may be alcohol-induced cerebrovasospasm, which is a local contraction of the muscle wall of blood vessels in the brain that can severely restrict or even block blood flow (Barbour et al., 1993). Altura and coworkers have suggested that the vasospasm induced by alcohol may result from a loss of cellular magnesium (Altura et al., 1993).

Specific recommendations:

(1) Determine the molecular changes that are associated with the development of alcoholic cardiovascular disease, using "gene-chip" array technology to screen for unknowns, targeted molecular approaches, and functional studies of specific components (particularly signal transduction, ion channels, and effectors of apoptosis).

(2) Determine the molecular and cellular mechanisms that underlie the protective effects of moderate alcohol consumption (including CAD, atherosclerosis, and recovery from acute myocardial infarction), focusing on expression and function of signal transduction pathways.

NIAAA PORTFOLIO ON CARDIOVASCULAR RESEARCH
(Thomas F. Kresina, Ph.D.)

Currently, the NIAAA Cardiovascular Research Portfolio supports 36 grants for a total of $5.5 million. Of these, 30 are basic research grants, five are career development awards and one award is a fellowship. (Table 1)

The NIAAA Cardiovascular Research Portfolio supports a wide range of research that includes both the apparent beneficial effects of moderate alcohol consumption and the toxic effects of chronic alcohol consumption on the cardiovascular system. As presented in Table 2, the scope of the portfolio comprises research addressing:

• The mechanisms of possible beneficial effects of moderate alcohol consumption on coronary artery disease and atherosclerosis
  
• The effects of moderate alcohol consumption on ischemia-reperfusion and myocardial infarction
  
• The effects of moderate alcohol consumption on stroke and thrombolysis

• The toxic effects of chronic alcohol consumption on the cardiovascular system

The NIAAA Cardiovascular Research Portfolio supports a balanced portfolio of research that includes both the possible beneficial effects of moderate alcohol consumption and the toxic effects of chronic alcohol consumption on the cardiovascular system. With regard to the mechanisms of possible beneficial effects of alcohol consumption, studies on coronary heart disease and atherosclerosis are investigating intracellular signaling pathways, lipid metabolism and adherence proteins. In ischemia-reperfusion /myocardial infarct studies, contractile function, Ca channels, cytoskeleton and cell surface receptors are targeted. In other studies, investigators are focused on fibrinolysis. Thus, the NIAAA has an active portfolio in all the areas of proposed cardioprotection: HDL cholesterol; platelet aggregation; reduced thromboxane synthesis; vasodilatation; LDL oxidation and free radical scavenging. In this area, recent studies have shown specific changes induced by alcohol in HDL constituents as well as in enzymes and transfer proteins that regulate HDL levels. Individuals who consume alcohol (1gm/kg/day) exhibit a significant increase in HDL-cholesterol levels.

In a study of chronic alcohol consumption and toxicity to the cardiovascular system, stress, hypertension, Ca signaling and cardiac function are being investigated. Recent studies have also shown that alterations in the concentration and composition of plasma lipids and lipoproteins arise with alcohol toxicity. With regard to ethanol-induced hypertension, tricuspid valve function has been shown to be impaired as well as mitral valve insufficiency due to alcohol consumption.

-  Further studies on mechanisms of possible beneficial effects of moderate alcohol consumption are needed. Once biological mechanism(s) are elucidated, then novel therapeutic approaches can be developed that provide cardioprotection.

-  Further studies are needed to elucidate the interaction of moderate drinking and cardiovascular medications

-  Understanding the mechanisms of alcohol-induced cardiomyopathy, hypertension and arrhythmia will lead to possible novel therapeutic interventions

Table 1. Grant Distribution by Funding Mechanism

Table 2. Number and Grant Support Level by Cardiovascular Category

Table 3. Grant Mechanism Distribution by Cardiovascular Category

State of Knowledge (Mary Ann Emanuele, M.D.)

In both males and females, the regulation of reproduction involves the hypothalamic-pituitary-gonadal (HPG) axis.

Acute or chronic ethanol ingestion suppresses HPG function resulting in, among other things, low serum testosterone, i.e., hypogonadism. While alcohol affects hypothalamic luteinizing hormone releasing hormone (LHRH) and pituitary luteinizing hormone (LH) in the adult (Cicero, 1982) and peripubertal male rat (Little et al., 1992), direct inhibition of testosterone steroidogenesis has been implicated as well (Johnston et al., 1981; Orpana et al., 1990). It is likely that ethanol is acting at more than one site of the testosterone synthetic pathway.

In testes, there are several potential, alcohol-influenced mechanisms for damage involving opioids (Emanuele et al., 1999), nitric oxide (Shi et al., 1998), the adrenergic system (Rivier, 1999), elevated pituitary prolactin and brain proinflammatory cytokines (Ogilvie et al., 1999), and perturbations in other hormonal systems that interact with the HPG axis.

The impact of ethanol exposure on hypothalamic LHRH in the male has been inconsistent and differs with the paradigm used. While secretion of LHRH has been reported to both be unaffected (Uddin et al., 1996) and reduced after ethanol (Ching et al., 1988; Hiney and Dees, 1991; Ogilvie and Rivier, 1997), the ability of the hypothalamus to synthesize LHRH appears to be unaltered by ethanol at any dose (Uddin et al., 1996).

Although there is a decrease in testosterone with ethanol exposure, the expected rise in serum LH does not occur, implying a central neuroendocrine effect (Emanuele et al., 1991). Studies in ethanol-fed rats have established that a decrease in LH blood levels results from impairment of both LH production and LH secretion. While there is less available data on FSH, the secretion of FSH does appear to be reduced by ethanol while FSH synthesis is unaffected (Emanuele et al., 1992).

Ethanol may increase opioids both directly and indirectly. While endogenous opioid peptides may mediate some of ethanol’s testosterone suppressive effects (Grattagliano et al., 1997), ethanol can also cause testicular oxidative injury and increase testicular apoptosis (Adams and Cicero, 1991).

Chronic ethanol exposure in the peripubertal age group decreases fecundity, which may be mediated by testicular oxidative injury leading to accelerated germ cell apoptosis in ethanol-exposed fathers (Little et al., 1992).

The major effect of chronic ethanol exposure in adult female rats is disruption of estrous regularity manifested mainly by a prolongation of diestrous (Eskay et al., 1981; Rettori et al., 1987). When proestrous occurs, it appears to be hormonally normal. Transient estradiol elevation (Lox et al., 1982), increase endogenous opioid peptides tone (Froehlich, 1993), and IGF-1 decline (Steiner et al., 1997) provide mechanistic bases for ethanol’s deleterious effects on female reproduction. In females, as in males, the onset of puberty is markedly disrupted by ethanol exposure, and one possible mechanism might be endogenous opioid peptides (Creighton-Taylor and Rudeen, 1991). Ethanol’s disruption of puberty may be in part owing to interference with the synthesis and secretion of IGF-I (Srivastava et al., 1995).

Acute ethanol exposure activates the HPA axis by inducing release of corticotropin-releasing factor (CRF) from the hypothalamus (Rivier, 1996). The HPA response to ethanol is dose-dependent, with activation at blood ethanol levels greater than 100 mg%, while at 75 mg% blunted ACTH and cortisol responses to exogenously administered CRF are noted, suggesting an attenuated ability of the HPA axis to respond to physiological stress (Wand, 1993). This impairment may result from ethanol-induced inhibition of arginine vasopressin, a secretogogue that potentiates the action of CRF on ACTH release (Wand and Schumann, 1998).

Chronic ethanol exposure is associated with increased as well as decreased HPA axis activity. The direct effects of chronic ethanol exposure on the HPA axis are difficult to assess due to concomitant problems, including malnutrition, depression, liver disease, and other stress factors. Individuals who are actively drinking and are non-depressed have been reported to have a two-fold increase in urinary cortisol levels and blunted ACTH and cortisol responses to CRF. These data support the contention that alcoholics have an abnormal HPA axis. In rodents, chronic ethanol exposure is not accompanied by elevations in ACTH and corticosterone, yet long-term influences in the HPA axis occur, including stress-related attenuation. Possible mechanisms include down-regulation of pituitary CRF receptors, increased corticosterone feedback, loss of responsiveness of nerve terminals, and hyperactivity of inhibitory neurons (Turnbull et al., 1999).

There is an increase in HPA axis activation during ethanol withdrawal; cortisol levels are increased as a result of elevation in cortisol burst amplitude and cortisol mass secreted per burst. Excess CRF, cortisol, and other neuroactive steroids (Devaud et al., 1996) enhance the magnitude of withdrawal symptoms, including seizure activity. Also disrupted is the normal circadian pattern of cortisol release and non-suppressability of the HPA axis to low doses of dexamethasone in chronic alcoholics during withdrawal. This hypercortisolism subsides over time with cortisol levels normalizing after 7-10 days of abstinence (Wand, 1993).

In early abstinence, hypo-responsiveness of the HPA axis is observed, with an attenuated hormonal responsiveness at each level of the axis. The attenuation of the HPA axis is still apparent after six months of abstinence.

A low-level cortisol response to ethanol has been shown to characterize individuals at high risk for alcohol abuse and dependence (Gianoulakis et al., 1996; Schuckit et al., 1988).

Chronic ethanol consumption is associated with increased prolactin release in men (Majumdar, 1979). Acute effects are more variable in women. Persistent hyperprolactinemia and associated menstrual cycle disturbances have been reported in heavily drinking women (Volpi et al., 1994). Inconsistent results on prolactin secretion have been observed in animals given ethanol. There is a marked inhibition of suckling-induced lactation in ethanol exposed post-partum female rats, associated with diminished pup growth (Subramanian et al., 1991)

Ethanol exposure is associated with suppression of the GH-IGF axis by lowering peripheral levels and altering the availability of these hormones (Srivastava et al., 1995), with younger animals being the most vulnerable (Steiner et al., 1997).

Acute and chronic ethanol abuse appears not to produce clinically relevant thyroid dysfunction (Garbutt et al., 1995). However, chronic alcoholics often display the nonspecific chemical features of the euthyroid sick syndrome, and resulting thyroid functions tests are often misleading, particularly in malnourished alcoholics with liver disease (Emanuele, 1997).

Although not a traditional neuroendocrine hormone, leptin has recently emerged as important to normal endocrine functioning, particularly in the reproductive arena. Ethanol may have alternating effects, acutely lowering leptin (Hiney et al., 1999) and chronically raising it (Lin et al., 1998). There may be a slightly different response in females since many studies have shown higher serum leptin levels in females than in males, a difference greater than can be accounted for by fat distribution alone.

Single bouts of ethanol exposure do not worsen and may improve glucose tolerance in diabetics. Some studies have indicated that isolated episodes of drinking with a meal may have a beneficial effect by slightly lowering blood glucose excursions (Swade, 1997). This potentially beneficial effect was observed in both men and women regardless of age. Studies of acute ethanol consumption in non-diabetic individuals have yielded variable results, with increases, decreases, or no change in glucose levels. However, daily drinking in moderate amounts (i.e., 0.5 to 1.0 mg/kg) clearly worsens diabetic control and increases the prevalence of impotence, retinopathy, and possibly peripheral neuropathy. The mechanisms underlying the hyperglycemia in chronically imbibing diabetics are still not fully known.

Specific recommendations:

(1) Study the role of oxidative injury and apoptosis as a common pathway in endocrine alteration.

(2) Examine differential sensitivity to humoral perturbations with age (adolescent to adulthood).

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NIAAA PORTFOLIO ON ENDOCRINOLOGY
(Thomas F. Kresina, Ph.D.)

