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.
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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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