# CYP1A1, mEH, and GSTM1 Polymophisms and Risk of Oral and Pharyngeal Cancer: A Spanish Case-Control Study

Authors: L. Varela-Lema; A. Ruano-Ravina; M. A. Juiz Crespo; K. T. Kelsey; L. Loidi; J. M. Barros-Dios
Journal: Journal of Oncology
 (2008)

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## Abstract

Background. Genetic polymorphisms of drug metabolizing enzymes involved in the detoxification pathways of carcinogenic substances may influence cancer risk. Methods. Case-control study that investigates the relationship between CYP1A1 Ile/Val, exon 4 mEH, and GSTM1 null genetic polymorphism and the risk of oral and pharyngeal cancer examining the interaction between these genes, tobacco, and alcohol. 92 incident cases and 130 consecutive hospital-based controls have been included. Results. No significant associations were found for any of the genotypes assessed. The estimated risk was slightly elevated in subjects with the wild type of the mEH gene and the null GSTM1 genotype. For exon 4 mEH heterozygous polymorphism, the risk was slightly lower for heavy smokers than for light smokers. The inverse association was observed for the GSTM1 null genotype. Conclusions. The results suggest that exon 4 mEH and GSTM1 null polymorphisms might influence oral and pharyngeal cancer.


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## Body

## 1. Introduction

Oral and pharyngeal
cancers represent an important problem worldwide. The incidence and prevalence
rates for these tumors are double in men than in women. Cancers of the oral
cavity rank as the eighth most common cancer among men, being responsible for
3% of the cancers diagnosed in this gender [1]. Mortality rates are
substantially lower than incidence rates. According to the World Health
Organization (WHO) data, the standardized mortality rate for 2002 was 2.2
deaths per 100 000 population.Tobacco and alcohol
are the main risk factors for oral and pharyngeal cancers. In USA and Europe,
they are responsible for 75–80% of these
tumors [2, 3]. Epidemiologic studies performed in all continents have found an
increased risk in smokers, which seems to rise with daily consumption and
duration [4–7]. There is also
sufficient evidence to conclude that excessive consumption of alcoholic
beverages is associated with oral and pharyngeal cancers, causing in some cases risks higher than those found for smokers [8–10].The reason why some
individuals develop cancer and others do not can be attributed, at least partly,
to variations in genetic polymorphisms responsible for metabolizing
carcinogenic substances found in tobacco and alcohol. Although many genes have
been associated with metabolism of these compounds, some with the highest
rational of those being
involved are CYP1A1, mEH, and GSTM1.The CYP1A1 gene
belongs to the CYP1 subfamily and encodes for the enzyme aryl hydrocarbon
hydrolase, which is involved in the activation of many polycyclic aromatic
hydrocarbons and aromatic amines [11] and is present in oral tissue [12]. This
enzyme is implicated in the metabolism of benzo[a]pyrene, a potent tobacco
carcinogen. Various studies have shown that CYP1A1 catalyzes the initial
conversion of benzo[a]pyrene to 7,8 dihydrodiol-9,10-oxide [13, 14]. Two CYP1A1
polymorphisms have been related to different tumors, including head and neck
cancers. One of these is a single-base substitution of adenine to guanine at
position 2455 in the heme-binding region of exon 7, which induces an amino acid change in
isoleucine to valine at codon 462, known as the Ile/Val or exon 7 polymorphism
(Ile 462 Val) or CYP1A1*2C. This mutation has also been referred to as mutation
m2. The Ile/Ile genotype corresponds to the wild type, and Ile-Val and Val-Val
to the heterozygous and homozygous genotypes for the mutant allele,
respectively [15]. This mutation is rare in Caucasians, and is in complete
linkage disequilibrium with the CYP1A1 MspI mutation (CYP1A1*2B) [16].The microsomal form
of epoxide hydrolase is primarily associated with the metabolism of exogenous
xenobiotic compounds. Its interest in oral and pharyngeal cancers comes from
the fact that it has been detected in all
tissues including the aerodigestive tract and catalyzes the hydrolosis of arene, alkene, and aliphatic epoxides from
polycyclic aromatic hydrocarbons and aromatic amines [17]. Enzymatically, mEH catalyzes the hydrolysis of
epoxides to trans-dihydrodiols [18, 19]. Two polymorphisms in the mEH gene have been reported [20, 21].
The first polymorphism is produced as a
consequence of a substitution of C→T
within exon 3 of the gene and results in a substitution of His to Tyr in amino acid
position 113. This polymorphism is known as the “slow allele” since in vitro
studies show a 40–60% decrease in
enzyme activity in comparison to the wild type. This allele is also known as
HYL*2. In the second polymorphism, G substitutes A in exon 4, leading to an
emplacement of histidine for arginine in the amino acid position 139 (139 Arg→His). This polymorphism is known as the fast allele HYL*3
since it produces a 25% increase in enzyme activity in vitro. Tyr is the
predominant amino acid at the 113 position in Caucasian populations, and His is
the most predominant at position 139.The glutathione S
transferase (GST) comprises a family of phase II detoxifying enzymes that
catalyze a great number of detoxification reactions that take place between the
cytosolic glutathione and compounds containing an electrofilic centre [22]. The
GST substrates include acetaldehyde and several polycyclic aromatic
hydrocarbons found in tobacco smoke. The GSTM1 is involved in the
detoxification of benzo[a]pyrene-7,8-diol-9,10-oxide [23]. Metabolism of this
carcinogen involves a balance between the activation steps mediated by the
epoxide hydrolase and the cytochrome system and the detoxification steps,
involving GSTM1, that inhibit the activity of the DNA binding intermediates and
catalyze the conversion of the reactive electrophiles to inactive, water-soluble
conjugates that can be easily removed [13]. Even though frequencies of GSTM1
null genotypes vary among different ethnic groups, in white Caucasians, it is
deleted in approximately 50% of the population [24–33].In the present case-control
study, we aimed to examine the relationship between the CYP1A1 Ile/Val, exon 4 mEH
(139Arg→His), and GSTM1 null
genetic polymorphism and the risk of oral and pharyngeal cancers, investigating
also the association with smoking, drinking, and the gene-gene interactions.


