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Carcinogenesis Advance Access originally published online on October 4, 2007
Carcinogenesis 2007 28(12):2537-2542; doi:10.1093/carcin/bgm222
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Polymorphisms of GSTP1 and GSTT1, but not of CYP2A6, CYP2E1 or GSTM1, modify the risk for esophageal cancer in a western population

A. Rossini1, D.C.M. Rapozo1, S.C. Soares Lima1, D.P. Guimarães2, M.A. Ferreira2, R. Teixeira2, C.D.P. Kruel3, S.G.S. Barros4, N.A. Andreollo5, R. Acatauassú6, H.J. Matos7, R.M. Albano1 and L.F.Ribeiro Pinto1,2,*

1 Departamento de Bioquímica, IBRAG, Universidade do Estado do Rio de Janeiro, RJ 20551-013, Brasil
2 Serviço de Endoscopia Digestiva e Serviço de Pesquisa Clínica e Translacional, Coordenação de Pesquisa, Instituto Nacional de Câncer, Rio de janeiro, CEP: 20231-050, Brasil
3 Departamento de Cirurgia Gástrica, Faculdade de Ciências Médicas-Hospital da Clínicas de Porto Alegre, Rio Grande do Sul, CEP 90035-003, Brasil
4 Serviço de Gastroenterologia, Hospital da Clínicas de Porto Alegre, Rio Grande do Sul, CEP 90035-003, Brasil
5 Departamento de Cirurgia e Gastrocentro, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, CEP 13083-970, Brasil
6 Disciplina de Gastroenterologia, FCM-HUPE, Universidade do Estado do Rio de Janeiro 20551-013
7 Departamento de Tecnologia da Informação e Educação em Saúde, FCM, Universidade do Estado do Rio de Janeiro, RJ 20551-013, Brasil

* To whom correspondence should be addressed. Tel: +55 21 25876428; Fax: +55 21 25876136; Email: frpinto{at}oi.com.br


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Esophageal cancer is among the most common and fatal tumors in the world. Eighty percent of esophageal tumors are esophageal squamous cell carcinoma (ESCC). Brazil is one of the high incidence areas in the West, where tobacco and alcohol consumption have been associated with ESCC. However, polymorphisms in xenobiotic metabolizing genes may also contribute to the risk. Therefore, in this study, we analyzed the risk of ESCC associated with tobacco and alcohol consumption and with polymorphisms of CYP2A6 (CYP2A6*2), CYP2E1 (CYP2E1*5B, CYP2E1*6), GSTP1 (Ile105Val), GSTM1 and GSTT1 null genotypes in 126 cases and 252 age- and gender-matched controls. Data on the amount, length and type of tobacco and alcohol consumed were collected, and DNA was extracted from blood lymphocytes from all individuals. Polymorphisms were analyzed by polymerase chain reaction (PCR)–multiplex (GSTM1 and T1), PCR–Restriction Fragment Length Polymorphism (CYP2E1*5B and *6 and GSTP1 Ile105Val) or allele-specific PCR amplification (CYP2A6*2). Risks were evaluated by multivariate conditional regression analysis. As expected, tobacco [odds ratio (OR) = 6.71, 95% confidence interval (95% CI) 3.08–14.63] and alcohol (OR = 16.98, CI 7.8–36.98) consumption, independently or together (OR = 26.91, CI 13.39–54.05) were risk factors. GSTP1 Ile105Val polymorphism was an independent risk factor (OR = 2.12, CI 1.37–3.29), whereas GSTT1 wild-type was an independent protective factor for ESCC (OR = 0.37, CI 0.16–0.79). There was ~80% statistical power to detect both results. There was no risk associated with CYP2A6, CYP2E1 and GSTM1 polymorphisms. In conclusion, this study suggests an opposite role of GSTP1 and GSTT1 polymorphisms for the risk for ESCC.

Abbreviations: CYP, cytochrome P450; 95% CI, 95% confidence interval; ESCC, esophageal squamous cell carcinoma; GST, glutathione S-transferase; OR, odds ratio; PCR, polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Cancer of the esophagus is the eighth most common cancer in the world, being responsible for ~4% of new cancer cases every year. Although there has been an increase in the incidence of esophageal adenocarcinoma in developed countries in the last decades (1), 80% of esophageal cancer cases still occur in developing countries, predominantly as esophageal squamous cell carcinoma (ESCC) (2). Despite much effort that has been done in improving treatment and diagnosis, esophageal cancer prognosis is still poor, making it the sixth most fatal malignancy in the world (2). Therefore, it is important to understand the mechanisms involved in esophageal cancer so that more effective preventable measures can be implemented.

The geographical variation in incidence for esophageal cancer is probably larger than for any other type of cancer, suggesting the influence of environmental factors (2). In the East and in Africa, the main etiological factors involved have been a diet contaminated with nitrosamines and mycotoxins and deficient in anti-oxidants (3–5). Tobacco smoking and chewing in the Indian subcontinent and tobacco smoking in Transkei have also been shown to increase the risk (6). In the West, the main etiological factors involved are alcohol consumption and tobacco smoking (4,7).

Brazil presents one of the highest incidence areas for this malignancy in the West (2), where esophageal cancer is the fourth most fatal malignancy among men and the sixth among women, being responsible for >7000 deaths each year (8). The high-incidence areas are in the south and in the southeast regions, where 96% of esophageal tumors are ESCC (8).

