Carcinogenesis vol.28 no.6 pp.1247–1253, 2007 doi:10.1093/carcin/bgm016 Advance Access publication January 21, 2007

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 Ahn
1
, Carmen Rodriguez
2
, Michael J.Thun
2
and
Eugenia E.Calle
2
Department of Cancer Prevention and Control, Roswell Park Cancer Institute,
Buffalo, NY 14263, USA,
1
Nutritional Epidemiology Branch, Division of
Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD
20892, USA and
2
Epidemiology 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]ginia.gov
NOS3 and MPO genes encode endothelial nitric oxide synthase
and myeloperoxidase (MPO), respectively, which generate nitric
oxide and reactive oxygen species. Because cigarette smoking gen-
erates reactive species, we hypothesized that NOS3 and MPO
polymorphisms could influence susceptibility to breast cancer,
particularly among smokers. We examined the associations be-
tween 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 50.67, 95% CI 50.45–0.99), but were associated with high-
er risk among ever smokers (OR 51.59, 95% CI 51.05–2.41) and
2-fold increase in risk for those who smoked .10 cigarettes per
day (OR 52.19, 95% CI 51.21–3.97). 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 as-
sociated with lifetime estrogen exposure, age at first full-term preg-
nancy, 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 micro-
environment such as those related to oxidative stress and genes me-
tabolizing 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 oxi-
dative mechanism and carcinogen metabolism (3,4).
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 (BH
4
), an essen-
tial eNOS cofactor (5–7). NO has multiple physiological functions,
including vasodilatation, neuronal transmission, smooth muscle re-
laxation 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 relax-
ation, 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 metal-
loproteins and preventing lipid peroxidation (15). At high concentra-
tions, NO and its derivatives can cause DNA strand breaks and impair
the tumor suppressor function of p53, which may lead to carcinogen-
esis (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 reduc-
tion 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 sub-
stantially alleviated after treatment with antioxidants such as vitamin
C (27,29). Smoking is well known to increase oxidative stress. Oxi-
dative damage measured by 8-hydroxydeoxyguanosine (8-OH-dG),
protein carbonyls and isoprostanes, markers for damage to DNA, pro-
teins 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 de-
composed (33–35). Cigarette smoking can also deplete tetrahydro-
biopterin (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, includ-
ing 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 dif-
fering susceptibility to cleavage in eNOS enzyme in transfected cells
and human endothelial cells (37), related to reduced basal NO pro-
duction (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, partic-
ularly 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 poly-
morphism (51).
Myeloperoxidase (MPO) is a lysosomal enzyme abundant in neu-
trophils, 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 het-
erocyclic 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
Abbreviations: CI, confidence intervale; NOS, endothelial nitric oxide syn-
thase; MPO, myeloperoxidase; NO, nitric oxide; OR, odds ratio; ROS, reactive
oxygen species.
ÓThe Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]g 1247

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 associ-
ated 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 associ-
ated with reduced breast cancer risk among pre-menopausal women
who consumed higher amount of fruits and vegetables, but with a non-
significant 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 Nu-
trition 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 question-
naire that included demographic information, cigarette smoking his-
tory, 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 par-
ticipants every 2 years beginning in 1997 to update exposure infor-
mation 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 sam-
ple, 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.
Laboratory methods
DNA was extracted from buffy coat, and genotyping assays were per-
formed using TaqMan (Applied Biosystems, Foster City, CA). Ge-
netic 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 Infor-
mation 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 mod-
els 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 re-
gression 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 veg-
etable 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 po-
tential modification effects on associations between genetic polymor-
phisms 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 512.0)
as 10 versus .10 cigarettes and pack-years of smoking by 10
(median 511.7) 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 Pvalue and Pfor 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 initi-
ation did not change the results. When cigarette smoking was taken
into account, the association with NOS3 was modified by tobacco
smoke exposure (P50.01 for interaction test). Among never smok-
ers, 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 50.45–0.99, Pfor trend 50.10), although of borderline
significance; in contrast, an increase in risk was observed among ever
smokers with T alleles (adjusted OR 51.59, 95% CI 51.05–2.41,
Pfor trend 50.02) compared with those with GG genotypes
(Table II). No significant interaction was found between MPO gene
polymorphism and smoking status (P50.43 for interaction test)
(Table II).
J.Yang et al.
1248

