Vol. 7, 1013-1018, November 1998 Cancer Epidemiology, Biomarkers & Prevention /0/3

Susceptibility to Esophageal Cancer and Genetic Polymorphisms in

Glutathione S-Transferases T 1 , P1 , and M 1 and

Cytochrome P450 2E1

Dong-Xin Lin, Yong-Ming Tang, Qiong Peng, Shi-Xin Lu,Christine B. Ambrosone, and Fred F. Kadlubar’

Department of Chemical Etiology and Carcinogenesis, Cancer Institute,

Chinese Academy of Medical Science and Beijing Union Medical College,

Beijing 100021, People’s Republic of China [D-X. L., Q. P., S-X. LI, and

Division of Molecular Epidemiology, National Center for Toxicological

Research, Jefferson, Arkansas 72079 [Y-M. T., C. B. A., F. F. K.l

Abstract

Genetic polymorphisms in enzymes involved incarcinogen metabolism have been shown to influencesusceptibility to cancer. Cytochrome P450 2E1 (CYP2E1)is primarily responsible for the bioactivation of many lowmolecular weight carcinogens, including certainnitrosamines, whereas glutathione S-transferases (GSTs)are involved in detoxifying many other carcinogenicelectrophiles. Esophageal cancer, which is prevalent inChina, is hypothesized to be related to environmentalnitrosamine exposure. Thus, we conducted a pilot case-

control study to examine the association betweenCYP2E1, GSTM1, GSTT1, and GSTP1 geneticpolymorphisms and esophageal cancer susceptibility.DNA samples were isolated from surgically removedesophageal tissues or scraped esophageal epithelium fromcases with cancer (n = 45), cases with severe epithelial

hyperplasia (n 45), and normal controls (n 46) froma high-risk area, Linxian County, China. RFLPs in theCYP2EJ and the GSTPJ genes were determined by PCRamplification followed by digestion with RsaI or DraI andA1w261, respectively. Deletion of the GSTM1 and GSTTJ

genes was examined by a multiplex PCR. The CYP2E1polymorphism detected by RsaI was significantly differentbetween controls (56%) and cases with cancer (20%) orsevere epithebial hyperplasia (17%; P < 0.001). Personswithout the RsaI variant alleles had more than a 4-6-foldrisk of developing severe epithebiab hyperplasia (adjustedodds ratio, 6.0; 95% confidence interval, 2.3-16.0) andcancer (adjusted odds ratio, 4.8; 95% confidence interval,1.8-12.4). Polymorphisms in the GSTs were notassociated with increased esophageal cancer risk. Theseresults indicate that CYP2E1 may be a geneticsusceptibility factor involved in the early events leading

to the development of esophageal cancer.

Received 5/29/98; revised 8/25/98; accepted 9/1/98.

The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed. at Division of Molecular

Epidemiology, National Center for Toxicological Research, 3900 NCTR Drive,

Jefferson, AR 72079-9502.

Introduction

Cancer of the esophagus is one of the most common fataldiseases in certain regions of China such as Linxian County,where the mortality rate is as high as 150/100,000 (1). The risk

for esophageal cancer in these areas has been associated with

exposure to environmental carcinogens, particularly nitro-samines (2-4). However, even in high-risk regions, only certainindividuals develop esophageal cancer. Most people live a

normal life span, suggesting that host susceptibility factors mayplay an important role in cancer development. Most chemicalcarcinogens require metabolic activation for DNA-damaging

capabilities, a step widely believed to be essential in carcino-genesis (5, 6). On the other hand, carcinogens may also bedetoxified before damaging DNA by in vivo metabolic detox-ifying systems (7, 8). Thus, it has been suggested that individ-

uab differences in carcinogen metabolism, which may be heri-

table or due to sustained environmental exposure to agents thataffect the expression of enzymes involved in carcinogen acti-vation or detoxification, may determine the susceptibility to

chemically induced cancers. The major enzymes involved inthe metabolic activation of chemical carcinogens are CYPs2

(4), a multigene superfamily of enzymes. Of the 25 or moreCYP enzymes known to be expressed in human tissues,CYP2E1 is believed to be involved in the activation of most

carcinogenic nitrosamines (9-12). Furthermore, this enzymehas the ability to metabolically activate many low molecularweight carcinogens (9) and to produce reactive free radicalsfrom ethanol (13, 14), which also might be of importance in

cancer etiology.CYP2E1 represents a major CYP isoform in the human

liver and is also expressed in extrahepatic tissues (15-17).Although the enzyme can be induced by certain chemicals such

as ethanol, large interindividual variations have been observed

before and after induction (18-20), suggesting that the varia-

tion may be due to genetic polymorphisms. RFLPs of thehuman CYP2EJ gene have been identified, and a polymorphismin the upstream region of the gene detected by RsaI has beenshown to be associated with the transcriptional regulation of

gene expression (21). Another genetic polymorphism that isdetectable with DraI is located in intron 6 (22), and this allelehas been found to contain a number of functional mutationsaffecting protein expression and catalytic activity (23). Asso-ciations between these genetic polymorphisms in CYP2EJ andthe susceptibility to some types of cancer have been reported in

case-control studies (22, 24-29), although some negative re-sults have been reported (30-40). To date, one study in Japanhas evaluated the role of the RsaI polymorphism in relation to

2 The abbreviations used are: CYP. cytochrome P450: GST, glutathione S-

transferase; OR, odds ratio: CI, confidence interval: dNTP, deoxynucleotide

triphosphate.

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= �

1014 CYP2E1 and Esophageal Cancer Susceptibility

3 W. Tan, Z. Li, and P. Lin, unpublished observations.

esophageal cancer and found no association (36). However, anincreased risk of both oral (41) and nasopharyngeal (42) cancer

has been associated with the prevalence of this variant allele inTaiwan.