## 2. Patients and Methods

### 2.1. Design, Subjects, and Settings

The present
hospital-based case-control study was conducted at the Santiago de Compostela
University Hospital Complex (Galicia,
NW Spain) between January 1996 and January 2000. Data was collected on a total
of 92 incident Caucasian male cases with histopathologically confirmed
diagnosis of primary oral or pharyngeal cancers. The study was restricted to
newly diagnosed patients over 20 years of age without a prior history of
cancer. For study purposes, tumors of the lip were excluded.A total of 130 consecutive
controls were included from patients attending the Hospital Complex
Preoperative Unit for nonsmoking- and nonalcohol-related trivial surgery. The
inclusion criterion for controls was absence of prior history of cancer. Subjects
under 20 years of age were excluded. The types of surgical procedures controls were scheduled to undergo mainly
comprised inguinal hernias, cataracts, and orthopedic surgery. Informed consent
was obtained from all study subjects prior to the interview and the extraction
of total blood. Only 1 case and 6 controls refused to participate in the study.
The study protocol was approved by the Galician Ethical Research Committee.


### 2.2. Information Retrieval

All study subjects
were interviewed by a person purpose-trained to administer a structured
questionnaire addressing various aspects of lifestyle, with special emphasis on
smoking habit, alcohol consumption, occupation, and other activities related to the development of
oral and pharyngeal cancers. For cases, the interview was done as soon as
possible after cancer was detected and always within 15 days from diagnosis.
The questionnaire used was previously applied to a group of individuals
fulfilling the characteristics of the subjects going to be included and all
questions with difficult interpretation were duly amended.


### 2.3. Laboratory Methods

After cases and
controls were identified, whole blood samples of 6 mL were collected from each
subject in heparin-containing tubes. The samples were stored at 4°C and centrifuged at 2800 rpm
for at least 10 minutes within the next 24 hours. The three independent
fractions were isolated and stored at −84°C until analysis. DNA was extracted from
the buffy coat of middle layer containing monocytes. The cells were washed with
TE (10x) and centrifuged at 3000 rpm during 10 minutes several times until they
were cleaned. The pellet was then treated with lysis buffer and proteinase K in
1% SDS previous to the extraction of DNA with phenol-chloroform and ethanol
precipitation similarly as previously described [34]. The DNA was precipitated in the presence of high
concentrations of ammonium acetate (to further purify the DNA) and resuspended
in TE to approximately 300μg/mL.
The genotyping
assays were performed at the Molecular Medicine Unit of the Santiago de
Compostela University Hospital Complex.Genotyping for GSTM1
was carried out in the whole sample using a modified PCR method described
previously [35]. Reactions were carried out in a final volume of 12.5μL containing 20 mM Tris-HCl (PH 8.4), 50 mM KCl, 3 mM MgCl2, 0.25μM
of each GSTM1 primer, 0.25μM of each β-globin primer (internal control), 1 U Taq DNA
polymerase (Promega), 2μM
each dNTPs, and 600–900 ng of DNA.
The GSTM1 primers were 5′-GAACTCCCTGAAAAGCTAAGC and 5′-GTTGGGCTCAAATATACGGTGG.  A negative control was included in all
batches. The PCR conditions were 94° for 3 minutes, followed by 35 cycles
of  94°C for 30 seconds, 58°C for 30 seconds, 72°C for 45 seconds, and a
final extension of 72°C for 7 minutes.To analyse the exon
4 mEH polymorphism (EH139arg), a 357-bp fragment containing the
polymorphic site was amplified [36]. The PCR was performed in all subjects
using the sense primer 5′-GGGGTGCCAGAGCCTGACCGT-3′ and the antisense primer
5′-AACACCGGGCCCACCCTTGGC-3′ (Sigma-Genosys). The PCR cycling conditions were 95°C for 2 minutes, followed by 35 cycles at 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, with a
final step at 72°C for 7 minutes. After PCR amplification, 10μL of
the PCR product was digested overnight at 37°C with 10U of RsaI (Invitrogen). The wild-type
genotype (AA) produced 295-bp and 62-bp bands, the heterozygous genotype (AG)
yielded 295-bp, 174-bp, 121-bp, 62-bp; and the rare allele (GG) gave 174-bp,
121-bp, and 62-bp bands.The Ile-Val
polymorphism was analysed in only 158 of the 222 individuals included using an allele-specific
oligonucleotide-PCR procedure previously described by Hayashi et al. [16]. In
the same reaction mix, two primers with different terminal bases (1A1A or 1A1G),
which contained the polymorphic site at the 3′end, were added (1A1G :
5′-GAACTGCCACTTCAGCTGTCT-3′ and 1A1A : 5′-AAGACCTCCCAGCGGGCAAT-3′) in
conjunction with another strand of primer (1A1.1 : 5′-GAACTGCCACTTCAGCTGTCT-3′).
Two amplification reactions were necessary for each one of the subjects analysed,
one with the primers 1A1.1/1A1A which recognize the Ile462 allele and another
with the primers 1A1.1/1A1G which recognize the Val462 allele. PCR was carried
out at 30 cycles under the following conditions: 30 seconds at 95° for
denaturing, 1 minute at 60°C for primer annealing, and 1 minute at 72°C for primer extension.All the products
were electrophoresed on a 2% agarose gel stained with ethidium bromide and
visualized under UV light. To test for eventual contamination, negative
controls were introduced in each run.


