Polymorphisms of the CYP1B1 Gene as Risk Factors for Human Renal Cell Cancer

Renal cell carcinoma is the most common kidney tumor in adults (1, 2). This type of cancer occurs predominantly in men, although the reason is unknown (1, 2). Epidemiological studies have shown that environmental factors, such as smoking, diesel exhaust, and various dioxins, may be involved in the development of sporadic renal cell cancer (2, 3, 4, 5). Of these, chemical procarcinogenic compounds may be involved in one-third of all cases, but the overall impact of such factors is unclear (3, 4, 5). Chemical procarcinogenic compounds require metabolic activation by oxidative enzymes such as CYP1B1 to be transformed into potentially carcinogenic forms (6, 7). Several studies have shown that polymorphisms of CYP1B1 induce hyperactivation of proteins and increase the incidence of several cancers (8, 9). The human CYP1B1 gene is located in the 2p21–22 region and consists of three exons (one of which is noncoding) and two introns (7, 10). Several polymorphisms of the CYP1B1 gene have been described: intron1–13C→T; codon 48C→G; codon 119G→T; codon 432C→G; codon 449T→C; and codon 453A→G (10, 11, 12). Prior studies have shown that point mutations in the heme-binding and substrate recognition regions of CYP1B1, encoded by exons 2 and 3, result in higher catalytic activity (12, 13). The activities of variant enzymes, in particular those with a single-nucleotide polymorphism on codon 119 and 432, were 2- to 4-fold higher than the wild-type enzyme (12, 13). Because CYP1B1 polymorphisms are inherited, exposure levels to its carcinogenic metabolites would be high over a lifetime (3, 4, 5, 6, 13). Thus, inherited alterations in the activity of CYP1B1 may explain interindividual and gender differences in renal cancer risk (13). We hypothesize that polymorphisms of the CYP1B1 gene are risk factors for pathogenesis of renal cell cancer. The rationale for this hypothesis is that chemical procarcinogenic compounds require metabolic activation by oxidative enzymes such as CYP1B1 to be transformed into potentially carcinogenic forms. To test this hypothesis, a case-control study using 211 male and female renal cell cancer patients and 200 controls was designed to investigate six different polymorphisms of the CYP1B1 gene and their susceptibility to renal cell cancer.

A total of 211 sporadic renal cell cancer patients (147 males and 64 females) and 200 healthy controls (102 males and 98 females) were recruited at Kagoshima University Hospital (Kagoshima, Japan) from 1995 through 2000. Only Japanese patients who were diagnosed with histologically confirmed renal cell cancer were invited to participate in the study as cases. The participation rate was ∼95% among patients contacted. The age range among the renal cell cancer patients was 46–77 years, with a median age of 61.7 years. Two hundred healthy individuals, free of any disease, were randomly selected as controls from the same area in Japan during the same period. The age range among the healthy controls was 35–69 years, with a median age of 59.3 years. The participation rate among eligible controls was ∼80%. The case-control study methods have been described in our laboratory (14, 15). Patients and controls were given a questionnaire to record family history of any cancers. Because no other ethnic groups were recruited, this study was limited to a native Japanese population. Appropriate informed consent was obtained from the patients in accordance with local human ethical committee guidelines. There were no significant differences between patients and control groups with regard to race, family history of cancer, indices of body size (height, weight, and body mass index), or income.

For the analysis of CYP1B1 polymorphisms, a two-step PCR procedure was designed. In the first PCR, DNA (10 ng) from renal tissues was amplified using 1.5 mm MgCl₂, 0.8 mm deoxynucleotide triphosphate, 0.5 unit of Taq polymerase (Applied Biosystems Inc., Foster City, CA), and specific first PCR primer sets (Table 1). The first PCR consisted of 30 cycles of denaturation (94°C for 60 s), annealing (Table 1), and extension (72°C for 60 s) and was followed by a final incubation at 72°C for 8 min. In the second PCR, each polymorphic fragment from first PCR was amplified with 1.5 mm MgCl₂, 0.8 mm deoxynucleotide triphosphate, and 0.5 unit of Taq polymerase, using specific second PCR primer sets (Table 1). The second PCR consisted of 20 cycles of denaturation (94°C for 30 s), annealing (Table 1), and extension (72°C for 30 s) and was followed by a final incubation at 72°C for 8 min. Each of the second-PCR products was then run on 3% agarose gels. The bands on the gels were visualized by ethidium bromide staining. For confirmation of genotyping, the first-round PCR products were subjected to direct sequencing using first-round PCR primer and ABI 377 Sequencer and Dye Terminator Cycle sequencing kit [Applied Biosystems Inc. (14)].

The relative risk associated with each genotype was analyzed by calculating odds ratios with 95% confidence intervals. Subsequent analysis included logistic regression analyses adjusting for confounding factors. Homogeneity was tested as described previously (14, 15). Two-sided χ² and t tests were used to analyze data for statistical significance (14, 15).

