5. TRADITIONAL AND SUSPECTED RISK FACTORS FOR BREAST CANCER

5.1. Diet While ecological and animal studies indicate that dietary fat intake may increase risk, cohort and case-control studies generally do not supported this hypothesis (38). Investigators have also studied possible associations with other related variables, such as total calories, animal fat and meat consumption (10,39,40), with mixed findings. There have been somewhat consistent data to indicate that a diet high in fruits and vegetables decrease risk of breast cancer, and the data strongly suggest moderate alcohol intake is associated with increased risk (41,42). Whether or not associations between these factors and risk are of the same magnitude for all women, however, has not been established. It is likely that for some women, the deleterious or protective effects of diet are more pronounced than for other women, based on metabolic variability.

5.2. Animal Products, Dietary Fat, and Heterocyclic Amines Studies of the consumption of animal products, particularly meat, have yielded inconsistent results, although a meta-analysis of 5 cohort and 12 case-control studies by Boyd and colleagues revealed a summary relative risk of 1.54 [95% Confidence interval (CI) 1.31-1.82] associated with consumption of red meat (43). A more recent investigation, however, involving a pooled analysis of cohort studies found no association between meat consumption and breast cancer (40). The assessment of meat as a risk factor for breast cancer has focused primarily on its role as a source of dietary fat or animal protein. Dietary fat intake has long been hypothesized to be associated with breast cancer risk (44) based on animal studies (45), ecologic studies (46,47), and studies of migrants from areas with low fat intake to those with high fat intake (48). However, many analytic epidemiological studies have not shown an effect of fat, including the results of a pooled analysis of seven cohort studies (38). Recently, it has been suggested that diet in childhood and at the time of puberty may be of importance (49). Evidence from animal studies suggests that only fat intake before the first pregnancy affects risk (50). It is possible that the failure to identify an association of fat intake with breast cancer in epidemiological studies may be because intake early in life, rather than recent consumption, is most important. Failure to detect an association may also be due to fact that there is not enough variability in fat consumption within populations (i.e., there are too few individuals with low intakes) (51,52), or because of measurement error inherent in dietary questionnaires (52). It may also be that specific types of dietary fat are more important than total fat, and investigators have not been evaluating the proper variables.

Blood levels of lipoproteins have been investigated in relation to breast cancer etiology as a potential mediating factor on the relationship between dietary fat and risk and as an independent risk factor. The associations between serum and plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides have been widely studied, but results from these investigations are inconsistent.

The apoE protein plays an important role in lipid metabolism (53) and has three common isoforms (E2, E3, and E) coded by the alleles e2, e3, and e4. In general, compared to individuals with the e3 allele, levels of total and LDL cholesterol tend to be lower for those with the e2 allele and higher for those with the e4. The e4 allele has been associated with increased risk for coronary heart disease (54,55) and Alzheimer’s disease (56) and has been found to be underrepresented in elderly populations

(57), including elderly coronary heart disease patients (55) and elderly smokers (58). With respect to breast cancer, Moysich et. al. (59) reported that women with the highest serum triglyceride levels had an increase in risk compared to women with the lowest levels. This effect was not apparent among women with the e2 or e3 alleles, but much stronger among women with at least one e4 allele, suggesting that the apoE 4 genotype may modify the association between serum triglycerides and breast cancer risk.

If meat consumption does increase breast cancer risk, it may not be due to its fat content, but rather to other components. Three recent studies found that breast cancer risk was significantly increased by consumption of meat, after controlling for total fat or protein (39,60,61). It is possible that meat consumption may impact breast cancer risk as a result of mutagens and carcinogens, such as heterocyclic amines, which are formed in the cooking of meats and are potent mammary mutagens and carcinogens in rodent models (62,63). One of the most abundant heterocyclic amines, PhIP, has been detected in breast milk indicating direct exposure of ductal epithelial cells to this potent mutagen (64-66). Ultimate levels of heterocyclic amines depend on cooking method, cooking time, cooking temperature, and protein source (63).

