Genotoxic effects of alcohol in human peripheral lymphocytes modulated by ADH1B and ALDH2 gene polymorphisms

Ethanol is almost totally broken down by oxidative metabolism in vivo. Ethanol per se is considered to be neither carcinogenic, mutagenic nor genotoxic. However, during the metabolic conversion of ethanol to acetaldehyde and acetate, the organism is exposed to both ethanol and acetaldehyde and therefore ethanol is suspected to be co-carcinogenic. The genetic polymorphisms of alcohol dehydrogenase-2 (ADH1B) and acetaldehyde dehydrogenase-2 (ALDH2) influence the metabolism of alcohol. The ADH1B*1/*1 genotype encodes the low-activity form of ADH1B, and ALDH2*1/*2 and ALDH2*2/*2 genotype encode inactive ALDH2. The aim of this study was to test the hypothesis that polymorphisms of the ADH1B and ALDH2 genes are significantly associated with genotoxicity induced by alcohol drinking, measured using the cytokinesis-block micronucleus (CBMN) assay, an established biomarker of genome instability, in peripheral blood lymphocytes of 286 healthy Japanese men. There was a significant trend for the mean micronuclei (MN) frequency in habitual or moderate drinkers without a smoking habit to increase as the numbers of the *1 allele in ADH1B increased (P=0.039 or P=0.029) and the *2 allele in ALDH2 increased (P=0.019 or P=0.037). A logistic regression analysis showed that the number of subjects with MN frequency levels more than median value of MN (3.0) was significantly higher in the subjects with the ADH1B*1 allele as adjusted estimates (OR 2.08, 95% C.I. 1.24-3.48), when the OR for the subjects with the ADH1B*2/*2 genotype was defined as 1.00. The number of subjects with MN frequency levels more than median value of MN was also significantly higher in the subjects with the ALDH2*2 allele as adjusted estimates (OR 1.79, 95% C.I. 1.04-3.11), when the OR for the subjects with the ALDH2*1/*1 genotype was defined as 1.00. The results of this study have identified important novel associations between ADH1B/ALDH2 polymorphisms and genotoxicity in alcohol drinkers.     

Formation of acetaldehyde-derived DNA adducts due to alcohol exposure

Epidemiological studies have identified chronic alcohol consumption as a significant risk factor for cancers of the upper aerodigestive tract, including the oral cavity, pharynx, larynx and esophagus, and for cancer of the liver. Ingested ethanol is mainly oxidized by the enzymes alcohol dehydrogenase (ADH), cytochrome P-450 2E1 (CYP2E1), and catalase to form acetaldehyde, which is subsequently oxidized by aldehyde dehydrogenase 2 (ALDH2) to produce acetate. Polymorphisms of the genes which encode enzymes for ethanol metabolism affect the ethanol/acetaldehyde oxidizing capacity. ADH1B*2 allele (ADH1B, one of the enzyme in ADH family) is commonly observed in Asian population, has much higher enzymatic activity than ADH1B*1 allele. Otherwise, approximately 40% of Japanese have single nucleotide polymorphisms (SNPs) of the ALDH2 gene. The ALDH2 *2 allele encodes a protein with an amino acid change from glutamate to lysine (derived from the ALDH2*1 allele) and devoid of enzymatic activity. Neither the homozygote (ALDH2*2/*2) nor heterozygote (ALDH2*1/*2) is able to metabolize acetaldehyde promptly. Acetaldehyde is a genotoxic compound that reacts with DNA to form primarily a Schiff base N²-ethylidene-2′-deoxyguanosine (N²-ethylidene-dG) adduct, which may be converted by reducing agents to N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG) in vivo, and strongly blocked translesion DNA synthesis. Several studies have demonstrated a relationship between ALDH2 genotypes and the development of certain types of cancer. On the other hand, the drinking of alcohol induces the expression of CYP2E1, resulting in an increase in reactive oxygen species (ROS) and oxidative DNA damage. This review covers the combined effects of alcohol and ALDH2 polymorphisms on cancer risk. Studies show that ALDH2*1/*2 heterozygotes who habitually consume alcohol have higher rates of cancer than ALDH2*1/*1 homozygotes. Moreover, they support that chronic alcohol consumption contributes to formation of various DNA adducts. Although some DNA adducts formation is demonstrated to be an initiation step of carcinogenesis, it is still unclear that whether these alcohol-related DNA adducts are true factors or initiators of cancer. Future studies are needed to better characterize and to validate the roles of these DNA adducts in human study.     

