Japanese Journal of Clinical Oncology

INTRODUCTION

The incidence of prostate cancer (PCa), one of the most common neoplasms, has risen dramatically in the Western world (1,2). It varies greatly with race and geography. Although the incidence of clinical PCa in Japan is lower than in the Western world, Japanese emigrants exhibit an increased frequency (3,4), and the frequency of occurrence in Japan has also been increasing.

p53, a tumor suppressor gene, is mutated in many human cancers, and it has been demonstrated that the spectrum of p53 mutations provides clues to the etiology and molecular pathogenesis of neoplasia (5). In a previous study, we found that 11 out of 90 (12.2%) Japanese PCa patients had p53 mutations and that 55% of these mutations were a transversion type which can be induced by bulky compounds (6) and also by the 8-hydroxydeoxyguanosine which is produced by active oxygen species. This result suggested that exogenous carcinogens and inflammatory factors might possibly play an important role in PCa development in Japan. Several factors, including dietary habits, hormone levels, occupational exposure, drinking and smoking habits, have been studied with regard to PCa, but no definitive causes have been identified (7,8), although these studies did include the Japanese. Although prostate cancer is rarely induced by chemicals in experimental animals, it was recently found that PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), one of the carcinogenic heterocyclic amines present in cooked foods, can induce prostate adenocarcinomas in rats (9). So there is a possibility that chemical carcinogens are involved in the development of human PCa.

Differences in metabolic activity would therefore be expected to exert an influence on susceptibility. Recently a considerable number of studies have been made on the association between the genetic polymorphisms of xenobiotic-metabolizing enzymes and cancer susceptibility. However, no data are available on the relationship between these genetic polymorphisms of metabolic enzymes and PCa development.

In the present study, we therefore analyzed genetic polymorphisms of the xenobiotic-metabolizing enzymes, CYP1A1 and GSTM1, in 115 PCa patients and 204 control patients.

MATERIALS AND METHODS

A total of 115 PCa patients and 204 control patients with other urological diseases at Mie University Hospital, Yokohama City University Hospital and Chiba University Hospital between 1995 and 1996 were included in this study after giving informed consent. The mean age was 73.0±8.0 for the PCa patients, and 71.2±7.2 for the control patients. Serological (prostate specific antigen, prostatic acid phosphatase), physical and histological examinations were conducted on all control patients to exclude the possibility of PCa. Histological grading and clinical staging of PCas were according to the Japanese General Rules for Clinical and Pathological Studies on Prostate Cancer (10). Thirty four patients (29.6%) had well differentiated, 48 (41.7%) had moderately differentiated and 33 (28.7%) had poorly differentiated adenocarcinomas. As for clinical staging, 8 patients (7.0%) were in stage A, 30 (26.1%) in stage B, 26 (22.6%) in stage C and 51 (44.3%) in stage D.

Genomic DNA was isolated from peripheral blood (10-20 ml) by either phenol/chloroform extraction and ethanol precipitation or the sodium iodide method, dissolved in TE buffer (pH 7.4) and stored at -20°C. Genotyping of CYP1A1 was performed essentially as previously reported (11). One µl of DNA (>10 ng) was added to a PCR mixture containing 1.2 mM MgCl₂, 200 µM dNTP, 0.2 units of Taq DNA polymerase and 20 pmol of each primers, in a total volume of 10 µl. The primers used in the PCR reactions were generated according to the method of Jaiswal et al. (12). Two primers with different primary bases, 5[prime]-AAGACCTCCCAGCGGGCAAT-3[prime] and 5[prime]-AAGACCTCCCAGCGGGCAAC-3[prime] (corresponding to an adenine to guanine mutation in exon 7) were used together with another primer, 5[prime]-GAAAGGCTGGGTCCACCCTCT-3[prime]. PCR was performed with denaturation at 94°C for 4 min, followed by 30 cycles at 94°C for 60 s, 65 °C for 45 s and a final extension at 70°C for 60 s. The genotyping of GSTM1 was performed as follows (13,14). PCR was carried out in a total volume of 10 µl as described above, using 20 pmol of each primer, 5[prime]-GAAGGTGGCCTCCTCCTTGG-3[prime] and 5[prime]-AATTCTGGATTGTAGCAGAT-3[prime]. The conditions were as follows; denaturing at 94°C for 4 min, followed by 30 cycles at 94°C for 60 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s.

PCR products were electrophoresed through 2.0% agarose gels and visualized by ethidium bromide staining. The genotype was determined by the presence or absence of PCR products. All DNA samples were confirmed to be adequate for PCR analyses using [beta]-globin as a positive control (data not shown).

Odds ratio (OR) and 95% confidence interval (CI) values were calculated from 2X2 tables with the Mantel-Haenszel technique and adjusted for age among subgroups (<70/70[le] years old).