Cytochrome P450 (CYP) are Phase I enzymes responsible for activating most environmental pre-carcinogens, whereas glutathione S-transferases (GSTs) are Phase II enzymes capable of detoxifying the electrophile carcinogens that result from the action of CYP enzymes. CYP and GST enzymes are highly polymorphic with some of these polymorphisms affecting enzyme expression and/or activity. This difference in the capacity of activating and/or detoxifying environmental carcinogens could result in interindividual genetic differences in cancer susceptibility (9).

We have previously shown that CYP2A6 and CYP2E1, the main enzymes capable of activating nitrosamines and other carcinogens in humans, are expressed in esophageal mucosa of Brazilian patients, with a high degree of variation in expression (10). The CYP2A6 gene is located on chromosome 19 and >30 different alleles have been described (11). The CYP2A6 wild-type allele was termed CYP2A6*1A. There are different alleles that code for proteins that show no activity in vivo (alleles *2, *4 and *5), but only CYP2A6*2 is present at a frequency >1% in the Brazilian population (12,13). The CYP2A6*2 allele has a single point mutation (1799T>A) leading to an amino acid substitution (L160H) resulting in a catalytically inactive enzyme (14,15). The CYP2E1 gene is located in the long arm of chromosome 10, at 10q24.3-qter, and several CYP2E1 polymorphisms have been identified (11). The wild-type allele (CYP2E1*1A) has a RsaI restriction site in the regulatory region of the gene, at –1053, and a restriction site for DraI in exon 6, at 7632. The CYP2E1*5B, detected by resistance to digestion by RsaI, seems to be associated with changes at the transcriptional level (16,17). This allele shows a restriction site for PstI, characterized by a G > C substitution at –1293. A second allele, CYP2E1*6, shows a T > A substitution at 7632 and is not digested by DraI. However, its effect on CYP2E1 activity remains unclear (17,18).

GSTM1 is expressed in the normal esophageal epithelium (19). Although three variant alleles for GSTM1 have been described, only the gene deletion seems to be related to disease susceptibility since GSTM1A and GSTM1B encode proteins with catalytic activities that are similar to the wild-type (20). GSTT1 is also expressed in human esophageal epithelium (21,22). The GSTT1 gene is located at 22q11.23 and its polymorphism is characterized by a deletion of almost the entire gene (23,24), causing the absence of the protein GSTT1 (25). The GSTM1 and GSTT1 deletions may abolish the detoxication of some GSTM1 and GSTT1 substrates that are carcinogens or highly toxic compounds. GSTP1 is the main GST expressed in human esophagus (21,22). The GSTP1 gene is located on the long arm of chromosome 11, at 11q13 (24). GSTP1 is polymorphic and the polymorphisms that have been most widely studied to date are Ile105Val and Ala114Val polymorphisms. Four haplotypes have been identified: the wild-type GSTP1*A (Ile105, Ala114) and three variant haplotypes, GSTP1*B (Val105, Ala114), GSTP1*C (Val105, Val114) and GSTP1*D (Ile105, Val114) (26). The most studied polymorphic variant is Ile105Val, and it encodes a protein with altered catalytic activity (24,27).

Therefore, in this work, we analyzed the risk of Brazilians, a deeply admixed western population, to develop ESCC associated with polymorphisms present in CYP2A6, CYP2E1, GSTM1, GSTP1 and GSTT1 genes.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Case–control study
Cases (126 individuals) were comprised of patients with a histologically confirmed diagnosis of ESCC recruited from four hospitals from south and southeast regions of Brazil: Hospital Universitário Pedro Ernesto (HUPE-UERJ) and Instituto Nacional do Câncer (INCA), both located at Rio de Janeiro, Hospital de Clínicas (HCPA-UFRGS), located at Porto Alegre, Rio Grande do Sul, and Hospital de Clínicas-Gastrocentro (HC-UNICAMP), located at Campinas, São Paulo. Controls (252 individuals) were age- and gender-matched healthy individuals submitted to routine examination, not related to cancer, at the same hospitals and were representative of the population assisted in the hospitals studied. Cases and controls were matched taking a 5 ± 2-year interval and were recruited between September 2000 and November 2006. All individuals who took part in this study signed an informed consent and information was obtained by a standardized questionnaire, including data on tobacco smoking and alcohol drinking. Data on tobacco smoking were obtained on the amount of cigarettes smoked and the duration of the habit, and individuals were classified as never- or ever smokers (defined as smoking at least one cigarette per day and persisting for >1 year). Individuals were classified regarding alcohol intake as never- or ever drinkers (defined as drinking alcoholic beverages at least twice a week and persisting for >1 year), and data were also collected about the type of alcoholic beverage regularly consumed. Cases and controls were interviewed by the same group. Ethnic classification was assessed on skin color (white and non-white, with the latter being further classified as mulattoes and blacks). After the interview, all individuals who took part in this study donated 10 ml of peripheral blood collected in ethylenediaminetetraacetic acid-containing tubes and stored at –20°C. The study proposal and all ethical proceedings were approved by the Ethic Committees of the four hospitals.

