Cytochrome P450 1A1 genetic polymorphisms and risk of hepatocellular carcinoma among chronic hepatitis B carriers

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1 British Journl of Cncer (1999) 80(3/4), Cncer Reserch Cmpign Article no. bjoc Cytochrome P450 1A1 genetic polymorphisms nd risk of heptocellulr crcinom mong chronic heptitis B crriers M-W Yu 1,2, Y-H Chiu 2, S-Y Yng 2, RM Sntell 3, H-D Chern 4, Y-F Liw 5 nd C-J Chen 2 1 School of Public Helth nd 2 Grdute Institute of Epidemiology, College of Public Helth, Ntionl Tiwn University, Tipei, Tiwn; 3 Division of Environmentl Helth Sciences, School of Public Helth, Columbi University, New York, NY 10032, USA; 4 Grdute Institute of Phrmceuticl Sciences, College of Medicine, Ntionl Tiwn University, Tipei, Tiwn; 5 Liver Reserch Unit, Chng-Gung Memoril Hospitl nd Chng-Gung Medicl College, Tipei, Tiwn Summry Cigrette smoking hs been ssocited with incresed risk of heptocellulr crcinom (HCC) in some epidemiologicl studies. Cytochrome P450 1A1 (CYP1A1) is involved in the biotrnsformtion of tobcco-derived polycyclic romtic hydrocrbons (PAHs) into crcinogenic metbolites. The im of this study ws to determine whether CYP1A1 polymorphisms were relted to HCC risk mong chronic heptitis B virus (HBV) crriers. Genotypic vrints of CYP1A1 were determined using polymerse chin rection in 81 incident cses of HCC nd 409 controls nested in cohort study of 4841 mle chronic HBV crriers. No overll ssocition between CYP1A1 genotypes nd HCC ws observed. The presence of the MspI (odds rtio (OR) 3.15, P = ) or Ile-Vl (OR 1.99, P = ) vrint llele of CYP1A1 incresed HCC risk mong smokers, but posed no incresed risk mong non-smokers. The smoking-relted HCC risk ws most pronounced mong those who hd susceptible llele of the CYP1A1 nd deficient genotype of glutthione S-trnsferse M1, which detoxifies PAH electrophilic metbolites produced by CYP1A1. In the bsence of the Ile-Vl vrint llele, the MspI polymorphism ws still ssocited with smoking-relted HCC. This study suggests tht tobcco-derived PAHs ply role in HCC risk mong chronic HBV crriers, nd CYP1A1 polymorphism is n importnt modultor of the heptocrcinogenic effect of PAHs. The MspI nd Ile-Vl polymorphisms of CYP1A1 my hve different mechnisms for incresing susceptibility to smoking-relted HCC. Keywords: chronic heptitis B virus crriers; cigrette smoking; cytochrome P450 1A1; glutthione S-trnsferse M1; heptocellulr crcinom; nested cse-control study Heptocellulr crcinom (HCC) is reltively rre tumour in the USA nd Europe, but it is common in much of Asi nd sub-shrn Afric (Yu nd Chen, 1994). There re lmost chronic heptitis B virus (HBV) crriers in the world, with the highest prevlence in Southest Asi, sub-shrn Afric nd Greenlnd (Tiollis et l, 1985). Although chronic HBV infection is the mjor cuse of t lest 80% of HCC throughout the world (Yu nd Chen, 1994), only minority of chronic HBV crriers re expected to develop HCC in their lifetime (Besley, 1988). The fct tht HCC is not n inevitble consequence of chronic HBV infection hs stimulted the serch for other risk fctors. In ddition to HBV, mny other environmentl fctors hve been documented s risk fctors for HCC, including chronic infection with heptitis C virus (HCV), fltoxin exposure, cigrette smoking, lcohol drinking nd low vegetble intke (Tsukum et l, 1990; Chen et l, 1991, 1996; Yu et l, 1991; Yu et l, 1991, 1995b, 1997;Yu nd Chen, 1994). Among environmentl risk fctors of HCC other thn HBV, cigrette smoking is the most prevlent in the generl popultion in Tiwn. However, the Received 2 July 1998 Revised 1 October 1998 Accepted 20 October 1998 Correspondence to: C-J Chen, Grdute Institute of Epidemiology, College of