The ALDH2 genotype, alcohol intake, and liver-function biomarkers among Japanese male workers

A highly prevalent, atypical genotype in low Km aldehyde dehydrogenase (ALDH2) may influence alcohol-induced liver injury because of higher production of acetaldehyde in the liver. In the present study, we examined relationships between the ALDH2 genotype, alcohol intake, and liver-function biomarkers among Japanese male workers. Study subjects were 385 male workers in a metal plant in Japan, who were free from hepatic viruses and did not have higher aminotransferase activities (<100). The subjects completed a questionnaire on alcohol drinking habits and other lifestyles. The ALDH2 genotype was determined by the PCR method followed by restriction-enzyme digestion. In the moderately and heavily drinking groups, those with ALDH2*1/*2 exhibited significantly lower levels than those with ALDH2*1/*1 for all three parameters of liver function, whereas no such differences were observed in the least-drinking group. Multiple linear-regression analysis, adjusting for age, obesity, and smoking habits, revealed that aspartate aminotransferase activity was positively associated with alcohol intake only in those with ALDH2*1/*1. On the other hand, alanine transferase activity was negatively associated with alcohol intake only in those with ALDH2*1/*2. The present study indicates that effects of alcohol intake on liver-function biomarkers are likely to be modified by the ALDH2 genotype in adult males.     

Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse

A principal pathway of 2-methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2-/-) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype.     

Alcohol and aldehyde dehydrogenase genotypes and alcoholism in Chinese men.

The liver enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), which are responsible for the oxidative metabolism of ethanol, are polymorphic in humans. An allele encoding an inactive form of the mitochondrial ALDH2 is known to reduce the likelihood of alcoholism in Japanese. We hypothesized that the polymorphisms of both ALDH and ADH modify the predisposition to development of alcoholism. Therefore, we determined the genotypes of the ADH2, ADH3, and ALDH2 loci of alcoholic and nonalcoholic Chinese men living in Taiwan, using leukocyte DNA amplified by the PCR and allele-specific oligonucleotides. The alcoholics had significantly lower frequencies of the ADH2*2, ADH3*1, and ALDH2*2 alleles than did the nonalcoholics, suggesting that genetic variation in both ADH and ALDH, by modulating the rate of metabolism of ethanol and acetaldehyde, influences drinking behavior and the risk of developing alcoholism.     

Single nucleotide polymorphism genotyping of aldehyde dehydrogenase 2 gene using a single bacterial magnetic particle

A single nucleotide polymorphism (SNP) genotyping for aldehyde dehydrogenase 2 gene (ALDH2) has been developed by using a nano-sized magnetic particle, which was synthesized intracellularly by magnetic bacteria. Streptavidin-immobilized on bacterial magnetic particles (BMPs) were prepared using biotin labeled cross-linkers reacting with the amine group on BMPs. ALDH2 fragments from genomic DNA were amplified using a TRITC labeled primer and biotin labeled primer pair, and conjugated onto BMP surface by biotin-streptavidin interaction. PCR product-BMP complex was observed at a single particle level by fluorescence microscopy. These complexes were treated with restriction enzyme, specifically digesting the wild-type sequence of ALDH2 (normal allele of ALDH2). The homozygous (ALDH2*1/*1), heterozygous (ALDH2*1/*2), and mutant (ALDH2*2/*2) genotypes were discriminated by three fluorescence patterns of each particle. SNP genotyping of ALDH2 has been successfully achieved at a single particle level using BMP.     