Genotyping analysis
DNA was extracted from blood lymphocytes by proteinase K/sodium dodecyl sulfate digestion as described elsewhere (28,29). Genotyping for the CYP2A6*2 allele was performed as described by Oscarson et al. (30). The genotyping assays for the CYP2E1 polymorphisms were performed by polymerase chain reaction (PCR)–Restriction Fragment Length Polymorphism analysis. The allele CYP2E1*5B was analyzed as described by Hayashi et al. (17) with modifications. After initial denaturation at 94°C for 4 min, amplification was carried out for 35 cycles at 94°C for 60 s, 53°C for 80 s, 72°C for 90 s, followed by a final elongation at 72°C for 5 min. Ten microliters of the PCR product (410 bp) were incubated with 2.5 U of RsaI (Life Technologies, São Paulo, SP) and 8 µl were incubated with 3.0 U of PstI (Life Technologies), and the resulting fragments were separated on a 2.5% agarose gel stained with ethidium bromide. The genotypes were classified as CYP2E1*1A/1A (RsaI+: 360 and 50 bp and PstI–: 410 bp), CYP2E1*5B/5B (RsaI–: 410 bp and PstI+: 290 and 120 bp) or CYP2E1*1A/5B (RsaI+/–: 410, 360 and 50 bp and PstI+/–: 410, 290 and 120 bp). For the analysis of allele CYP2E1*6, the PCR conditions were those described previously (13). The amplicon (10 µl) was incubated with 2.5 U DraI (Life Technologies), and the products were separated on a 10% polyacrylamide gel and stained with silver (31). The genotypes were classified as CYP2E1*1A/1A (DraI+: 572, 302 and 121 bp), CYP2E1*6/6 (DraI–: 874 and 121 bp) or CYP2E1*1A/6 (DraI+/–: 874, 572, 302 and 121 bp).

The polymorphism characterization of GSTM1 and GSTT1 genes was performed by a PCR–multiplex procedure, with concomitant amplification of GSTM1 and GSTT1 from genomic DNA. The amplification of CYP1A1 was used as an internal control. PCR conditions were the same as described by Abdel-Rahman et al. (32) except for primers (7.5 pmol/25 µl) and DNA (50–100 ng/25 µl) concentrations. Amplification comprised of an initial cycle with denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 2 min, annealing at 59°C for 1 min and extension at 72°C for 1 min. This was followed by a final step at 72°C for 10 min. PCR products were visualized in ethidium bromide-stained 2% agarose gels. The absence of GSTM1- or GSTT1-specific fragments (215 and 480 bp, respectively) indicated the corresponding null genotype, whereas the CYP1A1-specific fragment (312 bp) corresponded to the positive control. The polymorphism of GSTP1 (Ile105Val) was studied by PCR–Restriction Fragment Length Polymorphism (27), with some modifications. Genomic DNA (50–100 ng) was used as a DNA template in 25 µl of total volume reaction. The PCR products were digested in 25 µl for 2 h at 37°C with 5 U Alw261 (MBI Fermentas, Vilnius, Lithuania). The digestion products were separated on a 10% polyacrylamide gel that was silver stained (31). The presence of a 176 bp fragment indicated the wild-type genotype (I/I), whereas 85 and 91 bp fragments indicated the homozygous polymorphic genotype (V/V). Heterozygous individuals had all three types of fragments.

Statistical analysis
Crude odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated to estimate the risk of esophageal cancer associated with the factors studied using the software GraphPad Instat (GraphPad Software, San Diego, CA). The same software was used to test differences between cases and controls related to age and amount of cigarettes consumed. Allele frequencies were calculated and tested for Hardy–Weinberg equilibrium within cases and controls using Genepop Software (Genepop web version 3.4, http://genepop.curtin.edu.au).

The model adopted for analyzing the interaction between demographic, social habits and polymorphisms with the risk of esophageal cancer was the conditional backward stepwise logistic regression model. The dependent variable was the positive diagnosis of esophageal cancer, and the independent variables included presence of polymorphisms, cigarette smoking, alcohol consumption, color of skin, gender and age. This model eliminates in a backward stepwise approach those variables that did not reach a 10% statistically significant level. The final risk was estimated by calculating adjusted OR and 95% CIs. This model was developed and all the ORs were measured by using the software SPSS version 12 (SPSS, Chicago, Illinois). For polymorphism analysis, heterozygous and homozygous mutant genotypes were combined.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
The characteristics of the individuals who took part in this study are shown in Table I. There was no significant difference in the mean age between cases (59.2 ± 10.3, range 33–88) and controls (57.8 ± 13.4, range 28–89). Eighty-three percent of the individuals were men, representing a male to female ratio of 4.7. The gender ratio in cases was higher among white individuals (5.8) than among non-white individuals (3.6). There was a slight prevalence of individuals classified as white among cases (60%) than among controls (56%). Table I also shows the crude OR for the risks associated with skin color, tobacco smoking and alcohol drinking. There was no risk associated with skin color (P = 0.583). Most of the cases (89%) were regular alcohol drinkers (ever drinkers) compared with 40% in the control group (OR = 12.86, 95% CI 6.86–24.10, P < 0.0001). Almost all the cases were also smokers (91%) compared with 55% in the control group (OR = 8.47, 95% CI 4.35–16.52, P < 0.0001). Most of the smokers among cases (89%) and all smokers among controls smoked industrialized tobacco. The vast majority of cases (85%), but not of controls (24%), consumed both tobacco and alcohol (OR = 21.45, 95% CI 8.79–52.32, P < 0.0001).