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 52.19, 95% CI 51.21–3.97, Pfor 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 51.15, 95% CI 50.62–2.15). For dura-
tion and pack-years of smoking, the relative risks were elevated but
were not substantially different between women with different dura-
tion 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 52.41, 95% CI 5
1.27–4.59, Pfor interaction 50.11) (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 be-
tween NOS3 Glu298Asp and MPO G-463A polymorphisms and post-
menopausal breast cancer risk. However, we found that NOS3
Glu298Asp polymorphism was associated with reduced risk of post-
menopausal breast cancer among women who never smoked and with
Table I. NOS3 Glu298Asp and MPO G-463A polymorphisms and risk of post-menopausal breast cancer, American Cancer Society, 1992–2001
Cases
a
(n5502) Controls
a
(n5505) OR (95% CI)
b
OR (95% CI)
c
Wald v
2c
Pvalue
c
Pfor trend
c
NOS3
GG 204 (48.8%) 199 (48.7%) 1.00 1.00 1.17 0.56
GT 168 (40.2%) 176 (43.0%) 0.94 (0.72–1.22) 0.96 (0.71–1.28) 0.61
TT 46 (11.0%) 34 (8.3%) 1.15 (0.73–1.81) 1.26 (0.77–2.06)
GT and TT 214 (51.2%) 210 (51.3%) 0.97 (0.75–1.25) 1.01 (0.76–1.33) 0.003 0.96
MPO
GG 238 (58.6%) 245 (62.5%) 1.00 1.00 2.56 0.28
GA 149 (36.7%) 135 (34.4%) 1.07 (0.82–1.41) 1.11 (0.82–1.50) 0.16
AA 19 (4.7%) 12 (3.1%) 1.55 (0.79–3.03) 1.81 (0.84–3.87) 1.10 0.30
GA and AA 168 (41.4%) 147 (37.5%) 1.11 (0.86–1.45) 1.17 (0.87–1.56)
a
Numbers in the cells are smaller than the total sample size due to missing values in the covariates.
b
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.
Table II. NOS3 Glu298Asp and MPO G-463A polymorphisms and risk of post-menopausal breast cancer by smoking status, American Cancer Society, 1992–
2001
Cases
a
(n5502) Controls
a
(n5505) OR (95% CI)
b
Wald v
2
Pvalue Pfor trend Interaction
Never smokers
NOS3
GG 117 105 1.00 4.34 0.11 0.10 v
2
58.96
GT 80 108 0.65 (0.43–0.97) P50.01
TT 17 19 0.78 (0.37–1.63)
GT and TT 97 127 0.67 (0.45–0.99) 4.11 0.04
Ever smokers
NOS3
GG 87 94 1.00 5.53 0.06 0.02
GT 88 68 1.49 (0.96–2.31)
TT 29 15 2.06 (1.01–4.18)
GT and TT 117 83 1.59 (1.05–2.41) 4.81 0.03
Never smokers
MPO
GG 120 130 1.00 1.05 0.59 0.60 v
2
51.70
GA 78 83 0.98 (0.65–1.49) P50.43
AA 10 9 1.65 (0.62–4.37)
GA and AA 88 92 1.04 (0.70–1.55) 0.04 0.84
Ever smokers
MPO
GG 118 115 1.00 3.51 0.17 0.07
GA 71 52 1.35 (0.85–2.14)
AA 9 3 2.84 (0.73–11.1)
GA and AA 80 55 1.44 (0.92–2.24) 2.52 0.11
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.
Risk of post-menopausal breast cancer
1249

