Effects of ALDH2 gene polymorphisms and alcohol-drinking behavior on micronuclei frequency in non-smokers

Mutation Research 541 (2003) 71–80

Effects of ALDH2 gene polymorphisms and alcohol-drinking behavior on micronuclei frequency in non-smokers Hitoshi Ishikawa a,∗ , Hidetaka Yamamoto b , Ying Tian a , Mitsuo Kawano c , Toru Yamauchi a , Kazuhito Yokoyama a a

Department of Public Health and Preventive Medicine, Mie University School of Medicine, Edobashi 2-174, Tsu 514-8507, Japan b Department of Forensic Medicine and Sciences, Mie University School of Medicine, Edobashi 2-174, Tsu 514-8507, Japan c Department of Microbiology, Mie University School of Medicine, Edobashi 2-174, Tsu 514-8507, Japan Received 16 May 2003; received in revised form 25 July 2003; accepted 25 July 2003

Abstract Alcohol abuse is a serious health problem, leading to life-threatening damage to most of the important organ systems. Genotoxic damage is used as an early effect indicator in the surveillance of human exposure to genotoxic substances. Intraand inter-individual variations of baseline frequencies of micronuclei (MN) in peripheral blood lymphocytes of human populations have been reported previously. Polymorphisms in a few metabolic enzyme genes seem to account for a proportion of this variability, but the impact of specific genetic variants on MN frequencies has not yet been clarified. In 42 healthy Japanese non-smoking men, we investigated the relationship between the MN frequency levels and genetic polymorphisms in three different genes: aldehyde dehydrogenase 2 (ALDH2), X-ray repair cross-complementing group 1 (XRCC1) and excision repair cross-complementing group 2 (ERCC2). Genotyping was performed by PCR–RFLP analysis. The ALDH2 variant (deficient-type) was significantly associated with increased MN frequency levels in subjects with drinking more than three times per week, whereas the XRCC1 and ERCC2 variants seemed to be unrelated to the MN frequency. The ALDH2-deficient habitual drinkers had an average MN frequency of 5.88 ± 0.58 (±S.E.) compared with 3.20 ± 0.80 in the ALDH2-proficient habitual drinkers (P < 0.05). The ALDH2-proficient non-habitual drinkers had the lowest MN frequency (1.56 ± 0.41). Furthermore, subjects with highest levels of mean MN frequency, who consumed more than 100 g of alcohol per week and more than three times per week, had A2 genotype of ALDH2. A significant odds ratio (12.25, P < 0.05) for the MN frequency levels above the 50th percentile value was observed for the ALDH2-deficient individuals versus the ALDH2-proficient individuals after adjustment for several confounders. These results strongly suggest that human early genotoxic effect studies based on the cytogenetic markers of MN should take into account both the individual ALDH2 polymorphism and the potential confounding effect of the drinking behavior. © 2003 Elsevier B.V. All rights reserved. Keywords: ALDH2; Habitual drinker; Micronuclei

Abbreviations: CAs, chromosomal aberrations; SCEs, sister chromatids exchanges; MN, micronuclei; ALDH2, aldehyde dehydrogenase 2; XRCC1, X-ray repair cross-complementing group 1; ERCC2, excision repair cross-complementing group 2; CB, cytokinesis-block; OR, odds ratio; CI, confidence interval; wk, week ∗ Corresponding author. Tel.: +81-59-231-5012; fax: +81-59-231-5012. E-mail address: [email protected] (H. Ishikawa). 1383-5718/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1383-5718(03)00179-7