The GSTs are a family of multifunctional enzymes that

play a central robe in the detoxification of toxic and carci-

nogenic ebectrophiles (43). Individuals who are homozygousfor the null GSTMJ or GSTTJ alleles lack the respectiveenzyme function (44, 45), and these genotypes have beenassociated with an increased risk of cancer at many sites (44,46-49). GSTP1, which is a major GST isozyme expressed inthe human esophagus (17, 50), has also been shown to be

genetically polymorphic (5 1,52). A mutation of A to Gwithin exon 5 results in an I to V change at position 105 in

the amino acid sequence of the protein, which alters the

specific activity and affinity for the electrophilic substratesof the enzyme (52, 53). Therefore, the GSTP1 polymorphism

may also have potential effects on cancer susceptibility (51).Whereas numerous studies have been undertaken to examine

the association between genetic polymorphisms in CYPsand/or GSTs and cancer susceptibility (vide supra), there is

limited information on their association with esophagealcancer (36, 54). In this study, we analyzed genetic polymor-

phisms in GSTMJ, GS77’l, GSTPJ, and CYP2EJ in subjectswith esophageal cancer and severe epithelial hyperplasia,which is a precancerous lesion, and frequency-matched con-trols from Linxian County, China, an area where dietary

nitrosamine exposures are known to be high (3).

Materials and Methods

Study Subjects. A pilot case-control study was designed to

evaluate the possible robe of genetic polymorphisms in carcin-ogen-metabobizing enzymes in the susceptibility to esophagealcancer. All subjects were residents of Linxian County, HenanProvince, China. Smoking status was not available; however,recent studies3 with a similar population indicate that smokingprevalence is comparable between cases and controls. The

diagnoses of esophageal cancer and epithelial hyperplasia wereconfirmed either histologically or cytologically. Cases weregrouped as follows: (a) those with advanced dysplasia or severe

hyperplasia (n = 45); (b) those with squamous cell carcinomaor adenocarcinoma (n = 45); and (c) those with squamous cell

carcinoma, adenocarcinoma, or advanced dysplasia (n = 62).DNA samples were isolated from surgically removed esopha-geal tissues using standard methods (55) or from epithelial cellsscraped from the esophagus. The yields of DNA varied with theamount of tissue available, and the analysis of each sample for

all genotypes was not possible.

GSTMJ and GSTTJ Genotyping. GSTMJ and GS17’l geno-typing for gene deletions was carried out by a multiplex PCR

using primer pairs 5’-GAACTCCCTGAAAAGCTAAAGC-3’and 5’-GTTGGGCTCAAATATACGGTGG-3’ for GSTMJ,

which produced a 219-bp product, and 5’-TTCCTTACTG-

I and 5’ -TCACCGGATCATGGC-CAGCA-3’ for GSJ7’J, which produced a 459-bp product.

Amplification of the albumin gene (5’-GCCCTCT-GCTAACAAGTCCTAC-3’ and 5’-GCCCTAAAAAGAAAA-TCCCCAATC-3’) was used as an internal control and pro-duced a 350-bp product (56). PCR was performed in a 50-�l

mixture consisting of sample DNA (0. 1 pg), dNTP (0.2 mrvt;Boehringer Mannheim, Indianapolis, IN), MgCb2 (2.5 mM),

M 1234567

GSTTI (459 bp)

Albumin (350 bp)

__�GSTM1 (219 bp)

Fig. 1. An ethidium bromide-stained gel of electrophoresed products of the

amplification of GSTMJ, GSTTI, and albumin genes. Lanes 1-7 correspond to

individual subjects. The absence of the PCR product indicates the null genotype.

M, the molecular weight markers.

each primer (1 .0, 0.3, and 0. 1 p.M for GSTMJ, GSTJ’l, andalbumin, respectively), Taq polymerase (1.25 units; Promega,

Madison, WI), and reaction buffer [10 msi Tris-HC1, 50 mt�tKC1 (pH 9), 1% Triton X-100, and 2% DMSO}. Thirty-fivecycles of amplification were performed at 94#{176}Cfor 1 mm(denaturation), 62#{176}Cfor 1 mm (annealing), and 72#{176}Cfor 1 mm(extension) using a GeneAmp 9600 thermal cycler (Perkin-Elmer Corp., Norwalk, CT). A 15: 1 dilution of the amplifiedproducts was visualized by electrophoresis in an ethidium-

bromide-stained 1.5% agarose gel (NuSieve 3: 1; AmericanBioanalytical, Natick, MA) in TBE buffer [89 nmi Tris-HC1,0.89 ms� boric acid, and 2 ms� EDTA (p1-I 8.0; Fig. 1)].

GSTP1 RFLP Analysis. The primer pair 5’-ACGCACATC-CTCTFCCCCTC-3’ and S ‘-TACTFGGCTGGTfGATGTC-

C-3’ was used to amplify exon 5 in the GSTPJ gene thatincludes the A!w261 enzyme recognition site (cf. Ref. 49). PCRreactions were carried out in a 50-gil mixture containing sampleDNA (0. 1 I.Lg), reaction buffer, dNTP (0.2 mrsi), MgCl2 (2.5mM), each primer (1.0 pM), and Taq polymerase (1.25 units).

Amplification, which resulted in a 440-bp fragment, wasachieved by 35 cycles of 30 s at 94#{176}C,30 s at 66#{176}C,and 2 mmat 72#{176}C.Ten pi of the PCR reaction products were subjected todigestion with 20 units of the A!w26I enzyme in the buffer, as

recommended by the manufacturer (Fermentas, Vilnius,Lithuania), for 2 h at 37#{176}C.The samples were then analyzed byelectrophoresis in 2% agarose gels. The presence of the poly-morphic A1w261 restriction site yielded two fragments of 213

and 227 bp (GSTPJ *B), whereas the absence of the polymor-phic site was determined by the presence of a 440-bp fragment(GSTPJ *A)

CYP2E1 RFLP Analysis. The RFLPs in the 5’-flanking re-gion and in intron 6 of the CYP2EJ gene were determined byPCR amplification followed by digestion with RsaI or DraI,

using the methods described previously (21, 22). The PCRprimers used for the 5’-flanking region (the RsaI site) were

5’-CC1TCTFGGTFCAGGAGAGG-3’ and 5’-AGACCTC-CACATFGACTAGC-3’ and produced a 459-bp product. The

predominant allele (cl) was sensitive to RsaI digestion andyielded two products at 201 and 258 bp. The c2 allele was

resistant to RsaI digestion. The PCR primers for intron 6

(the Dral site) were 5’-CTGCTGCTAATGGTCACTFG-3’and 5’-GGAGTFCAAGACCAGCCTAC-3’, which produced a686-bp product. Only the D allele was sensitive to DraI cleav-age, resulting in fragments of 335 and 351 bp. All PCR am-plifications were performed in a total volume of 50 �l. Themixture used reaction buffer and contained each primer (1.0

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M 1 2 3 4 5 6 7 8 9101112

� �-

- - � � - - �� ‘ � - �

100-

Fig. 2. The RFLPs of PCR-amplified fragments obtained using RsaI and sub-jected to agarose gel electrophoresis. Lanes 1-4, 9. ii. and 12, cl/cl homozy-

gotes; Lanes 6 and 8, c2/c2 homozygotes; Lane 5, cl/c2 heterozygote.