Public Helth, Ntionl Tiwn University, No. 1 Jen-Ai Rd. Sec. 1, Room 1547, Tipei 100, Tiwn reltive risk of HCC ssocited with cigrette smoking is low in the bsence of chronic HBV or HCV infection (Chen et l, 1991; Yu et l, 1991). While there is convincing epidemiologicl evidence nd widespred wreness of the role of cigrette smoke s cuse for cncer of the lung, upper erodigestive trct nd bldder (Interntionl Agency for Reserch on Cncer, 1986; Spitz et l, 1989; Tlsk et l, 1991; Nkchi et l, 1993; Wynder nd Hoffmnn, 1994; Musct et l, 1996), the root mechnism responsible for the ction of cigrette smoking in heptocrcinogenesis is not yet well understood. Crcinogenic compounds in cigrette smoke which hve been extensively reviewed include polycyclic romtic hydrocrbons (PAHs), N-nitrosmines nd romtic mines (Interntionl Agency for Reserch on Cncer, 1986). The orgnospecificity of these tobcco-relted crcinogens for the liver in humns is unknown. Most genotoxic chemicl crcinogens re not intrinsiclly rective but require metbolic conversion to DNA-binding intermedites (Guengerich nd Shimd, 1991). Our recent study showing significnt ssocition between genetic polymorphism in cytochrome P450 (CYP) 2E1, criticl enzyme in the metbolic ctivtion of vrious low-moleculr-weight procrcinogens, including tobcco-specific N-nitrosmines, nd HCC mong cigrette smokers suggests tht N-nitrosmines my be involved in the etiology of HCC (Yu et l, 1995). Activted metbolites of crcinogens re subjected to metbolic conjugtion nd other kinds of detoxifiction. N-cetyltrnsferse 2 is non-inducible 598

2 Genotypes of CYP1A1 nd HCC risk 599 liver enzyme tht dectivtes crcinogenic romtic mines vi N-cetyltion (Hein et l, 1993). Slow cetyltors expressing little or no N-cetyltrnsferse 2 enzyme ctivity re t incresed risk of HCC (Agundez et l, 1996). None of the PAHs consistently found in cigrette smoke hve been shown to be heptocrcinogens in lbortory nimls. No studies hve been crried out to investigte the ssocition between PAHs nd HCC in humns. CYP1A1 is involved in the biotrnsformtion of tobcco-derived PAHs such s benzo()pyrene (B()P) into rective diol epoxide metbolites which cn form covlent dducts with DNA (Shimd et l, 1992). Activted metbolites of B()P re subjected in prt to metbolic detoxifiction by glutthione S-trnsferse M1 (GSTM1) (Mnnervik nd Dnielson, 1988). The metbolic blnce between ctivtion nd detoxifiction determines the biologiclly effective dose of crcinogens, thereby substntilly influencing cncer risk. Two seprte point muttions in the CYP1A1 gene hve been reported: one within exon 7 tht cuses n isoleucine to vline substitution in the hem-binding region (Ile-Vl polymorphism), nd the other in the 3 non-coding region (MspI polymorphism). The two polymorphisms re more frequent in Jpnese thn in Cucsins (Nkchi et l, 1993; Sivrmn et l, 1994). Cigrette smoking Jpnese homozygous for the vrint genotype of the MspI or Ile-Vl polymorphisms who lso hd GSTM1 gene deletion, were found to be t remrkbly high risk of lung cncer (Nkchi et l, 1993). CYP1A1 ws generlly considered to be involved in extrheptic crcinogenesis becuse erly studies showed tht the expression of CYP1A1 ws low in humn liver (Guengerich et l, 1991). However, recent studies using more sensitive techniques for the detection of CYP1A1 messenger RNA hve demonstrted tht CYP1A1 is expressed in high proportion of humn liver tissues (McKinnon et l, 1991; Schweikl et l, 1993). The role of CYP1A1 genetic polymorphism in susceptibility to HCC hs not yet been investigted. This cse-control study ws conducted within lrge-scle cohort of men in Tiwn to test the reltionship between susceptibility to smoking-relted HCC nd genetic polymorphisms