Currently, the NIAAA Endocrinology Research Portfolio supports 31 grants for a total of $5.0 million. Of these 23 are basic research grants, two are career development awards and six awards are fellowships. (Table 1)

The NIAAA Endocrine Research Portfolio supports a wide range of research that includes the deleterious effects of alcohol consumption upon the endocrine system as well as energy regulation, metabolism, diet and oxidative stress. As presented in Table 2, the scope of the portfolio comprises:

• Research addressing the effects of alcohol on the hypothalamic-pituitary-adrenal (HPA) axis
  

• Research addressing the effects of alcohol upon the opioid system
  

• Research addressing the role of oxytocin on alcohol consumption
  

• Research addressing the effects of alcohol on the male and/or female reproductive system, the hypothalamic-pituitary-gonadal axis
  

• Research addressing endocrine-mediated immune suppression by alcohol
  

• Research on the effects of alcohol upon the endocrine mediators of osteogenesis and osteoporosis
  

• Research on the effects of alcohol and glucose transport
  

• Research on the stress axis comprising the HPA and sympatho-adrenal (SA) axes and alcohol
  

• Research on neuropeptides, energy regulation, lipid metabolism and alcohol

The NIAAA Endocrine Research Portfolio is predominantly comprised of R01 grants (Table 1) that support a broad range of research related to the integrated endocrine systems, including the HPA axis, the mesocorticolimbic system and the renin-angiotensin system (RAS). Alcohol's deleterious effects upon the endocrine system are pervasive. A variety of endocrine functions are affected, and the ensuing endocrinological imbalances range from metabolic and behavioral defects, to reproductive or immune dysfunction, to osteoporosis or development of cancer. Thus considerable effort is underway to identify the mechanisms by which alcohol disrupts particular components of the endocrine system, and to understand how the interrelationships between these pathways and their target organ systems are perturbed. The NIAAA has focused recent efforts on the neuroendocrine peptide regulation of alcohol consumption. The NIAAA sponsored a workshop on April 28, 1999 entitled "Neuropeptides and Alcohol Intake" which interfaced alcohol research, the opioids, the classical neurotransmitters and the newly described network of peptides and hormones that regulate food intake and energy metabolism. Although many actions of alcohol on neurotransmitters have been reported, the potential role of various peptides is a current area of research. Neuropeptide Y (NPY), a hormone that can function as a neurotransmitter in the brain, is known to stimulate appetitive behaviors. Recently, NIAAA-funded scientists reported that mice rendered NPY-deficient by elimination of the NPY gene consumed more alcohol than were NPY-intact mice. NPY-deficient mice also were less sensitive to the sedative effects of alcohol than controls. Both of these phenomena have been associated with increased risk for alcoholism in humans. Conversely, mice genetically altered to produce abnormally high levels of NPY showed a lower preference for alcohol and were more sensitive to alcohol’s sedative effects. These findings suggest that NPY is part of the neural circuitry involved in responses to alcohol.

Based on such data, a RFA was released in FY99 entitled "Peptide Regulation of Alcohol Intake" for support of grants in the current fiscal year. The Areas of Research of this RFA included the HPA axis, the opioid system, the RAS, food intake and energy homeostasis peptides and alcohol intake.

Expanding the focus nucleated by the alcohol/peptide RFA would be an appropriate future direction for the endocrine portfolio. A genetic approach to the study of peptides, the endocrine system and their action on alcohol intake would promote an ongoing institute initiative on the use of transgenic animals and knockout mice to further study alcohol addiction. Further initiatives can focus on endocrine dysfunction and alcohol consumption in the induction of disease pathogenesis such as obesity, diabetes or oncogenesis. This area of research is currently not addressed in the endocrine portfolio. Furthermore, the study of endocrine dysregulation due to alcohol consumption could be augmented by collaborative initiatives with the immunology/host defense portfolios where investigations involving interactions between endocrine and immune systems in the context of alcohol consumption can be pursued. For example, an initiative requesting research proposals on disease susceptibility to infectious pathogens could focus on alcohol/HPA/immune system interactions

Table 1. Grant Distribution by Funding Mechanism

Table 2. Number and Grant Support Level by Endocrine Category

Table 3. Grant Mechanism Distribution by Endocrine Category

State of Knowledge (Russell T. Turner, Ph.D.)

Alcohol consumption is generally considered a risk factor for osteoporosis based on the frequent finding of a low bone mass, decreased bone formation rate, and increased fracture incidence in alcoholics. Alcohol has also been shown to reduce bone formation in healthy humans and animals, and to decrease proliferation of cultured osteoblastic cells. On the other hand, it has been difficult to demonstrate alcohol-induced bone loss and increased fracture rate in population-based studies. Indeed, most studies have shown a positive association between alcohol and bone mass and no change or a decrease in fracture risk. Overall, the evidence generally supports a detrimental effect of chronic alcohol abuse on the skeleton of a sub-population of men and a neutral or generally beneficial effect of moderate alcohol consumption, especially in women. This latter putative beneficial effect may be due to a reduction in the age-related increase in bone remodeling associated with postmenopausal bone loss.

Specific recommendations:

(1)  Determine molecular mechanisms of action.

(2)  Develop interventions to prevent and/or reverse bone loss.

NIAAA PORTFOLIO ON OSTEOPOROSIS
(Vishnudutt Purohit, D.V.M., Ph.D.)

Osteoporosis afflicts about 20-25 million Americans and is linked to 1.5 million fractures every year. Women are more susceptible than men to this condition. National health care cost for osteoporotic women is estimated to be 5 billion dollars annually. Chronic heavy alcohol consumption is a risk factor for osteoporosis since it decreases bone mineral density, increases bone loss, and increases rate of bone fractures. On the other hand, moderate alcohol consumption appears to have beneficial effects as shown by increased bone mineral density in postmenopausal women. NIAAA-funded researchers are investigating the underlying mechanisms of the effects of moderate and heavy drinking on bones.

The National Institute on Alcohol Abuse and Alcoholism currently supports nine projects that investigate relationships between alcohol and osteoporosis. The portfolio consists of 7 R01s, 1 R03, and 1 R21 that can be divided into five broad categories: moderate alcohol–related, adolescent-related, pregnancy-related, cellular and molecular mechanisms, and stress-related studies (Table 1 and 2). In FY 1999, the total amount of funding for these projects was $1.3 million.

Moderate Alcohol Consumption-Related Projects: Two research projects focus on the relationship between moderate alcohol consumption and bone disorders. One project is investigating the effects of moderate alcohol intake on bone metabolism in women by measuring the levels of parathyroid hormone, collagen breakdown products, osteocalcin, vitamin D, and estrogen. The hypothesis being tested is that moderate alcohol consumption increases bone mineral density. The second project is evaluating relative risk/benefits of moderate alcohol consumption on osteoporosis. The hypothesis under consideration is that mineral content, density and biomechanical strength of bone will be greater, and biological markers and mediators of bone metabolism will be improved in monkeys consuming moderate amount of alcohol.

Animal Model of Osteoporosis: A rat model of osteoporosis is being developed to determine life long consequences of ethanol consumption on bone growth in the young, bone maintenance in middle age, and bone loss rate in aging animals. In addition, consequences of long term ethanol consumption on the severity and the rate of formation of ovariectomy-induced osteopenia are being investigated.

Adolescent-Related Project: Chronic ethanol consumption is known to suppress hypothalamic-pituitary-testicular axis that leads to decreased levels of plasma testosterone, an androgen required to maintain bone mineral density in males. In this project investigators hypothesize that suppression of reproductive axis by chronic ethanol in peripubertal period results in long term bone disorders including osteopenia.

Pregnancy-Related Project: This study investigates effects of alcohol on calcium metabolism in pregnant rats and their fetuses. Alcohol-induced perturbations in calcium metabolism during pregnancy may have significant implications for the development of osteoporosis.

Cellular and Molecular Mechanisms: Three active projects focus on the cellular and molecular mechanisms by which ethanol impairs bone cell proliferation and bone metabolism. Of these, one project is testing a hypothesis that ethanol inhibits osteoblast proliferation by interfering with a specific site in the tyrosine kinase phosphorylation pathway that leads to activation of Ras gene. The second project focuses on cellular and molecular mechanisms of alcohol’s effects on bone and mineral metabolism. A rat model is being developed to evaluate dose response for the long-term effects of alcohol on bone mass, bone cell number, and bone’s mechanical properties. In addition, alcohol’s effects on bone remodeling, recruitment of osteoblasts and osteoclasts, and expression of genes related to osteoblast-derived cell signaling peptides are being studied. Third project examines the hypothesis that alcohol alters bone metabolism indirectly by elevating the secretion of cytokines – IL-1 and TNF, which have been shown to be involved in postmenopausal osteoporosis. Both cytokines are known to mediate bone loss due to estrogen deficiency by increasing bone resorption and decreasing bone formation.

Stress-Related Project: This project examines the combined effects of chronic ethanol consumption and stress on bone quality, serum osteocalcin (a bone protein), and biosynthesis of enzymes of the catecholamine pathway. The results of this project may help to understand the mechanisms leading to osteoporosis in humans who are heavy alcohol consumers and subjected to environmental stressors.

1. Osteopenia caused by chronic alcohol administration in adolescent female rats is not completely reversible.

2. Chronic alcohol administration in adolescent, adult, and elderly female rats reduces bone density, and peak bone mass in both cortical and cancellous bones without affecting calcium regulating hormones. In adult female rats, chronic alcohol also decreased the amount of bone surface containing osteoblasts (bone forming cells) and wall thickness of tibia, a measure of osteoblast activity. These results confirm a direct deleterious effect of alcohol on bone forming cells.

3. Chronic alcohol is injurious to the adolescent male rat skeletal system and recovery is not complete after abstinence.

4. Impairment of osteoblastic phospholipase D signal transduction plays a critical role in mediating antiprolifertaive effect of ethanol on osteoblast.

5. As low as 3% of caloric intake of ethanol for four months impaired bone metabolism in adult female rats, and this effect was related to a decrease in the number of active osteoblasts.

Future Directions for Research

1. Examine the effects of moderate alcohol consumption on bone parameters in postmenopausal women with a consideration of a) patterns of drinking; b) types of alcoholic beverages; c) dietary supplements; and d) hormonal therapy. Are the reported beneficial effects mediated via hormones or growth factors?

2. Study the interactive effects of chronic ethanol intake and the hormones of hypothalamic-pituitary-gonadal axis on the skeletal system of human adolescents. In addition, investigate the mechanisms by which alcohol impairs bone formation in adolescent rats.

3. Investigate the interactive effects of growth hormone-IGF-1 axis and alcohol on bone remodeling process including effects on osteoblast proliferation and functions.

4. Identify the bone growth factors and bone cytokines that are directly affected by alcohol.

5. Identify signaling processes of osteoblast proliferation that are directly affected by alcohol.

6. Investigate the effect of alcohol on the functions of osteoclasts that are involved in bone resorption.

Table 1. Alcohol and Osteoporosis: Grant Distribution by Grant Mechanism

Table 2. Alcohol and Osteoporosis: Grant Distribution by Areas of Research

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CONSEQUENCES OF ALCOHOL CONSUMPTION ON
IMMUNE-ASSOCAITED DISEASES

State of Knowledge (Lynell W. Klassen, M.D.)

While the association of alcohol abuse with increased deaths from infections was made over 75 years ago, only in the past 15 years has serious investigative efforts been made to understand the role of alcoholic exposure on immune dysfunction. The more recent studies from both humans and animals suggest that alcohol produces significant alterations in immunoregulation. These abnormal immunoregulatory effects produce either immunodeficiency or autoimmune features and can lead to clinical disease. Complications associated with chronic alcohol abuse have a variety of other abnormalities, which can contribute to immune dysfunction, including malnutrition, vitamin deficiency, and advanced tissue damage such as liver cirrhosis.