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  		Table I. Descriptive characteristics of esophageal cancer cases and controls and risk for esophageal cancer associated with tobacco and alcohol consumption

 
Figure 1 shows that cases drank alcohol predominantly (89%) as cachaça (a sugar cane spirit), whereas among the control individuals, those that consumed alcohol did it mostly (75%) by drinking beer.


Figure 1
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  		Fig. 1. Type of alcoholic beverage usually consumed by cases and controls (as percentage of individuals).

 
Table II shows that cases smoked more cigarettes than controls, although this difference is not statistically significant (47.1 ± 32.6 pack-years against 35.2 ± 30.9 pack-years, P = 0.3). Most of cases (71%) but not of controls (30%) were heavy smokers (cigarette consumption > 20 pack-years), and there was a clear dose–response relationship between the amount of cigarettes consumed and the risk associated with esophageal cancer.


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  		Table II. Amount of tobacco consumption (in pack-years of cigarette smoked) among cases and controls and esophageal cancer risk

 
The polymorphisms of CYP2A6 (allele *2), CYP2E1 (alleles *5B and *6) and GSTP1 (Ile105Val) within cases and within controls were all in Hardy–Weinberg equilibrium. The genotype frequencies of CYP2A6 (allele *2), CYP2E1 (alleles *5B and 6), GSTM1 (null genotype), GSTP1 (Ile105Val) and GSTT1 (null genotype) and the respective crude OR for the risk associated with esophageal cancer are shown in Table III. The frequencies of the genotypes of the genes analyzed are similar to those previously seen in other studies (12,30,33–44). The frequency of individuals who carry the CYP2A6*2 allele in heterozygosis or homozygosis was ~2.5-fold higher among cases than among controls, and the presence of this allele resulted in a borderline risk for esophageal cancer (crude OR = 2.79, 95% CI 1.01–7.68, P = 0.055). Although the frequencies of individuals who carried at least one of the polymorphic alleles of CYP2E1 were higher among cases (13 and 17.5% for CYP2E1*5B and for CYP2E1*6, respectively) than among controls (7.3 and 13.1% for CYP2E1*5B and for CYP2E1*6, respectively), this difference did not result in a statistically significant risk (crude OR = 1.91, 95% CI 0.94–3.89, P = 0.085, for CYP2E1*5B and crude OR = 1.4, 95% CI 0.77–2.55, P = 0.274, for CYP2E1*6). The frequency of individuals who carried the GSTM1 null genotype was similar between cases and controls (40%). The polymorphic allele of GSTP1 was present at a higher frequency among cases (66.4%) than controls (54%) and conferred a statistically significant risk to develop esophageal cancer (crude OR = 1.69, 95% CI 1.08–2.63, P = 0.026). The frequency of individuals who had the GSTT1 null genotype among cases (12%) was half of that found among controls (24%), which resulted in a statistically significant protective effect associated with this genotype (crude OR = 0.44, 95% CI 0.24–0.81, P = 0.006).


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  		Table III. CYP and GST polymorphisms and esophageal cancer risk

 
All the results shown above were then analyzed using a conditional backward stepwise logistic regression model, as described in Materials and Methods. Age, gender, drinking and smoking status and CYP2A6, CYP2E1, GSTM1, GSTP1 and GSTT1 polymorphisms were included in the model. Table IV shows that gender (OR = 3.86, 95% CI 1.66–8.98, P = 0.002), alcohol drinking (OR = 16.98, 95% CI 7.8–36.98, P < 0.001) and tobacco smoking (OR = 6.71, 95% CI 3.08–14.63, P < 0.001) remained as statistically significant independent risk factors. Although not shown in Table IV, the interaction between tobacco smoking and alcohol drinking resulted in a higher risk (OR = 26.91, 95% CI 13.39–54.05, P < 0.001) than the addition of the individual risks. Table IV also shows that among the polymorphisms, only the polymorphism of GSTP1 remained as an independent risk factor (OR = 2.12, 95% CI 1.37–3.29, P = 0.015), whereas the GSTT1 null genotype also remained as an independent protective factor for esophageal cancer (OR = 0.37, 95% CI 0.16–0.83, P = 0.001).


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  		Table IV. Multivariate conditional logistic regression analyses on risk factors for esophageal cancer

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Esophageal cancer presents a marked variation in incidence rates around the world, suggesting a strong influence of etiological factors. In the West, where the main etiological factors associated with ESCC are the consumption of alcohol and tobacco (7,45), the highest incidence rates are seen in certain areas of Europe and South America (2).

However, polymorphisms in genes that code for drug-metabolizing enzymes may alter the risk associated with environmental factors (9). Therefore, in this study, we analyzed the risk to develop ESCC presented by tobacco smoking and alcohol consumption and also with polymorphisms present in CYP2A6 (allele *2), CYP2E1 (alleles *5B and *6), GSTM1 (null genotype), GSTP1 (Ile105Val) and GSTT1 (null genotype). The genes chosen are all expressed in the human esophagus and are involved in the biotransformation of environmental carcinogens. The polymorphisms analyzed were chosen based on the phenotype consequence and also for being at a frequency in the Brazilian population >1% (13,46).