Polymorphisms of alcohol metabolizing enzyme and cytochrome P4502E1 genes in mongolian population

Although the genetic polymorphism of the alcohol-metabolizing enzymes was extensively studied at the molecular level by many investigators, the genetic polymorphism studies for ethanolmetabolizing enzymes in Mongolians are very rare. The present study was therefore performed to determine the genetic distribution of various forms of alcohol-metabolizing enzymes such as alcohol dehydrogenase 2 (ADH2, currently accepted nomenclature ADH1B), ADH3 (ADH1C), aldehyde dehydrogenase 2 (ALDH2) and cytochrome P4502E1 (CYP2E1) in 300 healthy Mongolian males. Genetic polymorphisms were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. The allele frequencies ofADH2 *1 andADH2 *2 were 0.24 and 0.76;ADH3 *1 andADH3 *2 were 0.92 and 0.08;ALDH2 *1 andALDH2 *2 were 0.96 and 0.04; andCYP2E1 *C andCYP2E1 *D were 0.15 and 0.85, respectively. Compared to the results reported by other investigators, the allele frequencies ofALDH2 *2 andCYP2E1 *C among Mongolian subjects were much lower than among East Asians (Korean, Japanese, and/or Han-Chinese), while those ofADH2 andADH3 were more similar. Interestingly, this study shows that the ineffectiveALDH2 gene (ALDH2*2 allele) among Mongolians is not as common as among East Asians.     

乙醛脱氢酶2（ALDH2）基因研究进展及其与饮酒行为的关系

亚洲人群中普遍存在突变型的乙醛脱氢酶2（ALDH2*2）。此酶突变后活性缺失,导致乙醛在肝脏内大量累积使突变携带者在喝酒后会有脸红等不适反应,因此这可能影响他们的饮酒行为。由于ALDH2*2等位基因与饮酒行为相关,它也可能与酒精引起的肝脏损伤及某些癌症密切相关,而且,它在不同的亚洲人群中有不同的频率分布。近年来对ALDH2*2等位基因的序列结构、表达及其重要功能等有了更深入的了解,对ALDH2的多态性在研究方法、研究群体分布范围等都有很大进展。本文还讨论了不同地理分布、不同年龄结构、性别差异条件下,中国人群中ALDH2基因型频率与饮酒行为的关系。 Abstract: An atypical allele （ALDH2*2） in low Km aldehyde dehydrogenase (ALDH2), which is highly prevalent in Asian, may influence drinking behavior because of higher production of acetaldehyde in the liver. High alcohol sensitivity such as flushing after drinking has been shown to be mainly due to the atypical ALDH2 genotypes. The atypical allele is associated with alcohol-induced liver injury and some cancers. Recently, the researches on the polymorphisms not only in the gene itself but also its frequencies in different Asian populations have been made great progress. Three factors, including different sex, age and geography, were also analyzed with the genotypes of ALDH2 in Chinese populations.     

CYP1A1 *2B and *4 polymorphisms are associated with lung cancer susceptibility in Mexican patients

BACKGROUND: CYP1A1 is a gene involved in the high aryl hydrocarbon hydroxylase -inducible phenotype, which is a genetically-determined variation among individuals that has been associated with lung cancer risk. More specifically, CYP1A1 *2B and *4 polymorphisms have been associated with high susceptibility to lung cancer among cigarette smokers. MATERIALS AND METHODS: DNA was obtained from blood samples and we studied by PCR-RFLP the distribution of CYP1A1 *2B (n=248) and *4 (n=222) polymorphisms in healthy controls and 222 lung cancer patients from a Mexican population. RESULTS: Comparisons between groups showed an increased risk for lung cancer patients of *2B/*2B (18%; OR 7.6; 95% CI 3.0-19.2) and *4/ *4 genotypes (15%; OR 11.45; 95% CI 2.19-59.85) compared to the control group (1% for *2B/ *2B and 4.4% for *4/ *4). A significant association between lung cancer and homozygous *2B/ *2B passive smokers and *4/*4 ever (cigarettes) and passive smokers was also observed (p<0.05). Multivariate analysis revealed an increased risk for the *2B/*2B genotype (OR 6.83), as well as for *4/*4 (OR 28.8). CONCLUSION: The results of the study indicate a significant association between *2B/*2B and *4/*4 genotypes and the risk of developing lung cancer among Mexicans.     

