Superfund Research Program
Developmental Effects of Superfund Hydrocarbon Mixtures in Fundulus heteroclitus
Progress during the past year has been focused on three areas: (1) continued comparative analysis of killifish populations from the project’s Superfund site (Elizabeth River, VA – “ER”) and unpolluted reference site (Kings Creek, VA – “KC”), (2) studies addressing mechanisms underlying observed synergistic developmental toxicity of PAH mixtures, and (3) investigations of interactive effects of PAHs and hypoxia. Highlights from these areas are described below.
The research group’s population comparison studies have focused on two questions: (1) Are ER killifish, demonstrated to be highly resistant to the teratogenic effects of PAHs, more or less sensitive to PAH-mediated liver injury including cancer? and (2) Are ER embryos more or less sensitive to the teratogenic effects of other chemicals that are also influenced by the aryl hydrocarbon receptor (AHR)-cytochrome P450 (CYP1A) pathway? Recent work has demonstrated that ER killifish, exposed to benzo(a)pyrene (BaP) as larvae, are more resistant to liver injury including pre-neoplastic foci and tumors than KC fish. Current studies are addressing the involvement of altered BaP metabolism in this population difference. With respect to altered sensitivity to chemicals other than PAHs, the researchers hypothesized that ER fish would be more resistant to chemicals activated by the AHR-CYP1A (e.g., the organophosphate insecticide chlorpyrifos) but more sensitive to chemicals detoxified by this pathway (e.g., the pyrethroid insecticide permethrin). However, ER fish were observed to be significantly more resistant to both chemicals. As further hypothesized, CYP1A induction by exposure to the AHR agonist β-napthoflavone (BNF) increased the toxicity of chlorpyrifos and reduced the toxicity of permethrin in KC fish; BNF had no effect on the toxicity of these compounds in the AHR-unresponsive ER fish.
The researchers continue to explore mechanisms underlying their earlier unanticipated discovery that binary PAH mixtures that include an AHR agonist (e.g., BaP, BNF) and a CYP1A inhibitor (e.g., α-naphthoflavone [ANF] and fluoranthene [FL] produced marked synergistic teratogenicity, with the developing cardiovascular system the key target. They recently conducted two microarray experiments; these involved BNF-ANF exposures (singly and in combination) to both killifish and zebrafish embryos. For the killifish study, the group used the array designed and built by Co-PI Dr. Margie Oleksiak; for the zebrafish experiment they used a commercial array (Agilent). Data for both experiments are now being analyzed with support form the Functional genomics Research Core. The group also recently completed the mRNA extraction phase from a more complex experiment in which zebrafish embryos were exposed to BaP and Fl, alone and in combination. In addition to these treatments, 50% of the embryos were injected with the AHR2 morpholino and hearts from all animals were excised for gene expression analysis, again using the Agilent platform.
In addition to these microarray experiments, the researchers are performing targeted experiments to examine the roles of specific mechanisms. They recently completed a set of experiments to test the hypothesis that oxidative stress plays a role in the synergy. Consistent with this hypothesis, they observed synergistic upregulations of several antioxidant genes in BNF-ANF-exposed zebrafish. These genes included manganese and copper-zinc superoxide dismutases, a glutathione peroxidases, a glutathione S-transferase, and glutamine cysteine ligase catalytic unit. Furthermore, pretreatment with a morpholino to block transcription of the antioxidant transcription factor NRF2 blocked upregulation of these genes and exacerbated BNF+ANF toxicity. However, manipulation of other antioxidants including glutathione and vitamin E had no effects. Thus, a role for oxidative stress is supported by this work but not confirmed. Other studies are exploring the role of PAH metabolism in the synergy, and the importance of mitochondria as a subcellular target.
Finally, the group is exploring interactions between hypoxia and PAH exposure with both in vitro (cell culture) and in vivo (embryo) experiments. Their cell work has supported the hypothesis of cross-talk between the AHR and hypoxia pathways that may underlie interactions. Embryo exposures have shown that for some PAHs (BaP and FL), hypoxia exacerbates toxicity while for others (BNF and benzo(k)fluoranthene) it does not. These results do not support the hypothesis that AHR agonism is required for this interaction and indicates that multiple mechanisms are likely to occur, reflecting the complexity of the PAH chemical class.