The Brazilian population is vastly admixed, being composed mainly by people from European (Portuguese), African and South Amerindian origin. Mitochondrial DNA and Y chromosome analysis have shown that the Brazilian population results from the mixture of Portuguese men with African and South Amerindian women (47). Therefore, ethnic classification of Brazilian individuals is very difficult, if not impossible, and can result in spurious results as demonstrated in pharmacogenetic analysis (48). So, the risk associated with the polymorphisms was analyzed in the population as a whole, to avoid bias due to population stratification, and with the exception of GSTM1, their frequencies are not different between individuals from European or African origin. Nevertheless, the percentage of individuals classified as white in cases (60%) and controls (56%) is similar. The polymorphisms of CYP2A6 (allele *2), CYP2E1 (alleles *5B and *6) and GSTP1 (Ile105Val) were all in Hardy–Weinberg equilibrium, and the frequencies of all the polymorphisms among controls are in accordance to those previously seen in a larger number of individuals in our population (13,46).

Consistent with previous studies (6,7), the present study shows that consumption of alcohol (OR = 16.98, 95% CI 7.8–36.98, P < 0.001) and tobacco (OR = 6.71, 95% CI 3.08–14.63, P < 0.001) was independent significant risk factors for ESCC in Brazilian patients. Although we did not collect data on the amount of alcohol consumed, most of the cases, but not of controls, drunk cachaça, a sugar cane spirit that has a high concentration (~42%) of ethanol. This agrees with previous studies (7,49) that the risk is associated with heavy consumption of alcohol, particularly in the form of strong spirits. The mean consumption of cigarettes among cases was ~30% higher than that of controls, but this difference was not statistically significant (P = 0.3). Nevertheless, most of cases (71%) but not of controls (30%) were heavy smokers (cigarette consumption > 20 pack-years). The vast majority of cases (85%) were regular consumers of tobacco and alcohol (ever smokers and ever drinkers) (OR = 26.91, 95% CI 13.39–54.05, P < 0.001), whereas among controls, most of them were only ever smokers (31%) or did not have either habit (29%).

There was a risk to develop ESCC associated with GSTP1 polymorphism (crude OR = 1.69, 95% CI 1.08–2.63, P = 0.026), and this study had an almost 80% power to detect this risk. The risk remained statistically significant after adjusting for the variables analyzed in this study (OR = 2.12, 95% CI 1.37–3.29, P = 0.015). Although we also detected a statistically significant risk (OR = 2.22, 95% CI 1.4–3.53, P = 0.001) for the interaction of GSTP1 polymorphism with tobacco smoking, this study does not have sufficient statistic power to detect gene–environment interactions, and therefore further studies are necessary to analyze this association. Nevertheless, since the polymorphic GSTP1 possesses a decreased catalytic activity (21,50,51), patients carrying the polymorphic GSTP1 could have a diminished capacity to detoxify carcinogens. There have been 11 studies that analyzed the association between GSTP1 polymorphism with ESCC, and in only three there were >100 cases, all of them from China (52–54). None of these three studies detected a risk of ESCC associated with GSTP1 polymorphism, but the main etiological factors in China are different from those seen in western countries. There have also been three studies done with Caucasians, and none of them had >50 patients. Nevertheless, one (50) detected a significant risk, another (55) detected a risk, but it was not statistically significant, and the third one (56) did not detect any risk.

The GSTT1 null genotype was an independent protective factor for ESCC (OR = 0.37, 95% CI 0.16–0.83, P = 0.001), and this study had a 90% power to detect this association. There have been previous reports of the protective effect of GSTT1 null genotype for different tumors such as lung (57), breast (58) and bladder (59). A recent study in India showed that the GSTT1 null genotype was a protective factor for oral cancer, particularly in those that consumed tobacco (60). Recently, Garte et al. (61) showed that individuals who carried the GSTT1 null, but not the GSTM1 null genotype, had significantly lower levels of 8-oxoguanine when compared with their wild-type counterparts. 8-Oxoguanine is the main pre-mutagenic base formed from oxidative species, and the lower levels of 8-oxoguanine associated with GSTT1 null genotype were correlated with the exposure to specific polycyclic aromatic hydrocarbons, such as benz[a]anthracene and chrysene, but not to benzo[a]pyrene. Furthermore, individuals who had the GSTT1 null genotype were more resistant to DNA damage caused by polycyclic aromatic hydrocarbons than the wild-type GSTT1 individuals (61).

Although there seemed to be a risk associated with CYP2A6*2, this became non-significant after adjusting for all the other variables analyzed in this study. This result agrees with the only other study that analyzed an association between polymorphism of CYP2A6 and ESCC and that did not detect any risk (54). The CYP2A6*2 allele leads to an enzyme without activity (14,15), and therefore, individuals who carried this polymorphism could have a diminished capacity to activate nitrosamines in their esophagus. However, we have previously shown that patients that present a high expression of CYP2A6 in their esophagus can activate N-nitrosodiethylamine at rates comparable with the esophagus of the rat (10), which is the most susceptible species to esophageal tumor induction by nitrosamines. This susceptibility is due to the high capacity of the rat esophagus to activate nitrosamines, a result of the expression of CYP2A3 in this tissue (62). Therefore, if the CYP2A6*2 allele presents no change of risk for ESCC, this would imply that tissue-specific activation of nitrosamines would not be important for ESCC in humans. However, to detect an OR of 2.79 with a statistic power of 80%, we would need about twice as many cases and controls than were analyzed in this study, and therefore, we are not able to determine at the moment the significance of the expression of CYP2A6 in human esophagus for ESCC.