The present knowledge of immune abnormalities that occur following significant alcohol exposure suggests the following clinical consequences.

· Decreased host defenses

- increased susceptibility to and progression of infections

· Decreased immunosurveillance

- increased cancer induction - increased tumor growth and metastasis

· Increased autoimmune reactions

- enhanced organ destruction by cellular mechanisms - presence of autoantibodies to normal
    proteins

· Induction of neoantigen

- organ specific dysfunction/damage

· Altered inflammatory responses

- abnormal cytokine activity - non-specific tissue damage and constitutional effects (fevers, weight
    loss)

Experimental and clinical studies have demonstrated that alcohol has multiple effects along the entire pathway of immune recognition, activation, differentiation, proliferation, and final effector activities.

Aldehyde-protein adducts derived from alcohol metabolism can induce an immune response to both the adducted epitope as well as to normal protein epitopes that have not undergone chemical alteration. Circulating antibodies to these adducts have been detected following chronic alcohol consumption. The detection of such adducts in the livers of alcohol-fed rats suggest that these immune reactive proteins may be important in producing an autoimmune-like reaction that can cause damage. There is a clear potential for cellular cytotoxic mechanisms to be directed against these protein adducts, thereby enhancing liver damage. Similar protein-aldehyde adducts have been detected in other tissues as a product of lipid peroxidation (Hill et al., 1998). The finding of MAA adducts in atherosclerotic coronary arteries raises the possibilities that alcohol-induced bioreactive proteins may accelerate a variety of other disease processes.

Alcohol has been clearly shown to modulate the ability of monocytes to process antigens and express immunogenic peptides to T helper cells (Szabo et al., 1993). In vivo studies suggest that this effect is mediated primarily by alcohol-induced reduction of IL-1b and an increase in TGF-b . Studies of delayed type hypersensitivity (DTH) have demonstrated that the effect of alcohol on APC activity is genetically linked, reversible, and is the main cause of DTH suppression (Waltenbaugh and Peterson, 1997). The clinical significance of these observations is that both quantitative and qualitative changes in an immune response can result in sub-optimal destruction of infectious agents with resultant clinical disease. Thus, any decrease in antigen processing efficiency can lead to increased bacterial or viral burdens and clinical infections.

Recent findings suggest that chronic exposure to excessive alcohol induces TH2 excesses with increased IL-10, decreased IL-12, and decreased INF-g production (Peterson et al., 1998). The consequences of this TH2 functional excess is a polyclonal increase in immunoglobulins following chronic alcohol ingestion, development of selective immunodeficiencies to specific infectious agents, blunted response to primary infections, and altered cytokine milieu causing abnormal inflammatory and fibrinogenic responses. While these findings suggest that chronic alcohol consumption results in increased TH2 function and potential dysregulation, its significance in human disease is still untested.

Acute and chronic alcohol exposure inhibits antigen-specific T cell proliferation (Szabo, 1999), but produces minimal effects on total B cell number (Romagnani, 1991). However, in vivo studies suggest that B cell differentiation is altered by alcohol, particularly by a decrease in IL-4 levels, which can inhibit both B cell proliferation and immunoglobulin class switching (Aldo-Benson et al., 1992). Chronic alcohol administration is associated with reduced natural killer (NK) cell numbers and activity (Cook et al., 1997).

Although many studies have correlated chronic or acute alcohol exposure with various cytokine changes, it is still unclear which isolated change may be biologically important. While in vitro studies suggest that alcohol has minimal effect on the ability of T cells to produce IL-2, in vivo studies suggest that chronic alcohol administration affects T cell utilization of IL-2, decreasing proliferation and ultimately down-regulating cell mediated responses (Jerrells et al., 1990). Human studies have documented a decrease in serum IFN-a , IFN-g , and IL-2 levels following alcohol exposure (Vicente-Gutierez et al., 1991). Acute alcohol administration has been shown to stimulate the production of prostaglandin E2, which also down regulates T cell proliferation and differentiation. Elevation of TNF-a following alcohol exposure has been described (McClain et al., 1993).

One of the most consistent immune abnormalities in alcoholics is a significant increase in serum immunoglobulin levels. It is often difficult to determine whether the elevated immunoglobulins are a consequence of chronic alcohol exposure or the result of subsequent liver disease. However, alcoholics without liver disease typically have elevations in IgA levels, while those with alcohol liver disease primarily have elevations of IgG. While the effect of alcohol in B cell function is probably minimal, a selective decreased antibody response is probably secondary to abnormal T cell function (Romagnani, 1991). Chronic alcohol exposure results in a generalized polyclonal activation of antibody production while at the same time there is often a decrease in specific antibody response following vaccinations. This pattern is also seen in classic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. T cell function is diffusely decreased following high dose alcohol ingestion (Israel et al., 1986). Abnormal delayed type hypersensitivity has been demonstrated by a significant reduction in tuberculin and fungal skin tests, and T cell mitogenic stimulation is usually markedly reduced. Decreased cytotoxic T cell activity against viral infected cells has also been reported.

There is inadequate evidence to determine whether alcohol is a co-factor either in HIV susceptibility or in disease progression.

The consumption of alcohol has been linked with the clinical progression of chronic hepatitis C infection leading to progressive liver damage (Ostopowicz et al., 1998). Individuals who are both alcoholics and positive for hepatitis C infection have additive effects in the development of liver disease. However, it is unclear whether these are two independent processes, or whether both conditions may directly interact to produce progressive tissue damage.

Specific recommendations:

(1) Determine the mechanisms and significance of alcohol-induced TH1/TH2 polarization; including

- which intracellular signaling pathways are affected by alcohol and contribute to the TH1/TH2 change,

- which changes in cell surface receptors accompany alcohol exposure result in TH2 activation,

- what cytokine environmental milieu is inducted by alcohol to produce a TH2 effect,

- what modalities can reverse the TH1/TH2 phenotype (cytokines, cytokine inhibitions, anti-oxidants, etc.).

(2) Determine whether the development of immunoreactive products, e.g., aldehyde adducts, directly cause fibrinogenesis
     and liver damage and

- by which molecular mechanism,

- by which cytokines.

NIAAA PORTFOLIO ON IMMUNOLOGY AND HOST DEFENSE
(Leslie S. Isaki, Ph.D.)

Research Mechanisms

Descriptions of Projects Supported

• Majority (61%) of investigators examines the chronic effects of ethanol on host systems, whereas about 20% of researchers study the acute effects of ethanol. Another 20% of investigators study both chronic and acute effects of ethanol. Of this group, two projects examine the acute (or binge) effects within a chronic model of alcohol consumption in animals. (Table 3)
  

• Table 4 summarizes the grant distribution by organs targeted for study. These studies include both the direct and indirect effects of ethanol in promoting pathological conditions of the targeted organs and tissues.
  

• Clinical studies in human subjects are generally preclinical in nature and consist of isolation of peripheral blood cells and manipulation of these cells ex vivo.
  

• All grants in this portfolio focus on basic molecular mechanisms and include investigations into aspects of alteration of mediator profiles and function.

II. Summary of Research (Table 5)

Table 5 provides a summary of the scientific disciplines of the research projects within the Immunology portfolio. The table is divided first into two general areas: Innate and Adaptive defenses. Innate responses refer to non-immune host resistance which act as first-line defense whereas adaptive responses consist of acquired immunity involving antigen-specific lymphocytes.

Research grants examine the early phases of host defense mechanisms that are altered by ethanol exposure and include:

• Signaling changes in ethanol-induced phosphorylation of proteins that transduce signals for transcription and cell growth factors.
  

• Molecular targets of ethanol, which result in impairment of mechanical barriers of host, defense in the lung (surfactant and ciliary motility).
  

• Protein modification of ethanol-related alterations in the regulation and processing of proinflammatory cytokines and ribosomal proteins in hepatic mitochondria.

• The effects of ethanol on respiratory function following traumatic brain injury in a swine model.

Research projects investigate the ethanol-related impairments to mechanisms of host defense. The grants can be divided into two research classes: studies on cellular and molecular aspects of the function of the immune system and studies on the effects of alcohol exposure on the pathogenesis of infectious microorganisms.

• In both humans and mice, two types of CD4-bearing T helper lymphocytes (Th cells), termed Th1 and Th2, are defined both by the types of cytokine secreted and immune function. Alcohol-consumption is associated with impaired antigen-specific cell-mediated immunity, whereas antibody responses are unimpaired or enhanced in vivo after alcohol exposure. Grants that focus on ethanol-altered cellular immune processes examine the early events in antigen recognition, evaluate accessory cell function and mediator production, and elucidate the mechanism of polarization of the immune response toward Th2-driven humoral immunity and away from Th1-driven cell-mediated immunity.
  

• Identifying the cellular and molecular targets of ethanol are the goals of researchers who study infectious agents and alcohol exposure. Compromise of host immunity, by excessive alcohol consumption, is associated with impaired or altered mediator production and function. These dysregulated cytokine activities can result in altered recruitment of effector cells to sites of infection and diminished pathogen killing.
  

• Research grants on tissue and organ damage as a consequence of excessive alcohol consumption focus on mechanisms of ethanol-induced immunodeficiency and autoimmunity as primary factors that lead to tissue and organ injury. Similar to events in resolving infections, ethanol-induced injury is generally preceded by inflammation that is associated with parenchymal infiltration of leukocytes (neutrophils and lymphocytes) and increased release of reactive oxygen species.
  

• Investigators who examine the effects of alcohol exposure in utero on the developing immune system focus on both B and T cell lymphopoiesis.

Ethanol-induced impairments to host defense has progressed rapidly within the last decade. Although both cell-mediated and humoral (antibody) immune responses are affected by alcohol, the underlying mechanism(s) of alcohol's effects on immune responses are unknown. The emerging mechanisms are based primarily on dysregulation or disruption of cytokine interactions rather than on the direct effects of ethanol. Recent evidence indicates that the primary outcome of excessive alcohol consumption, whether it be infection or tissue injury, may have an etiological basis in exacerbation or prolonged inflammation. It is likely that there will be many targets that underlie the ethanol-induced alteration of host defense responses. Table 6 lists the research projects that target emerging areas of study.

Alterations in both innate and adaptive immune responses by alcohol are manifested in the increased frequency and severity of infections and in alcoholic liver injury seen in alcoholics. To better understand the effects of alcohol on the entire immune response, projects on innate host defenses and interactions of the innate and adaptive immune responses need to be significantly expanded. Innate immune recognition appears to be primarily mediated through Toll proteins which recognize conserved molecular patterns that are associated with microbial pathogens (such as lipopolysaccharide, LPS). Upon encounter with pathogens or after LPS stimulation, these pattern-recognition receptors activate intracellular signaling, most notably via the transcription factor NF-6 B, which results in the induction of a variety of effector genes. The role of Toll-like receptors in alcohol-induced tissue injury and immune system impairments are specific investigations that can be pursued to further understand the relationship between innate responses and initiation and progression of alcohol-related injury.

Trauma

Alcohol consumption is an integral causal factor in most types of traumatic injury. Not only does alcohol increase the frequency and severity of injury, but both the acute and chronic usage of alcohol impair the body’s normal physiological response to injury and significantly complicates medical management of the trauma patient. Basic science trauma research focussing on ethanol-impaired mechanisms that lead to dysregulation of cytokines, increase in immunosuppression, and exacerbation of complications arising from infectious agents, burns, and surgical procedures are important areas for the conduct of research.

Although the neurological and behavioral aspects of withdrawal are well studied, little is known about the immunology and host responsiveness to infectious agents and injury during alcohol withdrawal. Data from the research supported in this portfolio are mainly generated from either active drinking models (or during the period of ethanol exposure with in vitro models) or immediately after ethanol administration has been halted.

