Genetic polymorphisms of glutathione S-transferases M 1 and T 1 associated with susceptibility to aflatoxin-related hepatocarcinogenesis among chronic hepatitis B carriers : a nested case – control study in Taiwan

Chien-An Sun1,6, Li-Yu Wang2, Chien-Jen Chen3, Introduction Sheng-Nan Lu4, San-Lin You3, Lian-Wen Wang5, Primary hepatocellular carcinoma (HCC) is a tumor that occurs Qiao Wang5, Der-Ming Wu1 and Regina M.Santella5 at high frequencies in southeast Asia and tropical Africa (1). In Taiwan HCC is the leading cancer in males and the fifth in 1School of Public Health, National Defense Medical Center, Taipei, Taiwan, 2Institute of Aboriginal Health, Tzu Chi University, Hualien County, females (2). Etiologically HCC is a complex and multifactorial Taiwan, 3Graduate Institute of Epidemiology, College of Public Health, disease that is linked to both viral and chemical carcinogens National Taiwan University, Taipei, Taiwan, 4Liver Unit, Kaohsiung (3–7). On the basis of epidemiological evidence, 70–90% of Medical Center, Chang Gung Memorial Hospital, Kaohsiung County, HCC patients in Taiwan are seropositive for hepatitis B virus Taiwan, Republic of China and 5Division of Environmental Health Sciences, surface antigen (HBsAg) or antibodies to hepatitis C virus Mailman School of Public Health, Columbia University, New York, NY,

that risk factors other than viral infection also contribute to This study was conducted to investigate the modifying the development of HCC in Taiwan.effect of glutathione S-transferase (GST) M1 and T1 poly-Aflatoxin B 1 (AFB 1 ) is a hepatocarcinogen in several animal morphisms on aflatoxin-induced hepatocarcinogenesis species (8).In humans epidemiological studies using urine and among chronic hepatitis B virus surface antigen (HBsAg) blood samples banked several years prior to diagnosis have carriers.A total of 79 HBsAg-positive cases of hepatocellushown that the presence of aflatoxin metabolites in urine and lar carcinoma (HCC) diagnosed between 1991 and 1997 aflatoxin-albumin adducts was associated with a significantly were identified and individually matched to one or two elevated risk of HCC (4,(9)(10)(11).In Taiwan several epidemio-HBsAg-positive controls on age, gender, residence and date logical studies have noted a significant aflatoxin-liver cancer of recruitment from the same cancer screening cohort in link.Our previous case-control study nested in a cancer Taiwan.Blood samples were tested for hepatitis B and C screening program with 56 cases of HCC and 220 matched viral markers by enzyme immunoassay and for aflatoxin controls from the same cohort of Ͼ25 000 individuals found B 1 (AFB 1 )-albumin adducts by competitive enzyme-linked that in hepatitis B virus (HBV)-infected males there was an immunosorbent assay.GSTM1 and GSTT1 genotypes were adjusted odds ratio (OR) of 2.8 for detectable compared with determined by PCR.There was a statistically significant non-detectable aflatoxin-albumin adducts and 5.5 for high relationship between detectable levels of AFB 1 -albumin compared with low levels of aflatoxin metabolites in urine adducts in serum and risk of HCC among chronic HBsAg (10).A nested case-control study in another cohort in Taiwan carriers, with an adjusted odds ratio (OR) of 2.0 [95% observed a dose-response relationship between urinary levels of AFM 1 (the primary oxidative metabolite of AFB 1 ) and HCC confidence interval (CI) 1.1-3.7].In addition, the effect of in chronic HBV carriers (11).aflatoxin exposure on HCC risk was more pronounced Accumulating evidence indicates that susceptibility to cancer among chronic HBsAg carriers with the GSTT1 null genois mediated by genetically determined differences in the type (OR 3.7, 95% CI 1.5-9.3)than those who were noneffectiveness of detoxification of potential carcinogens (12).null (OR 0.9, 95% CI 0.