In addition, metabolism of heterocyclic and aromatic amines varies among individuals and depends, in part, on polymorphisms in genes involved in their metabolism, such as N-acetyltransferases NAT1 and NAT2 and cytochrome P4501A2 (CYP1A2) (67). Several polymorphic sites have been identified at the NAT2 locus, and result in decreased N-acetyl-transferase activity (68). Slow NAT2 acetylation of aromatic amines is associated with increased risk for bladder cancer (69) and may increase postmenopausal breast cancer risk associated with cigarette smoking (70). Heterocyclic amines appear to be poor substrates for N-acetylation at the liver, however, and rapid O-acetylation of the activated metabolites by NAT2 in the target tissue appears to be associated with increased risk of colon cancer related to consumption of red meat (71).

In a pilot study of colon cancer, Lang et al. found that individuals with rapid activation by CYP1A2 and rapid O-acetylation by NAT2 had almost three times the risk of colon cancer as those with slow phenotypes (72). More recently, LeMarchand et al. confirmed this finding in a population-based, case-control study in Hawaii (73). They found that well-done meat intake increases risk of colorectal cancer, particularly in people who inherited the rapid phenotype for both NAT2 and CYP1A2. This association was only observed among smokers, however, presumably since CYP1A2 is induced by cigarette smoking. Because heterocyclic amines appear to be mammary carcinogens, it is possible that rapid hepatic activation by CYP1A2 and further activation by NAT1 or NAT2, may be related to breast cancer risk (74-76). Thus, heterocyclic amines may be associated with increased breast cancer risk among women with rapid CYP1A2 and rapid NAT2 status. Findings from epidemiological studies, however, have been inconsistent.

We found no associations between meat consumption, NAT2, and breast cancer in a study of Caucasian women in western New York (77). However, the questionnaire used was not designed to evaluate heterocyclic amines per se, thus substantial misclassification was possible. Using Sinha’s questionnaire specifically designed for heterocyclic amine exposure, Zheng et al. (78) recently reported that consumption of well-done meats increased breast cancer risk in a dose-dependent manner. Deitz et al. also reported an elevated association between well-done meats and breast cancer risk among rapid/intermediate NAT2 acetylators (79). In a subsequent paper (80), Zheng et al. examined the role of NAT1 genetic polymorphisms and risk related to smoking and meat consumption. They reported that the NAT1* 11 allele, thought to result in rapid activation, resulted in a significant sixfold increase in breast cancer risk among women who were high consumers of red meat. Among women with low intake, there was a nonsignificant risk of less than three associated with that putative allele. Contrary to these findings, Gertig and colleagues (81) and Delfino et al. (79) did not report an increased risk with read meat consumption, and risk was not modified by NAT2 status.

5.3. Fruit and Vegetable Consumption There are fairly consistent data indicating that higher consumption of fruits and vegetables is associated with decreased breast cancer risk (82,83), although not all studies support such an association (84,85). Fruits and vegetables are sources of a number of nutrients, including antioxidant vitamins such as carotenoids, the tocopherols, and vitamin C. Several nutritional epidemiological studies have noted inverse associations between dietary antioxidants and breast cancer risk (86-88). The mechanistic relationship of these putative risk factors, however, has not been elucidated. One hypothesis is that dietary antioxidants affect oxidative stress and the production of reactive oxygen species (ROS) by altering the balance between prooxidant cellular activity and antioxidant defenses (89). Reactive oxygen species are produced by normal cellular respiration and as a result of inflammation and cellular stress (90). When ROS are the result of normal metabolism, and there is sufficient antioxidant power and repair capacity, there are presumably few harmful effects. Excessive production of ROS resulting from toxic agents, such as tobacco smoke, or from insufficient in vivo defense mechanisms, can result in oxidative stress, leading to damage to DNA and cell membranes, mitochondrion, and protein (91-96). It is also possible that a diet low in fruits and vegetables could contribute to excessive ROS and oxidative stress. Oxidative damage has been reported to be higher in women with breast cancer, compared to controls, although studies to date remain small (97-99), and these levels vary with the consumption of meats, vegetables, and fruits (100-102).

