**2. Metabolism of alcohol**

232 Pharmacology

comorbid conditions. The body adapts metabolically and neurally to repeated exposure of

Recent advances in the field of neurobiology have improved our understanding about associated risk factors and neurochemical mechanisms responsible for the development of alcoholism. Evidences suggest that there is large inter-individual variation in terms of development of alcohol dependence and treatment of alcoholism. People consume alcohol and respond to its effects in a number of ways e.g. some develop no side effects even in moderate to higher levels and some may develop problems even when consumed in smaller doses. This variation is the result of individual's genetic makeup directly influencing the

Genetic factors have been found to play a critical role in the etiology of alcoholism (Heath et al., 2001; Sloan et al., 2008; Kenneth et al., 2011). Researchers have suggested that 50-60% of alcohol dependence is determined by genetics (Goldman and Bergen 1998; McGue et al., 1999). Based on results of adoption, twin, and family studies it is now clear that the vulnerability to alcoholism is determined by genetic factors as well as by environmental factors (Moussas et al., 2009). However, it is difficult to determine the individual determinant of alcoholism (Flensborg-Madsen et al., 2007). The candidate gene approach has revealed a number of biomarkers, which are responsible for alcoholism. Certain variants of alcohol dehydrogenase and aldehyde dehydrogenase genes (genes encoding for alcohol metabolizing enzymes) have been found to alter the metabolism of alcohol in a dramatic way (Nurnberger et al., 2004). In addition to this, polymorphisms in neurotransmitter genes (target receptor genes) such as gamma amino butyric acid and opioid receptor genes have also been reported to be associated with marked risk of alcohol dependence (Strat et al., 2008). Current treatment approaches to alcoholism are moderately effective with perhaps as many as half of the patients receiving treatment due to abstinent or significantly reducing episodes of binge drinking (Group, 1997). Pharmacotherapy and behavioral therapy including psychosocial support are two main types of treatment in alcoholism. The pharmacological agents approved by FDA prescribed in the treatment of alcoholism are disulfiran (antabuse), naltrexone (revia), acamprosate (campral)

The major drawback of ineffectiveness of pharmacotherapy of alcoholism is inter-individual variation in response to medication (Radel and Goldman, 2001). There are individuals, showing lesser/no therapeutic efficacy of a drug prescribed, known as non-responders. Another group of individuals showing high therapeutic efficacy towards the same drug are

Recent advances in the area of molecular biology have increased our knowledge of understanding the influence of genetic variants on pharmacokinetic and pharmacodynamic profile of alcohol and neurobiology of alcoholism (Ray et al., 2010a). The unavoidable alcohol withdrawl symptoms, depression, unpredicted death, medical complications, socioeconomic repercussions of alcoholism suggest that the treatment strategies should be

Pharmacogenetics is a measure of predicting individual's genetic profile responsible for variable drug responses. The genetic analysis along with consideration of other factors of alcoholic patients can lead to the identification of clinical subtypes of patients with specific

alcohol so as to develop tolerance (Zaleski et al., 2004).

metabolism of alcohol (Strat et al., 2008).

and Vivitrol (Krishnan-Sarin & O'Malley et al., 2008).

improved with new and targeted approach of pharmacogenetics.

known as responders (McLeod et al., 2000).

Alcohol is generally taken orally, absorbed unchanged through the whole length of digestive tract. Almost 20% absorption takes place rapidly through stomach and 80% through small gut (Caballeria, 2003). The rate of absorption depends on volume, concentration, nature of alcoholic drink, presence and absence of food in stomach, permeability of gastric and intestinal tissues and genetic variation. After absorption into the blood-stream, alcohol is distributed quickly throughout the total body fluid (Pawan, 1972). The distribution of alcohol is accelerated by vascularization and blood flow e.g. organs rich in blood supply such as brain and lungs achieve the higher initial concentrations of alcohol.

Liver is the main site of alcohol metabolism. In hepatocytes three systems are involved in alcohol metabolism located in three different cellular compartments. These are alcohol dehydrogenase (*ADH*) located in cytosol, microsomal ethanol oxidizing system (*MEOS*) situated in endoplasmic reticulum and catalase in peroxisomes (Caballeria, 2003). These are involved in conversion of alcohol to acetaldehyde (Figure 1).

The metabolic pathway involves conversion of alcohol (ethanol) to acetaldehyde via oxidation catalyzed by *ADH* in cytoplasm of hepatocytes, a rate limiting step. The second reaction is catalyzed by aldehyde dehydrogenases (*ALDH*), acetaldehyde is converted to acetic acid and finally to carbon dioxide and water through citric acid cycle into circulation. Acetaldehyde plays central role in the toxicity produced by alcohol consumption as in liver it reaches to saturation point and escapes into blood circulation. Further it impairs mitochondrial functions and reactions leading to damage of hepatocytes. The rate of metabolism of alcohol differs from person to person because it is influenced by genetic variants of metabolizing enzymes mentioned above (Quertemont, 2004).