Cancer Epidemiology, Biomarkers & Prevention /0/5

Table 1 Demographi c characteristics o f the study subjects

ControlsHyperplasia

.and dysplasia

CancerCancer

.and dysplasta

Sample size

Age (yr)

Mean

Range

Gender

Male (%)

Female (%)

46

53.3

40-65

50

50

45

54.8

47-65

47

53

45

55.5

47-55

60

40

62

54.3

35-70

58

42

4-

3-

200�

�LM), dNTP (0.2 nirvi), MgC12 (2.5 m.si), and Taq polymerase

(1.5 units/SO �tl). The PCR conditions for amplifying the 5’-

flanking region for the RsaI site were 35 cycles of 30 s at 94#{176}C,30 s at 59#{176}C,and 2 mm at 72#{176}C;for amplifying imtrom 6 withthe Dral site, the PCR conditions were 35 cycles of 30 s at

94#{176}C,30 5 at 64#{176}C,and 2 mm at 72#{176}C.Fifteen p.1 of eachamplified product were digested with 10 units of RsaI or 10

units of DraI (New England Biolabs, Inc., Beverly, MA),respectively, at 37#{176}Cfor 4 h, and the products were analyzed by

electrophoresis in a 2.5% agarose gel.

Statistical Analysis. The f test was used to examine the

differences in the distribution of genotypes between cases andcontrols. ORs with 95% CIs were computed using uncondi-

tional logistic regression (SPSS Inc., Chicago, IL) and adjustedfor age and gender (57). DNA analyses of all of the genotypes

for all study participants were not possible due to samplelimitations; thus, the number of participants varies for each

polymorphism.

Results

The characteristics of study subjects are presented in Table 1.Age and gender distributions among cases with cancer, caseswith hyperplasia, and controls were very similar, reflecting the

matching criteria used in this case-control study. DNA samplessubjected to PCR and enzymatic digestion with RsaI revealed

the expected fragment lengths, resulting in three genotypes ofCYP2EJ (Fig. 2). The three genotypes of CYP2EJ recognized

by PCR and Dral restriction fragment length analyses werereadily discerned (Fig. 3). Distributions of the CYP2EJ geno-types identified by these two restriction endonucleases were

similar to those described previously (21, 22), as shown inTables 2 and 3 for controls, cancer cases, and hyperplasia cases.

The frequencies of the homozygous RsaI-sensitive allele (cl/

Fig. 3. The RFLPs of PCR-amplified fragments obtained using Dral and sub-

jected to agarose gel electrophoresis. Lanes 1, 3, 5. and 9, CC homozygotes;

Lanes 4, 6, and 7, DD homozygotes; Lanes 2, 8, and 10, CD heterozygotes.

cl) and the combined genotypes (cl/c2 + c2/c2) detected byRsaI were found to be 44 and 56%, respectively, in the control

subjects, and those for the CC genotype and the CD + DDgenotypes recognized by Dral were found to be 42 and 58%,respectively. As shown in Table 2, the frequency of variant

genotypes of CYP2EJ detected by RsaI was significantly higher

(x2 20.8; P < 0.001) in controls (56%) than in epithebialhyperplasia and dysplasia cases (17%) or in cancer cases(20%). Subjects with the cl/cl genotype had a 6-fold increased

risk of developing epithelial hyperplasia and dysplasia (OR,6.0; 95% CI, 2.3-16) and an almost S-fold increased risk for

cancer (OR, 4.8; 95% CI, 1.8-12.4) as compared to subjectswith variant genotypes. The frequencies of the CD and DD

genotypes detected by DraI were also slightly higher in controlsubjects (58%) than in cases with epitheliab hyperplasia anddysplasia (41%) or in cases with cancer (45%), although thedifferences were not statistically significant. When cases with

cancer and advanced dysplasia (n = 62) were analyzed to-gether, the results for the RsaI (OR, 6.0; 95% CI, 1.8-2.4) andDraI (OR, 1.5; 95% CI, 0.7-3.6) polymorphisms were similar

to that described for the cancer cases only.The distributions of the GSTMJ, GS17’l, and GSTPJ gen-

otypes in cases and controls are shown in Table 4. GSTMJ and

GS77’l gene deletion was common in both controls and cases.The distribution of the genotypes of the three GST genes did

not differ significantly between the controls and either group ofcases, although the frequencies of the GSTTJ gene deletion and

the frequencies of the GSTPJ ‘tB minor alleles were slightlylower in the cancer and hyperplasia cases than in the controls.

No interactions were observed for combined contributions ofGST genetic polymorphisms, together and with CYP2EJ, onthe risk of developing epitheliab hyperplasia and esophagealcancer.

Discussion

Because the etiology of esophageal cancer has been hypothe-sized to be associated with environmental nitrosamine expo-

sure, we investigated whether genetic factors that might mod-ulate the activation and/or detoxification of these carcinogenscould have an impact on the risk of developing this malignant

disease. We analyzed genetic polymorphisms at the CYP2EJ,

GSTMJ, GS77’l, and GSTPJ loci and found that the CYP2EJ

genotype was associated with susceptibility to esophageal can-cer. Subjects who were homozygous cl/cl for CYP2EI were at

a more than 4-fold excess risk of developing epithelial hyper-

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/016 CYP2E1 and Esophageal Cancer Susceptibility

Table 2 RsaI polymorphisms of CYP2EI in controls and cas

hyperplasia and esophageal cancer

es with severe

Genotype

Flyperplasia andControls (%) Cancer (%)

(n 45) dysplasia (%) (n 45)(n32)

Cancer and

dysplasia (%)

(n�58)

cl/cl

cl/c2

c21c2

20(44) 25(83) 36(80)

22(49) 7(17) 6(13)

3(7) 0(0) 3(7)

48(80)

6(13)

4(7)

OR (CI)” I .0 6.0 (2.3-16.0) 4.8 (1 .8-1 2.4) 4.0 (1.8-12.4)

‘� ORs and 95% CIs were calculated by logistic regression, with the c//cl

genotype as the reference category. adjusted for age and sex.