RESULTS

Genetic polymorphisms are summarized in Table 1. In the controls, the distribution of the CYP1A1 genotype was 62.7% for the homozygous wild type (Ile/Ile), 32.4% for heterozygotes (Ile/Val) and 4.9% for the minor valine substituted allele homozygote (Val/Val). The distribution of the CYP1A1 genotypes in the PCa patients was 52.2% for Ile/Ile, 36.5% for Ile/Val and 11.3% for Val/Val. The frequency of Ile/Val and Val/Val genotypes was higher than for the controls and a significant difference was found in the frequency of the Val/Val genotype with an OR of 2.6 (CI = 1.11-6.25). The Ile/Val genotype also appeared to increase the risk, but not significantly (OR = 1.4; CI = 0.86-2.29). When the Ile/Val and Val/Val genotypes were combined, the OR was 1.6 (CI = 0.99-2.50).

As for GSTM1, 42.6% of the controls showed complete deletion of the GSTM1 gene (0/0 genotype), as compared to 49.6% of the PCa patients, the OR being 1.3 (CI = 0.82-2.04).

Table 1. Distribution of polymorphisms of CYP1A1 and GSTM1 among controls and PCa patients

Genotype	Control (%)	PCa (%)	OR	95% CI
CYP1A1 Ile/Ile	128 (62.7)	60 (52.2)	1.0
CYP1A1 Ile/Val	66 (32.4)	42 (36.5)	1.4a	0.86-2.29
CYP1A1 Val/Val	10 (4.9)	13 (11.3)	2.6a	1.11-6.25
CYP1A1 Ile/Val or Val/Val	76 (37.3)	55 (47.8)	1.6a	0.99-2.50
GSTM1 [+]	117 (57.4)	58 (50.4)	1.0
GSTM1 (0/0)	87 (42.6)	57 (49.6)	1.3b	0.82-2.04
GSTM1 [+]
+CYP1A1 Ile/Ile	68 (33.3)	32 (27.8)	1.0
GSTM1F (0/0)
+CYP1A1 Ile/Ile	60 (29.4)	28 (24.3)	1.0c	0.53-1.80
GSTM1 [+]
+CYP1A1 Ile/Val or Val/Val	49 (24.0)	26 (22.6)	1.1c	0.60-2.15
GSTM1 (0/0)
+CYP1A1 Ile/Val or Val/Val	27 (13.2)	29 (25.2)	2.3c	1.18-4.48

^aRatio of frequencies versus the Ile/Ile type between PCa patients and controls.
^bRatio of frequencies of GSTM1 (0/0) versus GSTM1 (+) between PCa patients and controls.
^cRatio of frequencies versus Ile/Ile + GSTM1 (+) type between PCa patients and controls.

Table 2. Association between genotype and age at initial diagnosis

Age	CYP1A1			ORa	95% CI
	Ile/Ile (%)	Ile/Val (%)	Val/Val (%)
<= 69	24 (50.0)	20 (41.7)	4 ( 8.3)	1.7	0.89-3.17
70-79	27 (50.0)	18 (33.3)	9 (16.7)	1.7	0.92-3.08
80 <=	9 (69.2)	4 (30.8)	0 ( 0.0)	0.8	0.22-2.15
Age	GSTM1		ORb	95% CI
	[+] (%)	(0/0) (%)
<= 69	22 (45.8)	26 (54.2)	1.6	0.84-2.99
70-79	28 (51.9)	26 (48.1)	1.3	0.68-2/28
80<=	8 (61.5)	5 (38.5)	0.8	0.27-2.66

^aRatios of frequencies of Ile/Val and Val/Val versus Ile/Ile type between age specific PCa and total control patients.
^bRatios of frequencies of GSTM1 (0/0) versus GSTM1 [+] between age specific PCa and total control patients.

Table 3. Association between genotypes and clinical Stages

Stage	CYP1A1			ORa	95% CI
	Ile/Ile (%)	Ile/Val (%)	Val/Val (%)
AB	18 (47.4)	18 (47.4)	2 (5.3)	2.0	0.99-3.92
C	16 (61.5)	8 (30.8)	2 (7.7)	1.1	0.47-2.39
D	26 (51.0)	16 (31.4)	9(17.6)	1.7	0.97-3.04
Stage	GSTM1		ORb	95% CI
	[+] (%)	(0/0) (%)
AB	23 (60.5)	15 (39.5)	0.8	0.40-1.62
C	13 (50.0)	13 (50.0)	1.3	0.59-2.96
D	22 (43.1)	29 (56.9)	1.7	0.93-3.17

^aRatios of frequencies of Ile/Val and Val/Val versus the Ile/Ile type between PCa and control patients for each stage.
^bRatios of frequencies of GSTM1 (0/0) versus GSTM1 [+] between PCa and contol patients for each stage.

We next analyzed the combined effect of the CYP1A1 and GSTM1 genotypes on the risk of PCa development. Considering the risk of the CYP1A1 Ile/Ile genotype combined with the GSTM1 positive genotype as a baseline, the combination of the CYP1A1 Val allele (Ile/Val and Val/Val) with GSTM1 (0/0) resulted in an OR of 2.3 (CI = 1.18-4.48). On the other hand the combination of CYP1A1 Val allele and GSTM1 positive or CYP1A1 Ile/Ile and GSTM1 (0/0) genotypes did not increase the risk.

