Acetaldehyde, the toxic metabolite produced by enzymatic ethanol oxidation in the human liver, is further metabolized by ALDH in a NAD+-dependent reaction. These enzymes have broad substrate specificity for aliphatic and aromatic aldehydes, which are irreversibly oxidized to their corresponding carboxylic acids. The ALDH are cytosolic enzymes, expressed in a wide range of tissues [8]. A number of isoenzymes of ALDH coded by different gene loci have been detected in humans, which differ in their electrophoretic mobility, kinetic properties, as well as in their cellular and tissue distribution and show a certain degree of overlapping substrate specificity.
Genes coding for ALDH enzymes are divided into nine major families (Table 2); the major ones are family 1 corresponding to cytosolic ALDHs (ALDH1), family 2 to mitochondrial ALDHs (ALDH2), and family 3 which groups the major constitutive and inducible ALDH forms (ALDH3) found in human stomach, saliva, and hepatocarcinoma [8]. On the basis of kinetic properties and sequence similarities, the nomenclature for ALDH proteins has been recently revised; they have been tentatively classified as class 1 (low Km, cytosolic), class 2 (low Km, mitochondrial) and class 3 (high-Km ALDH, such as those expressed in tumors, stomach and cornea) [18].
There are multiple molecular forms of ALDH in human liver, but only class I and class II isozymes, encoded by ALDH1 and ALDH2 genes, respectively, are thought to be involved in acetaldehyde oxidation [6].
Both ALDH1 and ALDH2 are homotetrameric enzymes, characterized by isoenzyme specific subunits (MW ≈54 kD each) and by different catalytic properties. The lowest Km values for acetaldehyde has been measured for the mitochondrial ALDH2 (0.2-1 µM), although also ALDH1 has a relatively high affinity for the substrate (Km value around 30 µM), consistently with their relevant involvement in ethanol oxidation process. ALDH3 and ALDH4 show a lower affinity towards both acetaldehyde and propionaldehyde as substrates: indeed their Km values are in the millimolar range (≈11 mM). NAD+ is the preferred coenzyme for the low Km isoenzymes (ALDH1 and ALDH2), whereas the high Km isoenzymes (ALDH3 and ALDH4) can use either NAD+ or NADP+.
In addition to the above mentioned genes, a number of additional genes have been cloned and characterized in humans [20]; the major ones will be briefly listed in the following.
ALDH1B (ALDH5): it is expressed in various tissues including liver, brain, adrenal gland, testis, stomach, and parotid gland. ALDH1A6 (ALDH6): this isoenzyme is primarily expressed in the salivary gland, stomach, and kidney. The cDNA encodes 512 amino acid residues and shows a 70% sequence homology with ALDH1. ALDH3B1 (ALDH7): the isoenzyme is expressed mainly in the kidneys and lungs; the cDNA encodes 468 residues with a 52% positional identity with ALDH3. ALDH3B2 (ALDH8): the gene product shows 85% homology with of ALDH7. ALDH9A1 (ALDH9): the isoenzyme has a high activity for oxidation of gamma-aminobutyraldehyde and other amino aldehydes [8].
Genetic polymorphisms have been reported in a number of ALDH genes: nucleotide changes and effect on encoded proteins are listed in table 2. Due to the major role of mitochondrial ALDH2 in acetaldehyde oxidation [21], the genetic factor that most strongly correlates with reduced ethanol consumption and incidence of alcoholism is human ALDH2 functional gene polymorphism. The enzyme is encoded by two distinct alleles in chromosome 6: ALDH2*1 (wild type allele) and ALDH2*2, differing for the substitution glutamate-to-lysine at position 487 (E487K) due to a single point mutation (transition G⇒A). Although the difference between the two alleles appears to be minimal, the proximity in the primary structure between the mutation site and the region containing cysteine residues, very likely involved in the catalytic cycle, is compatible with the phenotypic decrease in ALDH2 activity, associated with the variant genotype. Indeed, individuals homozygous for the mutated ALDH2*2 allele are completely lacking ALDH2 activity, whereas heterozygous individuals showing the ALDH2*1,2 genotype maintain about 30–50% of the ALDH activity, shown by individuals carrying wild type gene. Blood acetaldehyde levels of ALDH2*2 homozygous individuals are 6-to-20 fold higher than in ALDH2*1 gene carriers, in which acetaldehyde is hardly detectable after ethanol consumption. The acetaldehyde blood concentrations reached in individuals homozygous for ALDH2*2 cause unpleasant side-effects (flush syndrome) which protects them from alcoholism. However, heterozygous individuals may become heavy drinkers or even alcoholics, thus experiencing the toxic effects due to acetaldehyde production [22].
