1 Division of Clinical Epidemiology, University Hospital, Geneva, Switzerland.
2 Division of Medical Genetics, University Hospital, Geneva, Switzerland.
3 Division of Radiooncology, University Hospital, Geneva, Switzerland.
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ABSTRACT |
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breast neoplasms; case-control studies; genes; tobacco; tobacco smoke pollution
Abbreviations: CI, confidence interval; NAT2, N-acetyltransferase 2 gene; OR, odds ratio; RFLP, restriction fragment length polymorphism.
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INTRODUCTION |
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Indeed, six studies (38
), recently reviewed by Wells (9
), have been conducted or reanalyzed according to this methodological recommendation. Five of them (3
7
) were based on incident cases and consistently show that women exposed to passive smoking are at increased risk of breast cancer relative to women never exposed to either active or passive smoke (10
). The association of breast cancer was of similar magnitude with passive smoking (odds ratio (OR) = 2.0, 95 percent confidence interval (CI): 1.5, 2.6) and with active smoking (OR = 2.2, 95 percent CI: 1.6, 2.9).
It has also been found that women who are active smokers and slow acetylators due to their N-acetyltransferase 2 (NAT2) genotype were at increased risk of breast cancer (11), suggesting that slow acetylation of aromatic amines may lead to a longer or greater exposure to some of the potentially carcinogenic compounds than fast acetylation. However, of the three studies (12
14
) that tried to reproduce the findings of Ambrosone et al. (11
), none showed higher relative risks among slow acetylators as clearly.
We had postulated that the NAT2-smoking interaction could explain the similar magnitude of effect of passive and active smoking on breast cancer risk; that is, women who develop breast cancer as a consequence of passive smoking would be more likely to be slow acetylators (15). We tested this hypothesis by obtaining buccal scrapes and isolating the desoxyribonucleic acid (DNA) from all surviving cases and from a random sample of age-matched controls drawn from the case-control study conducted in 19921993.
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MATERIALS AND METHODS |
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The present gene-smoking interaction study was conducted in 19961997. As no biologic material was collected in the original study (19921993) in view of a genetic study, we first identified the surviving cases from a computerized database indicating whether a person had remained a resident of Canton Geneva and, if applicable, whether the person was still living. We could identify 205 cases (84 percent) still alive and residing in Geneva. A group of frequency-matched controls were randomly selected so that the proportion of controls within each 10-year age group was similar to that of the cases. Of the 349 women selected, 287 (82.2 percent) were still residing in Geneva and eligible. A letter describing the aim of the study was sent to all eligible subjects. In case of nonresponse, we tried to contact every eligible person by telephone.
Participants were asked to come to the Division of Clinical Epidemiology of the Geneva University Hospital. All women signed an informed consent for DNA analysis. Cells were collected from the cheek mucosa using a cytologic brush. This approach minimized nonparticipation by eligible subjects.
Procedures at the laboratory
Buccal cells were sampled with two cytologic brushes per patient. After sample collection, the brushes were put immediately into 400 µl of 6 M sodium iodide. Samples were stored at 4°C before extraction. Total DNA was extracted by binding to a silica matrix (Prepagene; Bio-Rad, Hercules, California) in groups of 96 samples to allow manipulation in a microtiter plate format (QIAamp 96 DNA blood kit; Qiagen AG, Basel, Switzerland). DNA was extracted up to 6 months after sampling, with no apparent effect on efficiency. Two separate extractions were performed for each person. DNAs were stored in 10 mM Tris/10.1 mM ethylene diamine tetraacetic acid, at 4°C and at -80°C (duplicates).