increased risk among women who smoked or smoked at higher in-
tensity (.10 cigarettes per day). There were no significant relation-
ships 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 prop-
erty 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 micro-
environment and timing of production. In a recent review, Lancaster
et al. (3) noted that heterogeneity in the chemical process, biological
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), co-
lorectal cancer (67) and prostate cancer (68). For breast cancer, Ghi-
lardi 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
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
Cases
a
(n5502) Controls
a
(n5505) OR (95% CI)
b
Wald v
2
Pvalue Pfor trend Interaction
Duration of smoking
c
NOS3, 20 years
GG 41 47 1.00 2.39 0.30 0.14 v
2
50.34
GT 43 36 1.55 (0.81–2.98) P50.84
TT 16 9 1.79 (0.68–4.72)
GT and TT 59 45 1.61 (0.87–2.95) 2.31 0.13
NOS3, .20 years
GG 45 46 1.00 4.75 0.09 0.03
GT 43 31 1.73 (0.89–3.37)
TT 13 5 3.00 (0.92–9.84)
GT and TT 56 36 1.92 (1.02–3.60) 4.08 0.04
Cigarettes per day
d
NOS3, 10 CPD
GG 44 42 1.00 0.43 0.81 0.54 v
2
53.08
GT 33 30 1.08 (0.55–2.12) P50.21
TT 13 9 1.40 (0.51–3.83)
GT and TT 46 39 1.15 (0.62–2.15) 0.19 0.66
NOS3, .10 CPD
GG 42 51 1.00 7.73 0.02 0.005
GT 53 37 1.96 (1.05–3.64)
TT 16 5 3.87 (1.22–12.26)
GT and TT 69 42 2.19 (1.21–3.97) 6.65 0.01
Pack-years of smoking
NOS3, 10 PYS
GG 36 40 1.00 1.87 0.39 0.18 v
2
50.46
GT 36 32 1.29 (0.65–2.56) P50.79
TT 12 7 2.13 (0.69–6.58)
GT and TT 48 39 1.43 (0.75–2.72) 1.16 0.28
NOS3, .10 PYS
GG 50 53 1.00 5.38 0.07 0.02
GT 50 35 1.69 (0.92–3.11)
TT 17 7 2.76 (1.01–7.55)
GT and TT 67 42 1.88 (1.06–3.33) 4.62 0.03
Age at smoking initiation
e
NOS3, 18 years
GG 52 46 1.00 0.47 0.79 0.87 v
2
54.46
GT 43 43 0.90 (0.48–1.68) P50.11
TT 13 7 1.32 (0.44–3.95)
GT and TT 56 50 0.96 (0.53–1.74) 0.02 0.90
NOS3, .18 years
GG 34 47 1.00 7.41 0.02 0.009
GT 43 24 2.24 (1.12–4.48)
TT 16 7 3.01 (1.07–8.50)
GT and TT 59 31 2.41 (1.27–4.59) 7.16 0.007
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.
J.Yang et al.
1250

risk. In addition, studies on NOS3 Glu298Asp polymorphism and
tumor characteristics or prognosis have generally reported inconsis-
tent 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 con-
founding 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 concen-
trations (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, smok-
ing can oxidize tetrahydrobiopterin (BH
4
), 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 endog-
enous synthesis of NO, which may result in diminished endogenous
NO concentration and deprive smokers from the protective mecha-
nism 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 differenti-
ated at early ages and therefore are more vulnerable to tobacco carci-
nogens 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 anti-
estrogenic (75–79) and to inhibit endogenous NO (21,28,80), and
estrogen is capable of up-regulating eNOS and inducible NOS en-
zymes (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 be-
cause 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 possi-
ble that these findings are due to chance and further replication is
needed.
We did not observe a significant association between MPO geno-
types 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), blad-
der 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 asso-
ciated with increased risk of lung cancer among current smokers
(adjusted OR 52.38, 95% CI 50.9–6.2) 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 consump-
tion 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 sam-
ple size, study population, variable measurements and analytic strat-
egies 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 in-
crease 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.
In summary, we found that the Glu298Asp polymorphism in NOS3
was associated with reduced risk of post-menopausal breast cancer
among women who never smoked, but with increased risk among
women who ever smoked or smoked at a higher intensity. These data
support the hypothesis that there may be potential interactions be-
tween genetic status, smoking and breast cancer risk, and that women
whose genotypes favor the generation of ROS and activation of carci-
nogens may be susceptible to developing breast cancer when exposed
to tobacco smoke. Further investigations of relationships between
genes in oxidative stress pathways, smoking and breast cancer risk
are clearly warranted.
Acknowledgements
We are thankful to Ms Janet Hildebrand, Dr Yulin Li and Dr Ji-Yeob Choi for
their suggestions for data analysis.
Conflict of Interest Statement: None declared.
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