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Carcinogenesis Advance Access originally published online on July 7, 2007
Carcinogenesis 2007 28(9):1960-1964; doi:10.1093/carcin/bgm151
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Glutathione S-transferase polymorphisms, cruciferous vegetable intake and cancer risk in the Central and Eastern European Kidney Cancer Study

L.E. Moore*, P. Brennan1, S. Karami, R.J. Hung1,2, C. Hsu1, P. Boffetta1, J. Toro, D. Zaridze3, V. Janout4, V. Bencko5, M. Navratilova6, N. Szeszenia-Dabrowska7, D. Mates8, A. Mukeria3, I. Holcatova, R. Welch9, S. Chanock9, N. Rothman and W.-H. Chow

Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA
1 International Agency for Research on Cancer, Lyon 69008, France
2 University of California, School of Public Health, Berkeley, CA 94720, USA
3 Institute of Carcinogenesis, Cancer Research Centre, Moscow 115478, Russia
4 Department of Preventive Medicine, Faculty of Medicine, Palacky University, Olomouc 77515, Czech Republic
5 Institute of Hygiene and Epidemiology, First Faculty of Medicine, Charles University, Prague 12800, Czech Republic
6 Department of Cancer Epidemiology and Genetics, Masaryk Memorial Cancer Institute, Brno 65200, Czech Republic
7 Department of Epidemiology, Institute of Occupational Medicine, Lodz 90–950, Poland
8 Institute of Public Health, Bucharest 76256, Romania
9 Core Genotyping Facility at the Advanced Technology Center of the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Gaithersburg, MD 20877, USA

* To whom correspondence should be addressed. Tel: +301 496 6427; Fax: +301 402 1819; Email: moorele{at}mail.nih.gov


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
High consumption of cruciferous vegetables has been associated with reduced kidney cancer risk in many studies. Isothiocyanates, thought to be responsible for the chemopreventive properties of this food group, are conjugated to glutathione by glutathione S-transferases (GSTs) before urinary excretion. Modification of this relationship by host genetic factors is unknown. We investigated cruciferous vegetable intake in 1097 cases and 1555 controls enrolled in a multicentric case–control study from the Czech Republic, Poland, Romania and Russia. To assess possible gene–diet interactions, genotyped cases (N = 925) and controls (N = 1247) for selected functional or non-synonymous polymorphisms including the GSTM1 deletion, GSTM3 3 bp deletion (IVS6 + 22-AGG) and V224I G>A substitution, GSTT1 deletion and the GSTP1 I105V A>G substitution. The odds ratio (OR) for low (less than once per month) versus high (at least once per week) intake of cruciferous vegetables was 1.29 [95% confidence interval (CI): 1.02–1.62; P-trend = 0.03]. When low intake of cruciferous vegetables (less than once per month) was stratified by GST genotype, higher kidney cancer risks were observed among individuals with the GSTT1 null (OR = 1.86; 95% CI: 1.07–3.23; P-interaction = 0.05) or with both GSTM1/T1 null genotypes (OR = 2.49; 95% CI: 1.08–5.77; P-interaction = 0.05). These data provide additional evidence for the role of cruciferous vegetables in cancer prevention among individuals with common, functional genetic polymorphisms.

Abbreviations: CI, confidence interval; GST, glutathione S-transferase; ITC, isothiocyanate; OR, odds ratios; RCC, renal cell carcinoma


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
High consumption of cruciferous vegetables has been associated with reduced kidney cancer risks in several but not all studies (1). Host genetic factors may have contributed to the diverse results in different populations, but have not been assessed in epidemiologic studies.

