Carcinogenesis vol.28 no.6 pp.1247–1253, 2007 doi:10.1093/carcin/bgm016 Advance Access publication January 21, 2007 Relationships between polymorphisms in NOS3 and MPO genes, cigarette smoking and risk of post-menopausal breast cancer Jun Yang, Christine B.Ambrosone, Chi-Chen Hong, Jiyoung Ahn1, Carmen Rodriguez2, Michael J.Thun2 and Eugenia E.Calle2 Department of Cancer Prevention and Control, Roswell Park Cancer Institute, Buffalo, NY 14263, USA, 1Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892, USA and 2Epidemiology and Surveillance Research, American Cancer Society, Atlanta, GA 30329, USA To whom correspondence should be addressed at Virginia Department of Health, Office of Epidemiology, James Madison Building, Room 532A, 109 Governor Street, Richmond, VA 23219, USA. Tel: þ1 804 864 8140; Fax: þ1 804 864 8139; Email: [email protected] NOS3 and MPO genes encode endothelial nitric oxide synthase and myeloperoxidase (MPO), respectively, which generate nitric oxide and reactive oxygen species. Because cigarette smoking generates reactive species, we hypothesized that NOS3 and MPO polymorphisms could influence susceptibility to breast cancer, particularly among smokers. We examined the associations between NOS3 Glu298Asp and MPO G-463A polymorphisms and breast cancer risk by cigarette smoking among post-menopausal women in the American Cancer Society’s Cancer Prevention Study II Nutrition Cohort. Included in this analysis were 502 women who provided blood samples and were diagnosed with breast cancer between 1992 and 2001 and 505 cancer-free controls who were matched to the cases by age, race/ethnicity and date of blood donation. Genotyping for NOS3 and MPO was performed using TaqMan, and unconditional logistic regression was used to compute odds ratios (ORs) and 95% confidence intervals (CIs). No statistically significant relationships were found between NOS3 and MPO genotypes and breast cancer risk. When considering smoking, variant NOS3 genotypes (GT and TT) were significantly associated with reduced breast cancer risk among never smokers (OR 5 0.67, 95% CI 5 0.45–0.99), but were associated with higher risk among ever smokers (OR 5 1.59, 95% CI 5 1.05–2.41) and 2-fold increase in risk for those who smoked .10 cigarettes per day (OR 5 2.19, 95% CI 5 1.21–3.97). NOS3 genotypes appeared to be associated with risk of post-menopausal breast cancer among smokers, supporting the hypothesis that subgroups of women based upon genetic profiles may be at higher risk of breast cancer when exposed to tobacco smoke. Background Breast cancer is the most common cancer and the second leading cause of cancer death in women in USA (1). Identified risk factors for breast cancer include family history of breast cancer, factors associated with lifetime estrogen exposure, age at first full-term pregnancy, alcohol consumption, post-menopausal obesity and ionizing radiation. These factors, however, cannot fully explain the etiology and geographical variation of breast cancer (2). Heterogeneity in genetic predisposition, particularly in genes regulating cellular microenvironment such as those related to oxidative stress and genes metabolizing carcinogens, may be important in the etiology of breast cancer. Experimental studies have indicated a role for these genes in human carcinogenesis, including NOS3 and MPO, related to oxidative mechanism and carcinogen metabolism (3,4). Abbreviations: CI, confidence intervale; NOS, endothelial nitric oxide synthase; MPO, myeloperoxidase; NO, nitric oxide; OR, odds ratio; ROS, reactive oxygen species. NOS3 gene encodes endothelial nitric oxide synthase (eNOS), an isoenzyme of the NOS family that consists of eNOS, neuronal NOS and inducible NOS (5). eNOS generates low levels of the endogenous free radical nitric oxide (NO) during the transformation of L-arginine to L-citrulline at the presence of tetrahydrobiopterin (BH4), an essential eNOS cofactor (5–7). NO has multiple physiological functions, including vasodilatation, neuronal transmission, smooth muscle relaxation and immunity (5,8). The cellular effect of NO is complex and can be cytoprotective or cytotoxic, depending on its concentration and cellular microenvironment (3,9). At low concentrations, NO may exert cytoprotective function through cell homeostasis, muscle relaxation, neurotransmission and platelet function (10–12). Low levels of NO may also protect endothelial cells from exposure to risk factors such as reactive oxygen species (ROS) and tumor necrosis factor-a (13–15). The specific actions whereby NO protects against ROS may include interacting with metals to prevent the formation of oxygen species, scavenging oxidants, preventing the degradation of metalloproteins and preventing lipid peroxidation (15). At high concentrations, NO and its derivatives can cause DNA strand breaks and impair the tumor suppressor function of p53, which may lead to carcinogenesis (16–19). When exposed to tobacco smoke, a reduction in protein and mRNA levels of eNOS and a decrease in the bioavailability and bioactivity of NO in epithelial cells have been observed (20–22), which is correlated with impaired endothelial functions. The reduction of eNOS and NO and the impaired endothelial functions among smokers are probably due to the increased ROS from tobacco smoke, rather than nicotine or other non-specific toxicity (23–28), supported by the observation that the physiopathologic alterations could be substantially alleviated after treatment with antioxidants such as vitamin C (27,29). Smoking is well known to increase oxidative stress. Oxidative damage measured by 8-hydroxydeoxyguanosine (8-OH-dG), protein carbonyls and isoprostanes, markers for damage to DNA, proteins and lipids, is higher among smokers than non-smokers (30–32). Superoxide anion generated from smoking can react with NO to form peroxynitrite, which generates cytotoxic oxidant species when decomposed (33–35). Cigarette smoking can also deplete tetrahydrobiopterin (BH4), an essential cofactor of eNOS enzyme, resulting in the dysfunction of eNOS enzyme and the subsequent generation of superoxide instead of NO (29,36). Several polymorphisms in NOS3 gene have been identified, including a common missense variant at nucleotide position 894 (G894T) in exon 7, resulting in a substitution of glutamic acid to aspartic acid at amino acid position 298. This polymorphism is associated with differing susceptibility to cleavage in eNOS enzyme in transfected cells and human endothelial cells (37), related to reduced basal NO production (38). Because of the implication of endogenous NO and eNOS in human tumors including breast cancer (12,39), it is likely that NOS3 polymorphism may be important for breast cancer, particularly among those exposed to exogenous factors such as tobacco smoke, which contains mammary carcinogens and produces ROS (40–45). Previous studies have shown increased risk of coronary artery disease (46) and hypertension (47) associated with NOS3 Glu298Asp genotypes. In relation to breast cancer risk, the results have been inconsistent, with three studies reporting null association (48–50) and one showing increased risk associated with the Glu298Asp polymorphism (51). Myeloperoxidase (MPO) is a lysosomal enzyme abundant in neutrophils, primarily defending against microbial infection (52). MPO is capable of generating a variety of ROS and activating carcinogens such as polycyclic aromatic hydrocarbons, aromatic amines and heterocyclic amines, which are abundant in tobacco smoke (52,53). A G / A polymorphism at position 463 in the promoter region of MPO gene has been identified, with the A variant allele related to decreased Ó The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 1247 J.Yang et al. transcriptional activity and probably decreased MPO expression, which may be associated with reduced risk in human cancers (54). Several epidemiologic studies have observed decreased risk associated with this polymorphism in lung cancer (55–57), ovarian cancer (58), bladder cancer (59) and liver cancer (60). For breast cancer, we reported previously that the variant A allele was significantly associated with reduced breast cancer risk among pre-menopausal women who consumed higher amount of fruits and vegetables, but with a nonsignificant increased risk among post-menopausal women with low intake of fruits and vegetables (61). Lin et al. (62) did not