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1. Introduction Alcohol abuse is a serious health problem, leading to life-threatening damage to most of the important organ systems. Even though the toxic effects of alcohol are well documented, the mechanisms by which alcohol and alcoholic drinks affect the cancer risk are poorly understood. The toxic effects of alcohol may be mediated partially by genotoxic damage. In fact, alcoholics have a higher frequency of sister chromatid exchanges (SCEs), chromosomal aberrations (CAs) and micronuclei (MN) in their lymphocytes compared with non-alcoholics [1–5]. It has also been shown that alcohol can experimentally induce chromosome mis-segregation in Aspergillus nidulans, Drosophila melanogaster and mice [6–8]. Aldehyde dehydrogenase (ALDH2) is a mitochondrial enzyme, and one of the two main isoenzymes of aldehyde dehydrogenase (ALDH1 with high Km; ALDH2 with low Km). Together with alcohol dehydrogenase, ALDH2 is the main metabolizer of ethyl alcohol. ALDH2 is important for occupational workers, because it is involved in the metabolism of toluene, and also appears to be implicated in the metabolism of vinyl chloride monomers [9]. Approximately, half the Japanese population lack ALDH2 activity because of a structural point mutation in the ALDH2 gene (wild allele, ALDH2*1; mutated allele, ALDH2*2). This genetic polymorphism causes a transition of Glu to Lys, which results in the catalytic deficiency of aldehyde metabolism [10]. The same mutation has also been found in American and Caucasian populations, but at much lower frequencies than in Asian populations [11,12]. It is believed that the presence of at least one mutated allele leads to reduce catalytic activity (ALDH2*1/*2). Morimoto and Takeshita [13] indicated significantly increased frequencies of SCEs in the human lymphocytes from ALDH2-deficient habitual drinkers compared with those from ALDH2-proficient individuals. This means that, after alcohol intake, the metabolism of acetaldehyde is inefficient in ALDH2-deficient habitual drinkers and accumulates in the body, giving rise to genotoxic damage. The X-ray repair cross-complementing group 1 (XRCC1) gene plays a role in the base excision repair pathway, whereas the excision repair crosscomplementing group 2 (ERCC2) gene is involved in

the nucleotide-excision repair pathway (see [14] for review). It is considered that polymorphisms of the DNA repair genes may alter protein function and an individual’s capacity to repair damaged DNA. However, it is presently unclear whether polymorphisms in the DNA repair genes can affect individual genotoxic responses [15]. The MN assay is one of the most sensitive markers of DNA damage and has previously been used to investigate the genotoxicity of a variety of chemicals [16,17]. This MN test using interphase cells is more suitable as a cytogenetic marker because it is not limited to metaphases, and has the advantage of allowing rapid screening of large numbers of cells, compared with studies using SCEs or CAs [18]. MN are formed from acentric chromosome- or chromatid-type fragments and whole chromosomes that have lagged behind in cell division, and are left outside both daughter nuclei. Thus, MN analysis appears to be a good tool to investigate the effects of clastogens and aneuploidogens in occupational and environmental exposure. However, several factors have been associated with the intra- and inter-individual variation of MN [19,20]. These factors include not only gender and age, but also lifestyle habits such as smoking and drinking [4,21,22]. Furthermore, numerous epidemiological studies have indicated that several metabolic enzyme gene polymorphisms appear to have important roles as explanatory factors for the dispersion of the MN distribution [23–27]. However, no studies on the influence of metabolic enzyme gene polymorphisms on the frequency of MN in lymphocytes have yet given definitely positive results [28]. Thus, the identification and estimation of the relative contribution of metabolic enzyme gene polymorphisms are also necessary in the analysis of biomonitoring research fields. This study aimed to investigate the effects of alcohol-drinking behavior and DNA polymorphisms of the ALDH2, XRCC1 and ERCC2 genes on the MN frequency among non-smoking healthy Japanese subjects. 2. Materials and methods 2.1. Study subjects Studies were carried out on 42 healthy Japanese non-smoking men residing in Mie prefecture, to

