1 Departments of Gastrointestinal Medical Oncology,
2 Epidemiology,
3 Pathology and
4 Surgical Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA
Abstract
Previous studies by us and others have shown a significantly higher level of aromatic DNA adducts in normal adjacent breast tissue samples obtained from breast cancer patients than in those obtained from non-cancerous controls. The increased amount of DNA damage could be related to excess environmental carcinogen exposure and/or genetic susceptibility to such exposure. In the current study, we investigated the relationship between the levels of aromatic DNA adducts in breast tissues and polymorphisms of the drug-metabolizing genes cytochrome P4501A1 (CYP1A1), N-acetyltransferase-2 (NAT2), and glutathione S-transferase M1 (GSTM1), in 166 women having breast cancer. DNA adducts were measured using 32P-postlabeling and information on smoking status was obtained from medical records. When pooled data of smokers and non-smokers were analyzed by multiple regression analyses, no significant correlation was found between the level of total DNA adducts and age, race, or polymorphisms of CYP1A1, GSTM1, and NAT2. The only significant predictor of the level of DNA adducts in breast tissues was smoking (P = 0.008). When data were analyzed separately in smokers and non-smokers, however, a significant geneenvironment interaction was observed. Smokers with CYP1A1*1/*2 or *2/*2 genotypes had a significantly higher level of DNA adducts than those with the CYP1A1*1/*1 genotype. This effect was not seen among non-smokers. There was also a genegene interaction, as smokers with combined CYP1A1*1/*2 or CYP1A1*2/*2 genotypes and GSTM1 null had a much higher level of adducts than those with either CYP1A1 or GSTM1 polymorphism. Genetic polymorphisms of CYP1A1 and NAT2 were also significantly correlated with the frequency of certain types of DNA adducts. For example, a bulky benzo[a]pyrene (B[a]P)-like adduct was detected in 26% of the samples, the presence of which was not related to age, race, smoking status, or GSTM1 and NAT2 genotype. However, a significantly higher frequency of the B[a[P-like adduct was found in individuals having CYP1A1*1/*2 or *2/*2 genotypes than in those having the *1/*1 genotype (P = 0.04). In addition, individuals having slow NAT2 alleles had a significantly higher frequency of the typical smoking-related DNA adduct pattern, i.e. a diagonal radioactive zone (DRZ), than others did (P = 0.008). These findings suggest that polymorphisms of CYP1A1, GSTM1, and NAT2 significantly affect either the frequency or the level of DNA adducts in normal breast tissues of women having breast cancer, especially in smokers. Further large-scale studies are required to determine the exact role of these polymorphisms and types of DNA damage in breast cancer susceptibility.
Abbreviations: B[a]P, benzo[a]pyrene; CYP, cytochrome P450; DRZ, diagonal radioactive zone; GSTM1, glutathione S-transferase M1; NAT, N-acetyltransferase
Introduction
As with most other human cancers, the occurrence of breast cancer is probably determined by both environmental and intrinsic host factors. However, the role of environmental carcinogen exposure in breast cancer has not been well defined. Also, previous studies by us (1) and others (2,3) have shown that breast tissue samples obtained from women having breast cancer have a significantly higher level of DNA adducts than do those obtained from non-cancerous controls. While identification of the environmental factors that may influence the formation of these DNA adducts requires epidemiological investigation, in the current study, we explored the relationship between the DNA adduct profiles and polymorphisms of the drug-metabolizing genes, CYP1A1, GSTM1 and NAT2.
Polymorphisms of carcinogen-metabolism genes have been associated with an increased risk of several human cancers. Most studies of breast cancer have reported no increase in overall risk in patients having the variant CYP1A1 allele or GSTM1-null genotypes (4), but some have reported an increased risk in smokers among post-menopausal women who are carriers of the variant CYP1A1 or NAT2 alleles (5,6). Additionally, CYP1A1 enzyme has been found in breast tumors and other epithelial tissues (7,8), and the CYP1A1 MspI polymorphism has been associated experimentally with increased catalytic activity (9). Some studies of smokers among lung cancer patients have shown an increase in the effects on DNA adducts in those having the CYP1A1 variant alone (10) or in combination with deleted GSTM1 (1113). Also, the frequency of homozygous MspI alleles varies significantly among ethnic groups, with Caucasians having fewer variant alleles when compared with the Japanese population (14).
GSTM1 and NAT2 code for phase II detoxifying enzymes. About 50% of Caucasians and 30% of AfricanAmericans have the null genotype of GSTM1 (15). An increased level of DNA adducts and chromosome damage has been reported in people having GSTM1-null genotype (16). Furthermore, polymorphism of the NAT2 gene results in either slow or rapid acetylator phenotypes; individuals having homozygous low-activity alleles are slow acetylators while those carrying one or more high-activity alleles are rapid acetylators (17). In addition, individuals having slow NAT2 or rapid NAT1 acetylator genotypes have been shown to have a significantly increased DNA adduct level in bladder epithelium (18). Finally, Pfau et al. (19) reported a significant elevation of DNA adduct levels in breast tissue DNA obtained from women designated as slow NAT2 acetylators.