Scant data indicate that during the period of alcohol withdrawal, the host may still be vulnerable to the damaging effects of ethanol. Results from an alcohol binge model in animals suggest that elevated levels of oxygen-derived radicals detected during withdrawal may be a significant contributory factor to tissue injury. In a study of cardiovascular risk factors involving male alcoholics, cytokine profiles were altered compared to control subjects, and alcohol withdrawal induced potentially atherogenic changes in lipoprotein (a). Host responses during withdrawal have been observed for a period of several days to one year post-drinking, when differences in levels of proinflammatory cytokines were detected in groups of alcoholics with and without liver disease.

Table 1: Grant Mechanisms

* One active R01 grant in no-cost extension.
** One active R21 grant in no-cost extension.

Table 2. Grant Mechanisms of the Immunology (non-AIDS) Program

Table 3: Distribution by Duration of Alcohol Exposure

Table 4: Grant Distribution by Research on Target Organs

* Two active grants in no-cost extension.

Table 5: Grant Distribution by Alcohol-Related Impairments to Host Defenses

* One grant in no-cost extension.

Table 6: Grant Distribution by Targeted Areas of Research

NIAAA PORTFOLIO ON AIDS
(Thomas F. Kresina, Ph.D.)

1. Overview

Research Funding

• The role of alcohol in enhanced susceptibility to HIV infection through biological mechanisms such as depressed immunity or reduced barrier function
  

• The role of alcohol in the progression of HIV infection via augmented viral replication and/or viral load
  

• The role of alcohol in enhanced progression to AIDS through increased susceptibility to opportunistic infections
  

• The role of alcohol in the progression of morbidity due to opportunistic infections such as hepatitis C, tuberculosis or pneumococcus

• The role of alcohol in the modification of pharmacodynamics of drug treatment for HIV/AIDS

The Neuroscience and Behavior AIDS research portfolio supports a wide range of research that is both AIDS and HIV/AIDS related research where alcohol is a co-factor in the etiology or pathogenesis of neurological manifestations of HIV infection. The scope of the portfolio comprises research addressing:

• Alcohol use and cognitive and neurobehavioral dysfunction in HIV infection and AIDS
  

• Alcohol use and central nervous system morbidity
  

• Alcohol use and brain damage and neurological impairment in HIV infection and AIDS

• AIDS dementia complex and alcohol

The Biomedical and Neuroscience AIDS research portfolios address important questions related to alcohol consumption as a co-factor the etiology and pathogenesis of HIV/AIDS. For example: Is drinking harmful to individuals with HIV infection? Such medical management issues are increasingly important in the era of Highly Active AntiRetrovial Treatment (HAART). As successful treatment regimens progress, HIV infection is being transformed into a chronic asymptomatic viral infection where cofactors, such as alcohol consumption, play a larger role in disease burden. As such, alcohol and HIV biomedical and neurological research investigations are becoming important research issues. It is now important to investigate whether alcohol consumption will induce immune changes in individuals who are responders to HAART and undergoing immune reconstitution. The Biomedical and Neuroscience AIDS research portfolios are predominantly R01 research portfolios that supports research related to the broad issues of immunosuppression, viral enhancement, susceptibility and pathogenesis of opportunistic infections and alcohol.

Recent initiatives at NIAAA have promoted specific research areas of the portfolio. A workshop in March 30-31, 1999, entitled " Workshop on Alcohol and Immunology/AIDS" brought together experts in the fields to formulate research directions in the immunology of alcohol-HIV/AIDS. The NIAAA has cosponsored two RFA 's in the biomedical aspects of alcohol and HIV infection. The NIAAA cosponsored an NIAID RFA on Hepatitis C Centers that was open to HIV/alcohol/hepatitis C research investigations. A current RFA from NIDA entitled "Viral Hepatitis and HIV in Drug and Alcohol Users" provides a R01-based grant mechanism to support research in alcohol/HIV and hepatitis C research. A symposium entitled "Alcohol Use and HIV Pharmacotherapy" is scheduled for April 26-28, 2000 and will bring together pharmacologists, alcohol researchers and HIV experts to define issues in the field. This initiative promotes new collaborations for investigators in the field of alcohol research.

The Biomedical and Neuroscience AIDS research portfolios can be augmented by future initiatives supporting the broad research areas of the portfolio. For FY 2001, NIAAA is participating in an NIAID-sponsored Program Announcement entitled " Collaborations for Advanced Strategies in Complications of HIV Infection". This PA requests research applications on alcohol-induced metabolic complications, such as triglyceridemia, as well as the role of alcohol-induced immune modulation in the pathogenesis of opportunistic infections, such as tuberculosis. In neuroscience, an RFA is planned to augment the R01 research base.

Table 1. The NIAAA Extramural AIDS Allocation by OAR AIDS Area of Emphasis

Table 2. Biomedical & Neuroscience AIDS Grant Distribution by Funding Mechanism

Table 3. Scope of HIV/AIDS Research by Topic and Grant Mechanism

State of Knowledge (Stephen J. Pandol, M.D.)

Alcohol-related pancreatitis in humans accounts for about one half of the cases of acute pancreatitis and a majority of the cases of chronic pancreatitis.

Several animal models of acute pancreatitis have been investigated over the past three decades (Kaiser et al., 1995). In all of the models, there is development of an acute inflammatory response and digestive enzyme activation in the organ associated with cell death (necrosis and apoptosis) of parenchymal cells.

There have been two main areas of investigation of the mechanisms of acute pancreatitis. One area has been devoted to determining the mechanism of digestive enzyme activation in the pancreas (Steer and Meldolesi, 1987), and it is known that alcohol enhances the sensitivity of the pancreas to digestive enzyme activation from in vitro studies (Katz et al., 1996). The other major area of research involves the inflammatory response in the pancreas (Denham and Norman, 1999). It has been learned that acinar cells are capable of expressing a wide variety of proinflammatory ctyokines that are responsible for both mediating the inflammatory response as well as regulating the cell death responses of the parenchymal cells. Moreover, the processes of inflammation and intracellular digestive enzyme activation are linked. Preliminary data suggest that alcohol metabolites, acetaldehyde and fatty acid ethyl esters, regulate transcription factors involved in mediating the inflammatory response (e.g., NF-kB and AP-1).

Investigations into the mechanisms of chronic pancreatitis have been hampered by the lack of experimental models (Vaquero et al., 1999).

Knowledge about alcohol metabolism in the pancreas is limited and controversial. Little is known about the alcohol dehydrogenase (ADH) system that mediates oxidative metabolism in the pancreas (Wilson and Pirola, 1997). In contrast, a major pathway for alcohol metabolism in the pancreas is via non-oxidative metabolism (Wilson and Pirola, 1997). Although fatty acid ethyl esters may mediate toxic effects of alcohol on the pancreas, the enzyme systems that metabolize alcohol to non-oxidative metabolites are not fully characterized and there is controversy about whether they are regulated by chronic alcohol intake (Hamamoto et al., 1990; Laposata, 1998).

Pancreatic triglyceride accumulation has been observed in some animal studies after chronic alcohol administration (Iimuro et al., 1996). Furthermore, both reactive oxygen species and lipid peroxidation products have been demonstrated in the pancreas with alcohol administration (Iimuro et al., 1996). However, the roles of reactive oxygen species and lipid peroxidation products on the pathologic effects of alcohol in the pancreas have not been determined.

The greatest limitation to the investigation of the pathologic effects of alcohol on the pancreas is the lack of animal models of acute and chronic alcohol-induced disease.

One of the most puzzling aspects of alcohol-induced pancreatic disease is the individual susceptibility to alcohol-induced pancreatic disease (Wilson and Pirola, 1997). Although alcoholic pancreatitis incidence is proportional to the amount of alcohol consumed, only a small percentage of individuals who drink heavily develop alcohol-induced pancreatic disease. The factors responsible for this variability in susceptibility have not been found. It is possible that endogenous hormones and neurotransmitters or signals they regulate in the pancreatic acinar cell may be involved in the sensitization of the pancreas to the pathologic effects of alcohol.

Specific recommendations:

(1) Determine the metabolism of ethanol in the pancreas and the effects of ethanol on metabolic events in the pancreas, e.g., mitochondrial effects (methionine, SAM, and glutathione metabolism) and phospholipid effects.

(2) Determine the effects of ethanol and its metabolites in modulating the signals that regulate the processes of acute and chronic pancreatitis, e.g., inflammation, cell death, and fibrosis.

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NIAAA PORTFOLIO ON ALCOHOLIC PANCREATITIS
(Vishnudutt Purohit, D.V.M., Ph.D.)

Alcohol abuse is a major cause of pancreatitis, and up to 60-70% cases of pancreatitis are associated with alcoholism. Several factors have been suggested to mediate injurious effects of alcohol on pancreas including cholecystokinin (CCK), premature activation of pancreatic enzymes, oxidative stress, NF-kB activation, cytokines, chemokines, and inflammatory cells. However, the exact mechanisms of alcoholic pancreatitis are not clear.

The National Institute on Alcohol Abuse and Alcoholism currently supports three projects that investigate relationships between alcohol and pancreatitis. The portfolio consists of one R01 grant, and two center-grant components, with a total funding of about 435 K (Table 1).

Altered Pancreatic Enzyme Secretion: This project is testing a hypothesis that alcohol impairs pancreatic enzyme secretion by altering the gene expression of certain proteins in a subset of cells in pancreas, brain, and duodenum. The investigators have shown that chronic ethanol ingestion significantly elevates the mRNA levels of rat pancreatic monitor peptide (MP) which may contribute to pancreatic hypersecretion by stimulating the release of cholecystokinin. The expression of pancreatic cholesterol esterase mRNA is upregulated in rats by chronic ethanol ingestion. This may play a role in the pathogenesis of pancreatitis due to the ability of the esterase to metabolize cholesterol and form fatty acid ethyl esters.

Interactive Role of Alcohol and Cholecystokinin (CCK): The hypothesis of this project is to test that an ethanol diet alone or in combination with a low dose of CCK induces an activation of transcription factors that are involved in regulating the expression of proinflammatory cytokines and chemokines. The project is investigating the roles of transcription factors (NF-kB and AP-1), cytokines, and chemokines in the development of pancreatitis. The investigators have developed a rat model of alcoholic pancreatitis by administering alcohol and CCK, an intestinal hormone known to stimulate the secretion of pancreatic enzymes. Rats that were administered ethanol intragastrically for 2 or 6 weeks followed by intravenous infusion of CCK showed significant increases in serum amylase and lipase levels, number of inflammatory cells (neutrophils and macrophages), number of apoptic acinar cells, mRNA expression of inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2), mRNA expression of inducible nitric oxide synthase, and pancreatic NF-kB activity. Rats treated with either ethanol or CCK did not show significant changes in these parameters.

Role of Ethanol Metabolites on Pancreas: This project investigates the effects of both oxidative (acetaldehyde and acetate) and nonoxiadtive (fatty acid ethyl esters) ethanol metabolites on pancreas. It is hypothesized that these metabolites activate transcription factors (NF-kB and AP-1) which in turn activate the expression of proinflammatory cytokines, leading to inflammation, cell death, and fibrosis. This project has shown that NF-kB plays an important role in the development of CCK-induced pancreatitis.

Future Directions:

1. Determine mechanisms by which alcohol sensitizes pancreas to CCK effects.

2. Molecular mechanisms by which alcohol alters pancreatic enzyme secretion.

3. Role of acetaldehyde in initiating pancreatic injury.

4. Roles of cytokines, chemokines, adhesion molecules, and inflammatory leukocytes in the process of inflammation of the pancreas.

Table 1. Alcoholic Pancreatitis

State of Knowledge (Hide Tsukamoto, D.V.M., Ph.D.)

