Mutagenesis vol. 24 no. 4 pp. 303–308, 2009 Advance Access Publication 13 April 2009
doi:10.1093/mutage/gep008
Is habitual alcohol drinking associated with reduced electrophoretic DNA migration in peripheral blood leukocytes from ALDH2-deficient male Japanese?
Yuquan Lu and Kanehisa Morimoto* Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Osaka 565-0871, Japan
Alcohol drinking-derived acetaldehyde is believed to crosslink DNA and induce sister chromatid exchanges in peripheral blood lymphocytes. However, little population data are available to illustrate effects of alcohol-derived acetaldehyde on DNA migration as assayed by the comet assay in peripheral lymphocytes. In the present study, we investigated lifestyle behaviours, including alcohol consumption, in 150 Japanese males by questionnaire, determined their aldehyde dehydrogenase 2 (ALDH2) family genotypes by polymerase chain reaction and measured the DNA migration in peripheral blood leukocytes by the alkaline comet assay. The results showed that habitual alcohol drinking is significantly negatively associated with DNA migration in peripheral blood leukocytes (r 5 20.321, P 5 0.005) of ALDH2-deficient, but not of ALDH2proficient genotypes (r 5 0.048, P 5 0.683). The amount of pure alcohol consumed per time by the subjects showed a similar phenomenon (r 5 20.257, P 5 0.025 for the ALDH2-deficient, but r 5 20.061, P 5 0.606 for the ALDH2-proficient genotype). Further stepwise multiple regression analysis showed that alcohol drinking frequency was a significant predictor of DNA migration for subjects with ALDH2-deficient genotype, but not for subjects with ALDH2-proficient genotype. In summary, the present result suggests that frequent alcohol drinking is significantly associated with a reduced electrophoretic DNA migration in peripheral blood leukocytes from ALDH2deficient male Japanese subjects.
Introduction Chronic alcohol consumption is believed to associate with the development of cancers of the upper gastrointestinal tract due to the production of acetaldehyde, the first metabolite in alcohol degradation (1–3). It is also believed that acetaldehyde binds to DNA, thereby forming numerous DNA adducts, such as N2-ethylidene-deoxyguanosine, N2-ethyl-2#-deoxyguanosine (N2-EtdG) and N2-dimethyldioxane-deoxyguanosine (4,5), crosslinking DNA within DNA strands or with proteins (6,7), hindering DNA replication (8) and increasing the number of chromosome aberrations (9–11). In an in vitro study, the cross-linking effects of acetaldehyde were demonstrated by alkaline comet assay, as it dose dependently reduces electrophoretic DNA migration in lymphocytes in a similar manner to formaldehyde (12). In population studies, Fang and Vaca (13) found significantly higher levels of
acetaldehyde-derived DNA adducts such as N2-ethyl-3#-dGMP in leukocytes of alcohol abusers than that in control subjects, and Matsuda et al. (14) found that levels of acetaldehydederived DNA adducts, such as N2-EtdG, a-S-Me-c-OH-PdG and a-R-Me-c-OH-PdG, in white blood cells were significantly higher in alcoholics with aldehyde dehydrogenase 2 (ALDH2)deficient genotype (ALDH2*1/ALDH2*2 heterozygous, encoding mutated ALDH2 enzyme with ,6% of enzymatic activity) than in those with ALDH2-proficient genotype (ALDH2*1/ALDH2*1 homozygous, encoding full active ALDH2 enzyme). In addition, habitual alcohol drinkers were found to have more chromosome aberrations in their peripheral blood lymphocytes than infrequent drinkers and abstainers with ALDH2-deficient genotype (15,16). These data comprehensively suggest that frequent alcohol drinking results in accumulation of acetaldehyde in blood tissue, and the accumulated acetaldehyde may react with DNA and proteins, resulting in genotoxicity in leukocytes. However, no comet assay data have been reported for the acetaldehydederived genotoxicity of alcohol consumption in populations. We suppose that if alcohol drinking-derived acetaldehyde shows DNA cross-linking effects in peripheral leukocytes, frequent alcohol consumption would possibly retard DNA migration on electrophoresis among alcohol drinkers, particularly those with ALDH2-deficient genotype. As is widely known, the alkaline comet assay is a sensitive method to detect