3-2.4). The interaction between
Indeed, it has been recognized that the carcinogenic potential serum AFB 1 -albumin adduct level and GSTT1 genotype of AFB 1 may vary with the exposed individual's capacity was statistically significant (P ⍧ 0.03).For GSTM1 the to detoxify its mutagenic metabolite, aflatoxin 8,9 epoxide.effect of aflatoxin exposure on HCC risk in those with the Epidemiological studies have suggested that genetic polynull genotype was also greater (adjusted OR 2.8, 95% CI morphisms in AFB 1 metabolizing enzymes are a factor in 1.0-7.8)than in those with the gene present (adjusted OR individual susceptibility to aflatoxin-related HCC (13,14).1.8, 95% CI 0.8-4.5),but the difference was not significant Members of the glutathione transferase (GST) family, such as (P ⍧ 0.91).Notably, when the interaction between aflatoxin GST-µ (GSTM1) and GST-θ (GSTT1), are important candidates exposure and GSTT1 genotype was considered, aflatoxin for involvement in susceptibility to aflatoxin-associated liver exposure by itself was not a significant determinant of cancer because they may regulate an individual's ability to HCC risk among chronic HBsAg carriers.These results metabolize the ultimate carcinogen of aflatoxins, the exodemonstrate the importance of gene-environment interepoxide (15).To date the human cytosolic GST superfamily actions in the multifactorial development of HCC.
contains at least 16 genes subdivided into eight distinct classes designated α, Φ, µ, π, σ, θ, ζ and ω (16).A number of recent reviews have summarized the role of GST-µ (GSTM1) and among those who had non-null genotypes (14).In the present In this study serum specimens were grouped into case-control pairs and were assayed on the same day to minimize any effects of day-to-day laboratory work we have continued to follow up the cohort, expanding variation.Because specimens from cases and controls were treated similarly, the number of HCC cases analyzed for AFB 1 -albumin adducts differential losses should not have occurred, so case-control comparison on as well as determining the effects of GSTM1 and GSTT1 a relative scale should not be affected.In addition, laboratory personnel were genotypes on aflatoxin-related hepatocarcinogenesis among kept blind as to case or control status.chronic HBV carriers.
GSTM1 and GSTT1 genotypes GSTM1 genotyping for gene deletion was performed by PCR amplification with primers for exons 6 and 7, which produced a 210 bp band, according to

Materials and methods the method of Bell et al. (22). GSTT1 genotype was determined using the Study cohort
technique of Pemble et al. (19), with the modification that β-globin primers were added to the PCR.This nested case-control study was conducted within a cancer screening cohort of individuals who were between 30 and 64 years old and lived in Statistical methods seven townships in Taiwan.The cohort characteristics and methods of In the present report the serum level of AFB 1 -albumin adducts was analyzed screening and follow-up have been described previously (10,20).From July as a binary rather than continuous variable.Adjusted ORs and 95% confidence 1990 to June 1992 a community-based cancer screening project was carried intervals (CI), which were derived from conditional logistic regression models, out in seven townships.Makung, Huhsi and Paihsa Townships are located on were used to indicate the magnitude of the association between serum levels the Penghu Islets, an area with the highest mortality rate of HCC in Taiwan of AFB 1 -albumin adducts and HCC risk.The relationship between AFB 1 - (21).The other four townships, Sanchi, Chutung, Potzu and Kaoshu, are albumin adduct level and HCC risk was further examined through stratified located on the main Taiwan Island.There were 47 079 male and 42 263 analyses for different groups categorized by GSTM1 and GSTT1 genotype.female eligible individuals who were invited by letter to participate.A total The statistical significance for the interaction terms between serum AFB 1of 12 024 males and 13 594 females enrolled.All study subjects participated albumin adduct level and GSTM1 and GSTT1 genotype was also examined on a voluntary basis after giving informed consent.
through logistic regression analysis.All analyses were performed with SAS Participants were personally interviewed based on a structured questionnaire software (SAS Institute, Cary, NC). at recruitment.Blood samples were collected from each study subject.Aliquots In the present study four cases and nine controls were excluded from the of serum, buffy coat, plasma and red blood cells were separated and stored analysis because of inadequate blood samples for detection of AFB 1 -albumin at -70°C.Specimens were transported in dry ice to the central laboratory adducts in serum.Additionally, due to the failure of GSTM1 genotyping in at the National Taiwan University and were kept in deep freezers until 10 cases and 21 controls and of GSTT1 genotyping in 12 cases and 21 examination.
controls, these subjects were excluded from data analyses for the relevant In the program study subjects were first screened by serological markers, variables.Nevertheless, in their distributions by sex, age and residential area including alanine transaminase (ALT), aspartate transaminase (AST), αsubjects for whom specimens were available for study were similar to those fetoprotein (AFP), HBsAg and anti-HCV.Any subject who had an elevated for whom specimens were unavailable.level of ALT (ജ45 IU/ml), AST (ജ40 IU/ml) or AFP (ജ20 ng/ml), positivity for HBsAg or anti-HCV or a family history of HCC or liver cirrhosis among