There was no risk associated with either CYP2E1*5B or CYP2E1*6. There are other nine studies that analyzed the risk of ESCC associate with CYP2E1 polymorphisms, and most of them (eight) were done with Asians (who possess a higher frequency of these polymorphisms when compared with individuals of European origin) and analyzed the CYP2E1*5B allele. In only three of them, a significant risk was detected, and this was associated with the wild-type genotype (52,63). A recent study carried out in South Africa detected a risk associated with the CYP2E1*6 allele (64). The phenotype consequence of CYP2E1*5 is controversial since some studies related that it results in an increased transcription of the gene (16,17), whereas others related that it results in an enzyme with decreased activity (65,66). CYP2E1 is expressed in the esophagus of most individuals, with a several-fold variation in its messenger RNA and apo-protein expression (10,67,68), but it is not clear if this is due to polymorphisms in the CYP2E1 gene or to exposure to environmental compounds since CYP2E1 can be induced by several compounds, including ethanol (3).

There was also no risk associated with the GSTM1 null genotype. There are other 16 published studies that analyzed this association, and in only three of them (63,69,70), all done in China, there was a risk associated with the GSTM1 null genotype.

In conclusion, this study shows that the GSTP1 Ile105Val polymorphism confers a risk to develop ESCC, whereas individuals who carry the GSTT1 null genotype seem to have a lower risk to develop this cancer.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
CNPq: 473898/2004-7; Faperj: E26/170.561/2004.