Three polymorphisms of the arylamine-N-acetyltransferase 2 gene (NAT2) were analyzed by nested polymerase chain reaction and restriction digestion. A 900-base pair fragment containing the entire coding region of NAT2 was amplified (30 cycles, standard conditions); then 1 µl of this product was subjected to nested polymerase chain reaction to amplify a 740-base pair fragment containing the sites of the three functionally important polymorphisms 481 (KpnI restriction fragment length polymorphism (RFLP)), 590 (TaqI RFLP), and 857 (BamHI RFLP). The polymerase chain reaction primers were the following: first polymerase chain reaction, NAT2CL (GCC ATG GAG TTG GGC TTA GAG), NAT2R (GTG AGT TGG GTG ATA CAT ACA CAA G); nested polymerase chain reaction, NAT2DL (ATT TTT GAT CAC ATT GTA AGA AGA), NAT2CR (CAT ACA CAA GGG TTT ATT TTG TTC C). The polymorphic sites were analyzed by serial digestion with the appropriate restriction enzymes followed by electrophoresis on horizontal 12 percent polyacrylamide gels (GenePhor; Amersham Pharmacia Biotech, Piscataway, New Jersey). Samples were coded so that the laboratory was unaware of the case-control status. They were analyzed twice, and the gels were interpreted independently, with complete concordance. A total of 347 samples were given to the laboratory, of which 330 (95 percent) were successfully amplified. The failure to amplify 5 percent of samples was a consequence of using cheek scrapes as source material and was deemed acceptable. The restriction digestion results of 94 samples could not be unequivocally interpreted, and these samples were analyzed by direct sequencing of polymerase chain reaction products using Thermosequenase and an ALF Express DNA sequencer (Pharmacia). Unequivocal results were thus obtained for 322 samples (93 percent).
Statistical methods
Information concerning exposure to tobacco smoke was obtained from the original interview. Ever smokers had smoked at least 100 cigarettes in their lifetime. Among ever smokers, current smokers were women who reported having smoked regularly within the 2 years preceding interview. Passive smokers were defined as women who had been exposed at least 1 hour per day during 1 year or more either at home, at work, or during leisure time.
A woman was defined as a slow acetylator if she was homozygous for one, or heterozygous for two, of the three following NAT2 polymorphisms: 481C>T (KpnI RFLP), 590G>A (TaqI), and 857G>A (BamHI). This definition assumed that there could be no single allele with two or more of the tested mutations (11, 16
).
The odds ratios for developing breast cancer can be interpreted as relative risks of breast cancer. They were estimated using a logistic regression model that included as potential confounders the three variables that appeared to be related to breast cancer in the full sample, that is, age, education, and positive family history of breast cancer. Analyses were also stratified by menopause, defined as having had the last menses at least 1 year before interview or a bilateral ovariectomy.
A possible interaction among NAT2, smoking, and breast cancer was investigated using multiple logistic regression. Multiplicative interaction was assessed by adding a product term between NAT2 status and each smoking exposure. The interaction coefficient can be interpreted as the log of the ratio of the smoking by breast cancer odds ratios in fast relative to slow acetylators. Additive interaction was assessed by the relative excess risk due to interaction that was estimated (with its 95 percent confidence limits) according to the method of Hosmer and Lemeshow (17).
As DNA could not be analyzed for 25 women (10 cases and 15 controls), we performed a sensitivity analysis with four hypothetic scenarios: 1) all 25 missing subjects were fast acetylators; 2) all 25 missing subjects were slow acetylators; 3) missing cases were slow acetylators but controls were fast acetylators; and 4) missing controls were slow acetylators but cases were fast. Results of the four scenarios were highly consistent with those obtained without the 25 women.
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RESULTS |
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Of the randomly sampled subgroup of 287 control women, 177 (62 percent) participated. DNA was successfully analyzed in 160 cases and 162 controls (93 percent). Except for age matching and for a lower participation of women with primary education, characteristics of the present sample of controls were very similar to those of the nonparticipants.
Table 1 shows that the compared groups had the same age distribution. There were a reduction in risk of breast cancer with older age at menarche and an increased risk in women with age at first birth between 25 and 29 years and in women with a family history of breast cancer.