Table 3 Fr equencies of DraI CYP2EI genotypes among conwith severe hyperplasia and esophageal cancer

trols and cases

Genotype”

Hyperplasia andCancer (%)Controls (%)

(n - 45) dysplasia (%) (n 45)(n38)

Cancer and

dysplasia (%)

(n56)

CC

CD

DD

19(42) 21 (59) 25(55)

23 (51 ) 16 (39) 16 (36)

3(7) 1 (2) 4(9)

34(57)

16 (33)

6(10)

OR (Cl )“ I .0 2.0 (0.9-4.4) 1 .5 (0.7-3.6) 1 .5 (0.7-3.6)

‘, CC, homozygous for the common allele; CD, heterozygous; DD. homozygousfor the rare allele.

S ORs and 95% CIs were calculated by logistic regression, with the CC genotype

a.s the reference category and groups (CD + DD) combined, adjusted for age and

sex.

plasia and cancer of the esophagus. In view of the fact that

epithebiab hyperplasia of the esophagus is a precancerous lesion,our results indicate that CYP2E1 is a genetic susceptibility

factor involved in the early events of esophageal cancer. Be-cause CYP2E1 is a major enzyme that catalyzes the activationof various nitrosamines and other low molecular weight car-cinogens present in the diet, tobacco smoke, and the environ-ment (9-12), its involvement in esophageal carcinogenesis is

biologically plausible. Accordingly, it is expected that a lowerlevel of CYP2E1 activity would reduce individual susceptibil-ity to the cancer.

The mechanisms involved in CYP2EI expression and the

resulting amount and activity of the enzyme are not well char-acterized. Hu et a!. (23) recently identified several mutations in

the flanking regions in the DraI C allele using single-strandconformational polymorphism and found determinants ofmRNA stability in some motifs. The PstI or RsaI site is located

in the S’-flanking region and may affect gene transcription.Based on experiments with an in vitro expression system,Hayashi et a!. (21) and Watanabe et a!. (58) suggested that the

c2/c2 genotype detected by RsaI produced higher enzyme ac-tivity than the cl/cl genotype. However, this suggestion couldnot be confirmed in several in vivo and in vitro studies using

chborzoxazone and other specific substrates as probes for en-zyme activity (59-61). Moreover, in view of the role ofCYP2E1 in the metabolic activation of carcinogens and our

results showing a protective effect of the c2/c2 genotype against

esophageal cancer, we suggest that this genotype may result inless CYP2E1 activity toward esophageal carcinogens in Linx-ian County than the corresponding cl/cl genotype.

The basal expression of CYP2E1 seems to be relativelylow, but it can be highly induced. The polymorphism in theS’-flanking region might be expected to modify the inducibibityof the enzyme. Although the mechanism of CYP2E1 induction

Table 4 Genotypes of GSTM 1, GS’Il’l , and GSTPI in con

with severe hyperplasia and esophageal cancer

trols and cases

GenotypesHyperplasia and

Controls (%) . Cancer (%)dysplasta (%)

Cancer and.

dysplasia (%)

GSTMJ

+

-

n45 n45 n45

24 (53) 28 (62) 25 (56)

21 (47) 17(38) 20(44)

n=62

32 (52)

30(48)

OR (CIy’ 1.0 0.7(0.3-1.8) 1.0(0.4-2.3) 1.1 (0.5-2.4)

GSTJ’l

+

-

n45 n45 n45

22(49) 27(60) 26(55)

23(51) 18(40) 19(45)

n�62

34(55)

28(45)

OR (Cl)” 1.0 0.6 (0.3-1.5) 0.7 (0.3-1.5) 0.7 (0.3-1.6)

GSTPI*�4j*A

*�Aj*B

*B/*B

n36 n=31 n42

22(61) 27(60) 29(69)

II (31) 15(33) 12(29)

3(8) 3(7) 1 (2)

n57

39(68)

16(28)

1 (2)

OR (CI)” 1.0 0.9(0.5-1.5) 0.7(0.3-1.8) 0.7(0.3-1.7)

‘, ORs and 95% CIs were calculated by logistic regression, with the GSTMI null,

GSTTI null, and GSTPJ *�Af*A genotypes as the reference groups. GSTPJ *,4/*B

and GSTP/*B/B were combined for analysis, adjusted for age and sex.

remains controversial and seems to be mediated by differentmechanisms, transcriptional activation of the gene is likely tobe involved (62, 63). It is interesting that Lucas et a!. (20)

observed that chlorzoxazone metabolism did not differ foreither polymorphism across the genotypes for the basal rates

present in controls or in withdrawn alcoholics. However, afteralcohol induction, chborzoxazone metabolism was significantly

bower in heterozygous genotypes than it was in homozygous c 1genotypes detected by analysis using either RsaI or DraI. Theseresults indicate that the c2 genotypes might be associated with

a back of inducibility in CYP2E1 activity (20). To date, therehas been no evidence showing that enhanced CYP2E1 activityin vivo is related to the c2 genotypes.