   Acknowledgments
 
Dr L.F.R.P. and Dr R.M.A. are CNPq research fellows. We are thankful to Prof. Peter F. Swann for reviewing the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Blot WJ, et al. Continuing climb in rates of esophageal adenocarcinoma: an update. JAMA (1993) 270:1320.[Abstract/Free Full Text]
  2. Parkin DM, et al. Global cancer statistics. CA Cancer J. Clin. (2005) 55:74–108.[Abstract/Free Full Text]
  3. Yang CS. Research on esophageal cancer in China: a review. Cancer Res. (1980) 40:2633–2644.[Abstract/Free Full Text]
  4. Craddock VM. Cancer of the Esophagus. Approaches to the Etiology (1993) Cambridge, UK: Cambridge University Press.
  5. Dutton MF. Fumonisins, mycotoxins of increasing importance: their nature and their effects. Pharmacol. Ther. (1996) 70:137–161.[CrossRef][Web of Science][Medline]
  6. Parkin DM, et al. Cancer burden in the year 2000. The global picture. Eur. J. Cancer (2001) 37:S4–S66.[Web of Science][Medline]
  7. Castellsagué X, et al. Independent and joint effects of tobacco smoking and alcohol drinking on the risk of esophageal cancer in men and women. Int. J. Cancer (1999) 82:657–664.[CrossRef][Web of Science][Medline]
  8. Instituto Nacional de Câncer (INCA). Estimativas da Incidência e Mortalidade por Câncer no Ministério da Saúde, Brasil (2006) http://www.inca.gov.br, (14 May 2007 date last accessed).
  9. Nerbert DW, et al. The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis. Nat. Rev. Cancer (2006) 6:947–960.[CrossRef][Web of Science][Medline]
  10. Godoy W, et al. CYP2A6/2A7 and CYP2E1 expression in human oesophageal mucosa: regional and inter-individual variation in expression and relevance to mitrosamine metabolism. Carcinogenesis (2002) 23:611–616.[Abstract/Free Full Text]
  11. Ingelman-Sundberg M, Daly AK, Nebert W, eds. CYP Alleles Homepage. http://www.imm.ki.se/CYPalleles. (10 May 2007 date last accessed).
  12. Vasconcelos GM, et al. CYP2A6 genetic polymorphisms and correlation with smoking status in Brazilians. Pharmacogenomics J. (2005) 5:42–48.[CrossRef][Web of Science][Medline]
  13. Rossini A, et al. CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro. Braz. J. Med. Biol. Res. (2006) 39:195–201.[Web of Science][Medline]
  14. Haddidi H, et al. A single amino acid substitution (Leu160His) in cytochrome P450 CYP2A6 causes switching from 7-hydroxylation to 3-hydroxylation of coumarin. Food Chem. Toxicol. (1997) 35:903–907.[CrossRef][Web of Science][Medline]
  15. Oscarson M, et al. Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase. FEBS Lett. (1998) 438:201–205.[CrossRef][Web of Science][Medline]
  16. Watanabe J, et al. PstI and RsaI RFLPs in complete linkage disequilibrium at the CYP2E gene. Nucleic Acids Res. (1990) 18:7194.[Free Full Text]
  17. Hayashi S, et al. Genetic polymorphisms in the 5'-flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene. J. Biochem. (1991) 110:559–565.[Abstract/Free Full Text]
  18. Persson I, et al. Genetic polymorphism of cytochrome P4502E1 in a Swedish population. Relationship to incidence of lung cancer. FEBS Lett. (1993) 319:207–211.[CrossRef][Web of Science][Medline]
  19. Cobbe SC, et al. Alteration of glutathione S-transferase levels in Barrett's metaplasia compared to normal oesophageal epithelium. Eur. J. Gastroenterol. Hepatol. (2003) 15:41–47.[CrossRef][Web of Science][Medline]
  20. Strange RC, et al. Glutathione S-transferase: genetics and role in toxicology. Toxicol. Lett. (2000) 112–113:357–363.
  21. Peters WHM, et al. Glutathione and glutathione S-transferases in Barrett's epithelium. Br. J. Cancer (1993) 67:1413–1417.[Web of Science][Medline]
  22. De Bruin WCC, et al. Expression of glutathione S-transferase P1-1 and T1-1 in the human gastrointestinal tract. Jpn. J. Cancer Res. (2000) 91:310–316.[CrossRef][Web of Science]
  23. Kempkes M, et al. Comparative genotyping and phenotyping of glutathione S-transferase GSTT1. Arch. Toxicol. (1996) 70:306–309.[CrossRef][Web of Science][Medline]
  24. Strange RC, et al. Glutathione-S-transferase family of enzymes. Mutat. Res. (2001) 482:21–26.[Web of Science][Medline]
  25. Landi S. Mammalian class theta GST and differential susceptibility to carcinogens: a review. Mutat. Res. (2000) 463:247–283.[CrossRef][Web of Science][Medline]
  26. Ali-Osman F, et al. Molecular cloning, characterization and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. J. Biol. Chem. (1997) 272:10004–10012.[Abstract/Free Full Text]
  27. Harries LW, et al. Identification of genetic polymorphisms at the glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis (1997) 18:641–644.[Abstract/Free Full Text]
  28. Miller S, et al. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. (1988) 16:1215.[Free Full Text]
  29. Sambrook J, et al. Molecular Cloning - A Laboratory Manual (2001) Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  30. Oscarson M, et al. Identification and characterization of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism. FEBS Lett. (1999) 460:321–327.[CrossRef][Web of Science][Medline]
  31. Sanguinetti CJ, et al. Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques (1994) 17:915–919.
  32. Abdel-Rahman SZ, et al. A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies. Cancer Lett. (1996) 107:229–233.[CrossRef][Web of Science][Medline]
  33. Paschke T, et al. Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African Americans using a new one-step PCR-RFLP genotyping method. Toxicology (2001) 168:259–268.[CrossRef][Web of Science][Medline]
  34. Bourian M, et al. Genetic polymorphism of CYP2A6 in the German population. Toxicology (2000) 144:129–137.[CrossRef][Web of Science][Medline]
  35. Burim RV, et al. Polymorphisms in glutathione S-transferases GSTM1, GSTT1 and GSTP1 and cytochromes P450 CYP2E1 and CYP1A1 and susceptibility to cirrhosis or pancreatitis in alcoholics. Mutagenesis (2004) 19:291–298.[Abstract/Free Full Text]
  36. Nishimoto IN, et al. Cytochrome P450 2E1 polymorphism in gastric cancer in Brazil: case-control studies of Japanese Brazilians and non-Japanese Brazilians. Cancer Epidemiol. Biomarkers Prev. (2000) 9:675–680.[Abstract/Free Full Text]
  37. Stephens EA, et al. Ethnic variation in the CYP2E1 gene: polymorphism analysis in 695 African-Americans, European-Americans and Taiwanese. Pharmacogenetics (1994) 4:185–192.[Web of Science][Medline]