#### **2.1 Alcohol dehydrogenase system**

Alcohol dehydrogenase (ADH) occurs in multiple forms and is encoded by 7 different genes. These are ADH1A, ADH1B, ADH1C, ADH4, ADH5, ADH6 and ADH7. These genes are aligned along a small region of chromosome 4. ADH enzymes encoded by ADH gene function as dimers i.e. the active forms are composed of two subunits. On the basis of their similar amino acid sequences and kinetic properties, these seven ADH types have been divided into five classes. The class I genes ADH1A, ADH1B and ADH1C are closely related. These encode for α, β and γ subunits respectively, which form homodimers or heterodimers and account for most of the alcohol oxidizing capacity in liver (Hurley et al., 2002; Lee et al.,

Pharmacogenetics – A Treatment Strategy for Alcoholism 235

Apart from ADH which accounts for greater part of ethanol oxidation, a quantitatively small portion of alcohol is catalyzed by microsomal ethanol oxidizing system involving CYP2E1 (Edenberg, 2007). Studies have shown that CYP2E1 is induced by high ethanol concentration and by chronic intake of alcohol or ethanol (up to 10 fold) (Quertemont, 2004). It has been found that after chronic ethanol consumption CYP2E1 increases the rate of ethanol clearance and this may result in development of ethanol tolerance (Lieber et al., 1968; 1988; Takahashi et al., 1993; Tsutsumi et al., 1989). CYP2E1 induction may further lead to higher

Catalase oxidizes alcohol to acetaldehyde within the peroxisomes (Oshino et al., 1973). This reaction is hydrogen peroxide (H2O2) dependent. Under normal conditions catalase plays a minor role in ethanol oxidation. However, the functional activity of catalase is accelerated in the presence of reactive oxygen species and H2O2 (Quertemont, 2004). Zimatkin et al. (1997) have suggested that catalase may be one of alternative metabolic pathways for ethanol oxidation in brain where CYP2E1 and ADH appear to be of minor importance. However,

Apart from oxidative metabolism of alcohol, nonoxidative metabolism also takes place in organs lacking oxidative metabolism such as heart (Beckemeier et al., 1998). A minor extent of alcohol is metabolized by nonoxidative pathway using enzyme fatty acid ethyl synthases resulting in the formation of fatty acid ethyl esters (Caballeria 2003). Further, these esters have been found to be involved in alcohol-induced organ injuries (Beckemeier et al., 1998).

Genes encoding for alcohol metabolizing enzymes are supposed to have major influence on development of alcoholism. There are multiple ADH and ALDH enzymes encoded by different genes. Some of these genes have been reported to occur in several variants or alleles. The enzymes encoded by different alleles can differ in the rate at which they

Researchers have studied the genetic variants of ADH1B and ADH1C genes that result in the production of enzymes with different kinetic properties and have been implicated in the susceptibility to develop alcoholism. These genetic variants or SNPs and their effects have been widely studied in different populations and three different alleles have been reported which alter the amino acid sequence of the encoded beta subunit. ADH1B\*1 allele, (reference allele) encodes for β1 subunit that has arginine at positions 48 and 370. ADH1B\*1 is the predominant allele in most populations. ADH1B\*2 encodes for β2 subunit with histidine at position 48 and is commonly found in Asians. ADH1B\*3 encodes for β3 subunit that has cysteine at position 370 and is prevalent in people of African descent. In β2 and β3 subunits,

**2.3 Microsomal ethanol oxidizing enzymes** 

**2.5 Nonoxidative ethanol metabolism** 

metabolize ethanol (Edenburg, 2007).

**3.1 Genetic variants of alcohol dehydrogenase** 

**2.4 Catalase** 

concentrations of acetaldehyde leading to injuries in hepatocytes.

the precise role of catalase in brain ethanol oxidation is still not clear.

**3. Genetic variants affecting alcohol metabolism** 

2006). Further, ADH1A, ADH1B and ADH1C are mainly present in liver and linings of stomach. ADH4 encodes π-ADH which has been reported to contribute significantly to ethanol oxidation at higher concentration. The ADH5 gene encodes for χ-ADH, a ubiquitously expressed formaldehyde dehydrogenase, which has low affinity for ethanol. ADH6 mRNA is found in fetal and adult liver. Since the enzyme has not been isolated from tissues so far, therefore little is known about it. ADH7 encodes for σ-ADH, which oxidizes both ethanol and retinol (Edenberg, 2007).