Endogenous defenses against ROS include glutathione peroxidase, catalase, and superoxide dismutase (SOD) (90). There are three known forms of SOD: the cytosolic and extracellular copper/zinc SODs and the mitochondrial manganese SOD (MnSOD). MnSOD is synthesized in the cytosol and post-transcriptionally modified for transport into the mitochondrion (103,104). In the mitochondrion, it catalyzes the dismutation of two superoxide radicals, producing H2O2 and oxygen. MnSOD is induced with free radical challenge (105) and cigarette smoke (106). Recently, two genetic variants of MnSOD were identified. A nucleotide T to C substitution in the mitochondrial targeting sequence was found that changes an amino acid. The investigators who identified the polymorphism predicted that the resulting amino acid change would alter the secondary structure of the protein (104), and Rosenblum and colleagues (107) suggested that the alteration might affect the cellular allocation of the enzyme and mitochondrial transport of MnSOD into the mitochondrion. They further suggested that inefficient targeting of MnSOD could leave mitochondria without their full defense against superoxide radicals, which could lead to protein oxidation, as well as mitchondrial DNA mutations.

We hypothesized that the polymorphism in MnSOD would result in higher levels of ROS, and that in women whose diets were low in fruits and vegetables, this polymorphism would increase risk of breast cancer (108). Interestingly, we found this to be the case, particularly for premenopausal women (108). Those who were homozygous for the variant allele had a fourfold increase in breast cancer risk in comparison to those with who were homozygous or heterozygous for the common allele [odds ratio (OR) = 4.3, 95% CI, 1.7-10.8]. Risk was most pronounced among women below the median consumption of fruits and vegetables, and of dietary ascorbic acid and a-tocopherol, with little increased risk for those with diets rich in these foods. These findings were supported by Mitrunen et al. in their study of pre- and postmenopausal Finnish women (109).

These data support the hypothesis that MnSOD and oxidative stress play a significant role in breast cancer risk, particularly in premenopausal women. The finding that risk was greatest among women who consumed lower amounts of dietary antioxidants, and was minimal among high consumers, suggests that a diet rich in sources of antioxidants may compensate for the effects of the MnSOD polymorphism, thereby supporting public health recommendations for consumption of diets rich in fruits and vegetables as a preventive measure against cancer.

5.4. Alcohol The potential effect of alcohol consumption on breast cancer risk has been widely studied in the past decades. The importance of determining such an association has been emphasized due to the major public health problem associated with breast cancer as well as the notion that alcohol consumption is fairly common, yet modifiable (110). Based on findings from two meta-analyses, there appears to be a modest increase in breast cancer risk associated with daily consumption of alcoholic beverages (42,111). Several mechanisms for a role of alcohol consumption in breast carcinogenesis have been proposed, including increases of bioavailable estrogen and direct toxic effects associated with ethanol exposure (112,113).

Recently, efforts have been made to evaluate the role of alcohol metabolizing genes as a potential susceptibility marker for the adverse effect of alcohol consumption on breast cancer risk. The polymorphic alcohol dehydrogenase 3 (ADH3) gene is involved in the oxidation of ethanol to carcinogenic acetaldehyde and plays a rate-limiting role in the metabolic pathway for most human ethanol oxidation (114). The presence of the ADH31 allele, coding for the more rapid form of the ADH3 enzyme, has previously been associated with increased risk of cancer of the oral cavity and pharynx (115,116) and of hepatic cirrhosis and chronic pancreatitis (117).

In a population-based case-control study, Freudenheim et al. (118) observed an increased risk of premenopausal breast cancer among women with the highest self-reported alcohol consumption and at least one ADH31 allele. These findings are supported by a preliminary report (119) indicating that among women with at least one ADH31 allele, those who drank alcohol were at greater risk of breast cancer compared to those who abstained. Furthermore, there was also evidence for a risk elevation for women who drank and who carried the GSTM1 null genotype and at least ADH31 allele. Hines et al. conducted a prospective study on the effect of alcohol consumption, ADH3 genotype on plasma steroid hormone levels and breast cancer risk (120). While a modest association was seen for plasma hormones and alcohol consumption, no association was found between ADH3 genotype and breast cancer risk, regardless of alcohol consumption or menopausal status.