Cruciferous vegetables (Brassica) are rich in glucosinolates which undergo hydrolysis to form isothiocyanates (ITCs), thought to be responsible for the chemopreventative properties of this food group (2). Consumption of Brassica enhances glutathione S-transferase (GST) activity in humans via the antioxidant response element by inducing expression of metabolic and glutathione synthesis genes. Glutathione transferases, most notably GSTM1 and GSTT1, catalyze the conjugation of glutathione to ITCs before excretion by the kidneys (3). Polymorphic variants of the GSTM1 and GSTT1 genes include a deleted or ‘null’ allele. Individuals that are homozygous for the null allele do not produce an active enzyme (4,5). Therefore, individuals who are homozygous null for either or both genes accumulate higher ITC levels in the blood because the efficacy of ITC conjugation and excretion is reduced (6). The resulting longer half-life in blood and tissues allows circulating ITCs additional time to induce genes to improve xenobiotic metabolism (7). Support for this mechanism has been demonstrated in animal and human studies (8).

In one of the largest renal cell cancer studies with biological samples to date, we sought to determine whether polymorphisms in GST genes could modify associations between cruciferous vegetable intake and renal cell cancer risk (RCC). We hypothesized that inverse risks associated with consumption of cruciferous vegetables would be greater among individuals with genotypes that result in enzymes with lower functional activity.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
The Central and Eastern European Renal Cell Cancer Study was a hospital-based case–control study conducted in seven centers (Moscow, Russia; Bucharest, Romania; Lodz, Poland and Prague, Olomouc, Ceske Budejovice and Brno, Czech Republic) coordinated jointly by the US National Cancer Institute (NCI) and the International Agency for Research on Cancer (IARC). Cases were patients newly diagnosed with histologically confirmed renal cell carcinoma (RCC) (IDC-O-2 codes C64) between the ages of 20 and 79 years from August 1999 through January 2003. Trained medical staff reviewed hospital records to extract information on date and method of diagnosis, histologic classification, tumor location, stage and grade. Eligible controls were patients admitted to the same hospital as cases during the same time period for conditions unrelated to smoking or genitourinary disorders (except for benign prostatic hyperplasia) who were frequency matched to cases on age, sex and study center. No single disease made up more that 20% of the control group. A total of 1097 cases and 1555 controls were interviewed, yielding response rates ranging from 90 to 98.6% for cases and from 90.3 to 96.1% for controls. A subset of 925 cases and 1247 controls provided genomic DNA and were successfully genotyped for the selected genotyping panel. All study subjects provided written informed consent. This study was approved by the institutional review boards of all participating centers.

Interviews were conducted in person by trained personnel using standardized lifestyle and food frequency questionnaires as described previously. Details of the dietary intake assessment can be found elsewhere (9). Briefly, this component of the questionnaire comprised 23 food items, with frequency of consumption assessed for each item [never (0), < once per month (1), less than once per week (2), one to two times per week (3), three to five times per week (4) and daily (5)]. The questionnaire was repeated for two different time periods: the year prior to interview and prior to political and market changes in 1989 (1992 in Russia). A lifetime weighted average of intake for the two time periods was calculated by multiplying the score for each time period by the number of years alive during the time period, summing the time period scores and dividing by the subject's age. Frequencies of intake of related foods were summed to form food groups. The cruciferous vegetable group included Brussels sprouts and broccoli (which were rarely consumed) and cabbage, which was by far the most commonly consumed vegetable. Categories of consumption of food-specific items were as follows: low (less than once per month), medium (at least once per month but less than once per week) and high (at least once per week to daily).

Laboratory procedures
Blood samples were stored at –80°C and shipped to the National Cancer Institute (NCI) on dry ice. DNA was extracted using standard phenol–chloroform extraction. Genotyping was conducted at IARC and at the NCI's Core Genotyping Facility. Methods for all genotype assays can be found on the SNP500 web site (10). DNA from cases and controls were blinded and randomized on polymerase chain reaction plates to avoid any potential bias and duplicate genotyping performed for a randomly selected 5% of the total series for quality control. Polymorphisms were selected based on evidence of functional relevance or those leading to amino acid sequence changes. Call rates were >99% for the GSTM1 deletion, GSTM3 3 bp deletion and GSTT1 deletion assays and 98% for GSTP1 I105V and >96% for GSTM3 V224I assays. Concordance rates were >99% for all genes except for GSTM1 (97.4%). All genes analyzed in controls fell within the expected distributions of Hardy–Weinberg equilibrium.