observe a significant association between MPO G-463A polymorphism and breast cancer risk in a Chinese population. To date, the association between NOS3 and MPO genotypes and breast cancer risk has not been well studied, particularly in relation to cigarette smoking, a risk factor that has been confirmed for many human cancers but does not appear to be associated with breast cancer risk, for the most part. We hypothesize that polymorphisms in genes such as NOS3 and MPO, involved in pathways of oxidative stress and carcinogen metabolism, are associated with risk of breast cancer, particularly among women who smoke. Therefore, we conducted this nested case–control study to investigate the role of NOS3 and MPO polymorphisms and cigarette smoking in relation to post-menopausal breast cancer risk. Patients and methods Study population This report is based upon data and samples from the American Cancer Society’s Cancer Prevention Study II Nutrition Cohort, a prospective study including 184 000 US adults. The study design, recruitment, characteristics and follow-up of the Cancer Prevention Study II Nutrition Cohort have been described previously (63). Most participants were aged 50–74 years at the time of enrollment. Upon enrollment in 1992 or 1993, participants completed a self-administered questionnaire that included demographic information, cigarette smoking history, medical history, hormonal and reproductive history, diet and other lifestyle factors. Information on smoking history included age at smoking initiation, age at smoking cessation, duration of smoking, lifetime average number of cigarettes per day and years since quitting smoking. Follow-up questionnaires were sent to surviving cohort participants every 2 years beginning in 1997 to update exposure information and to ascertain occurrence of new cases of cancer. Incident cancers reported on the questionnaires were verified through medical records, linkage with state cancer registries or death certificates. From June 1998 to June 2001, blood samples were collected from a subgroup of 39 371 cohort members, including 21 965 women. After obtaining informed consent, a maximum of 43 ml of non-fasting whole blood was collected from each participant and separated into aliquots of serum, plasma, RBC and buffy coat. Samples were frozen in liquid nitrogen vapor phase at approximately 130°C for long-term storage. Among post-menopausal women who had provided a blood sample, we initially identified 509 women who had been diagnosed with breast cancer between 1992 and 2001 and had not been diagnosed with other cancers other than non-melanoma skin cancer. Most of the cases were diagnosed before they provided a blood sample. Sixty four percent of these prevalent cases provided a blood sample within 2 years of diagnosis, although the maximum time between diagnosis and blood draw was 7.6 years. For each case, we randomly selected one control from among post-menopausal women who had provided a blood sample and were cancer free at the time of the case diagnosis. Each control was individually matched to a case on birth date (±6 months), race/ethnicity (White, African American, Hispanic, Asian or other/unknown) and date of blood collection (±6 months). Seven of the cases and four of the controls initially selected were later excluded because on more detailed review, they were found to have not been truly post-menopausal, to not have had breast cancer (if a case) or to not have had a DNA sample available. A total of 502 cases and 505 controls remained for analysis. 1248 Laboratory methods DNA was extracted from buffy coat, and genotyping assays were performed using TaqMan (Applied Biosystems, Foster City, CA). Genetic polymorphisms at exon 7 of NOS3 gene (National Center for Biotechnology Information rs#1799983) and at 463 in the promoter region of the MPO gene (National Center for Biotechnology Information rs#2333227) were arrayed. Genotyping was performed by laboratory personnel blinded to case–control status and 10% blind duplicates were interspersed with the case–control samples to validate genotyping procedures. Concordance for the quality control samples was 100%. Exact tests for Hardy–Weinberg equilibrium among the controls were performed, which showed that both NOS3 and MPO genotype distributions were in Hardy–Weinberg equilibrium (P . 