H. Ishikawa et al. / Mutation Research 541 (2003) 71–80

exclude the contributions of gender and active smoking on the MN frequencies. The participation of each subject was voluntary and the subjects could withdraw at any time during the study (according to the Helsinki II declaration). All participants completed a questionnaire and provided a blood sample. The questionnaire elicited demographic data (age), medical history, smoking and drinking habits, and prior or current exposure to medication or environmental agents that could affect the MN assay (e.g. X-rays, anticancer chemotherapy, etc.). The samples carried code numbers, and information regarding the questionnaire data of the donors was not revealed before the analysis was completed. Written informed consents were obtained according to the ethical guidelines of the Japanese Government. This study was approved by the Committee for the Ethics in Mie University School of Medicine. 2.2. Frequencies and amounts of alcohol consumption With regard to the frequency of drinking, the intensity of drinking was evaluated as habitual drinking (4–7 times per week), non-habitual drinking (0–3 times per week) and never drinking. In the calculation of the mean MN frequency, non-habitual drinkers and ‘never’ drinkers were combined into a group as non-habitual drinkers because the numbers of these subjects were too small to detect any significant difference. Furthermore, average weekly alcohol consumption was calculated by participants themselves and self-reported as either alcohol consumption >100 and
100 g per week. Alcohol intake was converted to total alcohol consumption by using a standard method [29]. The cut-point among drinkers was approximately the median value of alcohol amount per week (Table 1). All subjects reported that they had not consumed drugs or alcohol on the previous day of blood collection. 2.3. MN analysis All chemicals used were purchased from Sigma– Aldrich Japan (Tokyo, Japan) unless otherwise noted. Venous blood was drawn from donors into sodiumheparinized vacutainers. Whole-blood lymphocyte cultures were set up according to the cytokinesis-block

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Table 1 Demographic characteristics of the study populations Number of subjectsa

42

Age (year) Mean ± S.D.

38.9 ± 12.7

No. of never drinker No. of non-habitual drinker (0–3 times per week) No. of habitual drinker (4–7 times per week) Alcohol amount per week (g) Mean ± S.D. Percentile (25th) Percentile (50th) Percentile (75th) Percentile (90th) MN frequency Mean ± S.D. Percentile (25th) Percentile (50th) Percentile (75th) Percentile (90th)

8 16 18

120.3 ± 119.1 15.8 89.3 168 294 3.29 ± 2.23 1.00 3.00 5.00 6.30

ALDH2 No. of ALDH2*1/ALDH2*1 No. of ALDH2*1/ALDH2*2 No. of ALDH2*2/ALDH2*2

19 20 3

XRCC1 No. of Arg/Arg No. of Arg/Gln No. of Gln/Gln

16 17 9

ERCC2b No. of Lyn/Lyn No. of Lyn/Gln No. of Gln/Gln

36 5 0

a b

All persons were healthy office workers. Genotype could not be determined for one person.

(CB) method [19] with minor modifications. Briefly, 0.8 ml of whole blood was added to a final volume of 10 ml of culture medium. The medium, RPMI 1640 with 24 mM HEPES buffer and l-glutamine, and 15% fetal bovine serum (Gibco), was supplemented with penicillin (100 units/ml, Meiji Seika, Japan), streptomycin (0.1 mg/ml, Meiji Seika, Japan), and 1.5% phytohaemagglutinin (PHA, Gibco). After 44 h of culture at 37 ◦ C in 5% CO2 with 98% relative humidity, cytochalasin B (3 ␮g/ml) was added to accumulate cells that had divided once only as binucleated cells. Cells were harvested by centrifugation at 72 h post-PHA stimulation, then exposed to 0.15 M

