Chronic hepatitis B carriers with null genotypes of glutathione S-transferase M1 and T1 polymorphisms who are exposed to aflatoxin are at increased risk of hepatocellular carcinoma

Chronic

Hepatitis

B

Carriers with Null

Genotypes

of

Glutathione

S-Transferase

Ml

and

Ti

Polymorphisms

Who

Are

Exposed

to

Aflatoxin

Are

at

Increased Risk of

Hepatocellular Carcinoma

Chien-Jen

Chen,'

Ming-Whei Yu,' Yun-Fan Liaw,2 Lian-Wen Wang,3 Sinnabhatr Chiamprasert,3

Farhan

Matin,3 Ari Hirvonen,4 Douglas A. Bell,4 and Regina

M.

Santella3

'InstituteofEpidemiology, College of Public Health, NationalTaiwan University,and 2LiverUnit,Chang-Gung Memorial Hospital, Taipei;

3DivisionofEnvironmental Health Sciences, School of Public Health, Columbia University, New York; and 4Laboratory of Biochemical Risk Analysis, National Institute ofEnvironmental Health Sciences, Research Triangle Park

Summary

This study was carried out toelucidate the effect of

gluta-thione S-transferase(GST) MlandTipolymorphismson

the aflatoxin-related hepatocarcinogenesis among chronic carriers ofhepatitis B surface antigen (HBsAg). A total of 32newlydiagnosedhepatocellularcarcinoma(HCC)cases and 73 age-matched controls selected from a cohort of 4,841 chronicHBsAgcarrierswhohadbeenfollowedfor

5 years were studied. The level of aflatoxin B1

(AFB1)-albumin adducts in their serum samples collected at the recruitment was examined by competitive enzyme-linked

immunosorbance assay, and genotypes of GST Ml and

Ti were determined byPCR.Therewas adose-response

relationship between serum level ofAFB -albuminadducts

andriskofHCC.Thebiological gradients betweenserum

AFB1-albumin

adducts leveland HCC risk were observed among chronic HBsAg carriers who had null genotypes of GST Ml and/or

Ti

but not among those who had

non-nullgenotypes.Themultivariate-adjustedodds ratios

ofdevelopingHCCforthosewho had lowandhighserum

levels of

AFB,

-albumin adducts compared with those who

had aundetectable adduct levelasthereferent (oddsratio

=1.0) were 4.1and12.4,respectively,forHBsAg carriers with null GSTMl genotype (P< .01, on thebasisofthe

significance

testfortrend);0.7and1.4forthosewith

non-null GSTMlgenotype (P= .98); 1.8and 10.2 forthose with null GSTTi genotype (P< .05); and 1.3 and 0.8

for those with non-null GSTTi genotype (P= .93). The interactionbetweenserum AFB -albumin adductleveland

polymorphismsof GST Ml andTiwasatmarginal statis-ticalsignificance levels (.05 < P < .10).

Received November 13, 1995; accepted for publicationApril 5,

1996.

Address forcorrespondenceand reprints:Dr.Chien-JenChen,

Insti-tuteofEpidemiology,College of Public Health, NationalTaiwan

Uni-versity, 1 Jen-Ai Road Section 1, Taipei 10018, Taiwan. E-mail:

[email protected]

©1996byThe American SocietyofHumanGenetics.Allrights reserved.

0002-9297/96/5901-0018$02.00

Introduction

Hepatocellularcarcinoma (HCC) is a highly malignant

disease with anextremelypoor prognosis. It is a major cancer, with 1,000,000 deaths annually in the world

(Bosch and Munoz 1989). Both viral andchemical car-cinogens are involved in development of HCC in hu-mans, and chronic hepatitis B virus (HBV) infection is the most important risk factor for HCC in Taiwan as inothercountries(YuandChen1994).Thereare almost

300,000,000 chronic hepatitis B carriers in the world,

with the highest prevalence in Southeast Asia,

sub-Sa-haran Africa, and Greenland (Tiollais et al. 1985).