Statistical analyses
Statistical analyses were performed using STATA, version 8.0 (Stata, College Station, TX). For all genotype frequencies, heterogeneity between countries was tested among controls using the standard Q-test. Frequencies of polymorphisms among controls were also compared with those observed among Caucasians in the SNP500 database. Hardy–Weinberg equilibrium was tested by the goodness of fit {chi}2 test for single-nucleotide polymorphisms in the GSTM3 and GSTP1 genes. For each polymorphism, we estimated odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression models adjusting for (i) sex, age at interview and study center and (ii) sex, age at interview and body mass index as continuous variables, study center, self-reported hypertension (no/yes) and smoking [never/former (having quit for at least 2 years)/current]. The additional variables did not modify ORs by >10% and were not associated with genotypes; therefore, only results from models adjusting for sex, age at interview and study center are presented. Comparison of regression models with and without interaction terms was conducted using a likelihood ratio test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Genotyping data were available for 925 (84.2%) cases and 1247 (80.2%) controls. Subjects without genotyping data were similar with respect to age and known RCC risk factors to those genotyped (data not shown). The majority of participants included in the current analysis were from Moscow and four centers in the Czech Republic (Table I). Cases and controls were comparable in age distribution; however, the proportion of men being genotyped was higher in controls than in cases (P = 0.01). Cases had higher body mass index at interview and prevalence of self-reported hypertension, but their smoking habits whether measured as ever/former/current smokers or pack-years of smoking were similar to that observed among controls. Consistent with results reported previously for the entire study population, individuals who consumed cruciferous vegetables less than once per month had a significantly higher risk of RCC compared with those who consumed them at least once per week after adjusting for center, age and sex (OR = 1.29; 95% CI: 1.02–1.62; P-trend 0.03).


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  		Table I. Frequency distribution of demographic variables among subjects genotyped in the Central and Eastern European Renal Cancer Study

 
None of the GST polymorphisms examined were significantly associated with RCC risk although associations with the GSTM1 null, GSTT1 null and GSTP1 GG polymorphisms were >1.0 (Table II). None of the genotypes modified associations between RCC risk and body mass index, sex, age, smoking or self-reported hypertension (data not shown).


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  		Table II. ORs for kidney cancer by GST genotypes

 
Associations between cruciferous vegetable intake and RCC risk were modified by GSTM1 and GSTT1 genotypes (Table III). Among all subjects, low cruciferous vegetable intake (less than once per month) was associated with increased cancer risk compared with those who consumed them at least once per week (P-trend = 0.03). The excess risk associated with low intake tended to be greater among individuals with null genotypes, and significant interactions were observed for GSTT1 (P-interaction = 0.05), but not for the GSTM1 gene (P-interaction = 0.78). Among GSTT1 null individuals, low cruciferous vegetable consumption was associated with having almost a 2-fold risk of RCC (OR = 1.86; 95% CI: 1.07–3.23). Among those with both GSTM1 and GSTT1 null genotypes, the RCC risk associated with low intake of cruciferous vegetables was further elevated (OR = 2.49; 95% CI: 1.08–5.77; P-interaction = 0.05). When examined as joint effects, modification by genotype was not apparent when intake was medium or high (supplementary Table 1 is available at Carcinogenesis Online).


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  		Table III. ORs for kidney cancer by cruciferous vegetable intake after stratification by GSTM1 and T1 polymorphisms

 
In Table IV, associations were examined after stratification by smoking status. All the controls within each stratum were included in the analyses to stabilize the control group and increase power. The higher risk of kidney cancer associated with low intake of cruciferous vegetables was only observed among current smokers with the GSTT1 null or both the GSTM1/T1 null genotypes compared with those with high intake. The interaction for the GSTM1/T1-combined genotype, smoking status (never/former/current) and cruciferous vegetable intake was also significant (P = 0.02). Significant interactions were not observed between cruciferous vegetable intake and the GSTM3 or GSTP1 genes (data not shown).