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KCL for 15 min at 37 ◦ C, and fixed three times in methanol:acetic acid (5:1). The resulting suspension was dropped onto microscope slides and stained with 3% Giemsa. MN were scored in 1000 T-lymphocyte binucleated cells according to the published criteria [30]. All slides were scored by one reader blinded to the status of the subjects. 2.4. Genotyping of XRCC1 and ERCC2 polymorphisms Genomic DNA was isolated from peripheral blood samples (300 ␮l) using a DNA Purification Kit (Promega, USA). All genotypings were done in duplicate and the validity of the method was confirmed by DNA sequencing of genotypes of ALDH2, XRCC1 and ERCC2 genes. XRCC1 and ERCC2 polymorphisms were determined using PCR-RFLP [31]. XRCC1: an Arg → Gln substitution in exon 10 (codon 399) was amplified to form an undigested fragment of 242 bp using the primer pair 5 -CCCCAAGTACAGCCAGGTC-3 and 5 -TGTCCCGCTCCTCTCAGTAG-3 . The amplifications were carried out with denaturing at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s, and extension at 72 ◦ C for 30 s. PCR products were digested with MSPI (New England Biolabs, Beverly, MA) at 37 ◦ C overnight and analyzed on 2% agarose gels. Arg/Arg genotypes were digested to form 94 and 148 bp fragments, whereas Gln/Gln genotypes had only a 242 bp fragment. ERCC2: a Lys → Gln in exon 23 (codon 751) was amplified to form an undigested fragment of 184 bp using the primer pair 5 -CCCCCTCTCCCTTTCCTCTG-3 and 5 -AACCAGGGCCAGGCAAGAC-3 . The amplifications were carried out with denaturing at 94 ◦ C for 30 s, annealing at 60 ◦ C for 1 min, and extension at 72 ◦ C for 30 s. PCR products were digested with MboII (New England Biolabs, Beverly, MA) at 37 ◦ C overnight and analyzed on 4% agarose gels. Lys/Lys genotypes were digested to form 94 and 148 bp fragments, whereas Lys/Gln genotypes had 94, 148 and 184 bp fragments. No individuals with Gln/Gln genotype were observed. 2.5. Genotyping of ALDH2 polymorphism ALDH2 polymorphism was determined by a modification of the methods of Harada and Zhag [32].

A Glu → Lys in exon 12 was amplified to form an undigested fragment of 135 bp using the primer pair 5 -CAAATTACAGGGTCAACTGCT-3 and 5 -CCACACTCACAGTTTTCTCTT-3 . The amplifications were carried out with denaturing at 93 ◦ C for 1 min 30 s, annealing at 56 ◦ C for 3 min, and extension at 72 ◦ C for 1 min 30 s. PCR products were digested with MboII at 37 ◦ C overnight and analyzed on 7% polyacrylamide gels. ALDH2*1 genotypes were digested to form 125 and 10 bp fragments, whereas ALDH2*2 genotypes had only a 135 bp fragment. 2.6. Statistical analysis The significance of the differences among genotypes for the MN levels was estimated by the Mann–Whitney U-test. Multivariate logistic regression analysis was carried out to calculate odd ratio (OR) adjusted for several possible confounders (age, drinking status and metabolic enzyme and DNA repair genes) using a dichotomous variable for the MN frequency levels (above/below 3 MN cells per 1000 binucleated cells), because the 50th percentile of MN was 3.00 (Table 1) among all samples. Correlation was analyzed by Spearman’s rank correlation test. Values were presented as the mean ± S.E. unless otherwise noted. The level of significance was P < 0.05. All statistical analyses were carried out with the StatView® 5.0J for windows (SAS Institute Inc.) software package.

3. Results 3.1. Sample characteristics The demographic characteristics of the study subjects are summarized in Table 1. All subjects were office workers, healthy and taking no medication on the day of blood collection, although there was no information about their family history possibly influencing MN frequency. The mean age of subjects was 38.9 ± 12.7 (±S.D.). Forty-two subjects had mean MN frequencies of 3.29. The mean alcohol amounts (g) per week for habitual drinker were significantly higher than that of non-habitual drinker (225.8 versus 41.3, P < 0.0001). The MN frequency was positively correlated with age (r = 0.57; P < 0.001).