in CYP1A1. The gene gene interction between CYP1A1 nd GSTM1 in the development of smoking-relted HCC ws lso investigted. SUBJECTS AND METHODS A cohort of 4841 mle chronic crriers of HBV surfce ntigen (HBsAg) nd 2501 non-crriers ged yers who were free of HCC ws recruited from the Government Employee Centrl Clinics nd the Liver Unit of Chng-Gung Memoril Hospitl in Tiwn from August 1988 to June At recruitment, ech study subject ws personlly interviewed to obtin informtion on sociodemogrphic chrcteristics (e.g. ge nd ethnicity), hbits of smoking nd lcohol drinking, dietry consumption, s well s history of mjor chronic diseses. Ech study subject ws sked bout the ethnicity of both prents. Dt on chronic liver disese, including chronic heptitis nd liver cirrhosis, dignosed by physicins were thus obtined. The dignosis of chronic heptitis in Tiwn ws minly bsed on sustined elevted serum minotrnsferse levels for 6 months. Only smll frction of the ptients with chronic heptitis were dignosed histologiclly. Liver cirrhosis ws dignosed by clinicl mnifesttion (including cogulopthy, hypolbuminemi, bnorml liver function tests, hemtologic evidence of hypersplenism, scites, jundice nd Cncer Reserch Cmpign 1999 portl hypertension with or without vricel bleeding), histologicl findings of liver biopsy nd/or ultrsonogrphy. Questionnire interview ws crried out by well-trined reserch ssistnts. Blood specimens, including serum nd white blood cells, from the study subjects were lso obtined nd frozen t 70 C until subsequent nlysis. All study subjects gve verbl or written informed consent for both the interview nd blood collection. Follow-up of the study subjects ws performed through vrious chnnels: nnul α-fetoprotein mesurement nd ultrsonogrphy exmintion, personl telephone interview, nd dt linkge with computer files of ntionl deth certifiction nd cncer registry systems. The dignosis of HCC ws bsed on (1) positive findings on cytologicl or pthologicl exmintion nd/or (2) positive imges on ngiogrm, ultrsonogrphy nd/or computed tomogrphy, combined with n α-fetoprotein level 400 ng/ml. There were 92 incident cses of HCC identified during the follow-up period from August 1988 to June A totl of 624 controls were rndomly selected from cohort subjects who were not ffected with HCC through the follow-up period. They were mtched with cses on ge (± 5 yers), dte of blood collection nd questionnire interview (± 3 months). Successful genotyping ws obtined for 84 of the 92 cse subjects nd 563 (154 HBsAg-negtive nd 409 HBsAg-positive) of the 624 control subjects. Ninetysix percent (81/84) of the HCC cses with vilble dt on CYP1A1 nd GSTM1 genotypes were positive for HBsAg. Genomic DNAs were isolted from peripherl leucocytes. For nlysis of the MspI restriction frgment length polymorphism in CYP1A1 the following two primers were used: 5 - TAGGAGTCTTGTCTCATGCCT-3 nd 5 -CAGTGAAGAGGT- GTAGCCGCT-3 (Nkchi et l, 1993). Amplifiction ws performed using initil denturtion t 94 C for 5 min followed by 30 cycles of 94 C for 30 s, 55 C for 30 s nd 72 C for 1 min, with finl extension t 72 C for 5 min. This generted 340 bp frgment tht ws then subjected to digestion with MspI (New Englnd Biolbs, Beverly, MA, USA) ccording to the mnufcturer s instruction. The digested product ws visulized on 2% grose gel, followed by ethidium bromide stining. The presence of the vrint llele (m2) ws indicted by bnds of 200- nd 140-bp, wheres no digestion of the wild-type llele (m1) occurred. A modified method, originlly described by Oym nd co-workers, ws used to identify the other CYP1A1 polymorphism resulting in the substitution of isoleucine for vline t residue 462 in the hem-binding region (Oym et l, 