Role of DNA methylation of AHR1 and AHR2 promoters in differential sensitivity to PCBs in Atlantic Killifish, Fundulus heteroclitus.

Atlantic killifish (Fundulus heteroclitus) inhabiting the PCB-contaminated Superfund site in New Bedford Harbor (MA, USA) have evolved genetic resistance to the toxic effects of these compounds. They also lack induction of cytochrome P4501A (CYP1A) and other aryl hydrocarbon receptor (AHR)-dependent responses after exposure to AHR agonists, suggesting an overall down-regulation of the AHR signaling pathway. In this study, we hypothesized that the genetic resistance is due to altered AHR expression resulting from hypermethylation of DNA in the promoter region of AHR genes in fish inhabiting New Bedford Harbor. To test this hypothesis, we cloned and sequenced AHR1 and AHR2 promoter regions and employed bisulfite conversion-polymerase chain reaction (BS-PCR) followed by clonal analysis to compare the methylation status of CpG islands of AHR1 and AHR2 in livers of adult killifish collected from New Bedford Harbor and a reference site (Scorton Creek, MA). No significant differences in methylation profiles were observed in either AHR1 or AHR2 promoter regions between NBH and SC fish. However, hypermethylation of the AHR1 promoter correlated with low expression of transcripts in the liver in both populations. In comparison to AHR1, hepatic mRNA expression of AHR2 is high and its promoter is hypomethylated. Taken together, our results suggest that genetic resistance to contaminants in NBH fish is not due to altered methylation of AHR promoter regions, but that promoter methylation may control tissue-specific expression of AHR genes in killifish.