Table 2 Mean (±S.E.) of MN frequency for several DNA polymorphisms according to classes of alcohol consumption Drinking frequency Non-habitual (24) (0–3 times per week)a

ALDH2 A1b A2c XRCC1 Arg/Arg Arg/Gln or ERCC2e Lyn/Lyn Lyn/Gln a

Gln/Gln

Habitual (18) (4–7 times per week)


100 g per week (22)

>100 g per week (20)

n (%)

Mean ± S.E.

n (%)

Mean ± S.E.

n (%)

Mean ± S.E.

n (%)

Mean ± S.E.

n (%)

Mean ± S.E.

19 (45.2) 23 (54.8)

2.42 ± 0.49 4.00d ± 0.44

9 (37.5) 15 (62.5)

1.56 ± 0.41 3.00d ± 0.40

10 (55.6) 8 (44.4)

3.20 ± 0.80 5.88d ± 0.58

8 (36.4) 14 (63.6)

1.63 ± 0.46 3.00d ± 0.43

11 (55.0) 9 (45.0)

3.00 ± 0.75 5.57d ± 0.60

16 (38.1) 26 (61.9)

3.44 ± 0.47 3.19 ± 0.48

10 (41.7) 14 (58.3)

3.50 ± 0.54 1.71 ± 0.27

6 (33.3) 12 (66.7)

3.33 ± 0.96 4.92 ± 0.73

10 (45.5) 12 (54.5)

3.40 ± 0.56 1.75 ± 0.31

6 (30.0) 14 (70.0)

3.50 ± 0.92 4.43 ± 0.71

36 (87.8) 5 (12.2)

3.14 ± 0.39 3.80 ± 0.58

21 (87.5) 3 (12.5)

2.29 ± 0.34 3.67 ± 0.88

15 (88.2) 2 (11.8)

4.33 ± 0.70 4.00 ± 1.00

19 (86.4) 3 (13.6)

2.32 ± 0.37 3.67 ± 0.88

17 (85.0) 2 (15.0)

4.06 ± 0.65 4.00 ± 1.00

Non-habitual includes never drinkers. A1 reveals ALDH2*1/ALDH2*1 genotype. c A2 includes ALDH2*1/ALDH2*2 and ALDH2*2/ALDH2*2 genotypes. d P < 0.05 by Mann–Whitney U-test. e Genotype could not be determined for one person. b

Alcohol intake

H. Ishikawa et al. / Mutation Research 541 (2003) 71–80

All subjects (42)

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H. Ishikawa et al. / Mutation Research 541 (2003) 71–80

3.2. ALDH2, XRCC1 and ERCC2 gene frequencies Genotype and allele frequencies for the three polymorphisms analyzed were calculated by direct counting and were in Hardy–Weinberg equilibrium. Allele frequencies were as follows: ALDH2*1/ALDH2*2, 0.69/0.31; XRCC1-399Arg/Gln, 0.65/0.35; ERCC2751Lyn/Gln, 0.94/0.06. The ALDH2*2 allele frequency (0.31) is similar to those reported previously for Japanese populations [32–34]. The XRCC1-399Gln allele frequency (0.35) and ERCC2-751Gln allele frequency (0.06) are also similar to those reported by Hamajima et al. [35]. The overall genotype frequencies are shown in Table 2. Since subjects with at least one ALDH2*2 allele have lower enzyme activity than those with the homozygous ALDH2*1 allele (A1) [36], those with at least one ALDH2*2 allele were grouped as ALDH2 variants (A2). The frequencies of the A1 and A2 groups of ALDH2 were 45.2 and 54.8%, respectively, whereas the frequencies of the Arg/Arg, and Arg/Gln or Gln/Gln of XRCC1 were 38.1 and 61.9%. The frequency of the ERCC2 Lyn/Lyn and Lyn/Gln genotype was 87.8 and 12.2%.