1995). Genomic DNA ws mplified by polymerse chin rection (PCR) using the primers 5 -GAACTGCCACTTCAGCTGTCT-3 nd 5 -GAAAGACCTCCCAGCGGTCA-3. The mplifiction rection ws t 94 C for 5 min to effect initil denturtion, followed by 30 cycles of 20 s t 94 C, 20 s t 55 C, nd 40 s t 72 C, with finl extension t 72 C for 5 min. Following mplifiction, PCR products were digested with HincII (New Englnd Biolbs, Beverly, MA, USA) t 37 C for 3 h. The digested products were electrophoresed on 3% grose gel nd visulized by ethidium bromide stining. PCR products of Vl polymorphism cn be cut between codon 462 (GTT) nd codon 463 (GAC) by HincII. The GSTM1 genotyping for gene deletion ws crried out by PCR mplifiction with primers for exons 6 nd 7 (Chen et l, 1996). All ssys were conducted nd interpreted blindly to cse-control sttus. The differences in the llele frequencies of the CYP1A1 gene between HBsAg crriers nd non-crriers were tested by multiple liner regression with simultneous control for ethnic groups nd British Journl of Cncer (1999) 80(3/4),

3 600 M-W Yu et l ge. Odds rtio (OR) nd confidence intervl (CI) were clculted by unconditionl logistic regression nd djusted for potentil confounding fctors. All P-vlues were clculted from two-sided sttisticl tests. RESULTS Distributions of CYP1A1 polymorphisms in HBsAgpositive nd HBsAg-negtive controls Ninety-eight percent of the study subjects reported tht their fther nd mother hd the sme ethnic group. Tble 1 shows the distribution of the CYP1A1 genetic polymorphisms in HBsAg-positive nd HBsAg-negtive control subjects by ethnic group. The ethnicity distribution presented ws bsed on fthers ethnic groups of the study subjects. The MspI vrint llele (m2) ws less frequent in HBsAg-positive controls thn in HBsAg-negtive controls, irrespective of ethnic group. Since the Ile-Vl polymorphism is closely linked to the MspI polymorphism (Nkchi et l, 1993), the Ile-Vl vrint llele (Vl) ws lso reduced in frequency mong HBsAg crriers in ech ethnic group. The overll frequencies of the MspI (P = ) nd Ile-Vl (P = ) vrint lleles were significntly lower in HBsAg crriers thn in non-crriers fter djustment for ethnic group nd ge. Since only three HCC cses with vilble dt on CYP1A1 nd GSTM1 genotypes were HBsAg-negtive, the only wy to control for the effect of HBsAg crrier sttus in strtified nlyses ws to restrict the nlyses to chronic HBV crriers. The following nlyses of CYP1A1 polymorphisms nd HCC risk were thus crried out mong chronic HBV crriers. CYP1A1 polymorphisms nd HCC risk in chronic HBV crriers The men ± stndrd devition (s.d.) of ges for the 81 HBsAgpositive HCC cses nd 409 HBsAg-positive control subjects in the nested cse-control study on CYP1A1 polymorphisms nd HCC were 53.1 ± 9.4 nd 51.9 ± 9.4 yers respectively. Tble 2 shows the frequencies of CYP1A1 genotypic vrints mong HBsAg-positive HCC cses nd controls. The MspI llele frequency for the m2 vrint type ws 43.2% in cses nd 39.2% in controls. The corresponding figures for the Ile-Vl vrint llele were 25.3% nd 23.2% respectively. Both MspI nd Ile-Vl lleles were found to be in Hrdy Weinberg equilibrium. There were no sttisticlly significnt differences in the distributions of CYP1A1 polymorphisms between cses nd controls. Interction between cigrette smoking nd CYP1A1 polymorphism in HCC After strtifying by sttus of cigrette smoking, we further exmined the ssocitions between CYP1A1 polymorphisms nd HCC risk mong chronic HBV crriers (Tble 3). Among non-smokers, there were essentilly no differences in the distributions of CYP1A1 polymorphisms between cses nd controls. Conversely, mong smokers, the presence of the MspI vrint llele significntly incresed HCC risk, showing multivrite-djusted