Several case-control studies have been conducted to ex-amine the associations between CYP2E1 polymorphisms andthe susceptibility to a number of cancers, although the results

are inconsistent. In a Swedish study, Persson et a!. (26) found

that the frequency of the c2 genotype was significantly lower inlung cancer cases than it was in controls and suggested that this

could constitute a protective factor against the disease. These

data were also supported by recent data from a study by Wu et

a!. (64). Yu et a!. (29) recently showed that the risk of devel-oping hepatocellular carcinoma in cigarette smokers in Taiwanwas significantly associated with the genotypes of CYP2E1

detected by PstI or RsaI, but not by DraI. They found that thefrequency of the c 1/c 1 genotype detected by PstI or RsaI washigher in cases than it was in controls, and this difference wasmore pronounced among smokers. The data reported herein are

consistent with these previous studies. Another case-controlstudy conducted in Taiwan on CYP2E1 genetic polymorphisms

and the risk of nasopharyngeal cancer, which is also hypothe-sized to be associated with exposure to nitrosamines, showed anincreased risk of the cl/cl genotype (42). However, in contrast

to our results and those reported by Yu et a!. (29) and Perssonet a!. (26), an excess risk of the cancer was associated withvariant genotypes detected by both RsaI and Dral (28). Inanother recent study in Taiwan (41), the c2 allele was found toincrease the risk of oral cancer among those who did not chew

betel quid. In a case-control study of breast cancer, Shields eta!. (27) also found the DraI polymorphism C alleles to beassociated with increased risk among premenopausal smokers.

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Cancer Epidemiology, Biomarkers & Prevention /0/7

A significant association of the variant genotype recognized byDraI with increased lung cancer risk was also reported in a

Japanese case-control study (22). In addition, contrary results

showing no association between CYP2EJ genotypes and cancer

risk in Caucasians were reported by other investigators (30, 3 1,33-35). The reasons for these discrepancies are not clear, andspecific exposures may need to be further addressed. However,the very limited statistical power in Caucasian studies due to theextreme rarity of the variant genotypes of CYP2EJ determined

by either RsaI or DraI in this population could make suchstudies difficult to evaluate. Interracial difference in CYP2E1activity may also account for the discrepancy. It is worthy to

note that CYP2E1 activity was lower in Japanese subjects thanit was in Caucasian subjects (61). Again, it is possible that the

risk associated with polymorphisms alone is not evident with-

out relevant exposure data.

GSTs, as an enzyme class, show activity against a broadrange of DNA-damaging chemical substrates. Although several

polycyclic aromatic hydrocarbon epoxides, such as benzo-(a)pyrene diol-epoxide, are known substrates for GSTM1 andGSTP1 (65), there is little information on the detoxification ofnitrosamines by these GSTs. It seems that genetic polymor-phisms of GSTM1 and GSTP1 are mainly associated with anincreased susceptibility for tobacco smoke-related cancers (44,46, 48, 51). GSTFl has significant activity in human erythro-

cytes and is implicated in the conjugation of natural and syn-thetic haloalkanes. The association between GSTT1 genetic

polymorphisms and cancer risk has not been studied as exten-

sively. Our results in the present study did not indicate anassociation between polymorphisms at the three GST genes and

susceptibility to esophageal cancer. On the basis of what isknown about substrates for human GSTM1, GSTP1, and

GSTf1 and given the present findings, we suggest that thecarcinogen(s) involved in the etiology of esophageal cancer in

this high-incidence region may not be substrates for theseGSTs. For example, previous epidemiobogical data alreadyshow that tobacco smoke plays a negligible role in the etiologyof esophageal cancer in this area (66, 67).

In summary, we report a significant role of CYP2EJ butnot GST genotypes in the development of esophageal cancer in

Linxian County, China. Although the number of individualswith the CYP2EJ c2 alleles was small, the differences in allele

frequencies between cases and controls were striking. Of par-

ticular note was the apparent similarity in the frequencies ofalleles in cases with esophageal cancer and in those with severe

epithelial hyperplasia, in contrast to those among controls.Because this is one of the few reports on the associationbetween genetic polymorphisms in carcinogen-metabolism en-

zymes and susceptibility to cancer of the esophagus, studies inlarger populations would be desirable to confirm the findings ofthis pilot study.

References1. Li, J. Y., Liu, B. Q., Li, G. Y., Chen, Z. J., and Sun, X. I. Atlas of cancermortality in the People’s Republic of China: an aid for cancer control and

research. Int. J. Epidemiol., 10: 127-133, 1981.

2. Lu, S. H., Yang, W. X., Guo, L. P., Li, F. M., Wang, G. J., Zhang, J. S., and

Li, P. Z. Determination of N-nitrosamines in gastric juice and urine and a

comparison of endogenous formation of N-nitrosoproline and its inhibition insubjects from high-and low-risk areas for esophageal cancer. IARC Sci. Publ., 84:

538-543, 1987.

3. Lu, S. H., Chui, S. X., Yang, W. X., Hu, X. N., Guo, L. P., and Li, F. M.

Relevance of N-nitrosamines to esophageal cancer in China. IARC Sci. Publ.,

105: 11-17, 1991.

4. Cheng. K. K., Day. N. E., Duffy, S. W., Lam, T. H., Fok, M., and Wong, J.

Pickled vegetables in the aetiology of esophageal cancer in Hong Kong Chinese.

Lancet, 339: 1314-1318, 1992.

5. Guengerich, F. P. Metabolic activation of carcinogens. Pharmacol. Ther., 54:

17-61, 1992.

6. Guengerich, F. P., and Shimada, T. Oxidation of toxic and carcinogenic

chemicals by human cytochrome P-450 enzymes. Chem. Res. Toxicol., 4: 391-

407, 1991.

7. Chasseaud, L. F. The role of glutathione and glutathione S-transferases in the

metabolism of chemical carcinogens and other electrophilic agents. Adv. Cancer

Res., 29: 175-274, 1979.

8. Coles, B., and Ketterer, B. The role of glutathione transfera.ses in chemical

carcinogenesis. Crit. Rev. Biochem. Mol. Biol., 25: 47-70, 1990.

9. Guengerich. F. P.. Kim, D. H., and Iwasaki, M. Role of human cytochromeP-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem.

Res. Toxicol., 4: 168-179, 1991.

10. Yang, C. S., Yoo, J. H., Ishizaki, H., and Hong, J. Cytochrome P45OIIEI:

roles in nitrosamine metabolism and mechanisms of regulation. Drug Metab.

Rev., 22: 147-159, 1990.

1 1 . Yang, C. S., and Smith, T. J. Mechanisms of nitrosamine bioactivation and

carcinogenesis. Adv. Exp. Med. Biol., 387: 385-394, 1996.

12. Hecht, S. S. Biochemistry. biology. and carcinogenicity of tobacco-specific

N-nitrosamines. Chem. Res. Toxicol., 11: 559-603, 1998.