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When pooling premenopausal and postmenopausal women, we found the adjusted odds ratio of breast cancer to be 3.1 (95 percent CI: 1.5, 6.0) for passive smokers and 3.3 (95 percent CI: 1.7, 6.5) for active smokers (not shown in a table). After stratification for NAT2 (table 2), both active and passive smoking increased breast cancer risk, with higher relative risks observed for the fast acetylation genotype. The one apparent exception, the current smokers of less than 20 cigarettes per day, may be an effect of the small size of this subgroup.
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We also categorized passive smokers according to the number of hours-per-day-years (3) of exposure, but these analyses did not lead to different interpretations (not shown). The median age of first exposure was 20 years for passive and 18 years for active smoking. We used these cutoffs to determine whether the effect of age at first exposure differed according to NAT2 status. Nonexposed women were not included in these analyses. The odds ratios for first exposure to passive smoking at age 20 or before (relative to after age 20) were 1.6 (95 percent CI: 0.7, 3.9) in slow acetylators and 0.6 (95 percent CI: 0.2, 1.7) in fast acetylators (p for interaction = 0.15). The corresponding odds ratios for first active smoking at age 18 or before (relative to after age 18) were 1.2 (95 percent CI: 0.6, 2.7) in slow and 0.4 (95 percent CI: 0.2, 1.1) in fast acetylators (p for interaction = 0.10).
In order to compare the present results with those of previous reports that lacked information on passive smoking (1114
), we repeated the analyses after grouping the nonexposed and the passive smokers in a single category. Among premenopausal women, there was no significant association for active versus never-active smokers in either slow (OR = 1.3, 95 percent CI: 0.5, 3.0) or fast (OR = 1.5, 95 percent CI: 0.6, 4.3) acetylators. In contrast, among postmenopausal women, active smoking was a risk factor for breast cancer in slow acetylators (OR = 2.5, 95 percent CI: 1.0, 6.2) but not in fast acetylators (OR = 1.3, 95 percent CI: 0.5, 3.3). The test for multiplicative interaction had p = 0.30.
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DISCUSSION |
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We expected to find a stronger association of smoking and breast cancer in slow acetylators (15), but we found instead more elevated relative risks for both passive and active smoking in fast acetylators, especially among postmenopausal women. If true, these results suggest that the carcinogenic substrate is not aromatic amines (which are found only as traces in tobacco smoke) but heterocyclic amines that are more concentrated in cigarette smoke than bicyclic aromatic amines and are at least 10 times more pres-ent in the sidestream than in the mainstream smoke. The ratio of sidestream to mainstream smoke is probably even greater in cigarettes with a perforated filter tip that is usually smoked by women. Heterocyclic amines, such as pyridine, are N-hydroxylated by cytochrome P450 1A2 and subsequently activated by O-acetylation by NAT2 to form electrophilic intermediates and DNA adducts that can initiate cancer (19
, 20
). If heterocyclic amines were the culprit, then fast acetylators would be most at risk. An analogy can be made here with colon cancer, which seems to be caused by dietary heterocyclic amines and for which a higher risk in fast acetylators has been consistently reported (21
). A relation of breast cancer to dietary heterocyclic amines is not well established (22
) but is strongly supported by the results of the Iowa Women's Health Study (23
).
Independently of the environmental agent involved, the equivalence of relative risk of breast cancer for passive or active smoking consistently reported (38
) remains counterintuitive. A priori, passive smokers are less exposed than active smokers who inhale their own and other people's cigarettes' sidestream smoke in addition to drawing puffs of concentrated smoke aerosols. Many compounds of intermediate tobacco combustion are more concentrated in the sidestream than in the mainstream smoke (when titrated by smoke collector devices), but the inhaled concentration is much diluted in the ambient air. It is, however, also important to stress that the magnitude of the exposure of the passive smoker is often underestimated. We have reported that the average lifetime exposure to passive smoke of the present study control population was equivalent to 30.8 hours/week during 10 years or 20.5 hours/week during 15 years (24
).