OR of 3.15 (95% CI ; P = ). Cigrette smokers with the Ile-Vl vrint llele were lso t incresed risk of HCC (djusted Tble 2 Frequency distributions of CYP1A1 genotypes in HBsAg-positive HCC cses nd controls Tble 1 Vrint CYP1A1 llele frequencies in controls by HBsAg sttus nd ethnic group Mrker (vrint llele) Totl HBsAg chromosomes Ethnic group sttus MspI (m2) Ile-Vl (Vl) tested % % Fukien Tiwnese Negtive Positive Hkk Tiwnese Negtive Positive Minlnd Chinese Negtive Positive Cses Controls Adjusted Genotypes No. (%) No. (%) odds rtio (95% CI) MspI polymorphism m1/m1 25 (30.9) 152 (37.2) 1.0 m1/m2 42 (51.8) 193 (47.2) 1.41 ( ) m2/m2 14 (17.3) 64 (15.6) 1.47 ( ) Ile-Vl polymorphism Ile/Ile 46 (56.8) 239 (58.4) 1.0 Ile/Vl 29 (35.8) 150 (36.7) 1.02 ( ) Vl/Vl 6 (7.4) 20 (4.9) 1.61 ( ) Adjusted for effects of ge, cigrette smoking, lcohol drinking, ethnic groups nd chronic liver disese Tble 3 CYP1A1 genetic polymorphisms nd HCC risk in HBsAg crriers by sttus of cigrette smoking Non-smokers Smokers Genotype Cses Controls OR (95% CI) Cses Controls OR (95% CI) MspI polymorphism m1/m m1/m2 nd m2/m ( ) ( ) b Ile-Vl polymorphism Ile/Ile Ile/Vl nd Vl/Vl ( ) ( ) c Adjusted for ge, lcohol drinking, ethnic groups nd chronic liver disese. b P = c P = British Journl of Cncer (1999) 80(3/4), Cncer Reserch Cmpign 1999

4 Genotypes of CYP1A1 nd HCC risk 601 Tble 4 Adjusted odds rtios of HCC ssocited with cumultive exposure to cigrette smoke in HBsAg crriers by combined genotypes of CYP1A1 nd GSTM1 Pck-yers of smoking No. of No. of CYP1A1 GSTM1 cses controls b 16.3 vs 0 > 16.3 vs 0 m1/m1 Non-null ( ) 0.59 ( ) m1/m1 Null ( ) 0.66 ( ) m1/m2 nd m2/m2 Non-null ( ) 1.52 ( ) m1/m2 nd m2/m2 Null ( ) c 1.54 ( ) Ile/Ile Non-null ( ) 0.81 ( ) Ile/Ile Null ( ) 0.48 ( ) Ile/Vl nd Vl/Vl Non-null ( ) 1.45 ( ) Ile/Vl nd Vl/Vl Null ( ) d 4.71 ( ) e Adjusted for effects of ge, lcohol drinking, nd chronic liver disese. b One control hd no vilble dt on quntity of smoking per dy nd two controls hd no vilble dt on GSTM1 genotype. c P = d P = e P = Tble 5 HCC risk ssocited with CYP1A1 MspI polymorphism in HBsAg crriers homozygous for the Ile/Ile genotype of the CYP1A1 Ile- Vl polymorphism Non-smokers Smokers Genotype Cses Controls OR (95% CI) Cses Controls OR (95% CI) m1/m m1/m ( ) ( ) m2/m ( ) ( ) P = OR 1.99, 95% CI ) compred with those without the llele; this ssocition ws mrginlly significnt (P = ). No significnt ssocition between cumultive exposure to cigrette smoke nd HCC ws observed when the ssocition ws exmined in strt of CYP1A1 genotypes. Becuse ctivted metbolites of tobcco-derived PAHs re subjected in prt to metbolic detoxifiction by GSTM1 (Mnnervik nd Dnielson, 1988), the ssocition between pck-yers of cigrette smoking nd HCC ws ssessed in ctegories simultneously strtified by genotypes of CYP1A1 nd GSTM1 (Tble 4). Genetic dt for GSTM1 were vilble for ll the 81 HBsAg-positive HCC cses nd 407 HBsAg-positive controls. Fifty-one percent of the cses nd 55% of the controls were the homozygotes for the GSTM1-null genotype which express no protein. After djusting for potentil confounders, the effect of smoking ws observed to be most pronounced in those with t lest one MspI (OR [95% CI] for smokers of 16.3 nd > 16.3 pck-yers vs non-smokers, 3.34 [ ] nd 1.54 [ ] respectively) or Ile-Vl vrint llele (OR [95% CI] for smokers of 16.3 nd > 16.3 pck-yers vs non-smokers, 9.04 [ ] nd 4.71 [ ] respectively) of CYP1A1 combined with deficient genotype of GSTM1; however, monotonic dose response reltion ws not observed. The MspI nd Ile-Vl vrint lleles of CYP1A1 re in linkge disequilibrium (Nkchi et l, 1993). It is likely tht the effect of the MspI polymorphism is essentilly scribed to the Ile-Vl