About one-fifth of chronic carriers are expected to de-velop HCC in their lifetime (Beasley 1988). The fact that HCC is not an inevitable consequence of chronic HBVinfectionhasstimulatedthesearch for otherHCC riskfactors.Inadditiontochronic carrier statusofHBV

surfaceantigen (HBsAg) and e antigen, cigarette

smok-ing,habitualalcoholconsumption, seropositivityof

an-tibodies against hepatitis C virus (anti-HCV), elevated

serum testosteronelevel,low

vegetable

consumption

fre-quencyand decreasedserumretinollevel, and aflatoxin

exposurehave beendocumentedasrisk factorsforHCC

inTaiwan (Chenetal. 1991, 1993; Lin et al. 1991; Yu

etal. 1991, 1995;Hatchetal. 1993; Yuand Chen1993;

Changet al. 1994).

AflatoxinB1(AFB1) isthemostpotent

hepatocarcino-gen in a variety of animal species (Dragan and Pitot

1994). It ismetabolizedbythemicrosomal

mixed-func-tion oxygenase enzyme system to various reduced and

oxidized derivatives,

including

an unstable reactive

AFB1-8,9-epoxide,whichcanbind

covalently

to

nucleo-philicsites of

biological

macromolecules, including

nu-cleic acids and proteins

(Gallagher

et al. 1994). The formationofAFB1 -guanineadducts

through

interaction

between

AFB1-8,9-epoxide

and

hepatic

DNA has been

shown to be critical for the carcinogenesis induced

by

AFB1 in animals (Kensler et al. 1986). AFB1 -guanine adducts are lost

rapidly

fromDNA and excreted inthe urine of AFB1 -treated animals.

A

significant

ecologicalcorrelation between aflatoxin

exposureand human HCC has been reported in Taiwan and other countries (Shank et al. 1972; Peers and Linsell 1973; Peers et al. 1976, 1987; Van Rosenburg et al. 1985; Yeh et al. 1989; Allen et al. 1992; Hatch et al. 1993). However, valid estimation of internal dose and biologically effective dose for individual exposure to

aflatoxin is still being developed. On the basis of an indirect immunofluorescence method (Zhang et al. 1991), AFB1-DNA adducts were detectable in liver tis-sues from 49 (64%) of 77 HCC patients in Taiwan (Zhang et al. 1991; Chen et al. 1992). A synergistic effect on HCC has recently been observed between uri-nary level of aflatoxins and HBsAg carrier status in Shanghai, China (Ross et al. 1992).

Because urinary aflatoxin level reflects intake on the previous day, it is an excellent marker for short-term exposure, but it may not reflect long-term intake by

individuals. While AFB1-guanine is the major DNA ad-duct, AFB1-albumin is the major protein adduct found in peripheral blood. Their use as a biomarker for afla-toxin exposure has several advantages: (1) aflatoxin-albumin adducts reflect DNA damage inhepatocytes,as does

aflatoxin-N7-guanine

in urine (Wild et al. 1986); (2) albumin adducts, at least in experimental animals, are as long-livedas albumin,which has ahalf-lifeof 21 d in humans, and thus provide a measure of exposure over aperiod of 2-3 mo(Sabbionietal. 1987); and (3) multiple measurements of urinary aflatoxin are required to reflect average exposure, but only a single measure-ment of albumin adducts is needed to provide a repre-sentative average exposure (Hall and Wild 1994). In other words, serumlevel ofAFB1 -albumin adducts is a better estimate of long-term biologically effective dose ofaflatoxin exposure than is urinary level of

aflatoxin-N7-guanine.

However, the association between serum AFB1 -albumin adductscontent and HCC risk at an indi-vidual level has never beenreported.

GlutathioneS-transferases (GSTs) are a unique group

of multifunctionalisozymes thatplayanimportantrole

intheconjugation and detoxification of various xenobi-otics, such asaflatoxins andpolycyclic aromatic hydro-carbons (Liu et al. 1991; Bell et al. 1992). GST Ml and

Ti

are polymorphic in humans, and deficiency in their enzyme activity is caused by the inherited homozygous absenceof the genes (Strange 1993; Pemble et al. 1994). Theproportion of GST Ml null genotype was reported toincrease inlung and bladder cancer patients compared with controls (Zhong et al. 1991; Bell et al. 1993; Strange 1993). GST Ml plays an important role in

de-toxifyingDNA reactive metabolites of AFB1 (Liu et al. 1991), but theeffectof GST

Ti

on aflatoxin detoxifica-tion remains unclear. It has been shown that the 100,000-fold difference between mice and rats in liver cancer response todoses of AFB1 is attributable to the difference in GST-mu activity between species, and the

resultant difference in detoxification of the AFB1 epox-ide, which is formed just as readily in mice as in rats (Eaton and Gallagher 1994). Whether the aflatoxin-re-lated HCC risk is also modified by genotypes of GST M1 and T1 in humans remains to be elucidated.