CYP1A1, CYP2D6, CYP2E1, NAT2, GSTM1 and GSTT1 polymorphisms or their combinations are associated with the increased risk of the laryngeal squamous cell carcinoma

Polymorphisms in the selected genes controlling carcinogen metabolism (CYP1A1, CYP2D6, CYP2E1, NAT2, GSTM1, GSTT1) considered separately or in different combinations, were investigated for an association with tobacco smoke-associated squamous cell carcinoma (SCC) of the larynx. The case-control study was performed in 289 patients with laryngeal SCC and in 316 cancer-free controls; all were Caucasian males from the same region of Poland and current tobacco smokers. The DNA samples were genotyped using PCR-RFLP and multiplex PCR. The variants' frequencies in both groups were compared; odds ratios and their 95% confidence intervals were calculated by logistic regression analyses. The CYP1A1*1/*4, CYP2D6*4/*4, NAT2*4/*6A genotypes, as well as the CYP1A1*4, CYP2D6*4 and NAT2*4 alleles, were found at significantly higher frequencies in cases than in controls indicating their role as "risk-elevating" factors in laryngeal SCC. Combined genotypes, characterized by the presence of the "risk-elevating" variants at more than one locus, often occurred together with the null variant of the GSTM1 gene and homozygous XPD A/A (Lys751Gln, A35931C) genotype. Furthermore, we identified some "protective" variants, found more frequently in controls than in cases, i.e. the NAT2*6A/*6A and NAT2*5B/*6A genotypes. A distribution of "risk" or "protection" genotypes/alleles seems to be connected with age as an occurrence or risk genes was more frequent in the group of "young" cases (< or = 49 years). Accumulation of certain alleles or genotypes of the CYP1A1, NAT2, GSTM1 and XPD seems to be associated with either increased or decreased risk to develop laryngeal SCC. Therefore, polymorphisms in these genes may play a role in the laryngeal cancer etiology.     

Evidence of association of the CYP2E1 genetic polymorphism with micronuclei frequency in human peripheral blood

Micronuclei (MN) are used as one of the cytogenetic biomarkers, and intra- and inter-individual variations in this frequency have been reported in human blood lymphocytes. Polymorphisms in a few metabolic enzyme genes seem to account for a proportion of this variability, but the impacts of specific genetic variants on the MN frequency have not yet been clarified. Here, we investigated the relationship between the MN frequency and several gene polymorphisms in 90 healthy Japanese men. The subjects with the CYP2E1(*)3 variant allele had a statistically lower mean MN frequency than subjects with the CYP2E1(*)1/(*)1 wild type. Furthermore, the adjusted odds ratio (OR) of the CYP2E1(*)3 variant with higher MN frequency levels was also significantly lower and calculated to be 0.25 (95% CI 0.07-0.83), when the OR for the subjects with the CYP2E1(*)1/(*)1 wild type was defined as 1.00. These data suggest that the CYP2E1(*)3 polymorphism may have the potential to influence the baseline frequency of MN.     

Polymorphism of aldehyde dehydrogenase and alcohol sensitivity

The metabolism of acetaldehyde has received considerable attention in the past years owing to its acute and chronic toxic effects in humans. Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of acetaldehyde in liver and other organs. Two major isozymes of hepatic ALDH (ALDH I or E2 and ALDH II or E1), which differ in their structural and functional properties, have been characterized in humans. The ALDH I with a low Km for acetaldehyde is predominantly of mitochondrial origin and ALDH II which has a relatively higher Km is of cytosolic origin. An inherited deficiency of ALDH I isozyme has been found among Japanese and Chinese which is primarily responsible for producing acute alcohol sensitivity symptoms (flushing response) after drinking mild doses of alcohol. Biochemical, immunochemical and molecular genetics data indicate a structural mutation in the ALDH I isozyme gene responsible for the loss in catalytic activity. Population genetic studies indicate a wide prevalence of this ALDH polymorphism among individuals of Mongoloid race. Flushing response to alcohol shows familial resemblances and preliminary family data from Japan, China and Korea hint to an autosomal codominant inheritance for ALDH I isozyme deficiency. The ALDH polymorphism is apparently responsible for the low incidence of alcoholism in Japanese, Chinese and Koreans. Alcohol-induced sensitivity due to ALDH isozyme deficiency may act as an inhibitory factor against excessive alcohol drinking thereby imparting a protection against alcoholism.     