The genetic polymorphism in cytochrome P4502E1 (CYP2E1) may also modify the association between alcohol consumption and breast cancer risk. Ethanol-inducible CYP2E1 is an enzyme of major toxicological interest because it metabolizes a wide range of environmental compounds to reactive metabolites (121). Shields et al. (122) found a smoking-associated risk elevation to be restricted to women with the CYP2E1 variant genotype, but did not investigate associations with alcohol consumption due to small numbers in groups with varying alcohol intake and variant alleles. In fact, molecular epidemiological studies on the CYP2E1 genetic polymorphism may pose substantial methodological challenges, due to the low prevalence of the CYP2E1 variant genotype in the general population.

5.5. Reproductive Factors and Hormones Because epidemiological studies indicate that key breast cancer risk factors are related to endogenous exposure to steroid hormones, intensive epidemiological research has been targeted at serum and urinary measurement of parent hormones and their metabolites in both case-control and cohort studies, yielding inconsistent results (123,124). However, measurement of serum levels of estrogens may reflect levels quite different from those hormone metabolites to which the target tissue is exposed. Many of the genes involved in the biosynthesis and metabolism of estrogen are polymorphic, and research attention has begun to focus on the impact of these variants on breast cancer risk. Investigating the distribution of functionally relevant genetic polymorphisms that alter the bioavailability of steroid hormones among persons with disease and persons without may provide more direct evidence for estrogen and estrogen metabolites as modifiers of human diseases, including breast cancer. A number of studies, to date, have evaluated relationships between breast cancer risk and genetic polymorphisms in CYP1A1, CYP17, CYP19, and COMT.

5.5.1. CYP1A1

Early studies of genetic polymorphisms in cytochrome P450 (CYP) 1A1 focused primarily on its role in lung cancer risk, since it activates polycyclic aromatic hydrocarbons, which are potent tobacco smoke carcinogens. However, CYP1A1 is also involved in the metabolism of estradiol. To date, four polymorphisms have been identified within this gene, one of which is specific to African-Americans (125). A number of studies have been conducted to evaluate associations between CYP1A1 and breast cancer risk, with mixed results (126-129). In a study with African-American and Caucasian women, Taioli and colleagues (128) noted that among African-American women, the m1 polymorphism significantly increased breast cancer risk (OR = 9.7, 95% CI: 2.0-47.9). Numbers in these stratified analyses, however, were quite small. In both the western New York study (129) and the Harvard study (129) it was found that while there were no main effects of CYP1A1 on breast cancer risk, the effects of CYP1A1 polymorphisms were modified by cigarette smoking. Women who were light smokers with variant alleles were at increased risk of breast cancer in the Ambrosone study (127), and those with variant alleles who began smoking before age 16 in the Ishibe study (129). Recently, Bailey et al. evaluated all four known CYP1A1 polymorphisms in relation to breast cancer risk, in a case-control study. None of these polymorphisms, including that specific to African-Americans, was associated with increased risk; smoking status “ever/never” did not modify risk. Furthermore, Basham et al. who combined data from their own study with those of four previously published, failed to observe an association between CYP1A1 genotype and breast cancer (130). No interactions were noted between genotype and alcohol or smoking habits.

5.5.2. CYP17

Another cytochrome P450 enzyme that has received much attention of late is the P45017a encoded by the CYP17 gene. This enzyme functions at key branch points in human steroidogenesis. The CYP17 polymorphism has also been evaluated by a number of groups; again, studies have had conflicting results. Feigelson and colleagues initially found that the variant allele conferred more than a twofold increase in risk among women with advanced disease (131). They also noted that late age at menarche was protective only among women who were homozygous for common allele. Several subsequent studies have not corroborated these findings (132-137), although analyses in the Nurses’ Health study (135) demonstrated that the protective effect of later age at menarche (>13 years) was only observed among women with the common allele and not among women carrying variant alleles. The recent meta-analysis involving data from 15 case-control studies (138) also showed that the variant allele in CYP17 acts as a weak modifier of breast cancer risk but is not an independent risk factor.