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  		Table IV. Kidney cancer risk, cruciferous vegetable consumption, GSTM1 and GSTT1 status, stratified by smoking status

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
In this study, cruciferous vegetable intake was inversely associated with RCC risk, and this risk was modified by GSTT1 and GSTM/T1-combined genotypes. Risk was only significantly elevated when intake was low. Modification by genotype was no longer apparent when intake was in the medium or high-frequency group. After stratification by smoking status, we observed that the effect was only significant among current smokers. The increased risk associated with low intake of cruciferous vegetables was highest among current smokers who had both GSTM1 and GSTT1 null genotypes compared with those with high intake.

These findings are similar to those previously observed for lung cancer in three independent studies (11–13). However, one study conducted in the USA reported that cruciferous vegetable intake was only associated with lung cancer risk among individuals with the GSTM1 present genotype (14). Differences in the frequency and amount of cruciferous vegetables consumed, which is generally lower in USA than Asian populations, could explain some of the differences across populations.

This is the first study to examine the association of RCC risk with GST genotypes and cruciferous vegetable consumption. In this population, we did not observe significant associations with GST genotypes and kidney cancer and there are currently no published studies with sufficient power to detect main effects of genes or gene–exposure interactions for comparison. However, there is toxicologic evidence demonstrating that GSTT1 is the most active glutathione transferase in the kidney (15,16) and that the highest expression levels are found in liver and kidney, followed by lung (15). Similarly, studies of genotype–phenotype relationships have shown that urinary ITC excretion differed significantly between subjects with active and null GSTT1 genotypes but not M1 genotypes, suggesting that the T1 transferase protein plays an important role in ITC conjugation compared with the other GSTs examined (17,18). GSTT1 also has higher enzymatic activity, ~10-fold higher than GST mu or phi classes (19). Considering the important role of GSTT1 enzyme activity in the kidney, the potential for GSTT1 to quickly conjugate and release ITCs for elimination, it seems likely that GSTT1 genotype could play an important role in modulating kidney cancer risk associated with cruciferous vegetable consumption. This effect could be stronger among smokers than non-smokers because one may expect higher levels of GST enzymes in response to the presence of tobacco-specific substrates. Therefore, the protective effect of cruciferous vegetables might not be modified by genotype among non-smokers, as they are in smokers because GSTs have not been induced.

The strengths of this study include high participation rates thus minimizing potential selection bias. The large sample size provided sufficient statistical power to detect relatively small associations between genotypes and risk, although the power to detect interactions was still limited. Also, although the food frequency questionnaire was limited, the high prevalence and frequent cabbage consumption in this population would tend to enhance recall and reporting of intake.

Alternatively, the food frequency questionnaire used in this study was not robust and only included asked questions concerning the frequency of consumption of 23 food items. Information on serving size quantities consumed and total caloric intake was not calculated. Therefore, it was impossible to estimate intake of specific micronutrients or control for total energy intake.

In summary, these findings suggest that genetic polymorphisms in GSTM1 and GSTT1 could play an important role in modifying the RCC risk associated with low cruciferous vegetable intake, particularly among current smokers. Low consumption of cruciferous vegetables increased cancer risk in individuals with inactive GSTT1 or inactive GSTM1/T1 genotypes, but not in individuals with active genotypes. Moreover, the increased risk associated with genotype was no longer apparent when frequency of cruciferous intake was increased. These data provide additional evidence for the emerging role of cruciferous vegetables in cancer prevention, particularly among individuals having one or both of these common, functional polymorphisms.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary Table 1 can be found at http://carcin.oxfordjournals.org/