Results
first degree relatives was referred for upper abdominal ultrasonography examination by board certified gastroenterologists.Subsequently, participants This study included 79 (66 male and 13 female) cases and who were found to be affected with liver cirrhosis by ultrasonography were intensively followed up every 3 months, whereas others were examined 149 (122 male and 27 female) controls (Table I).The mean annually.Any suspected HCC cases thus identified were referred to teaching age of both case and control subjects was 53 Ϯ 7 years.The  in Table II.HCC cases had a higher percentage of detectable genotype profiles on aflatoxin-related HCC risk among chronic HBsAg carriers AFB 1 -albumin adducts than controls (62.7 versus 45.7%), we selected 79 HCC cases who were chronic HBsAg carriers as the case group for this nested case-control study.On the basis of the availability of resulting in a statistically significantly adjusted OR of 2.0 CI 1.5-9.3)than those who had a non-null genotype (OR 0.9, produces liver tumors when administered to animals (23).
c Data not available for five cases and nine controls.
Human exposure to aflatoxin has been assessed in the context of epidemiological studies either by questionnaire or by laboratory measurements on biospecimens, such as urine or peripheral  as indicated by an OR of 1.4-1.9, was observed in previous c DNA samples were inadequate to do the GSTM1 genotyping in 10 cases and 21 controls and the GSTT1 genotyping in 12 cases and 21 controls.
epidemiological studies conducted in Taiwan (26,27).This weak association may result from the inadequacy of the questionnaire in measuring aflatoxin exposure.Subsequently, (95% CI 1.1-3.7).In contrast to the expected distribution, the frequencies of the GSTM1 and GSTT1 null genotypes were several nested case-control studies using biomarkers of aflatoxin exposure have been undertaken in Taiwan to examine significantly lower in HCC cases (37.7 and 44.8%, respectively) than in controls (60.2% for both the GSTM1 and GSTT1 null the relationship between aflatoxin exposure and incidence of HCC, and have confirmed the results of the Shanghai study genotypes).
To address the hypothesis that the effect of detectable levels (10,11).Urinary levels of AFM 1 were linked to the risk of HCC in a dose-dependent fashion (11) and high levels of total of AFB 1 -albumin adducts in serum on HCC risk might vary according to GSTM1 and GSTT1 genotype data were further urinary aflatoxin metabolites, as compared with low levels, were associated with a 5-fold increase in HCC risk in chronic examined within categories stratified by GSTM1 and GSTT1 genotype.As shown in Table III, the effect of aflatoxin HBV carriers (10).In addition, the presence of AFB 1 -albumin adducts in serum resulted in a 3-to 5-fold elevation in the exposure on HCC risk was more pronounced among chronic HBsAg carriers with a GSTT1 null genotype (OR 3.7, 95% risk of developing HCC (9,10).In the present study we found increased prostate cancer risk.Similarly, the current study observed that individuals who have presumably expressed and a statistically significantly adjusted OR of developing HCC functional GST-θ protein are at increased risk of HCC.These for detectable AFB1-albumin adducts (adjusted OR 2.0, 95% findings are consistent with the knowledge that some metabolic CI 1.1-3.7),which is comparable with previous studies using intermediates in the GST-θ pathway of glutathione conjugation, the same biomarker of exposure to aflatoxins (9,10,14).Taken i.e. dichloromethane, are mutagenic (28) and the production of together, this and previous nested case-control studies (9-11) mutagenic intermediates in the target site may be a mechanism using specimens banked before disease onset have provided promoting tumorigenesis.Another potential explanation the most reliable data on the role of AFB 1 in hepatocarcinoderives from a follow-up study by Kelada et al. (29).This genesis in Taiwan.
study looked at the interaction between smoking and GSTM1 While the present study and previous reports (4,(9)(10)(11)14) and GSTT1 genotype and risk of prostate cancer.While the have demonstrated that aflatoxin exposure is an important risk presence of a functional GSTT1 genotype was confirmed as a factor for HCC, several epidemiological observations have risk factor for prostate cancer, no main effect of smoking was implied that the carcinogenic potential of AFB 1 varies with observed; a significant increased risk was observed in men the exposed individual's capacity to detoxify its mutagenic who were both smokers and had a non-deleted GSTT1 genotype.metabolite, aflatoxin 8,9 epoxide (13,14).In 1995 McGlynn The authors suggest that in addition to the production of et al. (13) reported on a cross-sectional study and a casecarcinogenic metabolic intermediates, response to chronic control comparison in Ghana and China to assess serum levels inflammation associated with smoking may be modulated by of AFB 1 -albumin adducts and the development of HCC in the GSTT1 genotype.A similar inflammatory mechanism may relation to genetic polymorphisms of detoxifying enzymes take place in hepatocarcinogenesis.It has been documented involved in biotransformation of aflatoxins.The results support that chronic HBV infection induces recurrent liver injury and the existence of genetic susceptibility to the carcinogenic effect a resultant inflammatory reaction (30).It is known that the of AFB 1 .A subsequent study in Taiwan gave results consistent GSTs are, in general, mediators of the inflammatory response with the prior results from the HCC endemic areas of Ghana (31).Thus it is possible that upon exposure to aflatoxins of and China; a biological gradient between serum AFB 1 -albumin chronic HBsAg carriers a more vigorous or more chronic adduct levels and HCC risk was observed among HBsAg inflammatory response is mounted among individuals with chronic carriers who had GSTM1 and/or GSTT1 null genotypes non-deleted GSTM1 and GSTT1 genotypes, which paves the but not among those who had non-null genotypes (14).In this way for carcinogenesis (32).Finally, it has been noted that report we observed a significantly increased risk for aflatoxin-GST levels are regulated by a number of factors, including related HCC among chronic HBsAg carriers with the GSTT1 diet, in addition to inherited variants in genes coding for the null genotype but not among those who had a non-null GST enzymes (33,34).In recent literature efforts appear to genotype.However, while a similar trend was observed for concentrate on the interplay between dietary inducers of GST GSTM1, the results were not statistically significant.
Rather unexpectedly, a significant inverse relationship and GST genotypes in cancer risk.For example, in a prospective medical centers for confirmatory diagnosis by computerized tomography, distribution of residential area was similar for cases and digital subtracted angiogram, aspiration cytology and pathological examination.
controls.In addition, cases and controls had non-significant Therefore, a final diagnosis of HCC was based on either histological/ differences in frequency distributions for cigarette smoking cytological findings or elevated AFP levels Ͼ400 ng/ml combined with at least one positive image on an angiogram or by ultrasonography and/or (53.2 and 44.3%, respectively) and alcohol consumption(21.8computerized tomography.and 18.1%, respectively).Anti-HCV was detected in 10.8%Study subjects (8/74) of HCC cases and 7.9% (11/140) of controls.There were 99 confirmed cases of HCC, including 79 HBsAg-positive andThe risk of HCC associated with serum level of AFB 1 -20 HBsAg-negative individuals, identified during the follow-up period between albumin adducts and of GSTM1 and GSTT1 genotype is shown 1991 and 1997.To examine the modulatory effect of GSTM1 and GSTT1