13. lngelman-Sundberg, M., Johansson. I., Yin, H., Terelius, Y., Eliasson, E.,

Clot, P., and Albano, E. Ethanol-inducible cytochrome P4502E1 : genetic poly-

morphism, regulation, and possible role in the etiology of alcohol-induced liver

disease. Alcohol, 10: 447-452, 1993.

14. Albano, E., Tomasi, A., Person, J. 0., Terelius, Y., Goria-Gatti, L., Ingelman-

Sundberg, M., and Dianzani, M. U. Role of ethanol-inducible cytochrome P-450

(P45011E1) in catalysing the free radical activation of aliphatic alcohols. Bio-

chem. Pharmacol., 41: 1895-1902, 1991.

15. Wheeler, C. W., Wrighton, S. A., and Guenthner, T. M. Detection of human

lung cytochrome P450s that are immunochemically related to cytochrome

P45OIIE1 and cytochrome P450111A. Biochem. Pharmacol.. 44: 183-187, 1992.

16. de Waziers, I., Cugnenc. P. H., Yang. C. S., Leroux, J. P., and Beaune, P. H.

Cytochrome P450 isoenzymes, epoxide hydrolase and glutathione transferases in

rat and human hepatic and extrahepatic tissues. J. Pharmacol. Exp. Ther., 253:

387-394, 1990.

17. Nakajima, T., Wang. R-S.. Nimura, Y., Pin, Y-M., He, M.. Vainio, H.,

Murayama, N., Aoyama. T.. and lida, F. Expression of cytochrome P450s and

glutathione S-transferases in human esophagus with squamous-cell carcinomas.

Carcinogenesis (Lond.), 17: 1477-1481, 1996.

18. Guengerich, F. P., and Turvy, C. G. Comparison of levels of several human

microsomal cytochrome P-450 enzymes and epoxide hydrolase in normal and

disease states using immunochemical analysis of surgical liver samples. J. Phar-

macol. Exp. Ther., 256: 1 189-1 194, 1991.

19. Lucas, D., Berthou, F., Dreano, Y., Lozac’h, P., Volant, A.. Menez. J. F.

Comparison oflevels ofcytochromes P-450, CYPIA2. CYP2E1, and their related

monooxygenase activities in human surgical liver samples. Alcohol. Clin. Exp.

Res., I 7: 900-905, 1993.

20. Lucas, D., Menez, C., Girre, C., Berthou, F., Bodenez, P., Joannet, I.,

Hispard, E.. Bardou, L-G., and Menez, J-F. Cytochrome P450 2El genotype and

chlorzoxazone metabolism in health and alcoholic Caucasian subjects. Pharma-

cogenetics. 5: 298-304, 1995.

21. Hayashi, S., Watanabe. J., and Kawajiri. K. Genetic polymorphism in the5-flanking region changes the transcriptional regulation of the human cyto-

chrome P45011E1 gene. J. Biochem. (Tokyo), 1/0: 559-565, 1991.

22. Uematsu, F., Kikuchi, H., Motomiya, M., Abe, T., Sagami, I., Omachi, T.,

Wakui, A., Kanamaru, R., and Watanabe, M. Association between restriction

fragment length polymorphism of the human cytochrome P45OIIEI gene and

susceptibility to lung cancer. Jpn. J. Cancer Res.. 82: 254-256, 1991.

23. Hu, Y., Oscarson, M., Johansson, I., Yue, Q-Y., DahI, M-L., Tabone, M.,Arinco, S., Albano, E., and Ingelman-Sundberg. M. Genetic polymorphism of

human CYP2EI: characterization of two variant alleles. Mol. Pharmacol., 51:

370-376, 1997.

24. El-Zein, R. A., Zwischenberger, J. B., Abdel-Rahman. S. Z., Sankar, A. B.,

and Au, W. W. Polymorphism of metabolizing genes and lung cancer histology:

prevalence of CYP2E1 in adenocarcinoma. Cancer Lett., 112: 71-78, 1997.

25. Oyama, T., Kawamoto, T., Mizoue, T., Sugio, K., Kodama, Y., Mitsudomi,

T., and Yasumoto, K. Cytochrome P450 2E1 polymorphism as a risk factor forlung cancer: in relation to p53 gene mutation. Anticancer Res.. 17: 583-587,

1997.

26. Persson, I., Johansson, I., Bergling. H., DahI, M. L., Seidegard, J., Rylander,

R., Rannug, A.. Hogberg. J., and lngelman-Sundberg, M. Genetic polymorphism

of cytochrome P4502E1 in a Swedish population: relationship to the incidence oflung cancer. FEBS Leti., 319: 207-21 1, 1993.

27. Shields, P. G., Ambrosone, C. B., Graham, S.. Bowman, E. D., Harrington,

A. M.. Gillenwater, K. A., Marshall, J. R., Vena, J. E., Laughlin R., Nemoto. T.,

Association for Cancer Research. by guest on October 11, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

/018 CYP2E1 and Esophageal Cancer Susceptibility

and Freudenheim, J. L. A cytochrome P4502E1 genetic polymorphism andtobacco smoking in breast cancer. Mol. Carcinog.. /7: 144-150, 1996.

28. Ladero, J. M., Agundez, J. A., Rodriguez-Lescure, A., Diaz-Rubio, M., and

Benitez, J. RsaI polymorphism at the cytochrome P4502E1 locus and risk of

hepatocellular carcinoma. Gut, 39: 330-333, 1996.

29. Yu, M-W., Gladek-Yarborough, A., Chiamprasert, S., Santella, R. M., Liaw,Y-F., and Chen, C-J. Cytochrome P450 2E1 and glutathione S-transferase Ml

polymorphisms and susceptibility to hepatocellular carcinoma. Gastrocnterology.

/09: 1266-1273, 1995.

30. London, S. J., Daly, A. K., Cooper, J., Carpenter, C. L.. Navidi, W. C., Ding,

L., and Idle, J. R. Lung cancer risk in relation to the CYP2E/ RsaI genetic

polymorphism among African-Americans and Caucasians in Los Angeles

County. Pharmacogenetics. 6: 151-158, 1996.

31 . Kato. S., Shields, P. G., Caporaso, N. E., Sugimura, H., Trivers, G. E..