DNA strand breaks by electrophoresis under alkaline conditions (pH , 13). The DNA strand breaks caused by free radicals, alkali-labile sites, uncompleted DNA repairs, etc. will migrate to the anode under electrophoresis, forming a comet-like shape observed under the microscope. Generally, the more DNA fragments produced, the more DNA moves into the ‘tail’ of the ‘comet’ cell. As the nuclear DNA in living leukocytes generally keeps a dynamic balance between DNA damage and repair, a certain amount of DNA strand breaks can exist and a baseline DNA migration would be detected with the alkaline comet assay. However, when some high concentrations of chemicals temporarily react with DNA molecules and cross-link them within DNA strands or to proteins, the movement of DNA fragments would be retarded before the cross-links are completely removed. In the present study, we recruited 150 Japanese males, investigated their lifestyles, including frequency of alcohol consumption by questionnaire and amount of pure alcohol consumed per time, determined their ALDH2 genotypes by polymerase chain reaction (PCR), measured DNA migration from peripheral blood leukocytes by the alkaline comet assay and analysed the associations among alcohol drinking frequency, amount of pure alcohol consumed per time, ALDH2 genotype and electrophoretic DNA migration of leukocytes among Japanese subjects.
*To whom correspondence should be addressed. Tel: þ81 6 6879 3920; Fax: þ81 6 6879 3923; Email:
[email protected] Ó The Author 2009. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail:
[email protected].
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Materials and methods Study population The study population consisted of 150 healthy male volunteers, aged 21–63 years, recruited from a local hard-metal tooling factory in Japan. Volunteers were considered healthy based on self-reported health status and the absence of medication or clinical treatment. All information was gathered through questionnaires. Subjects were fully informed about the details of the study and provided written informed consent. The Medical Ethical Committee of Osaka University approved the protocol. Investigation of lifestyles Lifestyles of all participants were investigated by questionnaire that was mailed to each subject 1 week before blood sampling. Investigated items included age, height, weight, alcohol drinking frequency, amount of alcohol consumed per time by sorts and lifestyle factors such as physical activities, nutrition (nutritional balance, regularity of meals, breakfast habits, frequency of coffee drinking, consumption of black, green or other teas and snack eating habits), smoking habit [including cigarettes smoked per day, years of smoking and levels of daily exposure to cigarette tar (mg/day)], sleep habits (hours of sleep per day, sleep satisfaction and sleep soundness), occupation (years of employment, hours of work per day, work satisfaction, shift work and hours of overtime work in the previous week), mental stress situation [General Health Questionnaires 28 (GHQ28) (17), self-rating depression scale (18), life satisfaction and vacation satisfaction] and other factors (regular lifestyle, personal hobbies, marital status and educational background). In addition, several factors were evaluated before taking blood samples (cigarette smoking, alcohol drinking, consumption of fried or grilled meat, strenuous physical activity, sleep satisfaction over the previous 2 days and spiritual situation on the morning of blood sample collection). Each lifestyle item was divided into two options (yes or no) or three to six gradient choices, except quantitative data such as age, height and weight. We quantitatively estimated alcohol drinking frequency as times per month based on questionnaire investigation (1. almost everyday 5 six times a week 5 24 times/month; 2. three to five times a week 5 four times a week 5 16 times/ month; 3. once or twice a week 5 1.5 times/week 5 six times a month; 4. one to three times a month 5 two times a month; 5. 