5.5.3. CYP19

Aromatase or estrogen synthetase, encoded by the CYP19 gene, converts androgens to estrogens, and completes the pathway for estrogen biosynthesis from cholesterol (139). The conversion of testosterone to estradiol in adipose tissue is the main source of estrogens in postmenopausal women. A polymorphic tetranucleotide repeat (TTTA)n has been identified and although relatively rare, Kristensen et al. (140) noted a significant association with breast cancer risk in carriers of the longest repeat variant (TTTA)12, designated the A1 allele, in a case-control study with 366 cases and 252 controls. The A1 allele was present in less than 2% of the control population, but in almost 4% of cases. Siegelmann-Danieli (141) also evaluated this association and found increased risk with the variant A1 allele. Baxter et al. confirmed this association in a study of breast cancer in England (142). These findings were not confirmed, however, by Haiman et al. who evaluated CYP19 polymorphisms in relation to breast cancer and estrogen levels in the Nurses’ Health study (143).

5.5.4. Catechol-O-Methyltransferase Catechol-O-methyltransferase (COMT) is one of several phase II enzymes involved in the conjugation and inactivation of catechol estrogens (144). Because there is evidence that catechol estrogens, particularly the 4-hydroxy catechol estrogen, may bind to DNA and result in DNA damage (145), the possible role of lower activity in the enzyme in relation to breast cancer risk is important. Several groups to date, all with conflicting results, have evaluated the role of the COMT genetic polymorphism in relation to breast cancer risk. Lavigne et al. (146) found that women who were postmenopausal had a greater than twofold increase in risk with the low activity alleles, but inverse associations were noted for premenopausal women with the same genotype. Thompson et al. (147) performed similar analyses and observed that, among premenopausal women with breast cancer, those with at least one low activity allele showed significantly increased risk (OR = 2.4, CI, 1.4-4.3). In contrast to premenopausal women, there was an inverse association between low activity alleles and postmenopausal breast cancer. Mitrunen et al. (148) noted inverse associations for women with low activity COMT alleles in relation to premenopausal breast cancer risk, and elevated associations for postmenopausal women, particularly those using exogenous estrogens or early age at menarche. The authors hypothesized that there may be an opposing role of catechol estrogen metabolism in breast cancer etiology depending on the hormonal environment. Yim et al. (149) also reported that the low activity COMT allele was associated with increased risk of breast cancer among Asian women. Millikan (150) and Bergman-Jungestrom (151), however, found no associations with COMT genotypes and increased breast cancer risk for pre- or postmenopausal women. These discrepancies may be due to small sample sizes in the previous studies, or there may be biological factors that differentially impact risk associations.

5.6. Chemical Exposures Environmental factors have been implicated in breast cancer etiology, due to the steady increase in incidence over the last decades (152), regional and international differences in incidence, and observed changes in incidence rates in migrant populations (153).

5.6.1. Organochlorines One group of environmental exposures that has been examined in relation to breast cancer includes organochlorine compounds, such as 2,2-bis(4-chlorophenyl)-1,1-dichloroethane (DDE), the major metabolite of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT), and polychlorinated biphenyls (PCBs). Evidence from laboratory studies has demonstrated a complex diversity of biological effects associated with these compounds. DDE and some PCB congeners have been associated with induction of cytochrome P450 enzymes (154,155), which may or may not be associated with estrogenic (156-158) and antiestrogenic effects (158) shown in some investigations. Studies have also noted changes in immune responses (159) and tumor promoting effects (154,160,161).