Association of genetic polymorphisms of alcohol-metabolizing enzymes with excessive alcohol consumption in Japanese men

To evaluate the independent and interactive contributions of alcohol dehydrogenase-2 (ADH2), aldehyde dehydrogenase-2 (ALDH2) and ethanol-induced isozyme cytochrome P450-2E1 (CYP2E1) genes to alcohol consumption large enough to induce health problems, 643 healthy Japanese men aged between 23 and 64 years, recruited from two different occupational groups, were analyzed for genotype and drinking habits. The frequency of excessive alcohol consumers (EAC) who drank 90 ml or more alcohol more than 3 days a week was significantly higher in subjects possessing the ALDH2(1)/ALDH2(1) genotype than in those having ALDH2(1)/ALDH2(2) or ALDH2(2)/ALDH2(2) genotypes. A significant difference was also found in the different genotypes of CYP2E1. Moreover, a borderline significant interaction between the ALDH2 and CYP2E1 genotypes on excessive alcohol consumption was observed, i.e., the group of subjects having the c2 allele of CYP2E1 had a higher frequency of EAC than those having c1/c1 genotypes in the genotype subgroup ALDH2(1)/ALDH2(1), whereas these were not found in the heterozygote and homozygote subgroups of the ALDH2(2) allele. Neither the independent nor interactive genetic effect of ADH2 on excessive alcohol consumption was obvious. In conclusion, Japanese men with the ALDH2(1)/ALDH2(1) genotype and the c2 allele of CYP2E1 are at higher risk of showing excessive alcohol consumption.     

Genotypes of alcohol-metabolizing enzymes in Japanese with alcohol liver diseases: a strong association of the usual Caucasian-type aldehyde dehydrogenase gene (ALDH1(2)) with the disease

Genetic polymorphisms of two major alcohol-metabolizing enzymes-i.e., one of the class I alcohol dehydrogenase isozymes (ADH2) and the mitochondrial aldehyde dehydrogenase (ALDH2)-exist in Japanese and other Orientals but not in Caucasians. Liver ADH activity of about 90% of Orientals is much higher than that of most Caucasians, while approximately 50% of Orientals lack the ALDH2 activity. The genetic differences have been implicated in the high incidence of alcohol sensitivity observed in Orientals. We determined, by means of hybridization of genomic DNA samples with allele-specific synthetic oligonucleotide probes, genotypes of the ADH2 and the ALDH2 loci of Japanese with alcoholic liver diseases and of control subjects. No significant difference between the patient and control groups was found in the ADH2 genotypes. A remarkable genetic difference between the two groups was found in the ALDH2 locus. The frequency of the typical (Caucasian-type) ALDH1(2) gene was found to be .65 and that of the atypical (Oriental type) ALDH2(2) gene was .35 in the controls, while these were .93 and .07, respectively, in the patients. Thus, most (20 of 23) of the Japanese patients were homozygous Caucasian type ALDH1(2)/ALDH1(2), only three were heterozygous ALDH1(2)/ALDH2(2), and none of the patients were homozygous Oriental type ALDH2(2)/ALDH2(2). The results indicate that Japanese with the atypical ALDH2(2) allele are at a much lower risk in developing the alcoholic liver diseases than are those with homozygous, usual (Caucasian-type) ALDH1(2)/ALDH1(2), presumably owing to their sensitivity to alcohol intoxication.     

Interaction between the functional polymorphisms of the alcohol-metabolism genes in protection against alcoholism.