Although Lieber and colleagues (1975) suggested that progressive ALD proceeds despite adequate nutrition, compelling evidence exists for the role of nutritional deficiency in alcoholic liver disease (ALD) (Mendenhall et al., 1995). Moreover, it has been demonstrated that nutrients such as polyunsaturated fat or iron can have profound effects on alcoholic liver injury. Currently, ALD is thought to result from priming (Tsukamoto et al., 1999) and sensitizing (Colell et al., 1998) mechanisms produced by cross-interactions of primary mechanistic factors (acetaldehyde, oxidative stress, immune responses, hypermetabolism, membrane alterations, redox shift) and secondary risk factors (nutrition, genetic factors, gender, viral hepatitis, concomitant use of drugs). The multifactorial nature and complex interactions among primary mechanistic factors and between primary and secondary factors, appear to be the basis for the heterogeneous response that alcoholics exhibit for ALD.

Even though hepatocytes account for 65% of the total cell population and 85% of total volume of the liver, the five non-parenchymal liver cell types (endothelial cells, Kupffer cells/hepatic macrophages, hepatic stellate cells, bile duct epithelial cells, and pit cells/liver NK cells) possess distinct and important cellular functions in support of liver homeostasis and actively participate in pathologic processes.

Administration of antibodies against TNFa , the cytokine predominantly expressed by Kupffer cells, attenuates alcoholic liver injury (Iimuro et al., 1997), and the importance of TNFa is confirmed by the absence of alcoholic liver injury in TNF receptor 1 knockout mice (Yin et al., 1999). It is, however, unclear whether resident macrophages (Kupffer cells), newly recruited monocyte-derived macrophages, or both serve as the primary effector cell type for expression of TNFa in experimental ALD. Further, what primes hepatic macrophages for TNFa expression is not known.

Ethanol and LPS induce similar morphologic and functional changes in sinusoidal endothelial cells typically characterized by reduced fenestration and hyaluronan uptake (Sarphie et al., 1996; Deaciuc and Spitzer, 1996), both of which are preventable by elimination of Kupffer cells. Chronic ethanol intake impairs receptor-mediated endocytosis by sinusoidal endothelial cells at the level of internalization (Thiele et al., 1999). Intracellular adhesion molecule-1 (ICAM-1) expression by endothelial cells is unregulated in experimental ALD and correlates with plasma endotoxic, hepatic TNFa mRNA, and liver inflammation and injury (Nanji et al., 1995). Expression of a receptor component for the adhesion molecule such as CD18 (b 2-integrin) is unregulated on neutrophils of ethanol-fed rats (Bautista, 1997). However, ethanol-induced neutrophilic inflammation in the liver, the hallmark of alcoholic hepatitis, has been largely unsuccessful in experimental animals, suggesting the models are missing critical factor(s). Endotoxin challenge to ethanol-sensitized livers results in coagulative necrosis and accompanying neutrophilic inflammation, but most critical changes appear to occur initially at the sinusoidal endothelium, such as cellular swelling, blood cell aggregation, and microcirculation disturbance (McCuskey et al., 1995).

Knowledge of the mechanisms of hepatic stellate cell (HSC) activation in ALD is limited because of difficulties in both inducing diffuse liver fibrosis in ethanol-fed animals and isolating sufficient numbers of HSC from ethanol-fed rats. HSC show expected changes of cellular activation after exposure to ethanol, including increased collagen and DNA synthesis (Matsuoka et al., 1990), induced expression of a -smooth muscle actin, and depletion of retinyl palmitate (Tsukamoto et al., 1996). HSC isolated from rats fed diet high in polyunsaturated fat with or without ethanol, show increased responsiveness to hepatic macrophage-derived factors for stimulation of either collagen or DNA synthesis, providing the potential cellular basis for increased alcoholic liver fibrogenesis under the high polyunsaturated fat diet regimen (Matsuoka et al., 1990). Besides the paracrine mode of HSC stimulation involving soluble factors, oxidative stress may particularly be relevant to the mechanisms of HSC activation in alcoholic liver fibrogenesis. Although the role of oxidative stress in hepatocellular injury in ALD is convincing, the direct involvement of oxidative stress in HSC activation is still controversial.

Even though there is a close correlation between induction of CYP2E1 and experimental ALD (Nanji et al., 1994) and inhibitors for CYP2E1 ameliorate alcoholic liver injury (Morimoto et al., 1995), a recent study using CYP2E1 knockout mice demonstrated no suppression of alcoholic liver injury and a similar induction of other CYP families (CYP1A, CYP2A, CYP2B, and CYP3A) in ethanol-fed wild and knockout mice (Kono et al., 1997). Furthermore, gadolinium chloride blocks experimental alcoholic liver injury despite induction of CYP2E1 (Koop et al., 1997), demonstrating dissociation of CYP2E1 induction from liver injury. However, these latter two studies only examined the early stage of experimental ALD and whether CYP2E1 plays a role in progression of experimental ALD is yet to be tested.

Another important subcellular site that contributes to both priming and sensitizing effects of ethanol is mitochondria. Depletion of mitochondrial GSH is the most important sensitizing mechanism associated with ethanol exposure (Fernandez-Checa et al., 1991) to TNFa -induced cell death (Colell et al., 1998). This ethanol-induced defect is caused by impaired transport of GSH into mitochondria and is corrected by administration of S-adenosylmethionine but not by that of N-acetylcysteine (Colell et al., 1997). It is not known what molecular mechanisms underlie the ethanol-induced impairment in mitochondrial GSH transport except for the observation that fluidization of mitochondria with a fatty acid derivative restores their ability to transport GSH (Colell et al., 1997).

Ethanol is known to induce apoptosis in human primary hepatocytes and HepG2 cells in culture (Neuman et al., 1999), and mitochondria from rats chronically fed ethanol are more sensitive to induction of the mitochondrial permeability transition by ceramide, GD3 ganglioside, and Bax (Pastorino et al., 1999). Whether and how ethanol sensitizes hepatocytes for apoptosis are key questions that need to be pursued. What determines the type of cell death (apoptosis versus necrosis) in ALD is another important question, with the extent of ATP depletion perhaps being a critical factor. UCP-2 is expected to be upregulated in ALD, and this may make hepatocytes more vulnerable to cell death by compromising the mitochondrial membrane potential and ATP generation.

Ethanol exposure has been shown to inhibit calcium mobilization and DNA synthesis in hepatocytes induced by HGR (Saso et al., 1996) and TGFa -stimulated receptor autophosphorylation (Tuma et al., 1998). The HGF finding is consistent with the observation that improved recovery from ethanol-induced fatty liver followed the administration of HGF, the most potent mitogen for hepatocytes (Tahara et al., 1999). Ethanol also inhibits TNFa dependent increase in NF-K B binding in partially hepatectomized rat livers while not affecting IL-6 induced Stat-3 phosphorylation and DNA binding (Yang et al., 1998).

Specific recommendations:

(1)  Define/identify the missing secondary risk factors for development of progressive alcoholic liver disease. This will help improve animal models.

(2) Determine the mechanisms of PMN transmigration in alcoholic liver disease.

NIAAA PORTFOLIO ON LIVER DISEASE
(Vishnudutt Purohit, D.V.M., Ph.D.)

Alcoholic liver disease (ALD) is a major cause of illness and mortality in the USA. It is the fourth leading cause of death among adult men of 24-65 years residing in urban areas. While the early stages of the disease - fatty liver and hepatitis – are reversible, the end stage cirrhosis is irreversible and fatal. Cirrhosis is the tenth leading cause of death in this country. Of the 25,000 people who died of this disease in 1992, at least 12,000 deaths were attributed to alcoholic cirrhosis. NIAAA-funded researchers are investigating the underlying cellular, biochemical, and molecular mechanisms by which chronic alcohol consumption leads to the development of ALD. Results obtained from this research could be used to design strategies for the treatment and prevention of the disease.

Currently, NIAAA is supporting 54 research projects on ALD, with a total funding of $10.9 million (Table 1). The breakdown of the portfolio by grant mechanism is: regular research projects (R01), 64.8%; small grants (R03), 3.7%; developmental/exploratory grants (R21), 5.5%; MERIT awards (R37), 5.6%; center grant components (P50), 11.1%; independent/senior scientist awards (K02/K05), 5.5%; and fellowships (F30/F31), 3.7% (Table 1).

The program portfolio is classified into ten categories as presented in Table 2, and described below.

Role of Intestinal Macrophages and Permeability (Table 3)

Alcohol increases intestinal permeability to endotoxin that plays a significant role in the initiation of liver injury since endotoxin activates Kupffer cells which, in turn, can stimulate the inflammatory cascade. In addition, intestinal lamina propria macrophages may get activated by intestinal endotoxin that may lead to increased production of inflammatory cytokines. Currently, this portfolio has two projects with a total funding of $285 K.

Lamina propria macrophages: This project examines the role of bacteria and endotoxin in the activation of intestinal macrophages and associated production of TNF that can reach the liver and initiate the inflammatory cascade.

Intestinal permeability: Investigating the mechanisms by which alcohol and acetaldehyde increase permeability of intestine to endotoxin. The main focus of the study is the tyrosine kinase-dependent mechanism.

Role of Kupffer Cell Activation (Table 4)

Researchers are investigating the mechanisms of Kupffer cell activation that leads to the generation of various inflammatory mediators such as cytokines, oxidants, chemokines and adhesion molecules. In addition, researchers are developing molecular techniques for the suppression of the inflammatory cascade. At present NIAAA is supporting seven projects that are directly related to Kupffer cell activation by alcohol, with a total funding of $1.5 million. The portfolio consists of the following categories:

Endotoxin, TNF, MIP-2, CYP2E1, and ICAM cascade: Researchers are investigating the role of endotoxin and its receptor (CD14), and free radicals on the activation of Kupffer cells. The mechanisms by which Kupffer cells produce TNF, MIP-2, and ICAM-1 are being investigated. Knockout mice are being used to ascertain the role of endotoxin receptors, TNF receptors, and CYP2E1. In addition, antisense oligonucleotide targeting is used to suppress the generation of TNF and ICAM-1 in order to interrupt the inflammatory cascade.

NF-kB activation: These studies examine mechanisms of NF-kB activation in Kupffer cells that leads to expression of cytokines, chemokines, and adhesion molecules. The focus is on the sources of oxidative stress involved in NF-kB activation, including iron, NADPH oxidase, CYP2E1, xanthine oxidase, lipooxgyenase, and iNOS.

Cyclic AMP: This project is investigating the modulating role of cAMP on the activation and deactivation of Kupffer cells and associated impact on liver injury.

Lipid peroxidation: Investigating the mechanisms by which acetaldehyde and aldehyde products of lipid peroxidation alter normal Kupffer cell functions and provoke cellular activation.

Role of Leukocytes (Table 5)

Under the influence of chemoattractants and adhesion molecules, leukocytes migrate from the circulation to the hepatic parenchyma to produce their toxic effects by releasing cytokines, free radicals, and proteases. Currently, NIAAA is supporting three projects (610 K) to investigate the role of leukocytes in alcoholic liver disease.

Neutrophils: This project focuses on the role of neutrophillic proteinases which provoke critical changes in the hepatic perisinusoidal matrix, leading to hepatic stellate cell activation and development of fibrosis.

Lymphocytes: This project addresses the role of activated T lymphocytes in damaging hepatocytes either by direct cytotoxicity or through increased production of cytotoxic cytokines in ethanol treated mice.

Monocytes: Investigating the role of monocytes in ALD through activation of NF-kB, increased production of TNF and chemokine (MCP-1), and increased ICAM expression on endothelial cells.