The specific aim of this study is to assess the effect

of genotypes of GST Ml and T1 on the AFB1-related hepatocarcinogenicity in chronic HBsAg carriers. A dose-responserelationshipwasobserved between serum level of AFB1 -albumin adducts and risk of HCC. The biologicalgradientwasobserved for chronic HBsAg

car-riers who had null genotypes of GST Ml and/orTi,but not for those who had non-null genotypes atall.

Subjects and Methods Study Subjects

A cohort of 4,841 male, asymptomatic, chronic HBsAg carriers agedfrom 30 to 65 yearswas recruited

from theGovernment Employee Central Clinics and the Liver Unit of Chang-Gung Memorial Hospital in Tai-wan from August 1988 to June 1992.Theyall gave their consent toparticipateinthisstudyonavoluntarybasis. Atrecruitment, each study subjectwaspersonally inter-viewed according to a structured questionnaire, to ob-tain information ondemographic characteristics, habits ofcigarette smoking andalcohol drinking,dietary con-sumptionfrequency,and

personal

andfamily historyof variouschronic diseases. Both duration andquantity of cigarette smoking and alcohol drinking were queried.

"Having eversmokedcigarettes" was defined ashaving

smoked cigarettes :4 d/wk for n6 mo, "having ever drunk alcohol" as havingconsumedalcoholic beverage

¢ 1 dlwkfor

n6

mo. Questionnaire interview was car-ried outbypublichealth nurseswho were well trained to standardize their interviewtechniques.Bloodspecimens, including serum and white blood cellsfrom study

sub-jects, were collected, separated, and stored at -70°C until subsequent analysis. HBsAg and anti-HCV were

tested, respectively, by radioimmunoassay and enzyme immunoassay using commercial kits (Abbott).

Follow-up of study subjects was performed through variouschannels,including annual health examination,

personal telephone interviews, abstraction of medical

records, and data linkage with national death certifica-tionand cancerregistry systems. Thediagnosisof HCC was based on (1) positive findings on cytological or

pathologicalexamination and/or (2) positiveimages on angiogram, ultrasonography, and/or computerized

to-mography, combined with an alpha-fetoprotein level >400

ng/ml.

There were 37 new HCC cases identified

during the follow-up period. Controls were selected from the cohortof HBsAg carriers who remained unaf-fected throughout the period. They were matched with cases on the basis of age, recruitment clinic, and date

ofbiospecimen collection (within 3 mo) and randomly selected within matching strata. Because there werefive HCC cases and three matched controls who had no adequate biospecimens for the determination of serum

AFB1-albumin

adducts level and/or genotypes of GST, atotal of32 HCC cases and 73matched controls were included in this study.

Serum AFB1-Albumin Adducts Level

Albuminwas prepared fromplasmaessentially as

de-scribed by Wild etal. (1990a), and concentration was

determined with bicinchoninic acid (BCA Reagent Pierce). For the digestion, 2 mg albumin and 0.5 mg proteinase K were incubated 15 hat370C.Adducts were

isolatedby the procedure ofSheabaret al. (1993),

dis-solved in 0.5 ml phosphate buffer solution containing 1% FCS and 1 mM phenylmethyl-sulfonylfluoride, to inhibit residual protease activity, and assayed by com-petitive enzyme-linked immunosorbance assay (ELISA) using apolyclonal antiserum. Thedetection limit of se-rumAFB1-albumin adducts levelwas 0.01 fmol/jg.