### 2.4. Statistical Analysis

Logistic regression
was employed to analyse the effect of each of the genes studied. The wild type
gene was considered as the reference category for each gene in each analysis.
All the regressions were adjusted for age, tobacco consumption in pack-years,
and alcohol consumption in grams/week. For analyzing the effect of the
different polymorphisms across different categories of tobacco and alcohol
consumption, the cutpoint was established on the median creating two
categories, low and high consumption in order to achieve a higher statistical
power. In all analyses, the dependent variable was the status of case or
control and the risks were expressed as ORs with CI 95%. All the analyses were
performed with SPSS 11.5 statistical package.


## 2.1. Design, Subjects, and Settings

The present
hospital-based case-control study was conducted at the Santiago de Compostela
University Hospital Complex (Galicia,
NW Spain) between January 1996 and January 2000. Data was collected on a total
of 92 incident Caucasian male cases with histopathologically confirmed
diagnosis of primary oral or pharyngeal cancers. The study was restricted to
newly diagnosed patients over 20 years of age without a prior history of
cancer. For study purposes, tumors of the lip were excluded.A total of 130 consecutive
controls were included from patients attending the Hospital Complex
Preoperative Unit for nonsmoking- and nonalcohol-related trivial surgery. The
inclusion criterion for controls was absence of prior history of cancer. Subjects
under 20 years of age were excluded. The types of surgical procedures controls were scheduled to undergo mainly
comprised inguinal hernias, cataracts, and orthopedic surgery. Informed consent
was obtained from all study subjects prior to the interview and the extraction
of total blood. Only 1 case and 6 controls refused to participate in the study.
The study protocol was approved by the Galician Ethical Research Committee.


## 2.2. Information Retrieval

All study subjects
were interviewed by a person purpose-trained to administer a structured
questionnaire addressing various aspects of lifestyle, with special emphasis on
smoking habit, alcohol consumption, occupation, and other activities related to the development of
oral and pharyngeal cancers. For cases, the interview was done as soon as
possible after cancer was detected and always within 15 days from diagnosis.
The questionnaire used was previously applied to a group of individuals
fulfilling the characteristics of the subjects going to be included and all
questions with difficult interpretation were duly amended.