Stellate Cell Activation and Fibrosis (Table 6)

Liver fibrosis results from excessive deposition of extracellular matrix components, especially collagen, resulting in an imbalance between the amount of collagen produced and degraded in the liver. Hepatic stellate cells are the primary source of excessive extracellular components, regardless of the causal agent. Upon activation, quiescent stellate cells undergo distinct cellular changes leading to myofibroblastic transformation and increased production of matrix components. NIAAA currently supports ten research projects ($1.9 million) that investigate the role of various factors in the activation of stellate cells and increased production of collagen. The portfolio is briefly described below:

Role of Vitamin A: The Researchers in this field will characterize the catalytic and molecular properties of retinyl palmitate hydrolases and retinol dehydrogenases that are expressed during the activation of stellate cells when retinol esters are hydrolyzed. These studies may help to elucidate the role of retinol metabolism in stellate cell activation.

Role of Acetaldehyde: Acetaldehyde - an immediate metabolite of ethanol – has been implicated in the development of hepatic fibrosis by increased collagen production. Investigators are focussing on cellular and molecular mechanisms by which acetaldehyde stimulates type I collagen gene transcription in the liver. One group is investigating the role of interaction of acetaldehyde with cis-regulatory elements and trans-acting factors of a 2(I) collagen gene, transcription factors, and transforming growth factorb (TGFb ) in the up-regulation of a 2(I) collagen gene in human stellate cells. The other group is defining acetaldehyde and TGFb responsive elements, and investigating the roles of oxidative stress and c/EBPb on acetaldehyde and TGFb -mediated up-regulation of mouse a 1(I) collagen gene in hepatic stellate cells.

Transcription Factors: One group of researchers is examining the transcriptional regulation of a 1(I) collagen gene in stellate cells to understand the molecular mechanisms of increased collagen expression in activated stellate cells. The locations of cis-acting elements and trans-acting factors required for transcription of collagen genes are being determined. The other group is investigating the roles of NF-kB and tissue transglutaminase in the process of hepatic injury and fibrogenesis. Yet another group is testing the role of NF-kB and AP-1 transcription factors in the activation of quiescent stellate cells.

Cytokines: This group is investigating the molecular mechanisms by which IL-6, Il-1, and TNF modulate type I collagen gene expression in hepatic stellate cells.

Gene Manipulations: Researchers in this group are using TNFR1, ICAM-1 and protein kinase A RIIb subunit knockout mice, and antisense to TGFb to understand the role of specific proteins and enzymes in the development of hepatic fibrosis.

Treatment and Prevention: Researchers are investigating the mechanisms by which dilinoleoylphosphatidylcholine prevents alcohol-induced liver fibrosis.

Oxidative Stress and Antioxidants (Table 7)

Reactive oxygen species (ROS) have been implicated in the pathogenesis of alcohol-induced liver injury. Researchers have been working on the following aspects of ROS: 1) sources and mechanisms of ROS generation; 2) mechanisms by which ROS produce tissue injury; 3) mechanisms of the depletion of endogenous antioxidants by alcohol; and 4) potential use of antioxidants to prevent ROS-induced tissue injury. Currently NIAAA is funding 10 projects on oxidative stress, with a total funding of $1.9 million. The portfolio includes:

CYP2E1, NADH, NADPH, Iron: Researchers are investigating the role of CYP2E1, NADH, NADPH, and iron in the generation of ROS and associated tissue injury in liver using HepG2 cell line transfected with human CYP2E1.

Lipid Peroxidation: This portfolio addresses the role of lipid peroxidation products such as MDA, and 4-HNE in the pathogenesis of alcoholic liver injury. Three potential mechanisms by which these products may cause tissue damage are being investigated: a) inactivation of glutathione-S-transferase, a major hepatic antioxidant enzyme; b) adducts formation with proteins which results in cytotoxic autoantibodies; and c) effects on collagen-1 gene expression in stellate cells and fibrosis.

Free radicals from Fatty Liver used for Transplant: Investigating the role of SOD/Catalase insensitive free radicals from fatty liver, lipid peroxidation, and expression of adhesion molecules (selectin and ICAM-1) in transplant failure.

Mitochondrial glutathione-S-transferase: Examines the role of this enzyme in the detoxification of 4-HNE that inhibits cytochrome c oxidase, a key component of the respiratory chain in mitochondria.

Mitochondrial GSH transporter: Identifying the mechanism by which alcohol impairs GSH transporter and the role of defective transporter in the pathogenesis of alcoholic liver disease.

S-adenosyl methionine (SAM): Investigating the role of SAM deficiency in the development of ALD.

Gene therapy for ALD: Examines how to attenuate alcohol-induced liver injury by adenovirus-mediated delivery of genes for SOD and catalase.

Role of Ethanol-Derived Protein Adducts (Table 8)

Ethanol, acetaldehyde, and lipid peroxidation products can react with various proteins to form adducts which can be injurious to liver through immunotoxicity. This portfolio is comprised of four projects with a total funding of $729 K.

Acetaldehyde and malondialdehyde-protein adducts: Acetaldehyde and MDA can react together with proteins in a synergistic manner to form distinct hybrid adducts (MAA adducts). Researchers are trying to understand the chemistry and formation of these adducts, raising antibodies against these adducts, and investigating immune response. In addition, they are investigating mechanisms by which the adducts impair liver function and induce hepatotoxicity.

Ethanol-derived hydroxyethyl radicals-protein adducts: These studies investigate mechanisms of ethanol radical formation, covalent binding of ethanol radicals with mononuclear leukocyte proteins, identification of the adducted proteins, and correlation of adduct formation with liver injury.

Role of Chemokines (Table 9)

Investigating the role of C-X-C chemokines in the parenchymal migration and subsequent activation of neutrophils that leads to hepatitis. This project is funded at $223 K.

Diet-Ethanol Interaction: Animal Model of ALD (Table 10)

This portfolio examines the interactive role of diet and ethanol on the development of ALD. The focus is on the role of carbohydrate deficiency, increased CYP2E1 activity and associated increased ROS generation in liver injury. Findings from this research may contribute in the development of a better model of ALD. The funding of this portfolio is $401 K.

Impaired Functions of Hepatocytes (Table 11)

Chronic alcohol consumption may impair many functions of hepatocytes, rendering them susceptible to alcohol-induced cell damage that may eventually lead to the development of alcoholic liver disease. NIAAA is currently supporting eleven projects that target the effects of alcohol on various functions of hepatocytes. The current funding of this portfolio is about $2.4 million, and it can be categorized into the following areas:

Impairment of signaling system: This portfolio investigates the effects of alcohol on G-protein, protein kinase C, phospholipase C, and calcium signaling in the liver.

Altered secretory vesicle transport: Researchers are studying the molecular mechanisms by which alcohol disrupts the formation and transport of vesicles. The results of these studies may help explain mechanisms of alcohol-induced protein retention, hepatocyte swelling, and hepatomegaly.

Effects of ethanol on receptor-mediated endocytosis in hepatocytes: Ethanol induced- inactivation of asialoglycoprotein receptor leads to altered receptor function and impaired receptor-mediated endocytosis. Researchers are investigating molecular mechanisms of this phenomenon.

Proteolytic system- lysosomes-proteosomes: Chronic ethanol impairs lysosome biogenesis, inactivates proteosomes, and impairs hepatocyte capacity to degrade protein modified by ethanol metabolism. This can result in unwanted protein accumulation and liver injury. Mechanisms of this alcohol effect are being investigated.

Altered energy state of the liver-mitochondrial function: This portfolio is investigating the effects of chronic ethanol ingestion on mechanisms involved in maintaining the energy state of the liver and associated changes in hepatocyte structural and functional integrity. The studies include oxidative phosphorylation, mitochondrial ribosomes, oxidative damage to mitochondria, and relationship between oxidative stress and energy state.

Impaired mitochondrial function by obesity and ethanol: This project examines the combined effect of obesity and ethanol on hepatic mitochondria by evaluating mitochondrial UCP2 expression, vital mitochondrial parameters, ROS generation, and associated hepatotoxicity.

Mallory Body (MB) Formation: Researchers are seeking to identify the molecular mechanisms by which cytokeratin proteins aggregate in the cell that leads to MB formation.

Impaired Hepatic Regeneration (Table 12)

Chronic alcohol ingestion impairs liver regeneration by inhibiting the effects of various growth factors that are essential for proliferation and growth of hepatocytes. Currently NIAAA supports five projects that are investigating the molecular mechanisms by which alcohol inhibits the effects of hepatocyte growth factors. These projects fall into the following categories: 1) impairment of G protein–adenylyl cyclase-cAMP signaling process; 2) impairment of TNF-dependent hepatocyte proliferation; 3) impaired EGF signaling pathway; and 4) altered IL-6 and associated JAK-STAT signaling pathway. The current funding of this portfolio is about $967 K.

New model of ALD: A new rat model of ALD has been developed by intragastric administration of alcohol (9% calories) only once a day by using an oral syringe. In this model, rats are fed ad libitum a liquid diet containing 47% carbohydrate, 35% fat, and 18% protein calories. After 8 weeks of alcohol administration, rats developed severe fatty changes and mild necrosis and inflammation.

Role of TNF in ALD: Simultaneous administration of TNF antibodies and alcohol attenuated alcohol-induced liver injury in a rat model of ALD. Furthermore, in a mouse model of ALD, long term ethanol administration caused severe pathological liver injury (fatty liver, necrosis, and inflammation) in wild and TNF receptor 2 knock out mice, but failed to cause severe injury in TNF receptor 1 knock out mice. These results further confirm a central role of TNF in ALD.

Role of glutathione and TNF: Chronic ethanol administration to rats caused a selective mitochondrial glutathione depletion. Hepatocytes from these animals were more susceptible to TNF-induced toxicity. Administration of S-adenosyl methionine, but not glutathione, to ethanol fed rats prevented the mitochondrial glutathione defect, restored mitochondrial glutathione pool in vivo, and prevented the susceptibility of hepatocytes to TNF-induced injury in vitro.

Role of S-adenosylmethionine (SAM) and TNF: Administration of SAM to SAM-deficient rats attenuated serum TNF levels and LPS-induced liver injury. Furthermore, addition of SAM attenuated LPS-stimulated TNF synthesis and TNF mRNA expression in murine macrophage cell lines, suggesting a protective role of SAM in liver injury.

Role of TNF antisense in preventing ALD: Researchers have developed triple helix and antisense constructs that can effectively inhibit up to 90% of TNF production in Kupffer cells stimulated by LPS. Furthermore, they have developed a technique that preferentially delivers antisense nucleotide to Kupffer cells. Thus, use of antisense nucleotide may provide interventions for the prevention of ALD.

Role of SOD in attenuating ALD: Administration of adenovirus-associated superoxide dismutase gene to rats attenuated alcohol-induced liver pathology and serum AST levels.

Allopurinol prevents ALD: Administration of allopurinol – a xanthine oxidase inhibitor and free radical scavenger – to male rats significantly blunted alcohol-induced NF-kB activation, serum transaminase levels, and liver pathology, suggesting that allopurinol prevents early alcohol-induced liver injury by blunting oxidant-dependent activation of NF-kB.

Role of iron in ALD: Iron-mediated potentiation of alcoholic liver injury is associated with enhanced NF-kB activation, upregulation of NF-kB-responsive chemokine gene expression, and mononuclear cell infiltration. Recently, using an animal model of ALD, researchers have shown that increased iron storage in Kupffer cells is associated with potentiated NF-kB activation that can be normalized by administering an iron chelator. In addition, erythrophagocytosis by cultured Kupffer cells increases intracellular iron concentration and promotes LPS-induced NF-kB activation. These results suggest that iron primes Kupffer cells for NF-kB activation in ALD.