In order to prepare an antiserum for the detection

of AFB1-albumin adducts, human serum albumin and bovinegammaglobulinweremodified withAFB1 epox-idesynthesizedby the method of Baertschietal. (1988). Human serum albumin or bovine gamma globulin (9 mg in 5 ml 0.05 Mphosphate bufferpH 7.4) were mixed with 0.2 mg of theepoxidegeneratedfrom AFB1or[3H]

AFB1 (25 mCi/mmol; Moravic) in methylene chloride andincubatedovernightat roomtemperature. The

sam-plesweredialyzed extensivelyto removenonbound ma-terial.Modification levelsweredetermined from the ab-sorbanceat 450nm(e = 12,700) or thespecificactivity

andranged from0.6 to 2.3molAFB1/molprotein. New Zealandwhite rabbits were immunized by

intramuscu-lar injection at four sites with 1 mg of the modified

bovine gamma globulin (2.3 mol/mol) in complete

Freund's adjuvant, followed by monthly boostingwith 1 mg protein in incomplete adjuvant. Antiserum (#7) wascharacterizedbycompetitive ELISA. FortheELISA

standard curve, [3H] AFB1-human serum albumin was

digested

with proteinase K as described above for the

human samples and adducts isolated by Seppak C18 (Waters) extraction usingcartridgesthat had been pre-washed with 10 mlchloroform, 10mlmethanol, and10

ml water. Afterapplication of the samplein phosphate buffer and washing with 10 ml water and 5 ml 5%

methanol, the adducts were isolated with 5 ml 80% methanol. Adducts levels were determined from the spe-cificactivity.

Ninety-six microwell plates (Easywash, Corning) were coated with 3 ngAFB1-human serumalbumin by drying phosphate buffer solution. Nonspecific binding

was blocked by incubation for 1 h at 370C with 1% FCS in phosphate buffer solution containing 0.05%

Tween 20.Standards (50

gl

containing 3-100fmol

ad-duct) or human samples (equivalent of 100 jg) were then addedtoeachwell, followed bythe antiserum (50 ,l 1:200,000 dilution) and theplates incubated 90 min at 37°C. After washing, goat anti-rabbit IgG akaline phosphatase (1:500 dilution, Boehringer Mannheim) wasadded and theplates incubated at 37°C for 90 min. Afterwashing, 0.1 ml 1 mg/ml p-nitrophenyl phosphate in1 Mdiethanolamine (pH 8.6) was added to each well. Theabsorbance at 405 nm was measured on a Dynatech MR 2000 microplate reader (Dynatech Laboratories). This assay had 50% inhibition ofantiserum binding at 10-20 fmol AFB1 -adduct per well. The limit of sensitiv-ity (20% inhibition), when assaying the equivalent of 200 jg albumin/well, was 0.01

fmol/gg.

Samples were assayed by duplicate analysis induplicatewells; samples with <20% inhibition were considered undetectable. Twocontrol samples wereanalyzed with each batch of sera, apooled sample ofplasma fromnonsmokingU.S.

subjects, and a positive control of serum from a rat treated with 1.5 mg AFB1.

Genotypesof GST MI and Ti

Genotype of GST Ml was identified in leukocyte DNA after PCR amplification with primers to exons 6 and7, whichproduceda210-bpband(Belletal. 1993), while GST Ti genotype was determined by using the

technique of Pemble et al. (1994) with the additional

modification thatbeta-globinprimers were added to the PCR(Bell et al. 1993).

StatisticalAnalysis

Because it was notconsidered appropriate to assign avaluetothe undetectableserumlevelofAFB1 -albumin

adducts,theadducts levelwasanalyzedas acategorical, ratherthan continuous, variable. It wascategorizedinto three groups(undetectable, lowdetectable,andhigh

de-tectable),to examinethe

dose-response relationship

be-tweenaflatoxinexposure andHCCrisk. Theserumlevel of AFB1 -albumin adducts between HCC cases and

matched controlswas firstcompared. Oddsratioswith 95%confidenceintervals werecalculatedtoindicate the

magnitudeof associationsbetweenserumlevelof AFB1

-albumin adducts and HCC risk. Mantel-Haenszel X2 tests fortrend wereused toexamine the statistical

sig-nificance ofthe biological gradient.