polymorphism which ws ssocited with gene inducibility nd enzymtic ctivity (Kwjiri et l, 1993; Crofts et l, 1994). This hypothesis ws exmined in Tble 5. Among smoking chronic HBV crriers homozygous for the wild-type llele of the Ile-Vl polymorphism, the presence of the MspI vrint llele still incresed HCC risk. Adjustment for ge, lcohol drinking, ethnic groups nd chronic liver disese did not mterilly chnge the results. This suggests tht MspI nd Ile-Vl polymorphisms my hve different mechnisms for incresing susceptibility to smoking-relted HCC. DISCUSSION Our previous cse-control study found n excess risk of HCC in first-degree reltives of HCC ptients, even fter djustment for chronic HBV crrier sttus (Chen et l, 1991). Segregtion nlysis of fmilil HCC demonstrted n interction between HBV infection nd mjor genetic locus (Shen et l, 1991). These epidemiologicl findings suggest tht inherited predisposition my be component of HCC risk. While the reltionships between environmentl risk fctors nd HCC hve been ctively studied, the understnding of the genetic bsis of HCC remins primitive. Both virl nd chemicl crcinogens re involved in the humn heptocrcinogenesis. Previous epidemiologicl studies hve demonstrted tht fltoxin exposure nd cigrette smoking re importnt risk fctors for HCC (Tsukum et l, 1990; Chen et l, 1991, 1996; Yu et l, 1991; Yu et l, 1997). Afltoxins nd most genotoxic chemicl crcinogens in tobcco smoke re not intrinsiclly rective but require metbolic conversion to DNA-binding intermedites (Guengerich nd Shimd, 1991). While CYP1A2 is the P450 enzyme principlly responsible for the bioctivtion of fltoxin B 1 t low substrte concentrtions ssocited with dietry Cncer Reserch Cmpign 1999 British Journl of Cncer (1999) 80(3/4),

5 602 M-W Yu et l exposure (Gllgher et l, 1994), CYP1A1 is involved in the metbolic ctivtion of B()P nd other PAH constituents of cigrette smoke (Shimd et l, 1992). Polymorphisms ssocited with enzymtic ctivity of CYP1A1 my determine susceptibility to smoking-relted cncer. The frequencies of the CYP1A1 MspI nd Ile-Vl vrint lleles mong Chinese in Tiwn re similr to those described for Jpnese, but much higher thn those in Cucsins (Nkchi et l, 1993; Sivrmn et l, 1994). In this cse-control study nested within prospective study, we found tht the reltionship between CYP1A1 polymorphisms nd HCC in chronic HBV crriers depended on the sttus of cigrette smoking. The CYP1A1 MspI polymorphism ws significntly relted to HCC risk in cigrette smokers, wheres no significnt ssocition ws evident for those who hd never smoked. Becuse the Vl llele of the Ile-Vl polymorphism is much less frequent thn the m2 llele of the MspI polymorphism nd the limited smple size, the incresed risk of HCC ssocited with the presence of the Vl llele in smokers only reched the borderline significnce level. Electrophilic metbolites of B()P nd other PAHs ctivted by CYP1A1 re subjected, in prt, to metbolic detoxifiction by GSTM1 (Mnnervik nd Dnielson, 1988). Perhps individuls with GSTM1 non-null genotypes in combintion with the highrisk gene of CYP1A1 re ble to eliminte the ctivted metbolites of PAHs rpidly nd efficiently, nd thus tolerte crcinogenic insults. The HCC risk ssocited with cumultive exposure to cigrette smoke in this study ws found to be most pronounced mong chronic HBV crriers with t lest one vrint llele of the CYP1A1 MspI, or Ile-Vl polymorphism, who lso hd homozygous deletion of the GSTM1 gene. These results re comptible with the hypothesis tht CYP1A1 polymorphism is n importnt modultor of the heptocrcinogenic effect of cigrette smoke mong chronic HBV crriers. They lso indirectly support role for PAHs in cigrette smoke tht re ctivted by CYP1A1 