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Endothelial nitric oxide synthase (eNOS), encoded by the Nos3 gene, catalyzes the production of free radical nitric oxide (NO). NO is a mediator of follicle growth and ovulation, and plays a role in the release of endocrine hormones including LHRH. eNOS deficient mice exhibit late menarche, reduced ovulation, and early menopause. In humans, estradiol stimulates eNOS activity through an Akt/PKB dependent pathway and, in postmenopausal women, reduced endometrial expression of eNOS has been reported, which is reversible by hormone replacement therapy. eNOS overproduction is also involved in carcinogenesis. A common G894T polymorphism in exon 7 of Nos3 results in a Glu298Asp substitution that alters susceptibility to cleavage, leading to reduced NO levels. We hypothesized that the Glu298Asp polymorphism was associated with reduced breast cancer (BC) risk. A nested case-control study was conducted in the American Cancer Society Cancer Prevention Study II Nutrition Cohort involving 505 postmenopausal BC cases and 503 controls, matched on age, ethnicity, and date of blood collection. Information on demographics, reproductive history, diet, and other lifestyle factors was collected by a self-administered questionnaire prior to cancer diagnosis. DNA was extracted from buffy coats, and genotyped for the Glu298Asp variant using Taqman (Applied Biosystems, Foster City, CA). Unconditional logistic regression and χ2 tests were performed to examine associations between Nos3 genotype and cancer risk. We observed no association between the presence of at least one variant Asp allele and age at menarche, age at natural menopause, or number of full-term births or miscarriages. Allele frequencies did not differ between cases and controls. The presence of at least one variant Asp allele was not associated with BC risk before (50.4% vs. 51.3%; OR=0.96; 95% CI: 0.75-1.24; p=0.77) or after adjustment for body mass index, age, ethnicity, family history of BC, and history of benign breast disease (OR=0.99; 95% CI=0.77-1.29; p=0.95). The association between Nos3 genotype and BC risk may, however, be modified by smoking status (interaction χ2=3.34, p=0.07). Amongst never smokers, there was a borderline inverse association between Asp alleles and BC risk (OR=0.72; 95%CI: 0.51-1.02; p=0.07), while ever smokers showed a non-significant increase in risk (OR=1.30, 95%CI=0.90-1.89; p=0.17). Oxidation of BH4, an essential cofactor of eNOS, by free radicals in cigarette smoke can uncouple the eNOS enzyme and cause the enzyme to produce oxygen free radicals instead of NO, thereby increasing oxidative stress. This is supported by lower levels of NO in endothelial cells incubated with serum from smokers compared to non-smokers. In summary, we found no support for a main effect between the Nos3 genotype and reduced breast cancer risk, but our results suggest a biologically plausible interaction with smoking that should be examined in larger studies.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]