Multiple

logistic

re-gression analysis was used to estimate odds ratios of

developingHCCforserumAFB1 -albuminadducts level after adjustment for habitsofcigarette

smoking

and al-cohol drinking, which have been documented as risk factors for HCC (Chen et al. 1991, 1993). The dose-responserelationships betweenaflatoxin andHCC were

furtherexamined

through

stratification

analyses

for

dif-ferentgroups

categorized by

genotypes ofGSTMl and

be-Table 1

Serum LevelofAFB-Albumin Adducts in CasesAffectedwith HCC and Matched Controls

HCC CASES CONTROLS

SERUM AFB-ALBUMIN ADJUSTED ODDSRATIOa

LEVEL No. (%) No. (%) (95% confidenceinterval)

Undetectable 14 (43.7) 44 (60.3) 1.0 (referent)

Low 12 (37.5) 24 (32.9) 1.6(.6-4.0)

High 6 (18.8) 5 (6.8) 3.8(1.0-14.5)*

aAdjustmentforhabitsofcigarettesmokingand alcoholconsumptionthroughmultiple logisticregression

analysis.

*P-valuebased onthe statistical significancetestof odds ratio <.05; P-valuebasedonthetestfor trend

= .03.

tweenserumAFB1-albuminadducts level andgenotypes

of GSTMl andT1 wasalso examinedthrough logistic

regression analysis. SAS/STAT software (SAS Institute

Inc.) was used for the data analysis.

Results

Among 32 HCC cases, 12 were diagnosed within 2 years ofrecruitmentand20 >2yearsafterrecruitment.

The means ± SD ofageat recruitment for HCC cases

and matched controlswere 51.3 ± 9.7and 51.5 ± 9.9

years, respectively. Cases and controls had similar fre-quencydistributionsamong caseswho hadeversmoked

cigarettes (39% vs. 35%) or drunk alcohol (21% vs. 20%).

Table1 comparestheserumlevel of

AFB,

-albumin

ad-ducts betweenHCCcasesand healthy controls. HCCcases

had higher levels of serum

AFB,

-albumin adducts than

matched controls.Astatistically significant (P= .05,based

onMantel-Haenszel x2testfor trend) dose-response

rela-tionwas observed betweenserum level ofAFB1-albumin

adducts andHCCrisk. Thehigher theserum

AFB,

-albu-minadductslevel, the higher theHCCrisk. Thebiological gradient remained significant (P = .03) after adjustment for cigarette smoking and alcohol consumption through multiple logistic regression analysis.

Tables2 and 3show the biological gradient between

serumlevel ofAFB1 -albumin adducts and risk of HCC

stratifiedbygenotypes of GSTMl andTi,respectively. The dose-response relationship between aflatoxin expo-sureandHCCriskremainedstatistically significant with

increases inthemagnitude of oddsratiosamongchronic

HBsAgcarrierswithnullgenotypesof GST Ml and Ti.

However, no associations between aflatoxin and HCC were observedamongcarriers with non-nullgenotypes.

The monotonic increase in aflatoxin-related HCC risk for chronic HBsAg carriers with nullgenotypesofGST

Ml and Ti remained after adjustment for cigarette smoking and alcohol consumption. The statistical sig-nificance for theinteractiontermsbetweenserumAFB1

-albumin adductslevelandgenotypesof GSTMi andTi

wasmarginal, with P-values of .06 and .08,respectively,

after adjustment for cigarette smoking and alcohol drinking.

Table 2

BiologicalGradientBetweenSerumLevel of AFB-Albumin Adducts and Risk ofHCCbyGenotypesof

GlutathioneS-TransferaseMI

NULL NON-NULL

SERUMAFB-ALBUMIN Case Control Adjusted Odds Case Control Adjusted Odds

LEVEL (No.) (No.) Ratio (95% CI)' (No.) (No.) Ratio (95% CI)

Undetectable 5 27 1.0 (referent) 9 17 1.0 (referent) Low 8 14 4.1 (1.0-16.9)* 4 10 .7 (.2-3.2)

High 4 2 12.4 (1.7-92.7)** 2 3 1.4 (.2-10.9)

aAdjustmentforhabitsof cigarettesmoking andalcohol consumption through multiple logistic regression

analysis.

*P<.05.