Analogic thinking with the tobacco-lung cancer association also leads to expecting a huge relative risk differential between active and passive smokers. The lung cancer model may, however, not be relevant for the mammary gland that is not directly exposed to tobacco smoke. Carcinogens need to circulate in the blood before reaching the gland and seem to be able to remain for a very long time in the mammary epithelial cells (25, 26
). Environmental tobacco smoke particles are small and therefore poorly filtrated by the lung. The risk differential between passive and active smokers may therefore be smaller for breast than for lung cancer.
In addition, the relative risk observed for passive smokers may also be magnified by the low dose effect hypothesis formulated by Vineis and McMichael (21) for colon cancer: the modifying effect of a genotype or phenotype can be more evident at low dose. In the present study, it is reasonable to conceive that high levels of exposure to tobacco smoke saturate the NAT2 enzyme activity, resulting in formation of abundant DNA adducts from O-acetylation of hydroxylated human carbonic anhydrase in both slow and fast acetylators. In contrast, at low doses of exposure to tobacco, DNA adducts may accumulate only among fast acetylators, because, among slow acetylators, the N-acetoxy derivatives are either produced in too small a quantity or detoxified at the same rate they are produced.
Whether the first exposure to tobacco as a teenager determines the risk of breast cancer remains elusive (2730
). The present study shows a reduced risk of young age at first exposure to active and passive smoking among fast acetylators. Larger studies of gene-smoking interactions are needed in populations where it is not uncommon that women start to smoke before age 16.
An important observation from the present study for future research on the relation of smoking to breast cancer is that taking passive smoking into account had dramatic consequences on inference. It was intriguing to observe that, when active smokers were compared with never-active smokers, a reference category that includes both unexposed women and passive smokers, results suggested that postmenopausal slow acetylators were at higher risk of breast cancer if they smoked. This conclusion is consistent with the study of Ambrosone et al. (11). On the other hand, when nonactive, nonpassive smokers were used as the reference category, analyses led to the opposite conclusion; that is, postmenopausal fast acetylators were at higher risk of breast cancer if they smoked. We consider the latter results as being more valid, because it is logical to use the lowest level of exposure to tobacco as the reference category, especially in view of the epidemiologic evidence of a specific effect of passive smoking (3
8
).
Given the small number of cases, confidence intervals were wide, and only very large effects could be detected with sufficient statistical power. This limitation was particularly problematic for the analyses of interaction. Even in the multiplicative model for passive smoking, the low statistical power did not allow us to establish whether the interaction was quantitative (that is, association present in both fast and slow acetylators but with a difference of magnitude) or qualitative (that is, association only among fast acetylators). Thus, larger studies are needed to elucidate gene-smoking interactions with statistical confidence.
Other limitations include the unexpected reduction in risk of breast cancer observed in the more educated women (OR = 0.4, 95 percent CI: 0.2, 0.9) that is inconsistent with what is usually known about the etiology of this disease (31) and that can be attributed to the differential participation of educated women between cases and controls. This imbalance was taken into consideration in the analysis by adjusting the odds ratio for education and does not seem to have generated substantial bias, since the observed effects for smoking or other risk factors of breast cancer were consistent with our previous findings (3
) and with what is usually found by others (31
).
There could have been concerns about survival bias, since biologic material was collected among survivors of the original study. However, given the high survival rate (84 percent), it is unlikely that results would have been markedly different had the DNA specimens been collected at the time of the original study.
It has been reported that smoking may reduce the risk of breast cancer in women with mutant BRCA1 or BRCA2 (32). This finding is only marginally relevant for epidemiologic investigations in the general population because less than one in every 500 women in the general population carries these mutant genes (33
).
In conclusion, the risk of breast cancer related to smoking appears to be stronger in fast acetylators of aromatic amines. Separating passive smokers from the nonexposed has major implications on the inference about a possible NAT2-smoking interaction.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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