Alcoholism is a common disorder with a complex origin and outcome, since individuals react differently when exposed to comparable amounts of alcohol. Many epidemiological, biomedical and psychosocial studies support the hypothesis that some individuals suffer more severe adverse effects following alcohol use. Physiological features (such as age and gender) and socio-cultural/psychological factors may play a relevant role in determining the huge interindividual variability in the thresholds and lifetime prevalence of this disease. Indeed, social restrictions have been shown to have a huge influence on the risk for alcohol dependence, particularly in societies with a high prevalence of alcoholism [1]. Excessive and prolonged use of alcoholic beverages is the cause of serious social and medical disorders in a significant number of individuals associated with socioeconomic consequences for the rest of the population. Alcohol related pathologies are very often related to the deficient nutritional status of chronic drinkers, due to unbalanced diet and ethanol interference in the uptake and utilization of carbohydrates, lipids and vitamins, particularly vitamin A. Indeed, ethanol has been shown to inhibit the oxidation of retinol to retinoic acid (the active form of vitamin A) by competing for alcohol dehydrogenases [2]. As a consequence, the levels of retinoic acid, which is essential for growth and maintenance of normal epithelial function, are decreased in alcoholics. Ethanol has also been demonstrated to have teratogenic potential: alcohol consumption during pregnancy can potentially result in effects in the foetus ranging from transient outcome to a quite severe neurologic disorder known as foetal alcohol syndrome (FAS). The syndrome has been associated to both ethanol metabolism and related oxidative stress and to reduction in retinoic acid production during gestation.
Family studies on twins and adoptee estimated that individual risk for alcoholism can be equally addressed to environmental and genetic factors which show a high degree of interaction [3].
Ethnicity seems also to confer different susceptibility to ethanol toxicity, as suggested by studies showing that, when compared with African Americans or native Americans, Caucasians have higher and lower rates of ethanol elimination, respectively. Differences in liver mass may only partially explain ethnic and gender differences measured in alcohol clearance [4]. Indeed, recent molecular genetic research has assigned to functional polymorphisms at those genes encoding enzymes involved in ethanol toxicokinetics, the pivotal role in determining the differential susceptibility in alcohol-induced toxic effects. This genetic features may act in combination with environmental factors, such as nutrition, life-style and exposure to other xenobiotics, responsible for the acquired modulation (induction/inhibition) of the same enzymatic activities [5].
In the paper the main features of ethanol metabolism and the polymorphisms of the most relevant enzymes involved in determining the eventual different susceptibility to alcohol-induced effect will be briefly presented.
Ethanol toxicokinetics and metabolismEthanol-induced effects are due to both ethanol per se and to the products of its metabolism, including redox changes related to the production of acetaldehyde and acetate.
The time course of ethanol blood concentration after ingestion of alcoholic drinks is strictly dependent on its toxicokinetics, which determines the dose to the target organs and the toxicodynamic responses to ethanol [6].
After oral administration, ethanol is readily absorbed by the gastrointestinal tract; absorption takes place by passive diffusion through the stomach wall (about 20%), being the remaining 80% absorbed through the duodenum and small intestine wall [5]. The rate of absorption varies with the time of the day, the dosage form, the concentration of ethanol and the drinking pattern, mainly related to the gastric emptying status. After oral absorption of ingested doses
Elimination of absorbed ethanol occurs primarily through metabolism (95-98%), with small fractions of the administered dose being excreted unchanged in the breath (0.7%), sweat (0.1%), and urine (0.3%) [5, 7].
In adult nonalcoholic individuals, most ethanol biotransformation occurs in the liver (Fig. 1) mainly via oxidation catalyzed by alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and by a cytochrome P450 isoform (CYP2E1) [8, 9].
In the cytosol of hepatocytes, ethanol is oxidized to acetaldehyde, in a reversible reaction catalyzed by class I ADH, a high affinity (Km= 0.05-0.1 g/l) and low capacity enzyme, becoming saturated after only few drinks. Acetaldehyde is then oxidized in a non-reversible reaction to acetate, by the mithocondrial form of ALDH. Since the enzyme has a very low Km, the elimination of acetaldehyde is very efficient, so that the product of ethanol oxidation, which is highly toxic, is eliminated soon after its formation. It has been estimated that during ethanol intoxication only 1-2% of the acetaldehyde formed in the liver enter the bloodstream, giving rise to negligible peripheral venous levels (≈1 µmol/l) [10]. The activated form of acetate, acetyl CoA, may be further metabolized leading to ketone bodies, amino acids, fatty acids and steroids [8]; when it is oxidized in the Krebs cycle, CO2 and water are formed as the end-products of ethanol oxidation. Both ADH and ALDH utilize as the cofactor NAD+, which is reduced to NADH (Fig. 1): as a consequence, during ethanol oxidation the ratio NADH/NAD+ is significantly increased, altering the cellular redox state and triggering a number of adverse effects, related to alcohol consumption [5]. The hepatic NADH re-oxidation seems to be the rate limiting step of the process and together with the functional ADH and ALDH activities regulate the steady-state of ethanol oxidation rate.
When a moderate dose of ethanol is ingested, a small but significant amount (≈10%) is metabolized by the microsomal NADPH dependent-oxidation catalyzed by CYP2E1 [8, 9]. This enzyme is characterized by a lower affinity (Km= 0.5-0.8 g/l) with respect to ADH: its role in ethanol oxidation becomes relevant when large amounts of alcohol able to saturate ADH are ingested (>100 g per day). The capacity-limited elimination, due to CYP2E1 saturation, is counteracted by enzyme induction by ethanol itself, which is thus able to increase its own clearance in heavy drinkers and alcoholics.