The genes that encode the major enzymes of alcohol metabolism, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), exhibit functional polymorphism. The variant alleles ADH2*2 and ADH3*1, which encode high-activity ADH isoforms, and the ALDH2*2 allele, which encodes the low-activity form of ALDH2, protect against alcoholism in East Asians. To investigate possible interactions among these protective genes, we genotyped 340 alcoholic and 545 control Han Chinese living in Taiwan at the ADH2, ADH3, and ALDH2 loci. After the influence of ALDH2*2 was controlled for, multiple logistic regression analysis indicated that allelic variation at ADH3 exerts no significant effect on the risk of alcoholism. This can be accounted for by linkage disequlibrium between ADH3*1 and ADH2*2 ALDH2*2 homozygosity, regardless of the ADH2 genotypes, was fully protective against alcoholism; no individual showing such homozygosity was found among the alcoholics. Logistic regression analyses of the remaining six combinatorial genotypes of the polymorphic ADH2 and ALDH2 loci indicated that individuals carrying one or two copies of ADH2*2 and a single copy of ALDH2*2 had the lowest risk (ORs 0.04-0.05) for alcoholism, as compared with the ADH2*1/*1 and ALDH2*1/*1 genotype. The disease risk associated with the ADH2*2/*2-ALDH2*1/*1 genotype appeared to be about half of that associated with the ADH2*1/*2-ALDH2*1/*1 genotype. The results suggest that protection afforded by the ADH2*2 allele may be independent of that afforded by ALDH2*2.     

Genotype frequencies of polymorphic GSTM1, GSTT1, and cytochrome P450 CYP1A1 in Mexicans

The genotype frequencies of three metabolic polymorphisms were determined in a sample of a typical community in central Mexico. CYP1A1*3, GSTM1, and GSTT1 polymorphisms were studied in 150 donors born in Mexico and with Mexican ascendants; with respect to ethnicity the subjects can be considered Mestizos. PCR reactions were used to amplify specific fragments of the selected genes from genomic DNA. An unexpected 56.7% frequency of the CYP1A1*3 allele (which depends on the presence of a Val residue in the 462 position of the enzyme, instead of Ile) was found, the highest described for open populations of different ethnic origins (i.e., Caucasian, Asian, African, or African American). The GSTM1 null genotype was found with a frequency of 42.6%, which is not different from other ethnicities, whereas the GSTT1 null genotype had a frequency of 9.3%, one of the lowest described for any ethnic group but comparable to the frequency found in India (9.7%). The frequency of the combined genotype CYP1A1*3/*3 and the GSTM1 null allele is one of the highest observed to date (or perhaps the highest): 13.7% among all the ethnicities studied, including Caucasians and Asians, whereas the combination of CYP1A1*3/*3 with the GSTT1 null allele reached only 2.8%. The GSTM1 null allele combined with the GSTT1 null allele, on the other hand, has one of the lowest frequencies described, 4.24%, comparable to the frequencies found in African Americans and Indians. Finally, the combined CYP1A1*3/*3, GSTM1 null allele, and GSTT1 null allele genotype could not be found in the sample studied; it is assumed that the frequency of carriers of these combined genotypes is less than 1%. CYP1A1*3 and CYP1A1*2 polymorphisms were also evaluated in 50 residents in a community of northern Mexico; the CYP1A1*3 frequency was 54%, similar to that found in the other community studied, and the CYP1A1*2 frequency was 40%, which is high compared to Caucasians and Asians but comparable to the frequency found in Japanese and lower than the frequency found in Mapuche Indians. Haplotype frequencies for these CYP1A1 polymorphisms were estimated, and a linkage disequilibrium value (D) of 0.137 was calculated.     

Prevalence of CYP2C9 and VKORC1 alleles in the Argentine population and implications for prescribing dosages of anticoagulants

Dicumarinic oral anticoagulants have a narrow therapeutic range and a great individual variability in response, which makes calculation of the correct dose difficult and critical. Genetic factors involved in this variability include polymorphisms of genes that encode the metabolic enzyme CYP2C9 and the target enzyme vitamin K epoxide reductase complex 1 (VKORC1); these polymorphisms can be associated with reduced enzymatic expression. We examined the frequency of the most relevant variants encoding CYP2C9 (alleles *1, *2 and *3) and VKORC1 (SNP -1639A>G) in the Argentinian population. Molecular typing was performed by PCR-RFLP on a randomly selected sample of 101 healthy volunteers from the Hospital Italiano de Buenos Aires gene bank. Fifty-seven subjects were identified as homozygous for CYP2C9*1 and 14 for *2, while 24 and 5 were heterozygous for *2 and *3 alleles; one individual was a composite heterozygote (*2/*3). When we examined VKORC1, 21 subjects were AA homozygous, 60 were AG heterozygotes and 20 were GG homozygotes. This is the first analysis of genotypic frequencies for CYP2C9 and VKORC1 performed in an Argentinian population. These allele prevalences are similar to what is known for Caucasian population, reflecting the European ancestor of our patient population, coming mostly from Buenos Aires city and surroundings. Knowledge of this prevalence information is instrumental for cost-effective pharmacogenomic testing in patients undergoing oral anticoagulation treatment.     