## 2.3. Laboratory Methods

After cases and
controls were identified, whole blood samples of 6 mL were collected from each
subject in heparin-containing tubes. The samples were stored at 4°C and centrifuged at 2800 rpm
for at least 10 minutes within the next 24 hours. The three independent
fractions were isolated and stored at −84°C until analysis. DNA was extracted from
the buffy coat of middle layer containing monocytes. The cells were washed with
TE (10x) and centrifuged at 3000 rpm during 10 minutes several times until they
were cleaned. The pellet was then treated with lysis buffer and proteinase K in
1% SDS previous to the extraction of DNA with phenol-chloroform and ethanol
precipitation similarly as previously described [34]. The DNA was precipitated in the presence of high
concentrations of ammonium acetate (to further purify the DNA) and resuspended
in TE to approximately 300μg/mL.
The genotyping
assays were performed at the Molecular Medicine Unit of the Santiago de
Compostela University Hospital Complex.Genotyping for GSTM1
was carried out in the whole sample using a modified PCR method described
previously [35]. Reactions were carried out in a final volume of 12.5μL containing 20 mM Tris-HCl (PH 8.4), 50 mM KCl, 3 mM MgCl2, 0.25μM
of each GSTM1 primer, 0.25μM of each β-globin primer (internal control), 1 U Taq DNA
polymerase (Promega), 2μM
each dNTPs, and 600–900 ng of DNA.
The GSTM1 primers were 5′-GAACTCCCTGAAAAGCTAAGC and 5′-GTTGGGCTCAAATATACGGTGG.  A negative control was included in all
batches. The PCR conditions were 94° for 3 minutes, followed by 35 cycles
of  94°C for 30 seconds, 58°C for 30 seconds, 72°C for 45 seconds, and a
final extension of 72°C for 7 minutes.To analyse the exon
4 mEH polymorphism (EH139arg), a 357-bp fragment containing the
polymorphic site was amplified [36]. The PCR was performed in all subjects
using the sense primer 5′-GGGGTGCCAGAGCCTGACCGT-3′ and the antisense primer
5′-AACACCGGGCCCACCCTTGGC-3′ (Sigma-Genosys). The PCR cycling conditions were 95°C for 2 minutes, followed by 35 cycles at 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, with a
final step at 72°C for 7 minutes. After PCR amplification, 10μL of
the PCR product was digested overnight at 37°C with 10U of RsaI (Invitrogen). The wild-type
genotype (AA) produced 295-bp and 62-bp bands, the heterozygous genotype (AG)
yielded 295-bp, 174-bp, 121-bp, 62-bp; and the rare allele (GG) gave 174-bp,
121-bp, and 62-bp bands.The Ile-Val
polymorphism was analysed in only 158 of the 222 individuals included using an allele-specific
oligonucleotide-PCR procedure previously described by Hayashi et al. [16]. In
the same reaction mix, two primers with different terminal bases (1A1A or 1A1G),
which contained the polymorphic site at the 3′end, were added (1A1G :
5′-GAACTGCCACTTCAGCTGTCT-3′ and 1A1A : 5′-AAGACCTCCCAGCGGGCAAT-3′) in
conjunction with another strand of primer (1A1.1 : 5′-GAACTGCCACTTCAGCTGTCT-3′).
Two amplification reactions were necessary for each one of the subjects analysed,
one with the primers 1A1.1/1A1A which recognize the Ile462 allele and another
with the primers 1A1.1/1A1G which recognize the Val462 allele. PCR was carried
out at 30 cycles under the following conditions: 30 seconds at 95° for
denaturing, 1 minute at 60°C for primer annealing, and 1 minute at 72°C for primer extension.All the products
were electrophoresed on a 2% agarose gel stained with ethidium bromide and
visualized under UV light. To test for eventual contamination, negative
controls were introduced in each run.


## 2.4. Statistical Analysis

Logistic regression
was employed to analyse the effect of each of the genes studied. The wild type
gene was considered as the reference category for each gene in each analysis.
All the regressions were adjusted for age, tobacco consumption in pack-years,
and alcohol consumption in grams/week. For analyzing the effect of the
different polymorphisms across different categories of tobacco and alcohol
consumption, the cutpoint was established on the median creating two
categories, low and high consumption in order to achieve a higher statistical
power. In all analyses, the dependent variable was the status of case or
control and the risks were expressed as ORs with CI 95%. All the analyses were
performed with SPSS 11.5 statistical package.