3.3. Effects of DNA polymorphisms and drinking status on MN frequency The mean MN frequency for the A2 polymorphism of ALDH2 was significant higher than that of A1 polymorphism (4.00 versus 2.42) (Table 2). When we divided the drinking frequency into two categories, non-habitual and habitual drinkers, we found that the mean MN frequency for the A2 polymorphism was still significantly greater than that of A1 polymorphism in non-habitual (3.00 versus 1.56, respectively, P < 0.05) or habitual (5.88 versus 3.20, respectively, P < 0.05) drinkers. Habitual drinker had a higher MN frequency than non-habitual drinkers (4.39 versus 2.46, respectively, P < 0.05). Habitual drinkers with the A2 genotype had the highest mean MN frequencies, 5.88 (n = 8), whereas non-habitual drinkers with the A1 genotype had the lowest MN frequencies, 1.56 (n = 9). When we divided the amount of alcohol consumed into two categories,
100 and >100 g per week, and calculated the mean MN frequency, we found a significantly higher MN frequency in the A2 genotype compared to that of A1 genotype both in
100 and >100 g per week groups (Table 2). Furthermore, subjects with highest levels of mean MN frequency, who

Fig. 1. Association of mean MN frequency with alcohol consumption and polymorphisms of ALDH2 gene. MN were scored in 1000 T-lymphocyte binucleated cells. T1: 0–3 times per week; T2: 4–7 times per week; G1:
100 g per week; G2: >100 g per week.

H. Ishikawa et al. / Mutation Research 541 (2003) 71–80 Table 3 Distribution of 42 Japanese individuals for above/equal to or below the 50th percentile MN frequency levels for the wild-type allele homozygotes versus subjects with at least one variant allele for ALDH2, XRCC1 and ERCC2 genes A/Ba (n)

Crude OR (95% CI)

Adjusted ORb (95% CI)

5/14 14/9

1 4.36g (1.16–16.30)

1 12.25g (1.20–124.92)

9/7 10/16

1 2.10 (0.31–14.20)

1 0.97 (0.11–8.51)

ERCC2f Lyn/Lyn Lyn/Gln

15/12 3/2

1 0.49 (0.14–1.72)

1 0.34 (0.07–1.67)

Drinking status Non-habituale Habitual

7/17 12/6

1 4.86g (1.30–18.10)

1 14.60g (1.37–155.95)

ALDH2 A1c A2d XRCC1 Arg/Arg Arg/Gln or Gln/Gln

a A: no. of subjects with the more than the 50 percentile MN frequency level (3.00 per 103 binucleated cells). B: no. of subjects with the 50 percentile MN frequency level (3.00) or below. b Multivariate logistic regression: OR adjusted by age, metabolic enzyme and DNA repair genes and drinking status. c A1 reveals ALDH2*1/ALDH2*1 genotype. d A2 includes ALDH2*1/ALDH2*2 and ALDH2*2/ALDH2*2 genotypes. e Non-habitual includes never drinkers. f Genotype could not be determined for one person. g P < 0.05.

consumed more than 100 g of alcohol per week and more than three times per week, had A2 genotype of ALDH2 (Fig. 1). A significant odds ratio emerged by the comparison of A2 with A1 (crude OR 4.36, 95% CI 1.16–16.30). To take into account the effect of the age of the individuals, multivariate logistic regression analyses were also carried out. The OR values were still significant (adjusted OR 12.25, 95% CI 1.20–124.92) (Table 3). Similarly, significant ORs were observed by the comparison of habitual drinking with non-habitual drinking (crude OR 4.86, adjusted OR 14.60). No detectable influence of the XRCC1 and ERCC2 polymorphisms on the MN frequencies was observed in this study.

