McCarty et al. association between GST polymorphisms and PAH–DNA adducts among control women stratified by smoking status. Women who ever smoked and had the GSTM1 present genotype had a 35% reduced odds (OR, 0.65; 95% CI, 0.45–0.93) of having detectable PAH–DNA adducts compared with never smokers with the same genotype. We noted similar results for ever smokers with the GSTM1 geno- type compared with never smokers with the GSTM1 common genotype (OR, 0.64; 95% CI, 0.43–0.93). Ever smokers with the GSTT1 present genotype had a 27% reduced odds of PAH–DNA adducts (OR, 0.73; 95% CI, 0.53–0.99) compared with never smokers with the same genotype. Ever smokers with the common GSTA1 genotype had a reduced odds of PAH–DNA adducts compared with never smokers with the same genotype (OR, 0.67; 95% CI, 0.47–0.96; Table 5). We found no evidence of effect modification between any of the individual genotypes and smoking sta- tus on PAH–DNA adduct status on the mul- tiplicative scale (LRT p-values: GSTT1, 0.20; GSTM1, 0.75; GSTP1, 0.15; GSTA1, 0.40) or on the additive scale. We did not find statisti- cal evidence between number of variant alleles and smoking status on the odds of developing detectable PAH–DNA adducts on the addi- tive scale (one-variant allele: ICR, 0.28; 95% CI, –0.39 to 0.45; two-variant alleles: ICR, –0.091; 95% CI, –0.57 to 0.38; three or more variant alleles: ICR, 0.177; 95% CI, –0.23 to 0.59) or on the multiplicative scale (p-value from the LRT = 0.13). When considering the number of adducts as a continuous outcome (data not shown), we found no evidence of a main effect of the individual genotypes on the number of adducts in an age-adjusted model (GSTT1 p = 0.86, GSTM1 p = 0.54, GSTP1 p = 0.91, GSTA1 p = 0.86). Discussion To the best of our knowledge, this is the first epidemiologic investigation of breast can- cer to consider the joint effects of four GST polymorphisms and several measures of PAH exposure, including a PAH–DNA adduct biomarker. Despite the biologic plausibility of our hypothesis, we found no strong evi- dence for an interaction between the number of variant GST alleles with various measures of PAH exposure (including ever/never smok- ing status, pack-years of smoking, or detected PAH–DNA adducts) and breast cancer risk, nor did we find an association among the control women between number of variant GST polymorphisms and the outcome of detectable PAH–DNA adducts. We found some evidence for a reduced risk of detectable adducts among control women who were ever smokers regardless of whether they had the GSTM1 deletion polymorphism or the pres- ent genotype (Table 5). We also report for 556 the first time that among controls with the A*/A* GSTA1 genotype, ever smokers had lower odds of having detectable PAH–DNA adducts than did never smokers with the same genotype (OR, 0.62; 95% CI, 0.41–0.92). GST polymorphisms and breast cancer risk. A few previous studies (Helzlsouer et al. 1998; van der Hel et al. 2005) including our own have found that individual polymor- phisms in GSTA1 (Ahn et al. 2006), GSTM1 (Helzlsouer et al. 1998; Spurdle et al. 2007), and GSTT1 (van der Hel et al. 2005) and GSTP1 (heterozygote or homozy- gote valine) (Helzlsouer et al. 1998) are mod- estly associated with breast cancer, although others have reported no association with GSTT1 (García-Closas et al. 1999; Helzlsouer et al, 1998; Vogl et al. 2004), GSTP1 (Vogl et al. 2004), or GSTM1 (Ambrosone et al. 1995; García-Closas et al. 1999; Vogl et al. 2004). Effects of multiple combinations of GST polymorphisms on breast cancer risk have previously been conducted in smaller studies, with conflicting findings (Helzlsouer et al. 1998; Steck et al. 2007b; van der Hel et al. 2005; Vogl et al. 2004). Combined effect of all three GSTT1, GSTM1, and GSTP1 vari- ants have been reported to have greater than a 3-fold increase in breast cancer risk compared with women with the common genotype for all three polymorphisms (Helzlsouer et al. 1998; Steck et al. 2007b). In this analysis, in which we considered the combined effects of the GSTT1, -M1, -P1, and -A1 variants that are