Several recent epidemiologic studies have investigated the role of DDE and PCBs in breast cancer etiology (162-175), but results from these studies are inconsistent. While results from an earlier investigation pointed to a potential role of organochlorine exposure in breast carcinogenesis, most subsequent studies did not observe significant risk elevations among women with the highest blood or adipose levels of these compounds.

Some efforts have been made to examine the effect of environmental organochlorine exposure among susceptible subgroups, defined by reproductive or genetic factors. Moysich et al. (163) observed a significant increase in risk of breast cancer among parous postmenopausal women who never lactated with the highest serum PCB levels compared to those with the lowest levels. Organochlorine levels were also associated with age and serum lipids, but not fruit intake (176). It is possible that women who had lactated were less susceptible to the adverse effect of organochlorine exposure due to the fact that they had eliminated a substantial amount of organochlorine body burden at a biologically relevant period of time. Alternatively, lactation in itself may contribute to the terminal differentiation of the mammary epithelium, resulting in larger compartments of nonproliferating cells (2). It has also been suggested that organochlorine body burden may have been measured more accurately among women who had never breastfed an infant. Serum levels in this group may represent a more valid measure of chronic exposure, uninterrupted by elimination of these compounds through lactation. Based on the same study population, these investigators also attempted to determine whether or not the genetic polymorphism in the CYP1A1 gene affected the association between PCB exposure and risk (177).

In laboratory studies, PCBs are potent inducers of CYP1A1, a drug-metabolizing gene, involved in the activation of potentially genotoxic endogenous and exogenous substances (178,179). Their results indicated that postmenopausal women with the highly inducible CYP1A1 variant genotype and high PCB levels were at significantly increased risk for breast cancer compared to women with the CYP1A1 wild genotype and lower PCB levels. A potential mechanism for this finding relates to the PCB mediated enhanced induction of polymorphic CYP1A1, leading to increased activation of environmental carcinogens and subsequently resulting in the production of reactive intermediates and DNA damage. Thus, by inducing CYP1A1, PCBs, and other inducers can trigger the activation of xenobio-tics, such as those found in tobacco, into mutagenic compounds.

5.6.2. Cigarette Smoking and Breast Cancer Environmental contaminants other than organochlorines could also be associated with breast cancer risk, including aryl and heterocyclic aromatic amines, nitro- and polycyclic aromatic hydrocarbons, and N-nitroso compounds, all of which are known mammary mutagens and carcinogens. In addition to their presence in an industrialized environment, these carcinogens are present in cigarette smoke. Aromatic amines form DNA adducts in cultured human epithelial cells (180), and cause unscheduled DNA synthesis (180). In vivo activated aromatic amine metabolites have been shown to cause DNA damage in rodents (181,182) to transform mouse mammary glands (183), and to induce rodent mammary tumors (184,185). Polycyclic aromatic hydrocarbons are also likely human breast carcinogens. The PAHs benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene induce mammary tumors in rodents (186,187) and cause transformation in human breast epithelial cell lines in vitro (188).

The mutational spectrum of the p53 tumor suppressor gene also supports a role for chemical carcinogens in breast cancer risk. While the pattern of p53 mutations in breast cancer differs from the fingerprint mutations associated with smoking in lung cancer, they occur on sites that are suggestive of an unknown, environmental exposure (189,190). Furthermore, it is clear that chemical carcinogens reach the breast in laboratory animals and humans, and because they are lipophilic, they are stored in breast adipose tissue (191,192). Ductal epithelial cells are directly exposed to nicotine (193) and mutagenic compounds (194). Heterocyclic amines administered to nursing rat dams were found at high levels in the breast tissue, and were excreted in the milk (195). Three studies have identified DNA adducts in normal breast tissue from women with and without breast cancer (66, 196-199), some of which were putatively related to tobacco smoking. Therefore, the breast is certainly exposed to chemical carcinogens, and can be susceptible to the carcinogenic process.

