We attempted to build on prior studies of CBT and pesticide exposure by considering individual differences in the metabolism of insecticides that target the nervous system. We a priori designated a “high-risk” allele for each polymorphism based on the expected functional impact with respect to acetylcholinesterase (AChE) inhibition (OP and carbamate insecticides; PON1, BCHE, FMO1, FMO3, and GSTT1 polymorphisms) and ion channel stimulation [OC and pyrethroid insecticides; aldehyde dehydrogenase 3A1 (ALDH3A1) and GSTT1 polymorphisms]. Although some insecticides metabolized by these enzymes are ubiquitous in the environment or diet, ORs for CBT in relation to the hypothesized “high-risk” allele for the nine polymorphisms were close to the null, or fluctuated equally above and below the null. However, we observed interactions between genotype and chemical treatment of the home for insects during childhood for three functional SNPs located on different chromosomes: PON1C–108T, FMO1C–9536A, and BCHEA539T. The direction of these interactions was consistent and biologically plausible. Moreover, they were present in each of our two largest racial/ethnic groups.
variants are respectively associated with reduced in vivo
activity of PON1 (Brophy et al. 2001
; Chen et al. 2003
; Leviev and James 2000
) and the butyrylcholinesterase enzyme (BuChE) (Babaoglu et al. 2004
; Bartels et al. 1992
; Maetzler et al. 2009
). Both neutralize AChE inhibitors: PON1 hydrolyzes selected OPs, notably chlorpyrifos and diazinon (Furlong 2007
), and BuChE sequesters all OP and carbamate insecticides (Cokuğraş 2003
). In vitro
studies suggest that FMO1–9536A
materially reduces promoter activity (Hines et al. 2003
). Its product, flavin-containing monooxygenase 1 (FMO1), oxidizes the thioether sulfur of some OP and carbamate insecticides (Hajjar and Hodgson 1980
), and for some substrates (e.g., fenthion; Furnes and Schlenk 2004
) the resulting sulfoxide is a weaker AChE inhibitor than is its parent compound. FMO1 does not appear to oxidize other sulfur atoms in OP insecticides (Hajjar and Hodgson 1980
) (activate the parent compound to its oxon). Thus, our results are consistent with the possibility that children with a reduced ability to metabolize OP and perhaps carbamate insecticides might be at increased risk of CBT when sufficiently exposed. The apparent specificity of the results to AChE inhibitors is interesting but in part reflects our selection of polymorphisms. Also, even if our results suggest a biological impact of the SNPs and insecticides, it is unknown whether this is a result of AChE inhibition per se or to some other effect of AChE-inhibiting insecticides used residentially during the study period. For example, chlorpyrifos and diazinon induce neurotoxic effects in neonatal rats, even when administered at levels insufficient to inhibit AChE (Slotkin et al. 2008
The consistency of results across the three SNPs for which we observed an interaction with childhood insecticide exposure is compelling but nevertheless could represent chance associations. Our results were based on modest numbers, and these SNPs have not been studied in independent samples of brain tumor patients, making the probability of false positives high (Wacholder et al. 2004
). The interaction involving FMO1C–9536A
must be interpreted especially cautiously. Whether the net effect of FMO1 would be protective may depend on the insecticide (Buronfosse et al. 1995
; Furnes and Schlenk 2004
; Levi and Hodgson 1988
). Although children are exposed to FMO1 insecticide substrates, including disulfoton used residentially outdoors, we have not identified an FMO1-metabolized insecticide registered for residential use indoors. The interaction with home insecticide exposure in childhood is also puzzling because FMO1 enzyme levels in the brain and liver drop substantially after birth (Koukouritaki et al. 2002
; Zhang and Cashman 2006
). Still, FMO1 is not absent from these sites and is expressed at greater levels in the lung and small intestine (Zhang and Cashman 2006
), presumably relevant to inhaled and hand-to-mouth exposure, respectively.
The presence of interactions between genotype and insecticide exposure occurring during childhood, but generally not during pregnancy, deserves further comment. During prenatal development, maternal enzymes serve as a first line of defense against exogenous exposures, and without maternal biospecimens we were unable to directly examine the effect of this. Also, perhaps fetal expression of some enzymes is too low, regardless of genotype, to alter insecticide dose sufficiently to protect the brain; here again, maternal enzymes may be important. Our data do not suggest a lack of effect of insecticide exposure during this potentially sensitive period, but rather a lack of synergism with fetal genotype.
We did not observe interactions for other PON or FMO SNPs. None were in the promoter region of their respective genes. Also, the effect of the PON1Q192R
amino acid change is dependent on the substrate, and the R isoform may be protective for chlorpyrifos but not diazinon (Davies et al. 1996
; Li et al. 2000
; Mutch et al. 2007
). FMO1 metabolizes insecticides better than FMO3 (Furnes and Schlenk 2005
; Leoni et al. 2008
; Usmani et al. 2004
). Perhaps more important, given our results for FMO3E158K
, this coding region SNP is in linkage disequilibrium with promoter region polymorphisms that confer opposing effects on FMO3 enzyme activity (Phillips and Shephard 2008
The childhood insecticide–PON1C–108T
interaction was confined to children < 3 years of age. This polymorphism has a greater effect on PON1 levels in neonates than in adults (Chen et al. 2003
), and adult levels are reached before 3 years of age (Cole et al. 2003
). In addition, by this age diet is the main source of chlorpyrifos (Buck et al. 2001
; Clayton et al. 2003
), so in older children dietary exposure to chlorpyrifos and diazinon may have overwhelmed any interaction between PON1C–108T
and residential exposure.
Since the time when the children in our study may have been exposed to home insecticides, chlorpyrifos and diazinon have been phased out of residential use in the United States. Nonetheless, children remain exposed to these and other AChE inhibitors not only via the diet but also potentially via drift from use in agricultural areas, on golf courses, and for mosquito control. In the home, OP and carbamate insecticides remain, for example, in topical treatments for lice (malathion) and flea collars (tetrachlorvinphos, carbaryl, propoxur). Therefore, the present study may have had an increased ability to observe the reported interactions because of the greater residential use of AChE inhibitors, yet our results remain relevant.
Another strength of our study is the use of archived DBS, available for participants regardless of survival status. This makes it unlikely that a relationship between genotype and responsiveness to treatment could underlie the observed interactions. Other opportunities for selection bias were present, including during specimen collection (Searles Nielsen et al. 2008
). Although it is therefore difficult to rule out bias in main effects, gene–environment interactions are generally unaffected by selection bias (Morimoto et al. 2003
). Further, despite the potential for differential reporting of past exposures, this more likely attenuated than caused the interactions we report (Garcia-Closas et al. 1999
To date, this is the largest study of CBT and genetic polymorphisms. Studies with more participants are needed to clarify the reported associations. Inclusion of additional polymorphisms in FMO3 and BCHE, especially those in the promoter region, would be worthwhile. These have been less studied than coding region polymorphisms in relation to cancer, but they appeared to be critical here. Objective measurement of specific insecticides in environmental or biological specimens, and detailed interview data on the timing of exposure (e.g., during spermatogenesis, by pregnancy trimester, and by childhood age) also would be important. Although our results most strongly indicated the importance of exposures during early childhood, it is likely that other periods are also important, notably prenatal development. In studies that do consider exposures before birth, it would be useful to assess parents’ genotypes and levels of selected enzymes, including PON1 and FMO1 that are relatively stable over time in adults.