Our study is the first to evaluate interactions between pesticides and genetic variation in the NER pathway with respect to prostate cancer risk. We observed 17 interactions that were robust to multiple comparison adjustment based on FDR <0.2. Of these 17 interactions, three displayed a significant monotonic increase in prostate cancer risk with increasing pesticide exposure in one genotype group (e.g. TT carriers) and no significant association in the other group: fonofos × ERCC1 rs2298881 and carbofuran × rs11744596 and rs2932778, two highly correlated SNPs tagging CDK7. The remaining 14 interactions that met FDR <0.2 were the result of a positive association in one genotype group and an inverse association in the other (i.e. qualitative interactions), which could arise because of a chance effect of the exposure of interest in one population subgroup when there is no main effect of the exposure. We also presented interactions that displayed the stratified pattern of interest and had a P value for interaction <0.01, although the P value did not meet FDR <0.2 in our study population and thus should be interpreted with caution.
Our finding of a significant interaction for fonofos, an organophosphate insecticide that is no longer registered for use in the USA (since 1998) (33
), but was used by ~25% of the AHS prostate cancer nested case–control study participants, is consistent with previous AHS findings of increased prostate cancer risk with fonofos exposure among participants with a family history of prostate cancer (34
), which suggested a role of genetic susceptibility to carcinogenic effects of this chemical. Specifically, fonofos interacted with rs2298881, an intronic SNP located before the first coding exon in ERCC1
, such that men carrying at least one variant allele exhibited increasing prostate cancer risk with increasing fonofos exposure, whereas men who were homozygous wild-type at this locus did not exhibit a significant association. The ERCC1 protein forms a heterodimer with XPF endonuclease (encoded by ERCC4
), which catalyzes the 5′ incision in NER removal of DNA lesions and also plays a role in the repair of interstrand cross-links and double-stranded DNA breaks. Rs2298881 was moderately correlated with rs928911, located in an intronic region of the gene PPP1R13L
(also known as IASPP
), which is important in p53 inhibition and located <10 kb downstream of ERCC1
. However, only the interaction with rs2298881 remained significant when both interaction terms were included in the same model, suggesting that rs2298881 was the SNP driving the risk. It is also possible that a third SNP in linkage disequilibrium with these two SNPs might be driving our findings, and further research is needed to identify the SNP responsible for the observed effect.
There is some plausibility for a role of DNA damage in our finding of an interaction between fonofos and genetic variation in ERCC1
. Whereas one report did not find evidence of fonofos genotoxicity based on several in vitro
), another study observed positive results for two assays in bacteria and yeast (36
), suggesting that fonofos may induce some DNA damage. To our knowledge, no human biomonitoring studies have specifically evaluated fonofos in relation to DNA damage endpoints (e.g. DNA adducts and chromosomal aberrations). However, our fonofos interaction finding appears consistent with numerous human biomonitoring studies suggesting the genotoxicity of organophosphate insecticides as a group (5
). In addition, in a previous analysis in the AHS prostate cancer nested case–control study (32
), fonofos demonstrated a significant interaction with a SNP in the promoter region of the base excision repair gene NEIL3
. Although the NER pathway is predominantly involved in repairing relatively bulky helix-distorting DNA lesions and the base excision repair pathway in repairing smaller lesions with minimal helix-distorting effect, there is some overlap between the two pathways (14
Carbofuran (a carbamate insecticide) interacted with several highly correlated SNPs tagging CDK7 (rs6865178, rs11744596, rs2932778 and rs7706902), such that men who were homozygous wild-type exhibited increasing prostate cancer risk with increasing carbofuran exposure. Additionally, carbofuran interacted with another CDK7 tag SNP (rs17331590), such that men carrying at least one variant allele exhibited increasing risk with increasing exposure. Taken together with the low correlations between rs17331590 and the other CDK7 SNPs that interacted with carbofuran and the findings of haplotype analysis, our results suggest that there might be two separate carbofuran × CDK7 interaction signals, emphasizing the potential importance of this region in modifying the effects of carbofuran exposure. However, only the carbofuran interactions with the very highly correlated SNPs rs11744596 and rs2932778 met FDR <0.2 and did not appear to be qualitative. Rs11744596 is located in an intronic region of MRPS36, which encodes a 28S subunit of the mitochondrial ribosome and is located ~17 kb upstream of CDK7, and rs2932778 is located in the promoter region of CDK7. CDK7 encodes a protein that is a member of the cyclin-dependent kinase family and complexes with cyclin H and MAT1 to form a cyclin-dependent kinase-activating kinase. As a component of the transcription factor TFIIH, the cyclin-dependent kinase-activating kinase complex plays a role in DNA repair and DNA transcription. This complex is also important in the regulation of cell cycle progression.