Gender differences in ALD - Role of estrogen: In response to chronic ethanol administration, female rats developed liver pathology faster and more severe, exhibited greater plasma endotoxin levels and CD14 expression on Kupffer cells, and showed greater TNF mRNA expression and NF-kB binding in hepatic nuclear extract when compared to male rats. These results shed some light on why women are more susceptible to ALD. In another study, ovariectomy attenuated ethanol-induced increases in liver pathology, plasma endotoxin levels, CD14 mRNA expression, and Kupffer cell TNF levels. Estrogen replacement reversed the effects of ovariectomy, suggesting that sensitivity of rat liver to alcohol-induced injury is related to estrogen.

Mitochondrial damage by alcohol: Chronic ethanol impairs respiratory activity of hepatic mitochondria, selectively deplete mitochondrial glutathione, and stimulate mitochondrial free radical generation. Recently researchers have shown that chronic ethanol feeding for 11-13 months decreases hepatic mitochondrial DNA content, and increases mitochondrial single-stranded breaks. These alterations may have been caused by alcohol-induced mitochondrial oxidative stress.

Role of CYP2E1 and oxidative stress in fibrogenesis: CYP2E1-dependent oxidative stress can increase collagen production in rat hepatic stellate cell line transfected with human CYP2E1.

NF-kB and collagen gene transcription: Transfection of NIH 3T3 fibroblasts and hepatic stellate cells with NF-kB p50, p65, and c-Rel expression plasmids with collagen gene reporter gene significantly inhibited the transcription of collagen gene promoter. Nuclear run-on assay showed that NF-kB inhibited transcription of the endogenous a 1 (I) collagen gene. These results demonstrate that NF-kB decreases transcription of the a 1 (I) collagen gene.

Role of polyenylphosphatidylcholine (PPC) in preventing ALD: PPC prevents alcohol-induced hepatic fibrosis and cirrhosis in baboons, and carbon tetrachloride-induced fibrosis and cirrhosis in rats. Recently researchers have shown that PPC provides protection against liver injury by attenuating alcohol-induced oxidative stress, and apoptosis of hepatocytes.

Role of Kupffer cells in hepatic regeneration: Kupffer cell depletion significantly delayed hepatic regeneration after partial hepatectomy in rats. This was associated with complete depletion of hepatic mRNA expression of IL-6 and IL-10, and decreased mRNA expression of hepatic TNF, TGFb , and hepatocyte growth factor. This suggests that Kupffer cells play important role in hepatic regeneration via cytokine production.

Role of ethanol on TNF signaling during liver regeneration: During hepatic regeneration, TNF increased mitochondrial ROS production that is toxic to the cells. On the other hand, TNF promoted the activation of NF-kB, Jun N-terminal Kinase (JNK), and various mitochondrial membrane proteins, which may permit hepatocytes to escape apoptosis and oxidant stress. Prior ethanol exposure inhibited the normal regenerative induction of NFkB and JNK. These results suggest that ethanol compromises the balanced induction of toxic and trophic (beneficial) signals by TNF. Further, in liver regenerating mice, TNF-dependent increases in mitochondrial oxidant production is associated with increased expression of uncoupling protein-2.

FUTURE RESEARCH DIRECTIONS

Genetic susceptibility to alcohol-induced liver injury: Since only a small proportion of alcoholics develops cirrhosis, genetic differences in susceptibility need to be investigated.

Ethnic differences associated with the severity of alcohol-induced liver damage: Available data suggest that African Americans and Hispanics have higher rates of mortality from alcoholic cirrhosis than whites or Asian Americans. Research is required to determine whether factors such as genetics, diet, and pattern of drinking can help to explain the increased vulnerability of some ethnic groups to ALD.

Gender differences in the susceptibility to alcohol-induced liver injury: Women are more susceptible than men to alcohol-induced liver injury. Researchers have shown that female rat Kupffer cells are more sensitive than male cells to activation by alcohol administration, and this has been attributed to estrogen. Further studies are required to understand the mechanisms of gender differences at the level of stellate cell activation.

Mechanisms whereby alcohol increases intestinal permeability to endotoxin: Chronic alcohol administration leads to increased plasma levels of endotoxin that result in the activation of Kupffer cells and development of liver injury. Simultaneous administration of antibiotics attenuates endotoxin levels and associated liver injury. Further research is required to understand mechanisms by which alcohol increases intestinal permeability to endotoxins.

Mechanisms of oxidative stress in ALD: Although several types of free radicals have been implicated in the pathogenesis of ALD, further work is required to establish cause and effect relationship. An important source of free radicals is the induction of CYP2E1 that is associated with the pathogenesis of ALD. The role of CYP2E1 in alcohol-induced liver injury has been further confirmed using HepG2 cell line and rat hepatic stellate cells transfected with human CYP2E1. However, recently the role of CYP2E1 has been questioned since simultaneous administration of CYP2E1 inhibitors did not prevent the development of ALD. This raises a question about the sources of oxidative stress that are relevant to liver injury. In this context, roles of NADH, NADPH, lipid peroxidation, iron, and, iNOS need evaluation. This information is required before an effective antioxidant therapy can be developed for the treatment of ALD.

Mechanisms of leukocytic infiltration of hepatic parenchyma in ALD: Studies are required to understand the underlying mechanisms by which leukocytes infiltrate into hepatic parenchyma during the progression of ALD. In addition, the relative role of neutrophils, lymphocytes, and monocytes in the pathogenesis of ALD needs to be investigated.

Mechanisms of stellate cell activation: Research is required to understand the mechanisms by which alcohol administration leads to the transformation of quiescent stellate cells to collagen-producing cells. In addition, research is required to understand the molecular mechanisms whereby PDGF stimulates stellate cell proliferation, and acetaldehyde and TGFb stimulate collagen production.

Role of diet in the development of ALD: Diet plays an important role in modulating the course of ALD. While unsaturated fatty acids potentiate the development of ALD, saturated fatty acids attenuate it. In addition, while carbohydrate deficiency potentiates the development of ALD, adequate carbohydrate prevents it. Further research is required to understand the interactive effects of alcohol and diet on the pathogenesis of ALD.

Table 1. Alcoholic Liver Disease Portfolio: Grant Distribution by Grant Mechanisms

Table 2. Alcoholic Liver Disease Portfolio: Grant Distribution by Areas of Research

Table 3. Role of Intestine: Permeability and Macrophage

Table 4. Kupffer Cell Activation

Table 5. Role of Leukocytes

Table 6. Stellate Cell Activation and Fibrosis

Table 7. Oxidative Stress and Antioxidants

Table 8. Ethanol-Derived Protein Adducts

Table 9. Role of Chemokines

Table 10. Role of Diet-Ethanol Interaction

Table 11. Impaired Functions of Hepatocytes

Table 12. Hepatic Regeneration

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State of Knowledge (William Bosron, Ph.D. and Vijay A. Ramchandani, Ph.D.)

The pharmacokinetics of ethanol determines the time course of ethanol concentration in blood after the ingestion of an alcoholic beverage, and thus the degree of exposure of organs to its effects

After oral ingestion, ethanol is almost completely absorbed, primarily from the small intestine, by passive diffusion (Holford, 1987). Ethanol ingested on an empty stomach is very rapidly absorbed with peak concentrations occurring between 30 to 90 minutes. The rate of absorption after oral administration is greatly influenced by the nature and concentration of the alcoholic beverage (Dubowski, 1985), rate of ingestion ( O'Neill et al., 1983), fed or fasted state (Sedman A et al., 1976), nature and composition of food (Sedman et al., 1976), rate of gastric emptying (Kalant, 2000), as well as other psychological, genetic, and temporal factors.

The distribution of ethanol throughout the body is largely governed by the water content of various organs and tissues, especially at equilibrium, because ethanol is a small, polar, completely water-soluble molecule. The volume of distribution of ethanol is comparable to total body water (Holford, 1987). No plasma protein binding has been reported for ethanol.

Elimination of ethanol occurs primarily through metabolism with minute fractions of the administered dose being excreted in the breath (0.7%), sweat (0.1%), and urine (0.3%) (Holford, 1987). Alcohol elimination occurs mainly via oxidation in the liver and is governed by the catalytic properties of the alcohol metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), as well as the miscrosomal ethanol oxidizing system (MEOS). Alcohol metabolic rates show a considerable degree of inter-individual and ethnic variability, due to the expression of ADH and ALDH isozymes and polymorphic variants (alleles). The ADH1B and ALDH2 polymorphisms have been shown to increase the variability in alcohol metabolism among individuals. Additionally, a multitude of environmental factors can influence the metabolic regulation of alcohol metabolism, which results in a large 3- to 4-fold variability in the alcohol elimination rate in humans (Eckardt et al., 1998).

Gender and Body Composition

Women absorb and metabolize alcohol differently than men. In general, women have a lower proportion of body water than men of similar body weight, so that women achieve higher concentrations of alcohol in the blood after drinking equivalent amounts of alcohol (Chen et al., 1999). Some studies have reported that differences in peak concentrations following equivalent doses of ethanol administered to men and women could be due to differences in first-pass metabolism of ethanol in the gastrointestinal tract, which was significantly correlated with gastric alcohol dehydrogenase activity (Baraona et al., 1998). Some investigators concluded that females have lower gastric alcohol dehydrogenase activity resulting in a lower degree of first-pass metabolism and therefore in higher concentrations compared to males. However, other studies have demonstrated no differences in the first-pass metabolism of ethanol between males and females (Ammon et al., 1996).

Gender differences in the elimination of alcohol have also been reported. This may be partly due to the differences in blood alcohol concentrations achieved following alcohol consumption and the nonlinear nature of alcohol pharmacokinetics, particularly at higher doses (Rangno et al., 1981). Thomasson et al. (1995), have found a higher b ₆₀ (pseudo-zero-order disappearance rate calculated over 60 min) in women compared to men of comparable ADH1B genotype following oral administration of alcohol to achieve similar peak concentrations. The authors speculate that these differences could be due to differences in daily alcohol intake, influence of sex hormones (estrogen and testosterone), or differences in liver size relative to body weight or lean body mass.

Other gender-related studies, evaluating the effect of menstrual cycle and of oral contraceptives on alcohol pharmacokinetics were reviewed recently (Mumenthaler et al., 1999). The authors concluded that the menstrual cycle had no effect on alcohol pharmacokinetics, and could not account for gender differences in alcohol disappearance rates in women. Studies of the effects of sex hormones and oral contraceptives on alcohol pharmacokinetics in women have been less conclusive, with some studies showing that women taking oral contraceptives show lower peak BACs and slower elimination of women compared to women not taking oral contraceptives (Mumenthaler et al., 1999). Other studies have shown no effect of oral contraceptives on alcohol pharmacokinetics.

Food and Food Composition

Most studies evaluating the effect of food and food composition on alcohol pharmacokinetics show a decrease in the rate of absorption and peak alcohol levels (Jones et al., 1997). This is probably due to food-induced delays in gastric emptying (Rasmussen et al., 1996). However, possible mechanisms for increased alcohol elimination include food-induced increases in liver blood flow and increases in activity of alcohol metabolizing enzymes.

Ethnicity and Genetic Polymorphisms

Due to the genetic polymorphisms of the alcohol metabolizing enzymes, ADH and ALDH, and due to differences in prevalence of these polymorphic forms of the isozymes in different ethnic populations, ethnic differences in alcohol metabolism and the influence of the ADH and ALDH genotypes has been the subject of many studies. The isozymes coded by the polymorphisms have highly divergent catalytic properties in vitro (Bosron et al., 1993) and influence the exposure to alcohol in individuals. Progress in characterizing differences in alcohol metabolism in the different polymorphic genotypes has been substantial.

Genetics

Alcohol dehydrogenases are cytosolic, dimeric enzymes with 40 KDD subunits that catalyze the NAD⁺-dependent oxidation of primary and secondary alcohols. In humans, there are 7 genes encoding the medium chain ADHs. The progress in the characterization of the enzyme kinetic properties of ADH isoenzymes with ethanol and NAD⁺ has been excellent.