0.05). Statistical analysis We used both unconditional and conditional logistic regression models in the analysis of the association between genotypes and breast cancer risk, and observed consistent results with both approaches, without remarkable discrepancies. Because conditional logistic regression prevents stratified analyses where the matching is broken, we used unconditional logistic regression in this analysis. Age, race, body mass index, family history of breast cancer, age at menarche, age at menopause, parity, use of hormonal replacement therapy and vegetable and fruit consumption, which showed significant associations with breast cancer or were biologically related to breast cancer, were included as covariates in the multivariate regression models. The following smoking characteristics were examined for the potential modification effects on associations between genetic polymorphisms and breast cancer risk: lifetime smoking status (ever/never), duration of smoking, cigarettes per day, pack-years of smoking and age at smoking initiation. Pack-years of smoking were calculated as the multiplicative product of cigarettes per day and years of smoking. Based on the distributions among control smokers, we chose the median values or values close to median to categorize age at smoking initiation, duration of smoking, cigarettes per day and pack-years of smoking. Specifically, we categorized age at smoking initiation by 18 (median) as 18 versus .18 years old; duration of smoking by 20 as 20 versus .20 years; cigarettes per day by 10 (median 5 12.0) as 10 versus .10 cigarettes and pack-years of smoking by 10 (median 5 11.7) as 10 versus .10 pack-years. All analyses were two-sided tests at the significance level of 0.05. Odds ratio (OR), 95% confidence interval (CI), Wald chi-square value, global P value and P for trend were reported. All analyses were performed using SAS statistical package version 9.0 (SAS Institute, Cary, NC). Results Approximately 49, 41 and 10% of study subjects had GG, GT and TT genotypes in NOS3 gene, and 60, 36 and 4% of subjects had GG, GA, and AA genotypes in MPO gene, respectively, comparable with the genotype distributions reported in other studies (46,61). There were no significant associations between NOS3 Glu298Asp and MPO G-463A polymorphisms and post-menopausal breast cancer risk after controlling for confounders (Table I). Additional adjustment for duration of smoking, cigarettes per day and age at smoking initiation did not change the results. When cigarette smoking was taken into account, the association with NOS3 was modified by tobacco smoke exposure (P 5 0.01 for interaction test). Among never smokers, NOS3 T alleles were associated with reduced breast cancer risk among women with GT and TT genotypes combined (adjusted OR 5 0.67, 95% CI 5 0.45–0.99, P for trend 5 0.10), although of borderline significance; in contrast, an increase in risk was observed among ever smokers with T alleles (adjusted OR 5 1.59, 95% CI 5 1.05–2.41, P for trend 5 0.02) compared with those with GG genotypes (Table II). No significant interaction was found between MPO gene polymorphism and smoking status (P 5 0.43 for interaction test) (Table II). Risk of post-menopausal breast cancer Table I. NOS3 Glu298Asp and MPO G-463A polymorphisms and risk of post-menopausal breast cancer, American Cancer Society, 1992–2001 NOS3 GG GT TT GT and TT MPO GG GA AA GA and AA Casesa (n 5 502) Controlsa (n 5 505) OR (95% CI)b OR (95% CI)c Wald v2c P valuec 204 (48.8%) 168 (40.2%) 46 (11.0%) 214 (51.2%) 199 (48.7%) 176 (43.0%) 34 (8.3%) 210 (51.3%) 1.00 0.94 (0.72–1.22) 1.15 (0.73–1.81) 0.97 (0.75–1.25) 1.00 0.96 (0.71–1.28) 1.26 (0.77–2.06) 1.01 (0.76–1.33) 1.17 0.56 238 (58.6%) 149 (36.7%) 19 (4.7%) 168 (41.4%) 245 (62.5%) 135 (34.4%) 12 (3.1%) 147 (37.5%) 1.00 1.07 (0.82–1.41) 1.55 (0.79–3.03) 1.11 (0.86–1.45) 1.00 1.11 (0.82–1.50) 1.81 (0.84–3.87) 1.17 (0.87–1.56) P for trendc 0.61 0.003 0.96 2.56 0.28 1.10 0.30 0.16 a Numbers in the cells are smaller than the total sample size due to missing values in the