Although one study found no evidence of genotoxicity for carbofuran based on several assays (36
), other studies have observed increased mutations, chromosomal aberrations and micronuclei formation with carbofuran exposure in bacterial or animal systems (37
), suggesting some biologic plausibility for a role of DNA damage in our interaction findings for carbofuran. Another study observed increased mutations associated with the N
-nitroso derivative N
-nitrosocarbofuran, but not carbofuran itself, in a bacterial system (40
). Several human biomonitoring studies have observed increased genetic damage (based on micronuclei formation, chromosomal aberrations and damage detected by the Comet assay) among workers exposed to carbofuran (41
). However, these populations were often exposed to other chemicals as well, and there was generally limited exposure assessment, which precluded estimation of the specific contribution of carbofuran to the findings.
It is possible that mechanisms other than DNA damage repaired by the NER pathway could have contributed to our interaction findings. For example, CDK7
has other functions beyond DNA repair as described above. In addition, the SNP(s) driving our findings could act in part by affecting the expression or function of nearby genes. For example, ERCC1
overlaps with CD3EAP
, which has been proposed to play a role in T-cell activation (44
), among other activities. Although fonofos has not been specifically implicated, organophosphate insecticides in general have been associated with immunotoxicity in experimental animal studies and epidemiologic studies (45
). There are several genes located nearby CDK7
, including CCNB1
, which contributes to the regulation of mitosis by encoding a protein that complexes with p34 (cdc2) to form the mitosis-promoting factor. CCNB1
may be relevant to the carcinogenicity of carbofuran as exposure to carbofuran has been associated with mitotic inhibition in mice (37
Although our findings may be due to chance, we took several steps to help reduce false-positive results in our study. We used the FDR method to adjust interaction P values for multiple comparisons. Additionally, we highlighted interactions with a significant monotonic increase in prostate cancer risk with increasing exposure in one genotype group and no significant association in the other. However, we recognize that by focusing on this subset of interaction findings, we might have missed some true-positive results among our remaining findings.
Our study was limited in power, and we may have missed some interactions by excluding SNPs with MAF <10% due to power concerns. The number of participants often became small when stratifying by genotype, particularly for the homozygous variant group. To help reduce this problem, we chose to use the dominant genetic model for the interaction assessments; however, this choice could have resulted in a loss of power if another genetic model was more appropriate. Additionally, there were insufficient case numbers to evaluate interactions by prostate cancer stage or grade. However, to our knowledge, no other study has greater power to evaluate pesticide–gene interactions for individual pesticides with prostate cancer.
There are also several strengths of our study. We were able to evaluate a number of pesticides from a range of chemical and functional classes. Previous findings in the AHS have suggested that the effects for pesticides within a chemical class are heterogeneous (46
). Thus, our examination of effect modification of individual as opposed to grouped pesticides is a further strength. Furthermore, self-reported pesticide information in the AHS has been demonstrated to be reliable and consistent with the dates of introduction to the market (47
). We focused our analyses on the intensity-weighted exposure metric, which incorporates an intensity score that has shown moderate correlation with biomarkers of pesticide exposure in post-application urine samples (49
). Additionally, availability of genotyping data for a large number of tag SNPs across the NER pathway allowed us to comprehensively explore the hypothesis that NER genetic variation might modify pesticide-associated risk of prostate cancer.
In conclusion, although we did not observe highly significant NER SNP main effects, our interaction findings between SNPs tagging ERCC1 and CDK7 and fonofos and carbofuran, respectively, suggest the importance of the NER pathway in prostate cancer risk in the presence of certain pesticide exposures. While requiring replication, our interaction results are consistent with a pesticide mechanism of effect involving DNA damage. Additional studies among pesticide-exposed populations are needed to evaluate interactions between pesticides and genetic variation in DNA repair genes and to assess the genotoxicity of individual pesticides in humans. Investigation of the mechanisms of action, metabolism and bioavailability of the different pesticides may help clarify their relationship with cancer risk.