## 3. Results

The study population
covered a total of 222 subjects, comprising 92 cases and 130 controls. A
description of the sample characteristics is presented in Table1. The mean age
of the controls (59.4 years; CI 95% 57–61.8) was
slightly higher than that of cases (55 years; CI 95% 52.7–57.3). The 57.6% of
the tumors were located in the oral cavity (tongue 27, oral floor 7, palate 6, and
other parts 12) and 96.7% were squamous cell carcinomas. Smoking and alcohol
consumption were more
frequent among cancer cases than controls. A percentage of 98% of the cases
were smokers and 46.7% of these (n=43) were considered as heavy drinkers (more
than six glasses of wine/beers or two liquors a day), whilst in the control
group 62% of the individuals smoked and 10.8% (n=14) were heavy drinkers.Table 1
Description of the study individuals.
Variable consideredCasesControlsAge (mean, IC95%)55.0 (52.7–57.3)59.4 (57.0–61.8)Cancer locationOral53 (57.6%)Pharyngeal39 (42.4%)Cigarette smokingNever smoker2 (2.2%)49 (37.7%)Former smoker17 (18.5%)42 (32.3%)Current smoker73 (79.3%)39 (30%)Tobacco consumption (pack-years)Percentil 2533.00Percentil 5046.59.9Percentil 7567.543.8Drinking habitNo alcohol drinking2 (2.2%)20 (15.4%)Light drinker (≤2 glasses of wine/beers or 2 licors a day) (0–280 gm/week)6 (6.5%)50 (38.5%)Moderate drinkers (3–6 drinks/day)
(281–840 g/week)41 (44.6%)46 (35.4%)Heavy drinkers (>6 drinks/day) (>840 gm/week)43 (46.7%)14 (10.8%)Alcohol
consumption gm/weekPercentil 2529070Percentil 50560145Percentil 75840300CYP1A1Wild type (Ile/Ile)64 (97%)90 (97.8%)Mutation (Ile/Val, Val/Val)2 (3%)2 (2.2%)mEH (139Arg→His)Arg/Arg63 (68.5%)88 (67.7%)Arg/His25 (27.2%)41 (31.5%)His/His4 (4.3%)1 (0.8%)GSTM1Present (GSTM1 +)45 (48.9%)68 (52.3%)Absent (GSTM1 −)47 (51.1%)62 (47.7%)Total92130The frequency of
GSTM1 null genotype was 51.1% among oral and pharyngeal cancer patients and 47.7%
among controls. The exon 4 mEH (139Arg→His) heterozygous
polymorphism (His/Arg) was present in 31.5% of the control group and in 27.2%
of the case group. Only 4 subjects (4.3%) in the case group and 1 subject in
the control group (0.8%) presented homozygous mutations. There were no
differences in the frequency of mutations for the CYP1A1 exon 7 polymorphism.
The mutant alleles (Ile/Val, Val/Val) were observed in only 2 cases (3%) and 2
controls (2.2%).Table2 shows the odds
ratio estimates for combined oral and pharyngeal cancers associated with the GSTM1,
CYP1A1, and exon 4 mEH polymorphisms. To determine the effect of these
polymorphisms on different cancer sites, oral cavity and pharyngeal tumors were
evaluated separately. No statistically significant effect was observed for any
of the polymorphisms studied for any of the anatomic subtypes. The limited
number of subjects with mutant CYP1A1 alleles did not allow for a calculation
of the OR for oral cavity tumors.Table 2
Risks for oral and pharyngeal cancers associated with
polymorphisms in genes CYP1A1, mEH, and GSTM1.
GeneCasesControlsOR crude (CI 95%)*OR adjusted (CI 95%)+All cancersCYP1A1No mutated1.001.00Mutated0.72
(0.10–5.28)1.68
(0.18–15.70)mEH
(139His→Arg)His/His1.001.00His/Arg0.95
(0.52–1.75)0.81
(0.38–1.71)Arg/arg4.30
(0.46–40.07)4.45
(0.39–50.45)GSTM1Present1.001.00Absent1.16
(0.73–1.99)1.25
(0.65–2.40)Oral cancerCYP1A1No mutated5364
(97%)1.001.00Mutated02
(3%)——mEH
(139His→Arg)His/His39
(73,6%)88
(67,7%)1.001.00His/Arg12
(22,6%)41
(31,5%)0.74
(0.34–1.58)0.54
(0.21–1.41)Arg/arg2
(3,8%)1
(0,8%)3.44
(0.29–40.16)4.32
(0.32–58.63)GSTM1Present26
(49,1%)68
(52,3%)1.001.00Absent27
(50,9%)62
(47,7%)1.14
(0.60–2.19)1.20
(0.56–2.18)Pharyngeal cancerCYP1A1No mutated37
(94,9%)64
(97%)1.001.00Mutated2
(5,1%)2
(3%)1.74
(0.23–12.92)4.06
(0.43–38.24)mEH
(139His→Arg)His/His24
(61,5%)88
(67,7%)1.001.00His/Arg13
(33,3%)41
(31,5%)1.27
(0.58–2.80)1.07
(0.42–2.71)Arg/arg2
(5,2%)1
(0,8%)5.58
(0.47–66.10)5.74
(0.34–97.71)GSTM1Present19
(48,7%)68
(52,3%)1.001.00Absent20
(51,3%)62
(47,7%)1.20
(0.58–2.48)1.44
(0.61–3.37)*Adjusted for age.
+Adjusted for age, smoking, and alcohol intake.To assess dose-response