The structure of the CYP1B1 gene and the locations of six different polymorphic loci are shown in Fig. 1,A. Fig. 1,B shows the gel picture from representative samples of genotyping for intron 1 and codons 48, 119, 432, 449, and 453 of the CYP1B1 gene in renal carcinoma patients. Samples 1–3 are genotype C/C, C/T, and T/T at intron 1. Samples 4–6 are genotype C/C, C/G, and G/G at codon 48. Samples 7–9 are genotype G/G, G/T, and T/T at codon 119. Samples 10–12 are genotype C/C, C/G, and G/G at codon 432. Samples 13–15 are genotype T/T, T/C, and C/C at codon 449. Samples 16–18 are genotype A/A, A/G, and G/G at codon 453. The frequency of distribution of the six genetic polymorphisms of the CYP1B1 gene in renal cell cancer patients and the control group is shown in Table 2. The distributions of the six polymorphisms were consistent with Hardy-Weinberg equilibrium.

The frequency of genotype T/T on codon 119 was significantly higher in renal cell cancer patients compared with healthy normal controls (P < 0.001; Table 2). A total of 12.8% of renal cell cancer patients showed genotype 119T/T, whereas 5.5% of healthy controls showed this genotype. A total of 37.4% of cancer patients showed genotype 119G/T versus 19.5% of healthy controls. The relative risk of genotype 119T/T and 119G/T was calculated as 3.01 and 2.14, respectively, as compared with wild type. The frequency of genotype 432G/G was also significantly higher in renal cell carcinoma as compared with controls (P < 0.001). A total of 12.3% of cancer patients showed genotype 432G/G compared with 6.5% of healthy controls. A total of 32.7% of cancer patients showed genotype 432C/G versus 23.0% of healthy controls. The relative risk of genotype 432G/G and 432C/G was calculated as 2.17 and 1.52, respectively, as compared with wild type. The polymorphism on codon 453 was not detected in either the control group or the patient group, i.e., all individuals tested in this study were homozygous A (Table 2). Also, there were no differences of genotypic distribution between renal cell cancer patients and healthy normal controls at other loci (intron 1, codon 48, and codon 449). No difference was observed for the distributions of any polymorphic sites between men and women in the normal control group (0.75 < P for all polymorphic sites).

Table 3 shows the correlation of the polymorphism on codon 119 and codon 432 of the CYP1B1 gene with gender. The genotypic frequencies of codons 119 and 432 were significantly higher in male as compared with female renal cell cancer patients (P < 0.001; Table 3). The relative risk of genotype 119T/T was calculated as 3.95 in male patients and 1.92 in female patients. The relative risk of genotype 432G/G was calculated as 2.81 in male patients and 1.35 in female patients. No significant correlation was found between the six polymorphisms of the CYP1B1 gene and cancer grades, age, family history of cancer, indices of body size (height, weight, and body mass index), or income of the patients.

Environmental risk agents such as chemical procarcinogenic compounds require metabolic activation by enzymes such as CYP1B1 into potentially carcinogenic forms (6, 7). Thus, genetic polymorphisms at loci encoding these enzymes may result in interindividual variation in susceptibility to the carcinogenic effects of environmental chemicals (16, 17). Of the six polymorphisms in the CYP1B1 gene, amino acid replacements occur at codons 48, 119, 432, and 453, leading to the replacement of Arg→Gly, Ala→Ser, Leu→Val, and Asn→Ser, respectively.

In the present study, we observed a significantly higher frequency of the genotypes 119T/T and G/T in renal cell carcinoma patients as compared with healthy controls. A difference between renal cell cancer patients and healthy controls was also found in the allellic distributions of the codon 432 of CYP1B1. It is interesting to note that these point mutations in the heme-binding region of CYP1B1, especially polymorphisms on codons 119 and 432, show higher catalytic activity (7, 13).

Prior studies have shown that polymorphisms in another CYP gene, CYP1A1, enhance the susceptibility of renal cell cancer (16, 17, 18). In this regard, Longuemaux et al. (18) investigated several carcinogen-metabolizing enzymes and observed a polymorphism in the CYP1A1 genes to be significantly associated with renal cell cancer (relative risk, 2.1). These reports strongly suggest that carcinogenic substances are involved in renal cell carcinogenesis and implicate CYP genes as key players in the process; however, CYP1B1 exceeds CYP1A1 in its catalytic efficiency for various carcinogens (6, 7, 13, 17). Our present study shows that renal cell carcinoma patients have a higher frequency of 119T/T and 432G/G polymorphisms of the CYP1B1 gene, which may lead to higher activity of this gene in renal cell carcinoma.

We have also analyzed the relationship between CYP1B1 genotypes and gender of the patients. The distribution of genotypes on codons 119 and 432 was significantly different between male and female renal cell cancer patients. A male predominance of renal cell cancer is well known but unexplained (1, 2). In this regard, Rintala et al. (19) showed that the activities of CYP enzymes in male kidney tissue were higher than those in women, and this difference between the genders was statistically significant. Gender-related differences in carcinogen metabolisms by CYP enzymes are substantial (19, 20). It is possible that the higher metabolic activity rate of procarcinogenic substances in male kidneys could be an etiological factor explaining the greater occurrence of renal cell cancer among men (19, 20).

This is the first report demonstrating that polymorphisms at codon 119 and 432 in the CYP1B1 gene can be risk factors for renal cell cancer, especially in male patients.