If these compounds are human mammary carcinogens, one would expect to see an association between smoking and breast cancer risk. However, in the majority of epidemiological studies, an association between smoking and breast cancer risk has not been found (200-202). However, most previous studies combined passive smokers with non-smokers in the reference category. Sidestream smoke contains higher levels of aromatic amines than mainstream smoke, as much as 10 mg of aniline per cigarette, as well as many other aromatic amines [e.g., multiple isomers of toluidine, naphthylamine and aminobiphenyl (ABP)]. Thus, passive smoke exposure may result in different circulating levels of carcinogens than active smoking. The presence of passive smokers in the referent ‘non-smoking’ category would certainly dilute risk estimates. Studies that confined the referent group to those never exposed to passive smoke all found increased breast cancer risk for active and passive smokers (203-208). Using data from the Nurses’ Health study, however, Egan et al. did not find a positive association between passive smoking and breast cancer risk, but a small increase in risk was noted for smoking initiated at young ages (<17 years old) (201).

It has been suggested that some components of tobacco smoke may have antiestrogenic effects (209,210). For example, cigarette smoking induces CYP1A2, which decreases the level of circulating estradiol. It is possible that genetic variability in metabolism of chemical carcinogens may make some women more susceptible to their carcinogenic effects from ubiquitous exposure, dietary intake, and exposure through active and passive cigarette smoke. Other women may be more affected by the putative antiestrogenic effect of tobacco smoke. When these subgroups are grouped together, however, as in population-based studies, the effects of a particular exposure may not be observable above the background of other exposures and susceptibilities. In this case, the effects may be diluted and thus, not statistically significant. Several molecular epidemiological studies have been conducted to ascertain possible associations between smoking and breast cancer risk among women likely to be susceptible to their carcinogenic effects. By evaluating genetic polymorphisms for enzymes involved in the metabolism of classes of chemical carcinogens, subgroups of the population who may be susceptible to tobacco smoke carcinogens may be identified.

Aromatic Amine Metabolism: Aromatic amines are likely to be first metabolized in the liver via two competing pathways. They may be either activated by CYP1A2, or detoxified through N-acetylation by NAT2. We hypothesized that among women who had inherited mutations NAT2 encoding a less efficient form of the enzyme and were thus, ‘slow acetylators’; aromatic amines would be more likely to be activated by CYP1A2. In this scenario, activated hydroxylamines could be further activated either in the liver or in the breast, DNA adducts could form, and breast cancer could result. In a study of several hundred pre- and postmenopausal women in western New York (70), we found that neither smoking nor the slow NAT2 genotype impacted breast cancer risk. However, postmenopausal women who had slow NAT2 and smoked were at dose-dependent, increased risk. This hypothesis was subsequently explored by other groups, with mixed results (207,211,212).

Polycyclic Aromatic Hydrocarbon Metabolism: PAHs are metabolized by a complex of phase I and phase II enzymes. Those studied in relation to smoking and breast cancer include CYP1A1 and glutathione S-transferase Ml (GSTM1). CYP1A1 activates PAHs, and as mentioned previously, Ambrosone et al. found that the exon 7 polymorphism (m2) increased risk among postmenopausal women who were light smokers. This finding was supported by data from the Nurses’ Health study (m1 and m2) (129), but Bailey and colleagues found no associations with any of the polymorphisms (m1-m4) (125). Earlier, Taioli et al. (128) evaluated CYP1A1 polymorphisms (m1-m3) among Caucasians and African-Americans, and found that the m1 allele increased risk among African-American women. These data were not presented in relation to smoking, however.

Phase II metabolism includes detoxification of reactive metabolites by conjugation with glutathione, which is catalyzed by glutathione S-transferases. GSTM1 has a deletion that is present in approximately 50% of Caucasian populations, resulting in loss of the enzyme. A number of groups have evaluated the possible association of the GSTM1 polymorphism with breast cancer risk. For the most part, studies have found no association between the null allele and breast cancer regardless of smoking status (125,127,213-217). Helzlsouer et al., however, reported an increased risk of postmenopausal breast cancer associated with GSTM1 deletion (218). This association was not modified by exposure to tobacco smoke.