1 to 10 times a year 5 0.5 times/month; 6. never 5 0 times/month). The amount of pure alcohol consumed per time was calculated by multiplying the content of ethanol (%) and the amount of the alcoholic beverages reportedly consumed per time for Sake, beer, western liquor and Sh och u, respectively. Blood sampling Blood sampling was carried out with the annual physical examination. After confirmation and collection of the informed consent and the questionnaire coded with employee number by subjects, a 5-ml blood sample was taken from fasting subjects via a peripheral vein between 8:30 and 10:30 in the morning of two successive 3 days in early July, 2006 using coded heparinized vacuum tubes (VENOJECTÒII VP-H100, Terumo, Tokyo, Japan). Questionnaires and blood samples were separately collected. The connection list of questionnaire to blood sample was solely kept by a supervisor who did neither attend the comet assay nor ALDH2 genotype determination. Blood samples were kept at 4°C and were divided into two portions, one portion was used to conduct alkaline comet assay within 60 h (19), and the other portion was used to determine ALDH2 genotypes at Osaka University Graduate School of Medicine. The study was conducted double blindly, and no subject was sampled repeatedly. Determination of ALDH2 genotypes We extracted DNA from 1 ml of blood by using a Gene Catcherä gDNA 0.3–1.0 ml Blood Kit (Invitrogen Life Technologies, Carlsbad, USA) and determined ALDH2 genotypes by amplifying exon 12 of ALDH2 for 35 cycle of PCR (1 min at 94°C, 30 sec at 60°C and 45 sec at 72°C) in the T-Gradient Thermoblock (Biometra, Gottingen, Germany). Amplification primers were as follows: primer pair-1 for wild-type genotype, forward, 5#-CAAATTACAGGGTCAACTGCT-3# and reverse-N, 5#-CCACACTCACAGTTTTCTCTTC-3# and primer pair-2 for the mutant genotype, forward, 5#-CAAATTACAGGGTCAACTGCT-3# and reverse-M, 5#-CCACACTCACAGTTTTCTCTTT-3# (T is the mutant base). For ALDH2 wild-type genotype (ALDH2-NN), only primer pair-1 primers produced DNA fragments of exon 12 of ALDH2. For the ALDH2 hetero-mutant-type genotype (ALDH2-NM), both primer pair-1 and primer pair-2 primers produced DNA fragments of exon 12 of ALDH2, while for the ALDH2 homo-mutant-type genotype (ALDH2-MM), only primer pair-2 primers produced DNA fragments of exon 12 of ALDH2. By this method, ALDH2 genotypes were determined (ISOHAIR Jr. NIPPON GENE Co., Ltd, Tokyo, Japan).
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In the present study, ‘ALDH2-proficient genotype’ denotes the ALDH2-NN genotype, while ‘ALDH2-deficient genotype’ denotes the ALDH2-NM and ALDH2-MM genotypes. Alkaline comet assay All steps were performed in the dark or under a fluorescent lamp to filter out ultraviolet radiation that might further damage DNA. The alkaline comet assay was performed as described elsewhere (20). Briefly, we sandwiched the wholeblood leukocyte suspension layer (0.75% low-melting agarose) between a preloaded gel and another layer of 0.75% low-melting agarose and treated the slides as described by Singh et al. (21) and performed electrophoresis for 20 min at 25 V (0.8 V/cm) using an electrophoresis compact power supply (ATTO Corporation, Tokyo, Japan). Following electrophoresis, slides were neutralized with two 5-min washes in 0.4 M Tris–HCl (pH 7.4), fixed in 99.9% ethanol for 10 min and stored in the dark at room temperature until use. We quantified DNA damage in 100–120 consecutively selected leukocytes after staining with 2 lg/ml ethidium bromide for 1 min using a DHS-SCG imaging system (Keio Electronic Imaging, Osaka, Japan) attached to a fluorescent microscope (Olympus, Tokyo, Japan). We used the comet ratio (% DNA in tail) (22) to represent the electrophoretic DNA migration in leukocytes of all subjects. The final data were date adjusted according to the differences between the mean comet ratio of all 150 subjects and the means on each sampling day (23). Statistical analysis Lifestyle data, ALDH2 genotypes and DNA migration parameter were compared after the completion of all assays. We used the Kruskal–Wallis test, Pearson’s correlation, Spearman’s correlation and stepwise multiple linear regression, as applicable, for statistical analysis using SPSS version 13.0 (SPSS, Chicago, IL, USA). The level of statistical significance was set at P , 0.05. The Kolmogorov–Smirnov test was used to test for normal distributions. All statistical tests were two sided.