Very few studies of the relationship between the CYP1A1, GSTM1, and NAT2 genotypes and DNA adduct levels in adjacent normal breast tissue of breast cancer patients have been done. Therefore, the aim of this study was to demonstrate whether there is a geneenvironmental interaction by examining the relationship between profiles of aromatic DNA adducts and the CYP1A1, GSTM1, or NAT2 genotype in women having breast cancer.
Materials and methods
Study subjects and tissue samples
This study used adjacent normal breast tissue samples obtained from 166 women having newly diagnosed breast cancer undergoing surgical treatment at The University of Texas M.D. Anderson Cancer Center from 1996 to 2000. The use of human surgical tissue samples was approved by the Institutional Review Board. Information about the patients' smoking history was obtained from their medical records. In most cases, there was only a brief description on whether the patient was a smoker and no detailed information on smoking history was available.
DNA adduct analysis
DNA was extracted from the breast tissues using the conventional phenol/chloroform procedure. DNA adducts were detected using the nuclease P1-enhanced version of the 32P-postlabeling procedure. The chromatography conditions used were as previously reported (1). An internal standard DNA from mouse skin exposed to dibenzo[a,j]acridine was included along with each sample to monitor the adequacy of enzymatic digestion, radioactive labeling, and chromatography (1).
Genetic polymorphisms
Polymorphisms of the CYP1A1 (20), GSTM1 (21), and NAT2 (22) genes were determined by PCR and restriction fragment length polymorphism as reported previously. An internal control gene was amplified along with GSTM1 gene. When both GSTM1 and the internal standard were not amplified, the sample was considered as non-informative. At least 10% of the samples were analyzed in repeats to ensure quality control.
Statistical analysis
The mean values (±SD) of the DNA adducts were compared between smokers and non-smokers and between different genotypes using Student's t-test and regression analysis. Both chi-square and Fisher's exact test were used in the analysis of frequencies of polymorphism and specific types of DNA adduct. Two-tailed P values were calculated for the determination of statistical significance; the signification value was P < 0.05. Logistic regression was applied to calculate the odds ratio (OR) and 95% confidence interval (CI) for the frequency of polymorphism in different ethnic groups. The levels of the DNA adducts, which were analyzed as a continuous variable, were modeled as a function of genotypes and smoking status controlled for age and race in analysis of covariance by a general linear regression model.
Results
Study subjects
The average age of the 166 patients was 48.7 ± 11.5 years (range, 2386 years). There were 96 (58%) women at the age of younger than 50 (presumed pre-menopausal) and 70 (42%) women at the age of 50 and older (presumed post-menopausal). The ethnic composition was 71% Caucasians, 16% AfricanAmericans, 11% Hispanics and 2% others. Also, 27% (44/166) of the study subjects were ever-smokers while 64% (106/166) were never smokers; and 16 subjects with unknown smoking history.
DNA adduct profiles
Three types of DNA adduct were detected in breast tissue DNA: the previously reported B[a]P-like adduct, the smoking-related diagonal radioactive zone (DRZ) and some unidentified non-specific adducts (1). The B[a]P-like adduct was present in 24% of the study subjects, including 8 (18%) ever-smokers and 30 (28%) never-smokers. DRZ was detected in 17% of the subjects, including 22 (50%) ever-smokers and 5 (5%) never-smokers. Multiple regression analysis showed that the level of total DNA adducts including all three types of adduct was not significantly associated with any factors but smoking status. The P values for smoking status, race, age, CYP1A1, GSTM1, and NAT2 were 0.009, 0.770, 0.820, 0.250, 0.870, and 0.240, respectively. In general, the level of total adducts was 66% higher in ever-smokers than in never-smokers (P = 0.03) (Table I). Among the different ethnic groups, Hispanics and AfricanAmericans had a relatively higher mean adduct level among ever-smokers than Caucasians; Hispanics also had the lowest level of adduct among never-smokers (Table I
). The smoking effect on the level of total DNA adducts in breast tissues was seen in women at the age of 50 and older but not in those younger than 50 years of age (Table I
). Regardless of the subjects' smoking status, there were no significant differences in the level of adduct between the two age groups. Patients having either the B[a]P-like adduct or DRZ had a much higher level of total adducts than did those not having those types of DNA adducts (Table I
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There is a general consensus that the majority of human cancers are a consequence of geneenvironmental interaction. Conventional epidemiological studies have not conclusively implicated any environmental factors other than radiation exposure in breast cancer etiology. The high-penetrant genes, such as Brca1, Brca2, p53 germ line mutation, and Ataxia Telangietasia Mutated (ATM) heterozygotes, account for <510% of all breast cancer cases. On the other hand, the low-penetrant but widespread cancer predisposing genes, such as those encoding for drug-metabolizing and DNA repair enzymes, may contribute to more breast cancer cases in the general population than do the high-penetrant genes, especially among individuals experiencing special environmental carcinogen exposure (23,24). In the present study, we found that the frequencies of some specific DNA adducts as well as the levels of total DNA adducts were significantly associated with polymorphisms of CYP1A1, GSTM1 and NAT2 genes, especially among ever-smokers. Specifically, ever-smokers having CYP1A1*1/*2 and *2/*2 genotypes had a significantly higher level of adduct than those with CYP1A1*1/*1 genotype did. Smokers with both CYP1A1*1/*2 or *2/*2 and GSTM1-null genotypes had a higher level of adducts than those with polymorphism of either genes. A significantly higher frequency of the BP[a]-like adduct was seen among individuals with CYP1A1*1/*2 and *2/*2 genotypes. Those having slow acetylator alleles of the NAT2 gene also displayed a significantly higher frequency of smoking-related DNA adducts than did those having the rapid acetylator alleles. These observations provide direct evidence supporting a geneenvironmental interaction in breast cancer etiology.