( 25 )
. A non-significantly positive association between peanut b Four cases and nine controls had inadequate serum samples for detection of consumption derived from questionnaire data and risk of HCC, AFB 1 -albumin adducts.
/well.The limit of sensitivity (20% inhibition) when assaying the Accordingly, a report by Chen et al. documented that a equivalent of 200 µg albumin/well was 0.01 fmol/µg.Samples were assayed by duplicate analysis in duplicate wells; samples with Ͻ20% inhibition were biological gradient between serum AFB 1 -albumin adduct considered non-detectable.Two control samples were analyzed with each batch levels and HCC risk was observed among chronic HBsAg of sera, a pooled sample of plasma from non-smoking US subjects and a positive carriers who had null GSTM1 and GSTT1 genotypes but not control of serum from a rat treated with 1.5 mg AFB 1 .
C.-A.Sun et al. adduct

Table I .
Baseline characteristics of 79 cases with HCC and 149 matched 95% CI 0.3-2.4).The interaction between serum AFB 1 - a Data not available for one case.in human hepatocellular carcinogenesis, largely because it b Anti-HCV, antibodies against hepatitis C virus.

Table II .
Risk of HCC in relation to detectable levels of aflatoxin-albumin a Adjusted for sex, age and residence.

Table III .
OR of HCC in relation to levels of aflatoxin-albumin adducts among chronic hepatitis B virus carriers, stratified by GSTM1 and GSTT1 genotypes Four cases and nine controls had inadequate blood samples for detection of aflatoxin-albumin adducts in serum.bAdjusted for age, sex and residence.cDNA samples were inadequate to do the GSTM1 genotyping in 10 cases and 21 controls and the GSTT1 genotyping in 12 cases and 21 controls.betweenboth GSTM1 and GSTT1 null genotypes and risk of a

Table IV .
Multivariate analysis of risk determinants for HCC in chronic HCC was observed in the current study.This finding is in hepatitis B virus carriers conflict with the knowledge that individuals who are deletion homozygotes exhibit absence of enzymatic activity and are a Non-detectable ϭ 0, detectable ϭ 1. contrast, Rebbeck et al. (18) recently reported case-control b Non-null ϭ 0, null ϭ 1.study results that indicate that men with GSTT1 present are at c Non-null ϭ 0, null ϭ 1.