Results Table 1 shows the profiles of subjects with different ALDH2 genotypes. Of the items, per cent of drinkers, alcohol drinking frequency and millilitres of pure alcohol consumed per time showed a significant variation among ALDH2 genotypes (P , 0.001) with 74 ALDH2-NN subjects consuming alcohol 14.8 times per month and 52.3 millilitres per time, 67 ALDH2-NM subjects consuming alcohol 9.9 times per month and 43.9 millilitres per time and 9 ALDH2-MM subjects consuming alcohol 0.1 times per month and 6.9 millilitres per time. Numbers of cigarettes smoked per day and years of smoking also showed a significant variance (P , 0.05) among the three ALDH2 genotypes, but the levels of daily exposure to cigarette tar (mg/day) did not show a similar variance among ALDH2 genotypes. In addition, there was no significant variance in the electrophoretic DNA migration (comet ratio) among ALDH2 genotypes (P 5 0.634). Figure 1 shows Spearman’s correlation between alcohol drinking frequency and DNA migration of peripheral blood leukocytes. For subjects with the ALDH2-NN genotype (Fig. 1A), alcohol drinking frequency was non-significantly correlated with DNA migration (r 5 0.048, P 5 0.683). For subjects with the ALDH2-NM and ALDH2-MM genotype (Fig. 1B), alcohol drinking frequency was significantly and negatively correlated with DNA migration (r 5 0.321, P 5 0.005). Table 2 shows that DNA migration in peripheral leukocytes demonstrated a significantly negative correlation with total amount of pure alcohol consumed per time for ALDH2deficient genotype (r 5 0.257, P 5 0.025), but not for ALDH2-proficient genotype. Of all alcoholic beverages, beer was consumed most (53.9%) and solely significantly associated with DNA migration (r 5 0.360, P 5 0.001) among the ALDH2-deficient genotype subjects. Table 3 shows results of stepwise multiple linear regression analysis of DNA migration with lifestyle factors, including smoking, alcohol consumption, amount of pure alcohol
Genotoxic effects of alcohol drinking
Table I. ALDH2 genotypes and profiles of Japanese subjects in Osaka, Japan 2006a
Age Height (cm) Weight (kg) Body mass index Years of employment Cigarette smoking Smokers Cigarettes/day Years of smoking LECT (mg/day)c Alcohol drinking Drinkers Times/month Millilitres (pure alcohol)/time DNA migration Comet ratio (% DNA in tail)
Total (n 5 150)
ALDH2-NN (n 5 74)
ALDH2-NM (n 5 67)
ALDH2-MM (n 5 9)
45.5 169.6 68.3 23.7 23.9
45.6 169.4 67.6 23.5 24.2
45.0 169.9 69.5 24.1 23.1
48.6 170.0 65.0 22.5 27.1
48 6.7 8.9 43.7
(9.8) (5.6) (10.1) (3.2) (10.2) (32.0%) (11.2) (13.6) (109.7)
28 7.6 10.0 47.0
(10.1) (5.6) (10.0) (3.1) (10.4) (37.8%) (11.0) (14.0) (103.0)
15 4.8 6.2 36.7
(9.7) (5.6) (10.6) (3.4) (10.1) (22.4%) (10.6) (11.9) (115.2)
123 (82.0%) 11.7 (10.4) 45.8 (37.4)
70 (94.6%) 14.8 (9.9) 52.3 (32.8)
51 (76.1%) 9.9 (10.2) 43.9 (41.1)
43.6 (1.1)
43.6 (1.1)
43.7 (1.0)
P valueb
(7.0) (6.5) (7.6) (2.2) (9.4)
0.676 0.833 0.404 0.506 0.516
(55.5%) (14.8) (18.2) (129.1)
0.064 0.038 0.015 0.081
2 (22.2%) 0.1 (0.2) 6.9 (13.9)
,0.001 ,0.001 ,0.001
5 13.3 19.2 70.0
43.8 (0.7)
0.634
a
Data are presented as means (standard deviations) except for smokers, and drinkers which are presented as number (percentage). Kruskal–Wallis test. c LECT 5 tar (mg) per cigarette number of cigarettes smoked per day. b
Fig. 1. Spearman’s correlations between DNA migration in peripheral blood leukocytes and alcohol drinking frequencies among Japanese males. (A) Subjects with ALDH2-NN genotype; (B) subjects with ALDH2-NM and ALDH2-MM genotype.