The role of smoking in breast cancer etiology has been controversial according to epidemiological findings. Early studies found no increase in breast cancer risk with smoking (2527); however, later studies reported an increased risk in women who smoke (6,28,29). Also, several studies have explored the interaction between smoking, genetic polymorphisms of NAT1/2 and breast cancer risk (6,3032). Some studies found a positive association between the risk of breast cancer among smokers with polymorphisms of NAT1/2 (6,31,32). This finding pointed out the importance of geneenvironmental interaction in cancer etiology. The present study has provided further supporting evidence of the interactive effect of smoking and genetic susceptibility in breast cancer. However, while a conventional epidemiological study may not be able to demonstrate a subtle association between an environmental factor and a disease, a molecular epidemiological study may have the potential to identify high-risk individuals with the help of genetic markers.
The association between the CYP1A1, GSTM1, and NAT2 genotypes and breast cancer risk has been examined in a number of casecontrol studies with varying results (3338). The distribution of the CYP1A1 MspI polymorphism, GSTM1, and NAT2 genotypes among Caucasians, who constituted 72% of our study group, was consistent with the prevalence reported in those previous studies. We also observed a higher frequency of CYP1A1 variant genotypes and lower frequency of GSTM1 null and NAT2 slow acetylator alleles in Hispanics, but the sample size was small and it needs to be verified in a larger scale study.
In our previous study we reported the presence of a B[a]P-like adduct in breast tissues samples obtained from women having breast cancer but not in those obtained from non-cancerous controls (1). However, the origin and chemical structure of this adduct has not been identified. Because CYP1A1 plays an important role in the activation of polycyclic aromatic hydrocarbon (PAH) compounds, the finding of a significant association between the frequency of this adduct and the CYP1A1*1/*2 or *2/*2 genotypes supports the hypothesis that the B[a]P-like adduct is derived from exposure to PAH compounds.
The association between the GSTM1-null genotype and an elevated level of aromatic DNA adducts in human tissues, including the breast, has been reported in several studies (3943). However, the lack of association between the two markers has also been reported (4446). We did not find a significant effect of the GSTM1-null genotype on the level of DNA adducts in breast tissues in the current study. However, the interaction between the CYP1A1 MspI polymorphism and GSTM1 genotype with respect to the level of DNA adducts in ever-smokers was evident. Patients having the CYP1A1*1/*2 or *2/*2 and GSTM1-null genotypes experienced a 115% increase in mean DNA adduct levels compared with those having the active GSTM1 genotype. Patients having the CYP1A1*1/*1 and GSTM1-null genotype also experienced a significant increase (65%) in DNA adducts compared with those having the active GSTM1 genotype. This is in line with recent studies revealing a noticeable reliance of adduct levels on the CYP1A1 genotype, which is most evident in GSTM1-deficient smokers (4751).
Additionally, we observed a significantly higher frequency of smoking-related DNA adducts in individuals with NAT2 slow acetylator genotype compared with that in individuals having a rapid NAT2 acetylator genotype. This finding is consistent with the results reported by Pfau et al. (19) that NAT2 slow acetylators had a higher level of adducts than rapid acetylators. The higher frequency of smoking-related DNA adducts and higher level of DNA adducts in breast tissues among individuals with the slow NAT2 acetylator genotype suggests that among smokers, slow NAT2 acetylators are more susceptible to smoking-induced DNA damage, which may subsequently contribute to a higher risk of breast cancer. It is not known, however, what specific type of tobacco carcinogens was responsible for the formation of DRZ.
Taken together, our results show clear interaction between smoking and genetic polymorphisms of CYP1A1, GSTM1, and NAT2 in the formation of DNA adducts in human breast tissues. Even though polymorphisms of individual genes in carcinogen metabolism may not be significantly associated with breast cancer risk in general, they may play an important role in individuals having known carcinogen exposure, such as ever-smokers. Future studies of breast etiology should take consideration of both environmental exposure and genetic susceptibility.
Notes
5 To whom requests for reprints should be addressed at: Department of Gastrointestinal Medical Oncology, Box 426, The University of Texas, M.D. Anderson Cancer Center,1400 Holcombe Boulevard, Houston, TX 77030, USA Email: dli{at}mdanderson.org
Acknowledgments
This work was supported by NIH grant CA70264 and NIEHS Center Grant P30 ES07784.
References