   Acknowledgments
 
Conflicts of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

  1. IARC. IARC Handbook of Cancer Prevention. Cruciferous Vegetables, Isothiocyanates and Indoles (2004) Lyon: IARC Press.
  2. Lampe JW, et al. Brassica, biotransformation and cancer risk: genetic polymorphisms alter the preventive effects of cruciferous vegetables. J Nutr (2002) 132:2991–2994.[Abstract/Free Full Text]
  3. Kolm RH, et al. Isothiocyanates as substrates for human glutathione transferases: structure-activity studies. Biochem. J. (1995) 311:453–459.[Web of Science][Medline]
  4. Bruhn C, et al. Concordance between enzyme activity and genotype of glutathione S-transferase theta (GSTT1). Biochem. Pharmacol. (1998) 56:1189–1193.[CrossRef][Web of Science][Medline]
  5. Brockmoller J, et al. Genotype phenotype of glutathione S-transferase class isoenzymes mu and psi in lung cancer patients and controls. Cancer Res. (1993) 53:1004–1011.[Abstract/Free Full Text]
  6. Seow A, et al. Effect of glutathione S-transferase polymorphisms on the cancer preventive potential of isothiocyanates: an epidemiological perspective. Mutat. Res. (2005) 592:58–67.[Web of Science][Medline]
  7. Zhang Y. Cancer-preventive isothiocyanates: measurement of human exposure and mechanism of action. Mutat. Res. (2004) 555:173–190.[Web of Science][Medline]
  8. Hecht SS. Chemoprevention of lung cancer by isothiocyantes. Adv. Exp. Med. Biol. (1996) 401:1–11.[Medline]
  9. Hsu CC, et al. Dietary risk factors of renal cell carcinoma in eastern and central Europe. Am. J. Epidemiol. (2007) 166:62–70.[Abstract/Free Full Text]
  10. Packer BR, et al. SNP500 Cancer: a public resource for sequence validation, assay development, and frequency analysis for genetic variation in candidate genes. Nucleic Acid Res. Database (2006) D617–D621. http://snp500cancer.nci.nih.gov/home.cfm.
  11. London SJ, et al. Isothiocyanates, glutathione S-transferase M1 and T1 polymorphisms, and lung cancer risk: a prospective study of men in Shanghai, China. Lancet (2000) 256:724–729.
  12. Spitz MR, et al. Dietary intake of isothiocyanates: evidence of a joint effect with glutathione S-transferase polymorphisms in lung cancer risk. Cancer Epidemiol. Biomarkers Prev. (2000) 9:1017–1020.[Abstract/Free Full Text]
  13. Brennan P, et al. Effect of cruciferous vegetables on lung cancer in patients stratified by genetic status: a mendalian randomisation approach. Lancet (2005) 366:1558–1560.[CrossRef][Web of Science][Medline]
  14. Seow A, et al. Dietary isothiocyanates, glutathione S-transferase polymorphisms and colorectal cancer risk in the Singapore Chinese Health Study. Carcinogenesis (2002) 23:2055–2061.[Abstract/Free Full Text]
  15. Hayes JD, et al. Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology (2000) 61:154–166.[CrossRef][Web of Science][Medline]
  16. Landi S. Mammalian class theta GST and differential susceptibility to carcinogens: a review. Mutat. Res. (2000) 463:247–283.[CrossRef][Web of Science][Medline]
  17. Seow A, et al. Urinary total isothiocyanate (ITC) in a population-based sample of middle-aged and older Chinese in Singapore: relationship with dietary total ITC and glutathione S-transferase M1/T1/P1 genotypes. Cancer Epidemiol. Biomarkers Prev. (1998) 7:775–781.[Abstract]
  18. Fowke JH, et al. Urinary isothiocyanate excretion, Brassica consumption, and gene polymorphisms among women living in Shanghai, China. Cancer Epidemiol. Biomarkers Prev. (2003) 12:1536–1539.[Abstract/Free Full Text]
  19. Hayes JD, et al. Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. (2005) 45:51–88.[CrossRef][Web of Science][Medline]
Received February 26, 2007; revised May 22, 2007; accepted June 26, 2007.


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