Tucker, M. A., Trump, B. F., Weston, A., and Harris, C. C. Analysis of cyto-

chrome P450 2E1 genetic polymorphisms in relation to human lung cancer.

Cancer Epidemiol. Biomark. Prey., 3: 515-518, 1994.

32. Anwar, W. A., Abdel-Rahman, S. Z., El-Zein, R. A., Mostafa, H. M., and Au,W. W. Genetic polymorphism of GSTM1, CYP2E1 and CYP2D6 in Egyptian

bladder cancer patients. Carcinogenesis (Land.), /7: 1923-1929, 1996.

33. Kato. S., Shields, P. G., Caporaso, N. E., Hoover, R. N., Trump, B. F.,

Sugimura, H., Weston, A., and Harris, C. C. Cytochrome P45OIIE1 geneticpolymorphisms. racial variation, and lung cancer risk. Cancer Res., 52: 6712-

6715, 1992.

34. Hirvonen, A., Husgafvel-Pursiainen, K., Anttila, S., Karjalainen, A., and

Vainio, H. The human CYP2EJ gene and lung cancer: DraI and RsaI restriction

fragment length polymorphisms in a Finnish study population. Carcinogenesis

(Lond.), /4: 85-88, 1993.

35. Lucas, D.. Menez, C., Floch, F., Gourlaouen, Y., Sparfel, 0., Joannet, I.,

Bodenez, P.. Jezequel, J., Gouerou, H., Berthou, F., Bardou, L-G., and Menez,

J-F. Cytochromes P4502E1 and 450lAl genotypes and susceptibility to cirrhosis

or upper aerodigestive tract cancer in alcoholic Caucasians. Alcohol. Clin. Exp.

Res., 20: 1033-1037, 1996.

36. Morita, S., Yano, M., Shiozaki, H., Tsujinaka, T., Ebisui, C., Morimoto, T.,Kishibuti, M., Fujita, J., Ogawa, A., Taniguchi. M., Inoue, M., Tamura, S.,

Yamazaki, K., Kikkawa, N., Mizunoya, S., and Monden, M. CYPIAI, CYP2E1

and GSTM I polymorphisms are not associated with susceptibility to squamouscell carcinoma of the esophagus. Int. J. Cancer, 71: 192-195, 1997.

37. Jahnke, V., Matthias, C., Fryer, A., and Strange, R. Glutathione S-transferase

and cytochrome P-450 polymorphism as risk factors for squamous cell carcinomaof the larynx. Am. J. Surg.. /72: 671-673, 1996.

38. Kato. S., Onda, M., Matsukura, N.. Tokunaga, A., Matsuda, N., Yamashita,K., and Shields, P. G. Genetic polymorphisms of the cancer-related gene and

Helicobacter pylon infection in Japanese gastric cancer patients: an age and

gender matched case-control study. Cancer (Phila.), 77: 1654-1661, 1996.

39. Sugimura. H., Hamada, G. S., Suzuki, I., Iwase, T., Kiyokawa, E., Kino, I.,

and Tsugane, S. CYPIAI and CYP2EI polymorphism and lung cancer: a case-

control study in Riode Janeiro, Brazil. Pharmacogenetics, 5(Suppl.): S145-S 148,

1995.

40. Watanabe, J., Yang, J. P., Eguchi. H., Hayashi, S., Imai, K., Nakachi, K., and

Kawajiri, K. An RsaI polymorphism in the CYP2EI gene docs not affect lungcancer risk in a Japanese population. Jpn. J. Cancer Res., 86: 245-248, 1995.

41. Hung, H-C.. Chuang. J., Chien, Y-C., Chem, H-D., Chiang, C-P.,Hildesheim, A., and Chen, C-J. Genetic polymorphisms ofCYP2E1, GSTMI, and

GS’fl’l : environmental factors and risk of oral cancer. Cancer Epidemiol. Bi-

omark. Prey., 6: 901-905, 1997.

42. Hildesheim, A., Chen, C-J., Caporaso, N. E., Cheng, Y-J., Hoover, R. N.,

Hsu. M-M., Levine, P-H.. Chen, I-H., Chen, J-Y.. Yang, C-S., Daly, A. K., andIdle, J. Cytochrome P4502E1 genetic polymorphisms and risk of nasopharyngeal

carcinoma: results from a case-control study conducted in Taiwan. Cancer Epi-demiol. Biomark. Prey., 4: 607-610, 1995.

43. Hayes. J. D., and Pulford, D. J. The glutathione S-transferase supergene

family: regulation of GST and the contribution of the enzyme to cancer chemo-

protection and drug resistance. Crit. Rev. Biochem. Mol. Biol., 30: 445-600,

I 995.

44. Seidegard. J., Pero, R. W., Miller, D. J., and Beaflie, E. J. A glutathione

transferase in human leukocytes as a marker for the susceptibility to lung cancer.

Carcinogenesis (Land.), 7: 751-753, 1986.

45. Pemble. S.. Schroeder, K. R., Spenser. S. R.. Meyer. D. J., Hallier, E., Bolt,

H. M., Ketterer, B., and Taylor. J. B. Human glutathione S-transferase theta

(GSTTI): eDNA cloning and the characterization of a genetic polymorphism.

Biochem. J.. 300: 271-276. 1994.

46. Zhong, S., Wyllie. A. H., Barnes, D., Wolf, C. R., and Spurr, N. K. Rela-

tionship between the GSTMI genetic polymorphism and susceptibility to bladder,breast and colon cancer. Carcinogenesis (Land.). 14: 1821-1824, 1993.

47. Chenevix-Trench, G., Young, J., Coggan, M., and Board, P. Glutathione

S-transferase Ml and Tl polymorphisms: susceptibility to colon cancer and ageof onset. Carcinogenesis (Lond.), 16: 1655-1657, 1995.

48. Bell, D. A., Taylor. J. A., Paulson, D. F., Robertson, C. N., Mohler, J. L., and

Lucier, G. W. Genetic risk and carcinogen exposure: a common inherited defect

of the carcinogen-metabolism gene glutathione S-transferase Ml (GSTMI) that

increases susceptibility to bladder cancer. J. Natl. Cancer Inst., 85: I 159-I 164,

1993.