Role of polymorphisms in ethanol metabolismand ethanol-induced effectsThe important contribution of genetic factors to alcoholism is not explained by Mendelian inheritance of single genes, but there are strong evidences suggesting that this pathology is a genetically-influenced complex multifactorial disease. The increasing utilization of molecular technologies in the genetic research on alcoholism has focused the attention on the possible role of functional polymorphisms in genes encoding ethanol metabolizing enzymes. Those genetic variants produce enzymes with altered activity, changing the rate of toxic metabolites production or of their detoxication.
Thus, elucidation of the molecular mechanisms that control and influence elimination and metabolism of ethanol is important in understanding the biochemical basis of ethanol toxicity and alcohol abuse-related pharmacological and addictive consequences in humans. In addition, the identification of genetic polymorphisms and the characterization of their putative role in alcoholism vulnerability may help in improving prevention and treatment approach.
In the following the major genetic variants, giving rise to functional polymorphisms of enzymes involved both in ethanol metabolism (ADH, ALDH, CYP2E1) or in the onset of effects due to the alteration of the redox status (glutathione S-transferase enzymes), will be presented.
Alcohol dehydrogenaseAlcohol dehydrogenase (ADH) is a cytosolic enzyme able to metabolize ethanol and a wide variety of substrates, including other aliphatic alcohols, hydroxysteroids and lipid peroxidation products. ADH exists as a polygenic family of seven genes located on chromosome 4, translated in various human ADH forms (Table 1). They can be divided into five major classes or distinct groups (I-V), according to their subunit composition as well as their physicochemical properties [11].
Human ADH is a dimeric protein, resulting from the association of different subunits with a molecular weight of 40 kD each. Class I ADH (ADH1, ADH2 and ADH3) isoenzymes are formed by different combinations of subunits (α, β, γ), coded by genes from either the same locus or different loci. Homodimeric proteins are formed by identical subunits coded by the same locus, while heterodimeric proteins are formed by alleles coded from different loci (e.g., αβ, αγ) or by different alleles at the same locus (e.g., β1β2, γ1γ2).
Although 20 ADH isoenzymes are known, which vary in their catalytic properties, relevant functional polymorphism has been found only for the beta and gamma subunits forming ADH2 and ADH3 [12]. Up to now no allelic polymorphism has been reported in human populations for the α-, π- and χ-subunits of ADH [8]. It has been suggested that the ADH variants may be involved in different attitude to alcoholism, since allele frequencies differ between alcoholics and controls [13].
The ADH2 gene may be present as ADH2*1, ADH2*2 and ADH2*3 encoding for β1, β2, and β3 subunit, respectively, which differ by single nucleotide exchanges; however, the difference of a single amino acid determines in the protein quite different catalytic properties. The enzyme containing the β1 subunit has high affinity and low capacity for ethanol, whereas the β2 and the β3 forms show lower affinity and higher capacity: the Vmax of β2 homodimers is around 40-fold higher than that of β1 homodimers. As a consequence, the activities related to β2 and β3 subunits are not highly capacity-limited by large amount of ethanol ingestion.
ADH3 (encoding the γ subunits) is also polymorphic; however, the functional meaning of these variants is limited, since the γ1 homodimers (encoded by ADH3*1) have an only two-fold higher Vmax than the one measured for γ2 homodimers (encoded by ADH3*2) [14].
Different tissues show differentially measurable human ADH gene expression; liver contains a large amount of ADH (representing about 3% of total soluble proteins in the hepatocyte) and expresses the widest number of isozymes, mainly class I. ADH5 (χ-ADH) is ubiquitously expressed in all human tissues tested so far; ADH4 (π-ADH) is solely expressed in liver, while ADH7 (σ-ADH) is the only isoform expressed at low level in the liver [6], but present at significant amounts in gastrointestinal tissue, mainly in the gastric mucosa of Caucasian, but nearly absent in Asians [15]. Similarly, a low ADH activity has been demonstrated in the gastric mucosa of females of Caucasian origin [16]. This feature has been associated with the lower gastric first pass effect in the toxicokinetics of ethanol observed both in Asian populations and females, as well as with ethanol decreased clearance and consequently with increased alcohol blood levels which may contribute to the higher susceptibility of females to ethanol-induced effects.
The actual role of ADH in alcohol related pathologies has not been elucidated yet. It is not clear the mechanism according to which heavy drinkers with liver damage and other gastrointestinal disorders show a higher serum ADH activity [17], in spite of an equal rate of ethanol metabolism with respect to controls. Although some correlation between ADH polymorphisms and ethanol susceptibility has been observed, the effect of ADH variants on the risk of alcoholic liver disease could be complex: high activity-ADH variants decrease alcoholism risk in carrying individuals, but if they persist in drinking, the risk for hepatic injury might increase, resulting from high intrahepatic concentrations of acetaldehyde [18].
The prevalence of the variant forms of ADH vary in different ethnic populations: 95% of Caucasians have the β1 enzyme form (ADH2*1); the 90% Orientals have the β2 form (ADH2*2) which is present in Europe with frequency varying from 5–10% of the English up to 20% of the Swiss population; the β3 form (ADH2*3) is present in the 24% Africans and African-Americans [14]. Analogously for ADH3*1 and ADH3*2: 90% of Asians and 50% of Caucasians have the γ1 and γ2 form, respectively [19]. In Table 1, the molecular basis of polymorphic changes in various ADH alleles and the effect on enzyme activity are summarized.
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