Orientals show the presence of the inactive ALDH2 isoenzyme phenotype in approximately 50% of the individuals [17, 23], whereas no ALDH2-deficient Caucasian or Negroid have been identified so far [24]. This is the reason why Orientals exhibit intense facial flushing after a mild dose of alcohol as compared to Caucasians, thus affecting their drinking habits. The percentage of heavy and moderate drinkers is higher among Caucasians, while abstainers and infrequent drinkers are more frequent among the Chinese and Japanese [8].
About 40% of the South American Native Indians (Mapuche, Atacamen’s, Shuara tribes) showed the presence of ALDH2*2, which on the contrary has been detected only in a very small percentage of the North American Indians (Sioux, Navajo and Mestizo) [25].
Microsomal CYP2E1 After ingestion of low amount of alcohol, about 10% of ethanol is metabolized in the liver by the microsomal cytochrome P450 CYP2E1, which catalyzes its oxidation to acetaldehyde and then to acetate [26]. During the reaction, CYP2E1 generates reactive oxygen species (ROS) such as H2O2, superoxide anion (O2−), hydroxyl (•OH) and substrate-derived radicals (1-hydroxyethyl radical), which can cause oxidative stress, triggering lipid peroxidation, protein inactivation, increased cytokine production, mitochondria and DNA damage leading to cell death [27].
Liver damage associated to ethanol consumption is hypothesized to be due at least partially to oxidative stress associated to its metabolism. Indeed alcohol-induced liver disease (ALD) has been related to the increased production of free radicals as well as to the decreased availability of antioxidants and/or impaired activity of a number of enzymatic systems able to detoxify ROS and their by-products, including Glutathione S-Transferases (GST), superoxide dismutase, glutathione peroxidase and catalase.
The potential sources of ROS in ALD are compartmentalized to (i) microsomes, via CYP2E1 and cytochrome P450 reductase; (ii) mitochondria, via the electron transport chain; (iii) peroxisomes, via fatty acid oxidases; and (iv) cytosol, via xanthine oxidase and aldehyde oxidase. However, among all the potential hepatic sources of ROS, CYP2E1 has been a center of attention for its pathogenic role in ALD [28].
In addition to ROS, the 1-hydroxyethyl radicals produced by CYP2E1 during ethanol oxidation bind covalently to proteins forming adducts able to induce autoantibodies which have been found in human alcoholics [29, 30]. These antibodies may represent markers of the production of ethanol-derived free radical adducts and contribute to the hepatotoxicity of ethanol in promoting immune mechanisms of liver injury [30].
The CYP2E1 protein is regulated both transcriptionally and post-transcriptionally through a substrate-induced protein stabilization; chronic ethanol consumption leads to an increase in CYP2E1 protein, by decreasing its degradation, without affecting its mRNA. Beside ethanol consumption other xenobiotics (acetone), a fatty diet, diabetes, obesity or starvation may lead to CYP2E1 induction, contributing to modulate ethanol metabolism.
The induction of CYP2E1 hepatic content, beside increasing ethanol clearance, has been demonstrated to positively correlate with the generation of hydroxyethyl radicals and lipid peroxidation. Consistently, induction of CYP2E1 has been shown to result in enhanced hepatic injury, whereas inhibition of CYP2E1 was associated with an improvement of these lesions [22]. In addition, increased ethanol metabolism may contribute to the development of alcohol dependence: faster ethanol inactivation during long-term alcohol drinking may increase motivation to consume more alcohol in order to maintain a desired level of ethanol at target sites [31].