Table II. Alcohol consumption, electrophoretic DNA migration and ALDH2 genotype of Japanese male subjects in Osaka, Japan, 2006 Genotypes
ALDH2-NN
ALDH2-NM and -MM
Alcohol drinking
Sake Beer Western liquor Shochu Total alcohol Sake Beer Western liquor Shochu Total alcohol
Number of subjects
74 74 74 74 74 76 76 76 76 76
% of drinker
6.7 74.3 6.7 29.7 94.6 2.6 53.9 2.6 27.6 69.7
Pure alcohol consumed per time (ml) 3.3 31.3 1.9 17.5 52.3 1.1 20.6 1.9 16.3 25.7
13.4 23.9 7.7 31.1 32.8 6.9 20.6 14.0 31.8 22.2
Comet ratioa rb
P value
0.023 0.043 0.098 0.050 0.061 0.038 0.360 0.195 0.012 0.257
0.844 0.716 0.406 0.670 0.606 0.746 0.001 0.195 0.917 0.025
a
Comet ratio 5 % DNA in tail. Pearson’s correlation coefficient.
b
consumed per time, physical exercises and nutritional balance. For ALDH2-deficient subjects, predictors for DNA migration were alcohol drinking frequency and levels of daily exposure to cigarette tar (LECT) (YDNA migration 5 0.030Xalcohol þ 0.002XLECT þ 43.892, R2 5 0.167), while for ALDH2proficient subjects, predictors for DNA migration was GHQ28
anxiety/insomnia (YDNA migration 5 0.384XGHQ28 þ 43.26, R2 5 0.145). For ALDH2-deficient subjects, alcohol drinking frequency was negatively correlated with DNA migration (partial r 5 0.324) and LECT (partial r 5 0.305) were positively correlated with DNA migration. For ALDH2-proficient subjects, GHQ28 305
Y. Lu and K. Morimoto
Table III. Lifestyle factors associated with electrophoretic DNA migration of peripheral blood leukocytes of Japanese males with different ALDH2 genotypesa
ALDH2-deficient (n 5 76, R2 5 0.167) Alcohol drinkingd LECTe ALDH2-proficient (n 5 74, R2 5 0.145) GHQ28 (anxiety/insomnia)f
Partial rb
Bc
SE
P value
0.324 0.305
0.030 0.002
0.010 0.001
0.005 0.008
0.380
0.384
0.110
0.001
a
Stepwise multiple linear regression analysis. Items analysed include regular lifestyle, hobbies, physical exercise, frequency of alcohol drinking (times per month), millilitres of pure alcohol consumed per time, LECT (mg/day), sleeping hour, sleep satisfaction, sleep soundness, regular meals, nutritional balance, breakfast habits, coffee drinking, green tea drinking, eating snacks, controlling salty food, work hours, life satisfaction, work satisfaction, vacation satisfaction, GHQ28, self-rating depression scales, age, years of employment, educational level, occupation, shift work, marital status, hours of overtime worked, cigarette smoking before blood sample collection, consumption of fried or grilled meat within 3 days before blood sample collection, strenuous physical activity within 3 days of taking blood sample collection, consumption of large quantities of alcohol within 3 days of blood sample collection, sleep satisfaction on the night before blood sample collection, spiritual situation on the morning of blood sample collection. b Partial regression coefficient. c Regression coefficient. d Alcohol consumption: times per month. e LECT (mg/day). f GHQ28 anxiety/insomnia.