in the etiology of HCC. The frequency of the GSTM1-null genotype is high in humn popultions, pproximtely 50%, with reported rnge of 30 70% (Rebbeck, 1997). Intervention imed t reducing cigrette smoking my be importnt for the prevention of HCC in res where chronic HBV infection nd the high-risk gene of CYP1A1 re common. The frequencies of mny polymorphic genetic mrkers vry mrkedly between different popultions. One mjor difficulty tht often rises in cse-control studies of genetic mrkers nd disese is the choice of n pproprite control group for smple of cses drwn from mixed popultion consisting of different subgroups with diverse distributions of genetic mrkers. Tiwn is n endemic re of HBV infection with HBsAg crrier rte of 15 20%. Chronic HBV infection hs been demonstrted s the most importnt determinnt of HCC in Tiwn (Besley, 1988; Chen et l, 1991; Yu et l, 1991; Yu nd Chen, 1994). In this study, the CYP1A1 MspI nd Ile-Vl vrint lleles were observed to be less prevlent in HBsAg crriers thn in non-crriers. This gene frequency difference ws present in ech ethnic group nd ws sttisticlly significnt fter djustment for ethnic groups nd ge when ll the three ethnic groups were pooled in the nlysis. Since only three HCC cses were negtive for HBsAg in this study, we thus restricted the nlyses of CYP1A1 polymorphisms nd HCC to chronic HBV crriers. Although the mechnism responsible for the discrepncies in the llelic frequencies of CYP1A1 polymorphisms between HBsAg crriers nd non-crriers is uncler, this study rises the possibility tht the filure to tke into ccount HBsAg crrier sttus in nlysis of genetic mrkers nd HCC my cuse bised estimtes of ssocition, especilly in endemic res of HBV infection. The Ile-Vl polymorphism of the CYP1A1 gene results in the replcement of Ile by Vl t mino cid residue 462 in the hembinding region. The Vl vrint llele ws shown to be ssocited with incresed gene inducibility nd ctlytic ctivity (Kwjiri et l, 1993; Crofts et l, 1994). The MspI polymorphism is locted in the 3 non-coding region of the CYP1A1 gene. Although the MspI polymorphism hs been shown to co-segregte with the inducibility phenotype of the CYP1A1 enzyme in fmily study, the mechnism for this ssocition remins to be estblished (Petersen et l, 1991). Since the MspI nd Ile-Vl vrint lleles of CYP1A1 re in linkge disequilibrium (Nkchi et l, 1993), it is likely tht the ssocition between the MspI polymorphism nd HCC mong cigrette smokers observed in this study is essentilly scribed to the Ile-Vl polymorphism. In smoking chronic HBV crriers homozygous for the wild-type genotype of the Ile-Vl polymorphism, however, we found tht the MspI vrint llele ws still ssocited with incresed risk of HCC. This finding suggests tht the MspI nd Ile-Vl polymorphisms my be cting through different mechnisms for influencing susceptibility to smokingrelted HCC. ACKNOWLEDGEMENTS This study ws supported by grnts DOH86-HR-627 nd DOH87- HR-627 from the Ntionl Helth Reserch Institutes, Deprtment of Helth, Executive Yun; grnt NSC B from the Ntionl Science Council of the Republic of Chin; nd grnt ES05116 from the Ntionl Institutes of Helth, USA. REFERENCES Agundez JAG, Oliver M, Ldero JM, Rodriguez-Lescure A, Ledesm MC, Diz- Rubio M, Meyer UA nd Benitez J (1996) Incresed risk for heptocellulr crcinom in NAT2-slow cetyltors nd CYP2D6-rpid metbolizers. Phrmcogenetics 6: Besley RP (1988) Heptitis B virus: the mjor etiology of heptocellulr crcinom. Cncer 61: Chen CJ, Ling KY, Chng AS, Chng YC, Lu SN, Liw YF, Chng WY, Sheen MC nd Lin TM (1991) Effects of heptitis B virus, lcohol drinking, cigrette smoking nd fmilil tendency on heptocellulr crcinom. 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