#### **2.2 Aldehyde dehydrogenase**

These enzymes rapidly convert acetaldehyde to acetate using cofactor NAD+ via oxidation. ALDH is divided into nine major categories. Some of these are significantly involved in acetaldehyde metabolism, and others metabolize a variety of substrates. Two main ALDH enzymes reported to be involved in metabolization of acetaldehyde during the oxidation of ethanol are ALDH1 and ALDH2. ALDH1 encoded by ALDH1A1 gene is found in fluid filling cells (the cytosol) while ALDH2 is found in mitochondria and is encoded by the ALDH2 gene. The two genes are 52 kb and 43 kb in length and are present on chromosome 9 and chromosome 12 respectively. Both genes have a similar structure with 13 exons and the protein encoded by both the genes is 70% similar in sequence and structure (Hurley et al., 2002). ALDH1A1, ALDH1B1 and ALDH2 are mainly involved in acetaldehyde oxidation.

Fig. 1. Metabolism of alcohol in liver hepatocytes using 3 systems, (i) Alcohol dehydrogenase, (ii) Microsomal ethanol oxidizing enzyme (iii) Catalase and finally Aldehyde dehydrogenase converts acetaldehyde into acetate

#### **2.3 Microsomal ethanol oxidizing enzymes**

Apart from ADH which accounts for greater part of ethanol oxidation, a quantitatively small portion of alcohol is catalyzed by microsomal ethanol oxidizing system involving CYP2E1 (Edenberg, 2007). Studies have shown that CYP2E1 is induced by high ethanol concentration and by chronic intake of alcohol or ethanol (up to 10 fold) (Quertemont, 2004). It has been found that after chronic ethanol consumption CYP2E1 increases the rate of ethanol clearance and this may result in development of ethanol tolerance (Lieber et al., 1968; 1988; Takahashi et al., 1993; Tsutsumi et al., 1989). CYP2E1 induction may further lead to higher concentrations of acetaldehyde leading to injuries in hepatocytes.

#### **2.4 Catalase**

234 Pharmacology

2006). Further, ADH1A, ADH1B and ADH1C are mainly present in liver and linings of stomach. ADH4 encodes π-ADH which has been reported to contribute significantly to ethanol oxidation at higher concentration. The ADH5 gene encodes for χ-ADH, a ubiquitously expressed formaldehyde dehydrogenase, which has low affinity for ethanol. ADH6 mRNA is found in fetal and adult liver. Since the enzyme has not been isolated from tissues so far, therefore little is known about it. ADH7 encodes for σ-ADH, which oxidizes

These enzymes rapidly convert acetaldehyde to acetate using cofactor NAD+ via oxidation. ALDH is divided into nine major categories. Some of these are significantly involved in acetaldehyde metabolism, and others metabolize a variety of substrates. Two main ALDH enzymes reported to be involved in metabolization of acetaldehyde during the oxidation of ethanol are ALDH1 and ALDH2. ALDH1 encoded by ALDH1A1 gene is found in fluid filling cells (the cytosol) while ALDH2 is found in mitochondria and is encoded by the ALDH2 gene. The two genes are 52 kb and 43 kb in length and are present on chromosome 9 and chromosome 12 respectively. Both genes have a similar structure with 13 exons and the protein encoded by both the genes is 70% similar in sequence and structure (Hurley et al., 2002). ALDH1A1, ALDH1B1 and ALDH2 are mainly involved in acetaldehyde oxidation.

Fig. 1. Metabolism of alcohol in liver hepatocytes using 3 systems, (i) Alcohol dehydrogenase, (ii) Microsomal ethanol oxidizing enzyme (iii) Catalase and finally

Aldehyde dehydrogenase converts acetaldehyde into acetate

both ethanol and retinol (Edenberg, 2007).

**2.2 Aldehyde dehydrogenase** 

Catalase oxidizes alcohol to acetaldehyde within the peroxisomes (Oshino et al., 1973). This reaction is hydrogen peroxide (H2O2) dependent. Under normal conditions catalase plays a minor role in ethanol oxidation. However, the functional activity of catalase is accelerated in the presence of reactive oxygen species and H2O2 (Quertemont, 2004). Zimatkin et al. (1997) have suggested that catalase may be one of alternative metabolic pathways for ethanol oxidation in brain where CYP2E1 and ADH appear to be of minor importance. However, the precise role of catalase in brain ethanol oxidation is still not clear.

#### **2.5 Nonoxidative ethanol metabolism**

Apart from oxidative metabolism of alcohol, nonoxidative metabolism also takes place in organs lacking oxidative metabolism such as heart (Beckemeier et al., 1998). A minor extent of alcohol is metabolized by nonoxidative pathway using enzyme fatty acid ethyl synthases resulting in the formation of fatty acid ethyl esters (Caballeria 2003). Further, these esters have been found to be involved in alcohol-induced organ injuries (Beckemeier et al., 1998).