Single-nucleotide polymorphism analysis using fluorescence resonance energy transfer between DNA-labeling fluorophore,fluorescein isothiocyanate,and DNA intercalator,POPO-3, on bacterial magnetic particles

To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*1/*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system.     

Effect of epoxide hydrolase and glutathione S-tranferase genotypes on the induction of micronuclei and DNA damage by styrene-7,8-oxide in vitro

Styrene is one of the most important organic chemicals used worldwide. Its main metabolite, styrene-7,8-oxide (SO), is considered responsible for the genotoxic effects associated with exposure to styrene. SO is detoxified by hydrolysis catalyzed by epoxide hydrolase (EH), or, to a minor extent, by conjugation mediated by glutathione S-transferases (GSTs). The purpose of the present study was to investigate whether EH (exons 3 and 4), GSTP1 (exons 5 and 6), GSTM1 and GSTT1 polymorphisms have any influence on the genotoxicity of SO in human leukocytes. Peripheral leukocytes from 30 healthy donors were exposed to SO (50 and 200 micro M) and genotoxicity was evaluated by means of the micronucleus (MN) test and alkaline comet assay, using 1% DMSO as solvent control. When EH genotypes were classified in low, medium, and high with respect to the expected EH activity, an increase in induced comet tail length was observed with decreasing EH activity in SO-exposed cells. An increase was seen in induced MN frequency in EH low-activity donors. These findings are consistent with the detoxifying activity of this enzyme. In addition, increases in MN frequencies for GSTP1 *A/*B and *A/*C genotypes with regard to the wild-type homozygous *A/*A genotype were detected. This may be due to a low detoxifying activity as a consequence of altered SO affinity of the variant protein, but must be confirmed using homozygote variant individuals, not included in this study. No clear results were obtained for GSTM1 or GSTT1 genotypes, even when performing the analysis after grouping individuals with the same expected EH activity, probably due to the minor role that glutathione conjugation plays in styrene metabolism. The present in vitro findings using human leukocytes suggest that polymorphisms in EH, and, to a lesser extent, in GSTP1, may influence induction of cytogenetic and DNA damage by SO.     

Deficiency in mitochondrial aldehyde dehydrogenase increases the risk for late-onset Alzheimer's disease in the Japanese population

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) deficiency is caused by a mutant allele in the Mongoloids. To examine whether genetic constitutions affecting aldehyde metabolism influence the risk for late-onset Alzheimer's disease (LOAD), we performed a case-control study in the Japanese population on the deficiency in ALDH2 caused by the dominant-negative mutant allele of the ALDH2 gene (ALDH2*2). In a comparison of 447 patients with sex, age, and region matched nondemented controls, the genotype frequency carrying the ALDH2*2 allele was significantly higher in the patients than in the controls (48.1% vs 37.4%, P = 0.001). Logistic regression analysis indicates that carriage of the ALDH2*2 allele is an independent risk for LOAD of the epsilon4 allele of the apolipoprotein E gene (APOE-epsilon4) (P = 0.002). Moreover, the odds ratio for LOAD in carriers of the ALDH2*2 allele was almost twice that in noncarriers, irrespective of status with regard to the APOE-epsilon4 allele. Among patients homozygous for the APOE-epsilon4 allele, age at onset of LOAD was significantly lower in those with than without the ALDH2*2 allele. In addition, dosage of the ALDH2*2 allele significantly affected age at onset of patients homozygous for the APOE-epsilon4 allele. These results indicate that the ALDH2 deficiency is a risk for LOAD, synergistically acting with the APOE-epsilon4 allele.