Analysis of the patients by age at the initial diagnosis revealed that the frequency of the CYP1A1 Val allele was 50.0% in the young age group ([le]69 years old), 50.0% in the middle-age group and 30.8% in the old group (80[le] years old). The ORs for PCa in the young and the middle-age groups were both 1.7 (CI = 0.89-3.17 and 0.92-3.08, respectively) and in the old group, the OR was 0.8 (CI = 0.22-2.51). The GSTM1 (0/0) genotype showed a similar tendency, being more frequent in the young group (54.2%) than in the middle-age or old groups (48.1% and 38.5%). In the young group, OR was 1.6 with CI of 0.84-2.99 (Table 2).

Associations with these genotypes with clinical stages were further examined (Table 3). An increasing OR with advancing stage was noted for GSTM1. In stages A and B, the OR was 0.8 (CI = 0.40-1.62), in stage C, the OR was 1.3 (CI = 0.59-2.96) and in stage D, the OR was 1.7 (CI = 0.93-3.17). No such tendency was detected for CYP1A1 polymorphisms.

DISCUSSION

Chemical carcinogens generally require activation to electrophilic reactive forms to produce DNA adducts, this being mainly catalyzed by phase I enzymes of the CYP family (15). In contrast, phase II enzymes, such as the GST family, conjugate metabolic intermediates to water soluble forms which are then easily excreted (16). It can therefore be assumed that individuals with increased metabolic activity and decreased detoxifying activity are at higher risk of prostate cancer development.

CYP1A1 which acts on most carcinogenic polycyclic aromatic hydrocarbons shows a major genetic polymorphism, resulting in substitution of Val for Ile in codon 462 of exon 7 in the heme-binding region of CYP1A1 (17). The proportion of people with the CYP1A1 polymorphism varies with race and the frequency of Val substitution is considerably lower in Caucasians than in Japanese (18). Those with the mutant allele (Ile/Val and Val/Val) have a higher catalytic activity than the Ile/Ile homozygotes (19) and this polymorphism has been reported to influence the risk of lung (12,20), breast (21) and colon cancers (22). In this study, the Val type allele apparently caused significant increase of PCa risk. It has been reported that metabolic activity in individuals with the CYP1A1 Val allele is 3-3.5 fold higher than in Ile/Ile homozygotes (19). Our results point to a close relation with susceptibility regarding PCa.

Glutathione S-transferases (GSTs) are a family of phase II enzymes which comprise at least four classes, a, m, p and q (23). The GSTM1 gene is one of the m-class forms and is classified into three genotypes, GSTM1A, GSTM1B, and GSTM1 (0/0) (24). The GSTM1 (0/0) genotype due to inherited homozygous deletion of both alleles, has no enzymatic activity (25). Mutagenicity of urine from smokers with the GSTM1 (0/0) genotype is several-fold higher than from those with the GSTM1 [+] gene (26). About 50% of Japanese and Caucasians lack GSTM1 (27,28). The GSTM1 (0/0) genotype has been possibly implicated in elevated risks of lung (20,28,29) and urothelial cancer (24,27,30). However, in this study, we found that individuals with the GSTM1 (0/0) genotype showed a slightly higher risk as compared with GSTM1 [+] individuals with an OR of 1.3, indicating that any effect of GSTM1 on PCa development might be weak (Table 1).

We further analyzed the influence of the GSTM1 genotype in combination with the CYP1A1 genotype on PCa development and detected a significant elevated risk in individuals carrying the CYP1A1 Val allele either heterozygously or homozygously and the GSTM1 (0/0) genotype with an OR of 2.3 (Table 1). However, there was no elevation of risk in individuals with only one of the high risk genotypes, CYP1A1 Val and GSTM1 positive or CYP1A1 Ile/Ile and GSTM1 (0/0). These results suggested that increased metabolic activation and decreased detoxification of endogenous or exogenous carcinogens might increase the DNA adduct formation and influence PCa induction.

Effects of high risk genotypes on early onset of PCa were also analyzed. The higher frequency of the CYP1A1 Val and GSTM1 (0/0) genotype in individuals diagnosed at an age of less than 70 years (Table 2) are in line with data for breast cancers (21), suggesting that individuals with these polymorphisms might accumulate DNA damage at a younger age. The frequency of the GSTM1 (0/0) genotype was also higher with advanced stage, so that progression may also be influenced.

In conclusion, our data indicate that CYP1A1 and GSTM1 polymorphisms may play an important role in individual susceptibility with regard to PCa development. Since PhIP which induced prostate cancer in F344 rats is only activated slightly by CYP1A1 and is not detoxified by GSTM1, PhIP is not a major candidate risk factor that would be affected (31) and it remains unclear whether carcinogens might be playing an important role. Although the observed effects were weak, the increase in risk for the early onset and advanced stage group was clearly of interest. Further studies are warranted to address this question and confirm the effects of xenobiotic metabolizing enzymes. The relations between polymorphisms in CYP1A2 and PCa development also need to be classified with reference to PhIP-prostate-carcinogenicity in the experimental animal.

Acknowledgments

The study was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture, and the Foundation for Promotion of Cancer Research, Japan.