relationship, we have calculated pack-years of smoking (1 pack (20
cigarettes/day)×years of smoking). Smokers were classified as non-to-light/moderate
smokers (≤35 pack-years) and heavy smokers (>35 pack-years). Non smokers were analysed
together with light/moderate smokers because there were only 2 nonsmokers among
the cases and this did not allow for a separate analysis. The odds ratios associated
with tobacco consumption by the different genotypes analysed are shown in Table 3. This analysis was not performed for CYP1A1 due to the limited number of patients
with mutated alleles and the same happened for alcohol consumption. For the His/Arg
genotype of exon 4 mEH, it could be observed that the risk was slightly lower
for heavy smokers (OR 0.68; 95% CI 0.25–1.86) than for
light smokers (OR 1.08; 95% CI 0.37–3.13) but the
associations were not significant for any of the categories of tobacco
consumption. The GSTM1 null genotype revealed an inverse pattern, showing an OR
of 0.88 (95% CI 0.34–2.34) for light
smokers and an OR of 1.40 (95% CI 0.57–3.43) for heavy
smokers.Table 3
Risks for the different polymorphisms broken down by
smoking categories.
Genetic polymorphismCasesControlsOR crudeOR adjustedLight/moderate
smokers (≤35 pack-years)CYP1A1No mutated28411.001.00Mutated120.76 (0.06–8.90)1.12 (0.09–13.94)mEH (139His→Arg)His/His20581.001,00His/Arg8241.06 (0.40–2.78)1.08 (0.37–3.13)Arg/arg111.91 (0.11–33.10)2.82 (0.15–52.41)GSTM1Present17431.001.00Absent12400.74 (0.31–1.76)0.88 (0.34–2.34)Heavy smokers
(>35 pack-years)CYP1A1No mutated23621.001.00Mutated01——mEH (139His→Arg)His/His43301.001.00His/Arg17170.94 (0.39–2.26)0.68 (0.25–1.86)Arg/arg30——GSTM1Present28251.001.00Absent35221.50 (0.67–1.33)1.40 (0.57–3.43)To examine the interaction between these genotypes and
the drinking status, we carried out a second stratification analysis. Two
categories of alcohol consumption were established. Those that consumed 280 gm/week
of alcohol or less (≤2
glasses of wine/beers or 2 liquors a day) were considered as light drinkers,
and those that exceeded those values as heavy drinkers. Once again, due to the
low number of nondrinkers among the cases (n=2), these subjects were analysed
in combination with light drinkers. The risks for the two different categories
of alcohol consumption, light and heavy drinkers, are displayed in Table 4.
There seems to be a very slight nonsignificant negative association for the His/Arg
genotype of exon 4 mEH for both categories of alcohol consumption. The GSTM1
gene has a different effect, with its absence posing a slightly higher association
for light drinkers (OR 1.97; 95% CI 0.73–5.35) than for moderate/heavy
drinkers (OR 0.69; 95% CI 0.28–1.72).Table 4
Risks for the different polymorphisms broken down by
alcohol intake.
Genetic polymorphismCasesControlsOR crudeOR adjustedLight drinkers (≤280 gm alcohol/week)CYP1A1No mutated23431.001.00Mutated02——mEH (139His→Arg)His/His18641.001.00His/Arg5310.57 (0.19–1.72)0.71 (0.22–2.24)Arg/arg01——GSTM1Present10521.001.00Absent13441.55 (0.62–3.88)1.97 (0.73–5.35)Moderate/heavy
drinkers (>280 gm alcohol/week)CYP1A1No mutated67211.001.00Mutated20——mEH (139His→Arg)His/His45241.001.00His/Arg20101.28 (0.50–3.31)0.83 (0.30–2.30)Arg/arg40——GSTM1Present35161.001.00Absent34180.79 (0.34–1.86)0.69 (0.28–1.72)The interaction
between mEH and GSTM1 genes is shown in Table5. No significant effect was
detected for the interaction although the estimated risk was higher for those
subjects with the wild-type mEH gene and the null GSTM1 genotype (OR 1.45; 95%
CI 0.66–3.17). There was
no any apparent effect when both genes were mutated or absent (OR 1.07; 95% CI
0.39–2.92).Table 5
Combination
of polymorphisms mEH and GSTM1 and risk of oropharyngeal cancer.
Combination of genetic polymorphismsCasesControlsOR crudeOR adjustedmEH
(Arg/Arg)/GSTM1 present31481.001.00mEH (Arg/Arg)/GSTM1
absent32401.22 (0.63–2.36)1.45 (0.66–3.17)mEH mutated/GSTM1
present14201.15 (0.50–2.65)1,19 (0.43–3.27)mEH mutated/GSTM1
absent15221.18 (0.52–2.66)1.07 (0.39–2.92).