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4. Discussion To investigate whether the ALDH2, XRCC1 and ERCC2 genes polymorphisms and drinking status were associated with differences in chromosome breakage or loss, we compared the MN frequency levels in the lymphocytes from peripheral blood. Our study suggests that the ALDH2 variant genotype and a habitual drinking status could result in an increase in the MN frequency. It has been suggested that smokers with the ALDH2 variant genotype had significantly higher SCEs frequency, although there was no information about the drinking behavior of the subjects [9]. Furthermore, significantly increased SCEs were also observed in almost every day drinkers with the ALDH2 variant genotype [13]. In an in vitro study, acetaldehyde, the first metabolite of ethanol oxidation, caused a dose-dependent linear increase in the induction of SCEs in lymphocytes from German and Japanese populations [37]. Furthermore, MN arises in human peripheral lymphocytes after treatment of acetaldehyde [38]. From these studies, we strongly suspect a positive relationship between MN frequencies and the combined effects of the ALDH2 variant genotype and the drinking status. To our knowledge, our study is the first report that habitual drinkers with the ALDH2 variant genotype have significantly increased MN frequency levels. ALDH2 is an enzyme responsible for individual sensitivity to alcohol [11]. Since ALDH2-deficient genotypes (ALDH2*2/ALDH2*2 homozygous and ALDH2*1/ALDH2*2 heterozygous) have higher acetaldehyde accumulation in blood, they may be susceptible to the harmful influence of alcohol via toxic effects of acetaldehyde, leading to chromosomal DNA damage [39]. The MN test is one of the most sensitive markers of DNA damage and has been used to investigate the genotoxicity of a variety of chemicals. Recent evidence also suggests the usefulness of the MN test for screening carriers of specific mutations that evaluate cancer susceptibility [40,41]. Our findings might reveal that ALDH2 polymorphisms, as well as the frequency of alcohol intake and total amounts of alcohol consumed, are important factors in the mechanism(s) of alcohol-related diseases such as cancer.

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Several studies suggested that alcoholism is associated with reduced levels of tissue folate and Vitamin B12, and that MN frequency is negatively correlated with folate and Vitamin B12 plasma levels [42–44]. Folate and Vitamin B12 are required for the synthesis of methionine and S-adenosyl methionine, the common methyl donor required for the maintenance of methylation patterns in DNA that determine gene expression and DNA conformation [45]. Deficiencies in folate and Vitamin B12 therefore may lead to elevated DNA damage rate, which could increase MN frequency [45,46]. Subjects with at least one ALDH2*2 allele, which are common among Orientals [11] but not Caucasians [12], have lower enzyme activity than the ALDH2*1/ALDH2*1 homozygous genotype [36]. Study approaches using Oriental populations, as well as our study, should be further performed in order to detect other biological significances of the ALDH2 polymorphisms. In addition to ALDH2 polymorphisms, CYP2E1 polymorphisms may affect MN frequencies, because CYP2E1 is the major component of the microsomal ethanol oxidizing system. In fact, CYP2E1 RsaI and DraI allele polymorphisms are related with to the risk of developing alcoholism [47,48], indicating that further studies are needed to detect possible relationships between MN frequencies and CYP2E1 polymorphisms. No detectable influence of the XRCC1 and ERCC2 polymorphisms on the MN frequencies was observed in this study. No reports have mentioned a relationship between ERCC2 polymorphisms and any cytogenetic marker, whereas lymphocytes from subjects homozygous or heterozygous for the 399Gln allele of XRCC1 were found to be more sensitive than lymphocytes from Arg/Arg subjects to SCEs induction by the tobacco-specific nitrosamine NNK in vitro [49] or by smoking habits [50]. The small number of subjects in our study has precluded us from determining the effects of the XRCC1 and ERCC2 polymorphisms on the MN frequencies. These results still need further studies for confirmation. In the present report, we found a clear association between the ALDH2 polymorphisms and/or drinking behavior and MN frequencies in a sample of 42 healthy Japanese non-smokers. These results suggest that studies of human genotoxicity based on cytogenetic mark-

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