49. Rebbeck, T. R. Molecular epidemiology of glutathione S-transferase geno-

types GSTMI and GS17’/ in cancer susceptibility. Cancer Epidemiol. Biomark.

Prey., 6: 733-743, 1997.

50. Peters, W. H. M., Roelofs, H. M. J., Hectors, M. P. C., Nagengast, F. M., and

Jansen, J. B. M. J. Glutathione and glutathione S-transferases in Barrett’s epi-

thelium. Br. J. Cancer, 67: 1413-1417, 1993.

51. Harries, L. W., Stubbins, M. J., Forman, D., Howard, G. C. W., and Wolf,

C. R. Identification of genetic polymorphisms at the glutathione S-transferase irlocus and association with susceptibility to bladder, testicular and prostate cancer.

Carcinogenesis (Lond.), 18: 641-644, 1997.

52. Zimniak, P., Nanduri, B., Pikula. S., Bandorowicz-Pikula, J., Singhal, S.,

Srivastava, S. K., Awasthi, S., and Awasthi, J. C. Naturally occurring human

glutathione S-transferase GSTP1 ,l isoforms with isoleucine and valine at position

104 differ in enzymatic properties. Eur. J. Biochem., 224: 893-899, 1994.

53. Hu, X., O’Donnel, R., Srivastava, S. K., Xia, H., Zimniak, P., Nanduri, B.,

Bleicher, R. J., Awasthi, S., Awasthi, Y. C., Ji, X., and Singh, S. V. Active site

architecture of polymorphic forms of human glutathione transferase P1-1 ac-

counts for their enantioselectivity and disparate activity in the glutathione con-

jugation of 7�,8s-dihydroxy-9a,I0a-oxy-7,8,9,l0-tetrahydrobenzo(a)pyrene.

Biochem. Biophys. Res. Commun., 235: 424-428, 1997.

54. Nimura, Y., Yokoyama, S., Fujimori, M., Aoki, T., Adachi, W., Nasu, T., He,

M., Ping, Y-M., and lida, F. Genotyping of the CYPIA1 and GSTM/ genes in

esophageal carcinoma patients with special reference to smoking. Cancer (Phila.),

80: 852-857, 1997.

55. Blin, N., and Stafford, D. W. A general method for isolation of high

molecular weight DNA from eukaryotes. Nucleic Acids Res., 3: 2303-2308,1976.

56. Arand, M., Muhlbauer, R., Hengstler, J., Jager, E., Fuchs, J., Winkler, L., andOesch, F. A multiplex polymerase chain reaction protocol for the simultaneous

analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms.

Anal. Biochem., 236: 184-186, 1995.

57. Breslow, N. E., and Day, N. E. Statistical Methods in Cancer Research. I. The

Analysis ofCase-Control Studies. IARC Scientific Publ. No. 32, pp. 5-338. Lyon,France: IARC, 1980.

58. Watanabe, J., Hayashi, S., and Kawajiri, K. Different regulation and expres-

sion of the human CYP2E1 gene due to the RsaI polymorphism in the 5-flanking

region. J. Biochem. (Tokyo), 116: 321-326, 1994.

59. Carriere, V., Berthou, F., Baird, S., Belloc, C., Beaune, P., and de Waziers,

I. Human cytochrome P450 2E1 (CYP2E1): from genotype to phenotype. Phar-

macogenetics, 6: 203-21 1, 1996.

60. Kim, R. B.. O’Shea, D., and Wilkinson, G. R. Interindividual variability ofchlorzoxazone 6-hydroxylation in men and women and its relationship to

CYP2E1 genetic polymorphisms. Clin. Pharmacol. Ther., 57: 645-655, 1995.

61. Kim, R. B., Yamazaki, H., Chiba, K., O’Shea, D., Mimura, F., Guengerich,

F. P., Ishizaki, T., Shimada, T., and Wilkinson, G. R. In vivo and in vitro

characterization of CYP2EI activity in Japanese and Caucasians. J. Pharmacol.

Exp. Ther., 279: 4-1 1 , 1996.

62. Koop, D. R., and Tiemey, D. J. Multiple mechanisms in the regulation of

ethanol-inducible cytochrome P-45011E1. Bioessays, 12: 429-435, 1990.

63. Takahashi, T., Lasker, J. M., Rosman, A. S., and Lieber, C. S. Induction of

cytochrome P-4502E1 in the human liver by ethanol is caused by a corresponding

increase in encoding messenger RNA. Hepatology, 17: 236-245, 1993.

64. Wu, X., Amos, C. I., Kemp, B. L., Shi, H., Jiang, H., Wan, Y., and Spitz,

M. R. Cytochrome P450 2EI DraI polymorphism in lung cancer in minority

populations. Cancer Epidemiol. Biomark. Prey., 7: 13-18, 1998.

65. Hu, X., Xia, H., Srivastava, S. K., Herzog, R., Awasthi, Y. C., ii, X.,

Zimniak, P., and Singh, S. V. Activity of four allelic forms of glutathione

S-transferase hGSTP1-l for diol epoxides of polycyclic aromatic hydrocarbons.

Biochem. Biophys. Res. Commun., 238: 397-402, 1997.

66. Yu, Y., Taylor, P. R., Li, J. Y., Dawsey, S. M., Wang, G. Q., Guo, W. D.,Wang, W.. Liu, B. Q., Blot, W. J., Shen, Q., and Li, B. Retrospective cohort study

of risk factors for esophageal cancer in Linxian, People’s Republic of China.

Cancer Causes Control, 4: 195-202, 1993.

67. Li, J. Y., Ershow, A. G., Chen, Z. J., Wacholder, S., Li, G. Y., Guo, W., Li,

B., and Blot, W. J. A case-control study of cancer of the esophagus and gastric

cardia in Linxian. Int. J. Cancer, 43: 755-761, 1989.

Association for Cancer Research. by guest on October 11, 2020. Copyright 1998 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

1998;7:1013-1018. Cancer Epidemiol Biomarkers Prev   D X Lin, Y M Tang, Q Peng, et al.   P450 2E1.in glutathione S-transferases T1, P1, and M1 and cytochrome Susceptibility to esophageal cancer and genetic polymorphisms

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