Tissue-Specific Expression of ADHs

The human ADH genes are expressed in different tissues, which is a very important feature for the physiological consequences of alcohol metabolism in specific cells and tissues (Edenberg, 2000). Liver contains a large amount of ADH (about 3% of soluble protein) and expresses the widest number of different isoenzymes. There has been good progress recently in the characterization of ADH promoters and tissue-specific expression of isoenzymes.

Enzyme Kinetics

The enzymes encoded by the six human ADH genes (excluding ADH5) all prefer NAD⁺

as coenzyme and exhibit broad substrate specificity (Edenberg and Bosron, 1997). They exhibit highest catalytic efficiency with primary alcohols of 2 to 8 carbon lengths. The isoenzymes exhibit widely different affinities for ethanol and NAD⁺. These differences can be explained by specific amino acid substitutions in the active site. Progress in the characterization of the enzyme kinetic properties of ADH isoenzymes with ethanol and NAD⁺ has been excellent.

Considerable effort has been devoted to identification of true "physiological" substrates of ADHs (Yin et al., 1999). The broad substrate specificity, redundancy of enzyme forms and high expression in liver suggests that the family of ADHs play a general role in elimination of alcohols from virtually all organisms. There are, however, several important physiological alcohols metabolized by ADHs. Vitamin A is distributed throughout the body as RBP-bound retinol. It can be oxidized to retinal by alcohol dehydrogenases and to retinoic acid by aldehyde dehydrogenases. All-trans and 9-cis retinoic acids are important transcriptional activators that act through the RAR and RXR receptors. They play a key role in developmental biology and cell signaling. The role of ethanol substrate competition and inhibition of the oxidation of physiological alcohols by medium chain ADHs is a potentially important issue in alcohol-mediated toxicology.

In the past decade, there has been excellent progress in the determination of the 3-dimensional structures of human alcohol dehydrogenase isoenzymes by X-ray crystallography.

Knockout Animals and Transfected Cell Systems

Transgenic knockout mice having null mutations in Adh1, Adh3 and Adh4 have been prepared (Delatour et al., 1999; Delatour et al., 1999) and developed normally. The facts that the knockout mice developed normally and are not totally deficient in alcohol oxidation are consistent with the so-called "redundancy" in the ADH system where several of the broad substrate specificity isoenzymes can oxidize ethanol, retinol or other physiologically important alcohols. Hence, no single gene is solely responsible for oxidation of ethanol, retinol or other alcohols. Cell lines transfected with alcohol dehydrogenase have been successfully used to investigate the effects of ethanol metabolism on lipid metabolism key metabolic processes.

The term microsomal ethanol oxidizing system (MEOS) describes all enzymatic activity for ethanol oxidation in the subcellular endoplasmic reticulum (Lieber, 2000).

Genetics

There are at least three cytochrome P-450 proteins encoded by: CYP2E1, CYP1A2 and CYP3A4. The major enzyme system is CYP2E1 that catalyzes the NADPH- and O₂-dependent oxidation of ethanol to form acetaldehyde, NADP⁺ and water. There are as many as 13 different CYP2E1 polymorphisms. Polymorphic sites in the 5’-flanking region of CYP2E1 have been reported that are differentially expressed in different racial populations (Hayashi et al., 1991). The c1/c2 polymorphism was shown to influence gene transcription in vitro. However, the linkage of CYP2E1 expression polymorphisms to frequency of alcoholic liver disease remains to be controversial (Lieber, 2000).

Regulation of Microsomal Expression

While MEOS accounts for a much smaller fraction of ethanol oxidation than that through the cytosolic alcohol dehydrogenase system under normal circumstances (probably less than 10% in humans), MEOS represents a major adaptive response of alcohol metabolism with chronic ethanol consumption (Lieber, 2000). This is due to the direct effect of chronic ethanol consumption on the expression of hepatic CYP2E1.

Enzyme Substrate Specificity

CYP2E1, CYP1A2 and CYP3A4 exhibit broad substrate specificity and catalyze the oxidation of a variety of alcohol and non-alcohol substrates. There are no 3-dimensional structures for the microsomal ethanol oxidizing enzymes, in large part because of a lack of knowledge of the protein arrangements in microsomal membranes.

Knockout Animals and Transfected Cell Systems

Gene knockout mice have been prepared with disruptions in both the CYP2E1 and CYP1A2 genes (Gonzalez, 1998). HepG2 cell lines that are transfected with Cyp2E1 have been used to show that the substrates ethanol, acetaminophen and arachidonic acid are cytotoxic to the cells that over-express CYP2E1 but not to control cells lacking the oxidase (Chen et al., 1998). It is proposed that the generation of reactive oxygen species cause the cellular damage. In the case of cells preloaded with arachidonic acid the reactive intermediates initiate lipid peroxidation, which subsequently causes apoptosis and cellular damage (Chen et al., 1998).

Catalase is an enzyme that catalyzes the H₂O₂-dependent oxidation of ethanol yielding acetaldehyde and two waters. It is found in cytosolic and mitochondria but its main expression and function is in peroxisomes. Most investigators indicate that it contributes very little to total ethanol elimination, because of the limited availability of H₂O₂ (Lieber, 2000). However, the activation of peroxisomal catalase by increased generation of H₂O₂ via peroxisomal-oxidation leads to a hypermetabolic state and a swift increase in alcohol metabolism (Bradford et al., 1999). This state may contribute to alcohol-related inflammation and necrosis in alcoholic liver disease.

Genes and polymorphic variants

The human ALDH gene family is composed of 12 related genes (Yoshida et al., 1998). The whole eukaryotic gene family of 86 known gene or protein sequences had been divided into 18 families based on evolutionary analysis of the sequences and chromosomal mapping (Vasiliou et al., 1999). Only two of the genes, ALDH1A1 and ALDH2, encode enzymes that catalyze the NAD⁺-dependent oxidation of acetaldehyde to acetate. ALDH1 or ALDH1A1 in the new nomenclature (Vasiliou et al., 1999) is the cytosolic ALDH form found in almost all tissues that exhibits relatively low catalytic efficiency (k_cat/K_M) for acetaldehyde oxidation. ALDH2 is the mitochondrial enzyme that is highly expressed in liver. It exhibits high catalytic efficiency for acetaldehyde oxidation and is primarily responsible for acetaldehyde oxidation in vivo. There are as many as four ALDH2 polymorphic variants.

ALDH1A1 is ubiquitously distributed in tissues including brain, whereas ALDH2 is strongly expressed fewer tissues like liver and stomach (Yoshida et al., 1998). Polymorphisms in the proximal ALDH2 promoter that could affect ALDH expression were recently reported (Harada et al., 1999).

The cytosolic ALDH1A1 and mitochondrial ALDH2 exhibit similar V_max values for acetaldehyde oxidation (0.2 U/mg) but ALDH2 has a lower K_M value, 1 mM, versus ALDH1A1 with a K_M of about 50 mM (MacKerell et al., 1987). Hence, the mitochondrial ALDH2 isoenzyme is thought to be the major acetaldehyde-oxidizing enzyme in liver.

In the last 3 years, structures for the major acetaldehyde metabolizing enzymes, ALDH2 (Steinmetz et al., 1997) and ALDH1 (Moore et al., 1998) have been solved. They exhibit similar overall folds, which have unique characteristics among the dehydrogenases. They have a 3-stranded oligomerization domain that allows communication between subunits. The loops "embrace" each other in the dimer units and form part of the substrate-binding channel (Lamb and Newcomber, 1999).

The wild-type form of the mitochondrial ALDH2*1 and the ALDH2*2 "Oriental" variant were express separately and together in HeLa cell lines (Crabb et al., 1998). This cell system was used to examine the observation that individuals having both heterozygous and homozygous genotypes for ALDH2*2 exhibit the alcohol flush reaction and have low ALDH activity (Crabb et al., 1989). Hence, the ALDH2*2 allele has a dominant phenotype. The study confirmed that the co-expression of both alleles decreases the basal activity of the wild-type enzyme (Crabb and Xiao, 1998).

The first effect of ethanol on liver metabolism is a rapid decrease of NAD⁺/NADH ratio primarily due to an increased NADH concentration while ethanol is being oxidized by the NAD⁺-dependent alcohol and aldehyde dehydrogenases. This leads to changes in the ratios of intermediary metabolites like lactate/pyruvate etc. that are sensitive to NAD⁺/NADH ratio or NADH concentration through their respective NAD⁺-dependent dehydrogenases (Kitson, 1999). This reduced cytosolic NAD⁺/NADH ratio, in turn, causes a reduction in the mitochondrial NAD⁺/NADH ratio, which reduces the cellular ability to oxidize fatty acids and acetyl-CoA and leads to fatty liver. In addition to the disturbance of metabolites sensitive to NAD⁺/NADH, ethanol oxidation causes a rapid increase in acetate concentration, which in then exported to peripheral tissues for metabolism (Kitson, 1999).

Ethylation Reactions

Fatty acid ethyl esters are produced by the esterification of fatty acids with ethanol. A variety of hydrolases can catalyze the transesterification of fatty acid esters with ethanol including carboxylesterases and lipases (Brzezinski et al., 1994;Laposata, 1999). More recently there was evidence for the role of an acyl-CoA:ethanol O-acyltransferase catalyzing fatty acid ethyl ester synthesis in rat liver microsomes (Diczfalusy et al., 1999).

Acetaldehyde Adducts

Acetaldehyde is a highly reactive and cytotoxic metabolite. Acetaldehyde-protein adducts have been postulated to alter cellular function by direct toxicity as well as producing immune-mediated tissue damage. Acetaldehyde is also known to form adducts with amine groups of intermediary metabolites

Substrate Competition

Retinol oxidation by the medium chain ADHs can be substantially inhibited by physiological concentrations of ethanol (Yin et al., 1999).

Specific recommendations:

(1)  Determine the regulation of expression of alcohol metabolizing enzymes in specific cell types and conditions, using "metabolic chip assay", quantitative PCR, and polymorphisms.

(2)  Study pharmacokinetic models for alcohol metabolites, especially acetaldehyde adducts, ethylated metabolites, and competitive substrates.

The binding of a number of ethanol metabolites to cell constituents has been demonstrated, and it has been postulated that subsequent impairment of key functions may lead to tissue injury. The most thoroughly studied is the primary metabolite acetaldehyde. This compound is rapidly metabolized by the aldehyde dehydrogenase of hepatic mitochondria, and its circulating level during ethanol metabolism is hardly detectable, i.e., < 1 m M (although it is somewhat higher in chronic alcoholics). Despite numerous conjectures that acetaldehyde may be involved in extrahepatic tissue injury, experimental damage of any kind at low micromolar levels of acetaldehyde has yet to be demonstrated. Covalent binding of acetaldehyde to proteins has been described, especially to lysine residues, but correlation with altered hepatocyte functions is lacking.

The role of so-called stem cells in the development of chronic liver damage is not clear at this time. Most forms of experimental chronic injury, including the administration of hepatotoxins CCl4, ethionine, dimethylnitrosamine) and human viral and alcoholic liver injury, are characterized by the proliferation of so-called ductular or oval cells, which have been postulated to represent stem cells of the liver. In view of the fact that the liver develops from a bile duct anlage, it is intriguing that the connections between the biliary system and the bile canaliculi, names the canals of Hering, are the location of putative stem cells. Interestingly, older research has demonstrated a quantitative correlation between ductular proliferation and the deposition of collagen in the liver.

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Gianoulakis C, Krishhnan B, Thavumdayil J: Enhanced sensitivity of pituitary b -endorphin to ethanol in subjects at high risk of alcoholism. Arch Gen Psychiatry 1996;53: 250-257.

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