The CYP2E1 gene has been localized to chromosome 10 and consists of 9 exons and 8 introns, encoding a 493-amino acid protein. Ten polymorphic loci on human CYP2E1 gene have been reported so far, most of them in the promoter and intron regions. In addition, a tandem repeat was identified in CYP2E1 regulatory region [32]. CYP2E1 gene polymorphisms are listed in Table 3.
A RsaI restriction fragment length polymorphism (RFLP) has been reported in the 5’-flanking region of the CYP2E1 gene. The rare mutant allele (c2 allele) lacking the RsaIrestriction site has been found to be associated with higher transcriptional activity, protein levels and enzyme activity than the wild-type c1 allele. Moreover, the frequency of RsaI c2 allele varies in different populations: the highest frequency has been observed in the Taiwanese (28%) and Japanese populations (19-27%), while the frequency is much lower (1-5%) in Africans [8]. The enhanced transcriptional activity of CYP2E1 c2/c2 might play a role in the development of severe ALD [33]. In individuals carrying the ADH3*2 allele, the presence of the CYP2E1 c2 allele increases the risk of ALD, presumably reflecting increased metabolism of ethanol by CYP2E1. The relevance of combination of different genotypes in modulating the risk is suggested by the fact that in the absence of the CYP2E1, ADH3 genotype itself does not influence the risk of ALD [34].
The polymorphic CYP2E1*D has been associated with greater CYP2E1 inducibility and it has been suggested to contribute to the development of alcohol and nicotine dependence. CYP2E1*1D allele contains a repeat sequence in the 5’ flanking region of the gene that may disrupt negative regulatory elements. Homo- and heterozygous individuals for CYP2E1*1D gene were found to have greater CYP2E1 activity after ethanol consumption [32].
CYP2E1 is also involved in the metabolism of various other xenobiotics, including procarcinogens, industrial and environmentally relevant small molecular weight volatile organic chemicals. Therefore, chronic ethanol consumption, leading to CYP2E1 induction, may result in the increased conversion of known hepatotoxic agents to their toxic metabolites [22], possibly explaining the enhanced susceptibility of alcoholics to the adverse effects of industrial solvents [33].
The finding of CYP2E1-mediated bioactivation of xenobiotic in prenatal human brain tissue seems of extreme interest. Significant levels of activity and specific mRNA were detectable between gestational days 45 and 53, a period during which embryogenesis overlaps with organogenesis, taking place at 50-60th days of gestation. The mRNA levels increase up to days 80-84, then remain almost constant throughout the early foetal period [35] and may be increased by ethanol itself or by its strong effect on maternal nutritional status (i.e. altered fat or vitamin A and B intake). The presence of CYP2E1 during organogenesis in the brain, the target organ of alcohol teratogenesis, has been associated with the appearance of foetal alcohol syndrome (FAS), as a result of alcohol consumption during pregnancy. FAS is characterised by a particular pattern of facial anomalies, growth retardation and developmental abnormalities in the central nervous system that could result in mental retardation. Even if FAS is not evident, some evidences indicate that adults, who had been prenatally exposed to alcohol, frequently suffer from mental disorders and maladaptive behaviours and are prone to become alcoholics themselves. Damages in the foetal brain due to alcohol consumption by the mother during gestation has been associated to the presence of many polyunsaturated fatty acid side chains in the membranes of embryonic and foetal brain, making the tissue a highly susceptible target for ROS and radicals arising from CYP2E1-mediated ethanol metabolism in situ. The damages in the brain caused by lipid peroxidative processes triggered by ROS might be manifested as the central nervous system dysfunction after birth, described as FAS, although other factors such as decreased levels of retinoic acid may act concurrently.