## 4. Discussion

Even though previous
studies have been undertaken to examine the association between CYP1A1 Ile/Val,
mEH, and GSTM1 null polymorphisms, as well as oral and pharyngeal cancers, few
investigated the modification of risk associated with tobacco and alcohol
consumption, and to our knowledge, this is the first to analyse the gene-gene
interactions between these polymorphisms. Our results support the view that there is no significant association
for any of these polymorphisms in Caucasians but our data suggest that exon 4 mEH (139Arg→His) and GSTM1 null
polymorphism might modify the risk related to tobacco and alcohol consumption.We observed that the
exon mEH polymorphism 139Arg→His was mutated
in a very similar proportion in cases and controls and the frequencies found (32,3%
in controls versus 31,5% in cases) were consistent with the results of the
previous investigations that assessed the association between this polymorphism
in head and neck tumors in both sexes [32, 37–41]. In these
studies, mutated alleles were present in 29.7–39.8% of the
control population and in 28.9–39% of the cancer
patients. In the present analysis, the estimated risks were not significant for any of the
tobacco or alcohol consumption levels but the results were in agreement with
those of Wenghoefer et al. [37] that showed that the heterozygous allele (His/Arg)
of exon 4 mEH could modulate the risk of head and neck cancer in smokers (OR 0.57;
CI 95% 0.34–0.95). In our
study, the risk was lower in heavy smokers (OR 0.68; 95% CI 0.25–1.86) than in
light smokers (OR 1.08; 95% CI 0.37–3.13) but not
significant, maybe influenced by the small sample size.Enzymaticaly, mEH
catalyzes the hydrolysis of arene, alkene, and
aliphatic epoxides from polycyclic aromatic hydrocarbons and aromatic amines to
trans-dihydrodiols [19]. This reaction is usually regarded as a detoxifying
pathway because the majority of metabolites produced are less reactive and can
be easily excreted, but, in some instances, these initial trans-dihydrodiol
metabolites can be further activated by subsequent P450 catalysis to form
highly carcinogenic electrofilic intermediates that can bind covalently to DNA.
Such is the case of the 7,8-diol-9,10-epoxide, which is more carcinogenic than
the other benzo[a]pyrene diol epoxide formed [42]. Whilst the results of some
studies, including the present one, are compatible with the fact that a high or
intermediate activity might exert a protective effect in subjects exposed to
tobacco products [39, 43, 44], others carried out in various
aerodigestive tract cancers find no association or a significantly higher risk
for smokers with the exon 4 mEH variant allele [32, 36, 38, 39]; reasons for
these inconsistencies are unclear. It could be argued that high activity should
be assessed taking into account both the exon 3 and exon 4 mEH polymorphisms.
Some authors predicted mEH activity as
low, intermediate, or high based on the presence or absence of the two
polymorphisms but the results are also contradictory [37, 38, 40, 41, 45, 46]. The
study undertaken by Wenghoefer et al. [37] found an association for the single
genotypes in head and neck cancers but not the combination genotypes, raising
uncertainties in categorizing enzymes. Given the
dual role of mEH on the bioactivation/detoxification of carcinogens, it is
highly probable that other polymorphisms might influence the formation of
carcinogenic metabolites and that gene-gene interactions might exist. In this
investigation, we did not find a significant interaction between the GSTM1 and exon
4 mEH polymorphism.The frequency of the
GSTM1 null allele polymorphisms in oral and pharyngeal
cancers has been reported to vary greatly depending on the geographical regions
[47, 48]. Whilst in Europe and USA
the frequencies reported in control populations range from around 49–55.6% [24–32] in Asian and
South American countries, this allele is frequently present in less than 49% of
the control subjects [49–60]. In the
present study, 47.7% of the control population presented the GSTM1 null
polymorphism. This is slightly lower than the values found in other Caucasian
studies and could be partly due to the fact that only males were included. Our
study, like other previous reports on Caucasians [24–26, 28, 29, 30, 31, 32], failed to
find a significant association between the GSTM1 null polymorphism and oral and
pharyngeal cancers.
Several studies undertaken in Asian populations showed contradictory results [49, 56–60]. In a very
recent meta- and pooled analysis, it was observed that the GSTM1 null
polymorphism was significantly associated with risk in Asian and
African-American populations in the meta- (OR 1.5; 95% CI 1.3–1.8) and pooled
(adjusted OR 2.4, 95% CI 1.1–5.5) analysis,
respectively, but not in Caucasians [48]. This discrepancy might be attributed to differences in
lifestyle, environmental risk factors, and variations in the activity of other metabolizing
enzymes, which often displays
genetic polymorphisms that might differ in Caucasians and in other ethnic
groups [11, 41, 59, 61].We did not find a
significant interaction between the GSTM1 null polymorphism and tobacco smoking,
but we did observe that the risk was slightly higher in heavy smokers than in
light/moderate smokers. To verify that these differences were not due to the
fact that nonsmokers were analysed together with light/moderate smokers, we
carried out a separate analysis leaving out these subjects and found that the
variations in the odds ratios were minimum (data not shown). It has been hypothesised
that lack of GSTM1 enzyme activity increases cancer susceptibility as a result
of a decreased ability to detoxify reactive intermediates of tobacco
carcinogens such as benzo[a]pyrene-7,8-diol epoxide, the activated form of
benzo[a]pyrene [22] but the results of previous studies undertaken in oral and
pharyngeal cancers are inconsistent [26, 54, 56, 57]. Some authors find a lower
difference in risk among the genotypes at high dose levels and suggest a
dose-response relationship of the enzymatic reaction [48, 57]. In the present