anxiety/insomnia (partial r 5 0.380) was positively correlated with DNA migration. Discussion In the present study, we demonstrated for the first time that alcohol drinking frequency and amount of pure alcohol consumed per time is associated with reduced electrophoretic DNA migration in peripheral leukocytes of ALDH2-deficient, but not ALDH2-proficient, Japanese males. These data are consistent with results reported by Morimoto and Takeshita (16) that lymphocytes from ALDH2-deficient drinkers had significantly higher frequencies of sister chromatid exchanges (SCE) when compared with infrequent drinkers and abstainers. They also support Ishikawa’s suggestion (24) that frequent alcohol drinking increases micronucleus (MN) frequency in lymphocytes of Japanese with ALDH2-deficient genotype, as the cross-linking effects of acetaldehyde derived from alcohol drinking increase SCE and MN frequency in lymphocytes (6,7,9,11) and hinder electrophoretic DNA migration in the alkaline comet assay (12). Frequent alcohol drinking by ALDH2-deficient genotype would also produce more acetaldehyde-derived DNA adducts and cross-links in leukocytes (14). It is now clear that the low Km ALDH2 is the main enzyme for metabolizing acetaldehyde, and it has three genotypes. A single point mutation (G / A) in exon 12 of the active ALDH2 gene, resulting in replacement of glutamine with lysine at codon 487, inactivates ALDH2 enzyme. As ALDH2 is a tetramer enzyme, the randomized composition of a tetramer ALDH2 enzyme from a hetero-mutated genotype is estimated to maintain only 6% activity when compared to the wild-type ALDH2, while the homo-mutated genotype would give almost zero activity (25). Thus, for ALDH2-deficient genotypes, 306
acetaldehyde would easily accumulate in the blood after alcohol consumption and would be difficult to eliminate. As acetaldehyde is highly toxic, its accumulation in blood induces severe physical reactions, such as facial flushing, nausea, vomiting, low blood pressure, headache and weakness (26–29). These reactions would prevent further exposure to alcohol. In fact, ALDH2-NM genotypic individuals are usually exposed to alcohol less frequently than ALDH2-NN genotypic individuals, and most ALDH2-MM genotype refuses to consume alcohol at all (30–32). Our present results are consistent with this conclusion (Table 1). Although 50% of Japanese have ALDH2-deficient genotype, alcohol consumption is deeply involving in Japanese lifestyles, traditions and cultures. In the present study, 94.6% of ALDH2-NN, 75.4% of ALDH2-NM and 22.2% of ALDH2-MM genotypic individuals reported drinking alcohol. As problematic alcohol consumption in Japanese populations increased (33–36), alcohol-related health problems are gradually emerging (2,3,37–40). The retarded DNA migration in the alkaline comet assay may be the result of two possibilities when acetaldehyde was used to treat cells; one being DNA–protein cross-links and the other being DNA–DNA cross-links. Kuykendall and Bogdanffy (41) incubated 0.25 lg of [3H] DNA and 1 ng of histones with 50 mM acetaldehyde at temperatures of 37°C for 1 h and obtained 0.4 DNA–histone cross-links per plasmid (2.7 kbp), which is equivalent to 4.7 105 DNA–protein cross-links per mammalian cell. This amount of DNA–protein cross-links should be sufficient to retard DNA migration on comet assay (equivalent to about one DNA cross-link per 6.75 kbp). If proteins were removed from human lymphocytes in the comet assay, acetaldehyde-induced DNA strand breaks could be dose dependently released, rather than retarding DNA migration (42). In addition, Matsuda et al. (14) has recently demonstrated that levels of mutagenic DNA adducts (PdG) from acetaldehyde in leukocytes were significantly higher in alcoholics with inactive ALDH2 than alcoholics with active ALDH2. As PdG is a precursor lesion to DNA–DNA crosslinks as well as DNA–protein cross-links (43), it is possible that acetaldehyde-induced DNA–DNA cross-links also play some roles in retarding DNA migration as detected in the comet assay. In the present study, the comet assay imaging system categorized each comet image into seven grades according to its fluorescence strength. The top three were defined as comet ‘head’ and the remaining four as tail. The calculated % DNA in tail was thus 43%. This value varies greatly among different comet assay imaging systems because of the cut-off point set between head and tail, and is not used to compare with data obtained from different comet assay imaging systems. In addition, we categorized all subjects into manufacturing and office staff and compared their % DNA in tail, but found no statistically significant difference between them. In the present study, blood samples were taken between 8:30 and 10:30 in the morning and were kept at 4°C for 5–60 h before analysis by the alkaline comet assay. Thus, what we detected should have been the background of DNA breakage and migration in the alkaline comet assay. As acetaldehyde– protein reversible adducts can be maintained in peripheral blood in the human body for nearly 48 h (44) and acetaldehyde would hinder DNA repair (12,45), we postulate that, for ALDH2-deficient subjects, alcohol consumption within 36 h of blood sample collection (alcohol drinking at supper 1 or 2 days