coded based on their PAH-related biologic activity (rather than for their isothiocyanate- related activity), we did not find a significant association between the number of variant GST alleles and breast cancer risk. GST polymorphisms, PAHs, and breast cancer. Previous studies have differed on whether GST polymorphisms modify the association between cigarette smoking and breast cancer risk (Millikan et al. 2000; Terry and Goodman 2006; van der Hel et al. 2005; Vogl et al. 2004; Zheng et al. 2002). Smaller studies have reported that smoking did not modify associations between GSTT1 (Vogl et al. 2004), GSTM1 (van der Hel et al. 2005; Vogl et al. 2004), or GSTP1 polymorphisms (Vogl et al. 2004) and breast cancer, whereas former smokers with GSTT1 were found to have an increased risk of breast cancer com- pared with never smokers with GSTT1 present (van der Hel et al. 2005). For example, one recent breast cancer meta-analysis reported no interaction between individual GST poly- morphisms and smoking, whereas another meta-analysis reported positive associations between breast cancer risk and GSTT1 present and GSTM1 genotypes among smok- ers (Terry and Goodman 2006; Vogl et al. 2004). In contrast, we observed an increased risk of breast cancer with three or more variant GST alleles in a previous analysis (Steck et al. 2007b), but no increased risk with number of variant alleles as defined by low PAH- metabolizing activity, and no evidence of gene–environment interaction with PAH exposure. Our present findings are unique in that we investigated four GST polymorphisms simultaneously in a single large study. To the best of our knowledge, this is the first study to examine the interaction between PAH–DNA adducts and individual/com- bined effects of GSTA1, GSTM1, GSTP1, and GSTT1 genotypes on breast cancer risk. We found no pronounced adverse effect for the genotype–PAH exposure interaction, regardless of the PAH measure. Given that the genetic polymorphisms we examined are known to be functionally associated with deactivation of the active intermediates of PAHs, our findings are not easily interpreted. It is possible that the PAH–DNA adduct biomarker effect estimates already reflect any effects associated with meta- bolic variation; however, if this were true, then any apparent interaction between PAHs and GSTs should have been evident when examin- ing the joint effects with smoking. We found a reduction in risk among women with at least one variant allele and no detectable adducts, but we did not find a similar reduction among these same women in relation to no smok- ing. Thus, we do not report evidence that PAHs interacted with these polymorphisms to further increase risk. We acknowledge that expression of GSTs can be modulated by a variety of factors, including diet. There is extensive literature on animal studies demonstrating an interaction between isothiocyanates and PAHs on cancer risk, and the induction of phase II enzymes by isothiocyanates (Hecht 1996, 2000). The interaction between GSTs and isothiocyanates does not appear to interact to affect breast cancer risk in humans or the variations in the urinary metabolites of isothiocyanates in our data (Steck et al. 2007a, 2007b). Whether cruciferous vegetable intake would further interact with PAHs and GSTs to affect breast cancer risk is unclear. Unfortunately, this study was underpowered to explore such three way interactions despite a large sample size. Nevertheless, it may be theoretically possi- ble that breast cancer risk is influenced by a potential three-way interaction of PAH–DNA adducts, GST polymorphisms, and crucifer- ous vegetable intake, although our current available data do not support this hypothesis (Steck et al. 2007a, 2007b). GST polymorphisms, smoking, and PAH– DNA adducts among control women. We considered the PAH–DNA adduct outcome as both a continuous and a dichotomous vari- able (detected/not detected), although the lat- ter may be more informative given the lack of a dose–response relationship between adducts ovlume 117 | number 4 | April 2009 • Environmental Health Perspectives