study, the relationship between tobacco exposure and these polymorphisms is
difficult to assess because there were only 2 nonsmokers among the cases and 6 subjects
that smoked for less than 20 years, forcing us to create a category that was
light/moderate smokers. Studies with larger number of patients are needed to
properly assess this dose response relationship taking into account that ethnic
and geographical differences might exist due to the different forms of tobacco
consumption and diet intake. In Asian and South American
countries, tobacco is usually smoked as “bidis” and the carcinogenic substances
found in this form of preparation are different to that in cigarettes, implying
that other enzymes different than
GSTM1 and CYP1A1 might be involved in the detoxification [28].Our study suggests
that the effect of the GSTM1 null polymorphism is more noticeable among light
drinkers, although this association was nonsignificant. This relationship was
maintained when we took out the nondrinkers from the analysis (data not shown).
This differential effect could be explained by the fact that the GSTM1
isoenzyme, together with the alcohol dehydrogenase, is involved in the
oxidation of ethanol to acetaldehyde [30]. Even though the exact mechanisms by
which ethanol may exert an influence in oral cancer is still unknown, it has
been suggested that acetaldehyde, a known carcinogenic agent, [62] could be
responsible for some DNA changes that could lead to cancer [63]. Individuals
with a null GSTM1 and high drinkers would not convert ethanol in acetaldehyde
and then the absence of the gene would confer them a protective effect for
oropharyngeal cancer. Another explanation for these finding could be that
ethanol increases the permeability of tobacco carcinogens, such as
nitrosonornicotine, across the oral membrane when it is present at low
concentrations. At concentrations higher than 50%, no further permeabilization
is noted, probably due to the fixative effect of ethanol on the mucosa [64]. It
should be highlighted that the alcohol consumption in the Galician population is
very high, as other studies have reported [65], and this is a limitation that does
not allow for a proper analysis of the interaction.Even though the
CYP1A1 mutation has been shown to increase microsomal activity for converting
procarcinogens, including PAH aromatic amines, the results of various reports
on smoking-related cancers are inconsistent [11, 32, 47, 66–70]. It has
been suggested that the DNA damage may depend on the link of CYP1A1 to other
polymorphisms that can affect the CYP1A1 transcription levels, such as
polymorphisms for promoter genes, AHR (Ah receptor) genes, or metabolic genes
such as GSTM1 [69, 70]. In our study, only 4 of the analysed subjects showed an
Ile/Val mutation. Although these frequencies are in accordance with those found
by Hahn et al. in a Caucasian population [25], it made it impossible to draw
any conclusion for this polymorphism. Confidence intervals obtained were very
wide and the distribution of this polymorphism was very similar between cases
and controls. In any case, neither the previous meta- and pooled analysis on
CYP1A1 and risk of head and neck cancer [47] nor the recently published meta
and pooled analysis on oral and pharyngeal cancer found
a significant association between  CYP1A1
(Ile/Val) polymorphism and oral and pharyngeal cancer [48].This paper has
several limitations. The main one is the small sample size included. This is especially
important when analyzing polymorphisms that have a very low frequency in the
population, such as CYP1A1 Ile/Val, but also limits the power to identify
gene-environmental and gene-gene interactions in any polymorphisms
investigated. Nevertheless, it has to be taken into account that for cancers
with a low incidence such as oral and pharyngeal ones, 92 cases can be a
relatively good number. Another limitation could be the fact that our study was
hospital-based and this could result in selection bias. Even though it has been
suggested that studies with hospital controls can provide lower risk estimates,
since diseases of controls could be associated with the polymorphisms under
study, previous meta- and pooled analysis that assessed these polymorphisms on
head and neck cancers found no differences for hospital-based studies in
relation to population-based studies [47, 66]. The fact that not all the sample
was analysed for CYP1A1 polymorphisms limited the power to detect a significant
risk.The present study
has also some advantages. One of them is that the participation of cases and
controls was very high. All the cases belonged to the same catchment area,
which has a unique reference hospital, and were collected consecutively, making
them representative of the cases in that area. The controls also belonged to
the same area and did not have any symptom or disease related to alcohol or tobacco
consumption. Another advantage is the fact that the genes analysed here were of
phase I and phase II, which adds value to the results obtained. When studying
susceptibility genes in many occasions, both types of genes are not studied and
individuals can have susceptibility in type I compensated with the activity of the other type such
as it was shown in  Park et al.
investigation [29].As a conclusion, it
seems that even though none of the three genotypes analysed have a significant
association with the risk of oral and pharyngeal cancer in this population, the
risks of tobacco and alcohol consumption might be modified by GSTM1 null and
exon 4 mEH polymorphisms. The small number of nonsmoking and nondrinking subjects
in our population limited our analysis, so we propose that further studies are carried
out to clarify this question. Even though we found no significant interaction
between the GSTM1null genotype and the exon 4 mEH genotype, the GSTM1 absence
did show a slight rise in risk so this should also be investigated taking into
account other polymorphisms including the CYP1A1.



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*Source: 741310-2008-09-22.xml*