covariates. Controlled for age. c Controlled for age, race, smoking status, body mass index, family history of breast cancer, age at menarche, parity, age at menopause, history of hormonal replacement therapy use, and vegetable and fruit consumption. b Table II. NOS3 Glu298Asp and MPO G-463A polymorphisms and risk of post-menopausal breast cancer by smoking status, American Cancer Society, 1992– 2001 Never smokers NOS3 GG GT TT GT and TT Ever smokers NOS3 GG GT TT GT and TT Never smokers MPO GG GA AA GA and AA Ever smokers MPO GG GA AA GA and AA Casesa (n 5 502) Controlsa (n 5 505) OR (95% CI)b Wald v2 P value P for trend Interaction 117 80 17 97 105 108 19 127 1.00 0.65 (0.43–0.97) 0.78 (0.37–1.63) 0.67 (0.45–0.99) 4.34 0.11 0.10 v2 5 8.96 P 5 0.01 4.11 0.04 87 88 29 117 94 68 15 83 1.00 1.49 (0.96–2.31) 2.06 (1.01–4.18) 1.59 (1.05–2.41) 5.53 0.06 4.81 0.03 120 78 10 88 130 83 9 92 1.00 0.98 (0.65–1.49) 1.65 (0.62–4.37) 1.04 (0.70–1.55) 1.05 0.59 0.04 0.84 118 71 9 80 115 52 3 55 1.00 1.35 (0.85–2.14) 2.84 (0.73–11.1) 1.44 (0.92–2.24) 3.51 0.17 2.52 0.11 0.02 0.60 v2 5 1.70 P 5 0.43 0.07 a Numbers in the cells are smaller than the total sample size due to missing values in the covariates. Controlled for age, race, body mass index, family history of breast cancer, age at menarche, parity, age at menopause, history of HRT use, and vegetable and fruit consumption. b Given the significant interaction between NOS3 genotypes and smoking status on breast cancer risk, we performed more detailed analysis of relationships between NOS3 genotypes and breast cancer risk (Table III). Although none of the interaction tests was significant, for cigarettes per day, there was a suggestion of increasing risk with number of cigarettes smoked among women with NOS3 variant T alleles (GT and TT genotypes). Among women who smoked .10 cigarettes per day, NOS3 T alleles were significantly associated with a 2-fold increased risk of breast cancer compared with those with GG genotypes (adjusted OR 5 2.19, 95% CI 5 1.21–3.97, P for trend 5 0.005); whereas among women who smoked 10 or ,10 cigarettes per day, NOS3 variant T alleles were not associated with increased risk of breast cancer (adjusted OR 5 1.15, 95% CI 5 0.62–2.15). For duration and pack-years of smoking, the relative risks were elevated but were not substantially different between women with different duration or pack-years of smoking. For age at smoking initiation, we observed that the NOS3 T alleles were significantly associated with an increased risk among women who started smoking after they were 18 years of age compared with those who smoked before age 18 (smoking after 18 years of age: adjusted OR 5 2.41, 95% CI 5 1.27–4.59, P for interaction 5 0.11) (Table III). The same analyses for MPO genotypes did not show any significant interaction with the above smoking measures on post-menopausal breast cancer risk (data not shown). Discussion In the present study, we did not observe significant relationships between NOS3 Glu298Asp and MPO G-463A polymorphisms and postmenopausal breast cancer risk. However, we found that NOS3 Glu298Asp polymorphism was associated with reduced risk of postmenopausal breast cancer among women who never smoked and with 1249 J.Yang et al. Table III. NOS3 Glu298Asp polymorphism and risk of post-menopausal breast cancer by duration of smoking, cigarettes per day, pack-years, and age at smoking initiation, American Cancer Society, 1992–2001 Duration of smokingc NOS3, 20 years GG GT TT GT and TT NOS3, .20 years GG GT TT GT and TT Cigarettes per dayd NOS3, 10 CPD GG GT TT GT and TT NOS3, .10 CPD GG GT TT GT and TT Pack-years of smoking NOS3, 10 PYS GG GT TT GT and TT NOS3, .10 PYS GG GT TT GT and TT Age at smoking initiatione NOS3, 18 years GG GT TT GT and TT NOS3, .18 years GG GT TT GT and TT Casesa (n 5 502) Controlsa (n 5 505) OR (95% CI)b Wald v2 P value P for trend Interaction 41 43 16 59 47 36 9 45 1.00 1.55 (0.81–2.98) 1.79 (0.68–4.72) 1.61 (0.87–2.95) 2.39 0.30 0.14 v2 5 0.34 P 5 0.84 2.31 0.13 45 43 13 56 46 31 5 36 1.00 1.73 (0.89–3.37) 3.00 (0.92–9.84) 1.92 (1.02–3.60) 4.75 0.09 4.08 0.04 44 33 13 46 42 30 9 39 1.00 1.08 (0.55–2.12) 1.40 (0.51–3.83) 1.15 (0.62–2.15) 0.43 0.81 0.19 0.66 