**P< .01 (basedonthe statisticalsignificance testof oddsratio);P-value basedon the testfortrend

Table 3

Biological Gradient between SerumLevel of AFB-Albumin Adducts and Risk ofHCC,byGenotypesof Glutathione S-Transferase T1V

NULL NON-NULL

SERUM AFB1-ALBUMIN Case Control AdjustedOdds Case Control AdjustedOdds

LEVEL (No.) (No.) Ratio(95% CI)b (No.) (No.) Ratio(95% CI)

Undetectable 3 19 1.0(referent) 10 23 1.0 (referent)

Low 4 14 1.8 (.3-9.7) 5 9 1.3 (.3-5.3)

High 5 3 10.2(1.3-78.2)* 1 2 .8 (.1-12.0)

aGSTT1 genotype was not available for four cases and three controls.

bAdjustment forhabitsof cigarettesmoking and alcohol consumptionthrough multiple logisticregression

analysis.

*P < .05 (based on thestatistical significance test of odds ratio); P-value based on the test for trend

<.05 for null set and =.93fornon-null set.

Discussion

Aflatoxins are toxic metabolites produced by various Aspergillus species. They are common contaminants of humanfoodstuffs such as maize, corn, peanuts, and rice, especially in sub-Saharan Africa and Southeast Asia (Wild et al. 1990a) where both HBV infection and hepa-tocellular carcinoma are also prevalent. A nested case-control study carried out in Shanghai has shown a syner-gisticeffecton thedevelopmentof HCCbetweenHBsAg carrier status and urinary aflatoxin biomarkers includ-ingunmetabolized aflatoxins, hydroxylated and

demeth-ylated metabolites, and aflatoxin-N7-guanine adducts. This interaction between chemical and viral hepatocar-cinogens provides important clues for prevention of HCC among chronic HBsAg carriers. But no dose-re-sponserelationship between urinaryaflatoxin level and

HCCrisk was examinedinthese two reports.

Because urinary aflatoxin level reflects intake on the

previous day, it may notreflect long-term intake by

indi-viduals unless theirexposures toaflatoxin arerelatively

constant. The serum level of the albumin adducts is a biomarker of choice for biologically effective dose of

long-termexposure to aflatoxin. Onthe basis of such a representative biomarker for average long-term expo-sure to aflatoxin, we found for the first time a statisti-cally significant dose-response relationship between aflatoxin and HCC, despite the fact that the numbers of cases and controls includedinthisstudy were slightly smaller than those inprevious studies inShanghai(Ross etal. 1992; Qian et al. 1994). Since welimitedourstudy

on casesand controlswho were chronic HBsAg carriers, it is not possible for us to elucidate the interaction be-tween HBsAg carrier status andaflatoxin exposure.

Animal experiment has shown a striking species dif-ference in liver-cancer response toAFB1,which isreadily

explained by the species difference in GST-mu activities

(Eatonand Gallagher 1994). We consistently found evi-dencethat theaflatoxin-related HCC risk may be

modi-fied by genotypes of GST Ml and Ti in humans. The biological gradient between serum

AFB,-albumin

ad-ducts level and HCC risk was significant among chronic HBsAg carriers with null genotypes of GST Ml and Ti but not among carriers withnon-nullgenotypes. Inother words, those who had no detoxifying enzymes such as GST Ml and Ti are at a considerably greater risk of developing HCC once they are exposed to aflatoxin. The lackof an association in the non-null group could be a consequence of the small sample size. The interaction

terms between serum

AFB,

-albumin adducts level and genotypesofGSTMiand Ti were atmarginal statisti-calsignificancelevels (.05< P < .10), possibly resulting from the small sample size in this study. However, the observed significant effect on the development of

afla-toxin-associatedHCCsuggests that HBsAg carrierswho

have a high aflatoxin exposure and no GST M1 and!

or Ti enzymes will have a 10-fold increased risk of developingHCC.Thegene-environment interaction be-tween aflatoxin exposure and GST polymorphism de-servesfurther elucidation based on alarger sample.The effects of genetic polymorphisms of cytochrome P450 enzymes on the development of HCC should also be

investigated.

Acknowledgments

Thisstudywassupported bygrantsfromthe National Sci-ence Council (NSC83-0412-B002-256) and Department of Health (DOH-H7707, H7903, H8003, H8103,andH8203),

ExecutiveYuan,Republic of China,andaNationalInstitutes

of Health grant(ES05116).

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