Genotoxic effects of alcohol drinking
before blood sampling) would maintain sufficient levels of DNA cross-links for comet assay. In our present study, 26.9% of ALDH2-NM genotype reportedly drank alcohol almost every day, and 14.9% of ALDH2-NM genotype drank alcohol two to four times a week (data not shown). This frequency would ensure the existence of acetaldehyde-derived cross-links in blood when we were taking blood samples. With regard to whether storage time at 4°C influenced the amount of DNA cross-links, future studies are required. Confounding lifestyle factors are known to affect the comet assay. To comprehensively analyse associations between lifestyle factors and electrophoretic DNA migration in this population, we performed stepwise multiple linear regression analysis for both ALDH2-deficient subjects and ALDH2proficient subjects. For ALDH2-deficient subjects, the significant predictors for electrophoretic DNA migration were alcohol drinking frequency and levels of daily exposure to cigarette tar (LECT). For ALDH2-proficient subjects, the significant predictor for electrophoretic DNA migration was GHQ28 anxiety/ insomnia. Of all the predictors, only alcohol drinking frequency was a negative predictor. As for the possibility that subjects with different ALDH2 genotypes might have different risk factors for DNA migration, we are planning further investigations in future studies. Cigarette smoking has long been confirmed to induce DNA single-strand breaks in cultured human cells and human leukocytes or lymphocytes (46–48). DNA strand breaks induced by cigarette smoking mainly result from the effects of free radicals derived from cigarette tar (48). However, as the existence of confounders in epidemiological study, effects of cigarette smoking are usually diminished by some unknown factors. For the ALDH2-proficient subjects, effects of general health situation, especially the anxiety/insomnia demonstrated a significant association with comet ratio (% DNA in comet tail). This result is in line with that of Irie et al. (49) who reported that the psychosocial factors can be associated with the levels of 8-hydroxydeoxyguanosine, a biomarker of cancer-related oxidative DNA damage and with some reports that anxiety could increase active oxygen species in peripheral leukocytes and lymphocytes (50–52). As the GHQ28 anxiety/insomnia reflects mental stress situations, we suggest paying more attention to this subpopulation in future studies. Although we showed here statistical correlations between alcohol consumption, ALDH2 genotypes and DNA migration in peripheral leukocytes and suggested a possible toxicological role of acetaldehyde in our investigation, we noticed that a small amount of alcohol consumption might be beneficial to human health (53). It is also reported that alcohol at low dose might induce an adaptive cytoprotection effect in human colonocytes (54). However, to individuals with different ALDH2 genotypes, the health effects of alcohol exposure at the same levels might vary greatly (55). As the complexity of lifestyle, we wish this association could get confirmation in future studies. In this study, to our surprise, of the four sorts of alcoholic beverages we investigated, only beer showed a significant association with DNA migration among ALDH2-deficient subjects. One plausible explanation might be that beer was most widely consumed by the subjects (Table 2) and it represented the exposure level of subjects to alcohol (or acetaldehyde); another possibility might be the antioxidant effects of other contents of beer, which is reported to prevent DNA damage and strand breaks (56,57). However, as a similar
phenomenon did not appear in ALDH2-profecient group, it is difficult to ascribe the decrease in DNA migration simply to the antioxidant effects of some components in beer. In summary, the association between alcohol consumption and reduced electrophoretic DNA migration in peripheral blood leukocytes from Japanese males is ALDH2 genotype dependent, and one of the most plausible explanations for such a phenomenon might be that the accumulation of acetaldehyde, a DNA cross-linking agent in blood, plays an important role in retarding the migration of DNA fragments in leukocytes of alcohol drinkers who possess an ALDH2-deficient genotype. In addition, other unknown factors, such as additional components in beer, should be also paid attention in future studies. Funding JSPS Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (No. 17590513). Acknowledgements We would like to thank the volunteers for their enthusiastic participation, the Association for Preventive Medicine of Japan for their assistance in collecting the blood samples and Kunio Nakayama for his help with the lifestyle investigations. Conflict of interest statement: None declared.
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