42 53 16 69 51 37 5 42 1.00 1.96 (1.05–3.64) 3.87 (1.22–12.26) 2.19 (1.21–3.97) 7.73 0.02 6.65 0.01 36 36 12 48 40 32 7 39 1.00 1.29 (0.65–2.56) 2.13 (0.69–6.58) 1.43 (0.75–2.72) 1.87 0.39 1.16 0.28 50 50 17 67 53 35 7 42 1.00 1.69 (0.92–3.11) 2.76 (1.01–7.55) 1.88 (1.06–3.33) 5.38 0.07 4.62 0.03 52 43 13 56 46 43 7 50 1.00 0.90 (0.48–1.68) 1.32 (0.44–3.95) 0.96 (0.53–1.74) 0.47 0.79 0.02 0.90 34 43 16 59 47 24 7 31 1.00 2.24 (1.12–4.48) 3.01 (1.07–8.50) 2.41 (1.27–4.59) 7.41 0.02 7.16 0.007 0.03 0.54 v2 5 3.08 P 5 0.21 0.005 0.18 v2 5 0.46 P 5 0.79 0.02 0.87 v2 5 4.46 P 5 0.11 0.009 CPD, cigarettes per day; PYS, pack-years of smoking. a Numbers in the cells are smaller than the total sample size due to missing values in the covariates. b Controlled for age, race, body mass index, family history of breast cancer, age at menarche, parity, age at menopause, history of HRT use, and vegetable and fruit consumption. c Additional adjustment for cigarettes per day. d Additional adjustment for duration of smoking. e Additional adjustment for duration of smoking and cigarettes per day. increased risk among women who smoked or smoked at higher intensity (.10 cigarettes per day). There were no significant relationships between MPO genotypes, smoking and breast cancer risk. Previous studies have indicated that NO may have both pro- and antitumor functions, under varied conditions in the course of tumor initiation, development, and progression (9,64). The pro-tumor property may be related to DNA damage, mutation of tumor suppressor gene p53, promotion of angiogenesis and stimulation of tumor cell blood flow (9,16–18,64,65), whereas the antitumor effects may be associated with the induction of apoptosis, activation of antioxidant defense, cytostatic effect and immunity (9,15,64). The effective NO functions depend, to a great extent, on its concentration, local microenvironment and timing of production. In a recent review, Lancaster et al. (3) noted that heterogeneity in the chemical process, biological 1250 production of NO and cellular responses to NO, among other factors, might render different biological outcomes related to NO. Several studies have investigated the associations between NOS3 Glu298Asp polymorphism and the risk of human cancers. In general, no significant associations were found with ovarian cancer (66), colorectal cancer (67) and prostate cancer (68). For breast cancer, Ghilardi et al. (48) did not observe an association between NOS3 Glu298Asp polymorphism and breast cancer risk in 71 patients and 91 controls in Italy. The studies by Royo et al. (50) and Lu et al. (49) also did not find associations between NOS3 and breast cancer risk, even after taking smoking status into account (49). However, in a case–control study involving 269 Caucasian breast cancer patients and 244 controls in Austria, Hefler et al. (51) reported that NOS3 Glu298Asp polymorphism was associated with a 2-fold increase in Risk of post-menopausal breast cancer risk. In addition, studies on NOS3 Glu298Asp polymorphism and tumor characteristics or prognosis have generally reported inconsistent results (48,66,69–72). It is probably that the complicated NO functions, as well as study-related issues such as heterogeneity of study population, sample size, selection of cases and controls and confounding may underlie the inconsistent results across studies. In the present study, we did not observe a significant relationship between NOS3 Glu298Asp and breast cancer risk, but we did find that this polymorphism was associated with the risk of post-menopausal breast cancer among subgroups of women defined by smoking status or intensity of smoking. Experimental studies have shown that eNOS produces low levels of endogenous NO (5), and NOS3 Glu298Asp polymorphism is related to reduced basal NO production (38). Thus, non-smoking women with variant NOS3 genotype might experience the protective effects of endogenous NO at low or moderate concentrations (10–12,15). However, among smokers, the cellular effects of NO may be mediated by a number of plausible mechanisms. First, smoking can generate a large amount of exogenous NO which, when inhaled, may immediately dilate pulmonary