Glutathione S-transferases Glutathione S-transferases (GST) are phase II xenobiotic metabolizing enzymes, acting as a highly efficient detoxification system. They catalyze the conjugation of harmful electrophilic compounds with reduced glutathione (a tripeptide present at relatively high concentrations in the cytosol), to produce less toxic or readily excreted metabolites. Moreover, these enzymes have a strong antioxidant function and protect cells from the natural by-products of lipid peroxidation and oxidative stress [36].
Since the implication of ROS, generated during ethanol metabolism and by ethanol-induced cell damage, has been postulated in the etiology of alcohol-induced pathologies, GST activity may play a central role by detoxifying both ROS and other ethanol-derived free radicals, as suggested by the alteration of GST expression in the liver of patients with ALD [37].
The cytosolic GSTs are dimeric proteins with each subunit having 22-28 kD MW. On the basis of their amino acid sequence, in humans 8 families of the cytosolic forms have been identified and named with greek symbols, each class consisting of various isoenzymes; GSTs mainly involved in ROS detoxication belong to Alpha, Mu, Theta and Pi families [38].
The α-GST are very abundant hepatic homo- or heterodimers, in humans accounting for about 85% of the total GST protein. The dimerization of two distinct subunits (A1 and A2) gives rise to GSTA1-1, GSTA1-2 and GSTA2-2. Other α-GST have been localized in extrahepatic tissues, such as GSTA3 and GSTA5, mainly expressed in the skin.
Alpha, Mu and Pi GST can detoxify harmful α,β-unsaturated carbonyl, such as 4-hydroxynonenal generated by lipid peroxidation and the products of oxidative DNA damage mediated by free radicals. The alpha-GST, as well as the microsomal membrane bound GST, exhibits glutathione peroxidase activity, suggesting an additional defense mechanism against lipid peroxidation associated with ethanol consumption.
In humans, genetic polymorphisms have been described in GSTM1, GSTT1 and GSTP1 genes. Among the described polymorphisms at the GSTM1 locus on chromosome 1p13.3, the most studied encodes for a gene deletion (GSTM1 null genotype), resulting in a complete absence of GSTM1 enzyme activity. The frequency of the GSTM1 null genotype ranges from 23 to 62% in different populations and is approximately 50% in Caucasians [38].
For GSTT1 locus, located on chromosome 12q11.2, one polymorphism has been described. The GSTT1 null genotype represents a gene deletion and is associated with the absence of functional enzyme activity. The frequency of these null genotypes ranges from 16 to 64% in different populations being approximately 20% in Caucasians [39].
Although the absence of an active GST isoform may be of relevance for the total detoxifying capacity of the cell, compensation mechanisms due to the overlapping substrate specificities exhibited by different GST can limit the consequent functional impairment. However, individuals with the homozygous GSTM1 or GSTT1 null genotypes express no protein of two major human GST isoforms, highly express in the gastric and intestinal mucosa, are expected to have a reduced ability to detoxify reactive metabolites resulting from ethanol metabolism.
At least four different polymorphisms have been described at the GSTP1 locus on chromosome 11, the most important of which encoding for an enzyme with altered activity. Polymorphisms of the GSTP1 gene consists of an A-to-G transition of nucleotide 313 in exon 5 (GSTP1*B) and a C-to-T transition of nucleotide 341 in exon 6 (GSTP1*C), involving the substitution of two amino acids in the enzyme active site, Ile ⇒Val and Ala ⇒Val. These allelic variants appear to influence GSTP1 activity, therefore modulating the risks of damage [36]. The amino acid 105 is proximal to the enzymatic active site, therefore it is not surprising that this residue can modulate the catalytic activity. The same transition may occur also in position 114, but the functional consequences of this mutation have not been clarified yet.
The GSTP1Val105/Val105 polymorphism is very common and may result either in reduction or increase of the enzyme activity of the compared to the wild type form (Ile105), dependent on the electrophilic structure of the substrate [38]. As an example GSTP1 Val105/Val105 genotype has been shown to be protective against asthma symptoms, since the mutated gene is more efficient in scavenging ROS formed during the chronic inflammation process associated with the pathology, thus protecting lung cells from damages produced by oxidative stress [38].
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