  38. Neuhaus T, et al. Association of cytochrome P450 2E1 polymorphisms and head and neck squamous cell cancer. Toxicol. Lett. (2004) 151:273–282.[CrossRef][Web of Science][Medline]
  39. Arruda VR, et al. Prevalence of homozygosity for the deleted alleles of glutathione S-transferase mu (GSTM1) and theta (GSTT1) among distinct ethnic groups from Brazil: relevance to environmental carcinogenesis? Clin. Genet. (1998) 54:210–214.[Web of Science][Medline]
  40. Bailey LR, et al. Breast cancer risk and CYP1A1, GSTM1, and GSTT1 polymorphisms: evidence of a lack of association in Caucasians and African Americans. Cancer Res. (1998) 58:65–70.[Abstract/Free Full Text]
  41. Hatagima A, et al. Glutathione S-transferase M1 (GSTM1) polymorphism in two Brazilian populations. Genet. Mol. Biol. (2000) 23:709–713.
  42. Watson MA, et al. Human glutathione S-transferase P1 polymorphism: relationship to lung tissue enzyme activity and population frequency distribution. Carcinogenesis (1998) 19:275–280.[Abstract/Free Full Text]
  43. Zhang H, et al. Glutathione S-transferase Ti and Mi genotypes in normal mucosa, transitional mucosa and colorectal adenocarcinoma. Int. J. Cancer (1999) 84:135–138.[CrossRef][Web of Science][Medline]
  44. Hatagima A, et al. Glutathione S-transferase polymorphisms and oral cancer: a case-control study in Rio de Janeiro, Brazil. Oral Oncol. (2007) in press.
  45. Launoy G, et al. Oesophageal cancer in France: potential importance of hot alcoholic drinks. Int. J. Cancer (1997) 71:917–923.[CrossRef][Web of Science][Medline]
  46. Rossini A, et al. Frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms in a Brazilian population. Genet. Mol. Res. (2002) 1:233–240.[Medline]
  47. Carvalho-Silva D, et al. The phylogeography of Brazilian Y chromosome. Am. J. Hum. Genet. (2001) 68:281–286.[CrossRef][Web of Science][Medline]
  48. Suarez-Kurtz G. Pharmacogenomics in admixed populations. Trends Pharmacol. Sci. (2005) 26:196–201.[CrossRef][Medline]
  49. Tuyns AJ. Epidemiology of alcohol and cancer. Cancer Res. (1979) 39:2840–2843.[Abstract/Free Full Text]
  50. Van Lieshout EMM, et al. Polymorphic expression of the glutathione S-transferase P1 gene and its susceptibility to Barrett's esophagus and esophageal carcinoma. Cancer Res. (1999) 59:586–589.[Abstract/Free Full Text]
  51. Tijhuis MJ, et al. Glutathione S-transferase phenotypes in relation to genetic variation and fruit and vegetable consumption in an endoscopy-based population. Carcinogenesis (2007) 28:848–857.[Abstract/Free Full Text]
  52. Tan W, et al. Impact of genetic polymorphisms in cytochrome P450 2E1 and glutathione S-transferases M1, T1, and P1 on susceptibility to esophageal cancer among high-risk individuals in China. Cancer Epidemiol. Biomarkers Prev. (2000) 9:551–556.[Abstract/Free Full Text]
  53. Cai L, et al. Dietary selenium intake and genetic polymorphisms of GSTP1 and p53 genes on the risk of esophageal squamous cell carcinoma. Cancer Epidemiol. Biomarkers Prev. (2006) 15:294–300.[Abstract/Free Full Text]
  54. Wang Z, et al. Etiological study of esophageal squamous cell carcinoma in an endemic region: a population-based case control study in Huaian, China. BMC Cancer (2006) 6:287–295.[CrossRef][Medline]
  55. Casson AG, et al. Associations between genetic polymorphisms of phase I and II metabolizing enzymes, p53 and susceptibility to esophageal adenocarcinoma. Cancer Detect. Prev. (2003) 27:139–146.[CrossRef][Web of Science][Medline]
  56. Abbas A, et al. GSTM1, GSTT1, GSTP1 and CYP1A1 genetic polymorphisms and susceptibility to esophageal cancer in a French population: different pattern of squamous cell carcinoma and adenocarcinoma. World J. Gastroenterol. (2004) 10:3389–3393.[Medline]
  57. Raimondi S, et al. Meta- and pooled analysis of GSTT1 and lung cancer: a HuGE-GSEC review. Am. J. Epidemiol. (2006) 164:1027–1042.[Abstract/Free Full Text]
  58. Garcia-Closas M, et al. Glutathione S-transferase mu and theta polymorphisms and breast cancer susceptibility. J. Natl Cancer Inst. (1999) 91:1960–1964.[Abstract/Free Full Text]
  59. Kim WJ, et al. GSTT1-null genotype is a protective factor against bladder cancer. Urology (2002) 60:913–918.[CrossRef][Web of Science][Medline]
  60. Devasena A, et al. Susceptibility to oral cancer by genetic polymorphisms at CYP1A1, GSTM1 and GSTT1 loci among Indians: tobacco exposure as a risk modulator. Carcinogenesis (2007) 28:1455–1462.[Abstract/Free Full Text]
  61. Garte S, et al. Role of GSTT1 deletion in oxidative damage by exposure to polycyclic aromatic hydrocarbons in humans. Int. J. Cancer (2007) 120:2499–2503.[CrossRef][Web of Science][Medline]
  62. Luis F, et al. Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis. Carcinogenesis (2001) 22:1877–1883.[Abstract/Free Full Text]
  63. Lu X-M, et al. Relationship between genetic polymorphisms of metabolizing enzymes CYP2E1, GSTM1 and Kazakh's esophageal squamous cancer in Xinjiang, China. World J. Gastroenterol. (2005) 11:3651–3654.[Medline]
  64. Li D, et al. Association of cytochrome P450 2E1 genetic polymorphisms with squamous cell carcinoma of the oesophagus. Clin. Chem. Lab. Med. (2005) 43:370–375.[CrossRef][Web of Science][Medline]
  65. Le Marchand L, et al. Associations of CYP1A1, GSTM1, and CYP2E1 polymorphisms with lung cancer suggest cell type specificities to tobacco carcinogens. Cancer Res. (1998) 58:4858–4863.[Abstract/Free Full Text]
  66. Huang YS, et al. Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug induced hepatitis. Hepatology (2003) 37:924–930.[CrossRef][Web of Science][Medline]
  67. Smith TJ, et al. Characterization of xenobiotic-metabolizing enzymes and nitrosamine metabolism in the human esophagus. Carcinogenesis (1998) 19:667–672.[Abstract/Free Full Text]
  68. Lechevrel M, et al. Characterization of cytochrome P450 expression in human oesophageal mucosa. Carcinogenesis (1999) 20:243–248.[Abstract/Free Full Text]
  69. Roth MJ, et al. Association between GSTM1*0 and squamous dysplasia of the esophagus in the high risk region of Linxian, China. Cancer Lett. (2000) 156:73–81.[CrossRef][Web of Science][Medline]
  70. Gao C, et al. Interaction between cytochrome P-450 2E1 polymorphisms and environmental factors with risk of esophageal and stomach cancers in Chinese. Cancer Epidemiol. Biomarkers Prev. (2002) 11:29–34.[Abstract/Free Full Text]
Received June 18, 2007; revised August 22, 2007; accepted September 7, 2007.


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