vessels and facilitate the delivery and absorption of tobacco carcinogens (73). Secondly, smoking can oxidize tetrahydrobiopterin (BH4), an essential cofactor of eNOS, which causes the enzyme to produce ROS instead of NO leading to increased oxidative stress (7,29). Finally, smoking-induced oxidative stress can damage eNOS enzyme and thus decrease endogenous synthesis of NO, which may result in diminished endogenous NO concentration and deprive smokers from the protective mechanism of endogenous NO (22,24). In this study, we unexpectedly observed an association between NOS3 variant alleles among women who started smoking at a later, rather than younger, age. Some previous studies have shown that smoking at an earlier age is associated with increased breast cancer risk, possibly because breast epithelial cells are not fully differentiated at early ages and therefore are more vulnerable to tobacco carcinogens and DNA damage (74). Although the mechanisms are unclear, it is possible that the findings are related to an interplay of smoking, estrogen and NO at an earlier age. Smoking is known to be antiestrogenic (75–79) and to inhibit endogenous NO (21,28,80), and estrogen is capable of up-regulating eNOS and inducible NOS enzymes (12,81,82). Women who smoked at an earlier age may thus be exposed to increased smoking-related carcinogenesis but decreased estrogen-related carcinogenesis. Women with variant NOS3 genotype may also experience the protective effect of the endogenous NO because it may be maintained at a low to moderate level under the influence of smoking and estrogen. Women with the variant genotype who smoked at a later age may be more susceptible to developing breast cancer because estrogen-driven carcinogenesis may dominate during the early ages without being repressed by smoking. The high levels of estrogen at early ages may also significantly up-regulate eNOS and inducible NOS, leading to high concentrations of NO and potential carcinogenic effects (16–19). However, it is also possible that these findings are due to chance and further replication is needed. We did not observe a significant association between MPO genotypes and post-menopausal breast cancer risk, nor modification by cigarette smoking. Previously, the variant A allele has been associated with reduced risk of lung cancer (55–57), ovarian cancer (58), bladder cancer (59) and liver cancer (60). However, a large nested case– control study in a Finnish clinical trial involving 29 133 Caucasian male smokers did not support an association between the variant A allele and lung cancer risk (83), and another case–control study showed that the homozygous variant genotype was marginally associated with increased risk of lung cancer among current smokers (adjusted OR 5 2.38, 95% CI 5 0.9–6.2) but not among former and never smokers (84). We (C.B.A) have reported previously that the variant A allele was significantly associated with a reduced risk of breast cancer among pre-menopausal women with higher consumption of fruits and vegetables, but with a non-significant increased risk among post-menopausal women at low levels of fruit and vegetable consumption (61). However, these findings were not observed in a Chinese population (62). It is probably that the discrepancies in sample size, study population, variable measurements and analytic strategies may partly explain the inconsistent findings. Furthermore, the heterogeneous findings may imply that phenotypic effects of MPO may vary depending upon levels of exposure to factors that may increase or decrease cancer risk. Brockstedt et al. (85) measured the levels of DNA adducts in human breast tissue and found that DNA adducts were significantly higher in women with MPO A alleles than in women with homozygous GG genotypes. In contrast, Van Schooten et al. (86) found that the levels of DNA adducts in the bronchoalveolar lavages were significantly lower in smokers with A alleles than in smokers with GG genotypes. Although these laboratory findings need additional confirmation, they imply that MPO activity might vary in different target tissues at different levels of carcinogen exposure. 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