UC Riverside

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can enter aquatic environments through runoff, atmospheric deposition, accidental discharge, and oil spills. These compounds can be oxidized photochemically or biologically into oxygenated PAHs (oxy-PAHs) which have been shown to be more toxic compared to parent PAHs. The polar properties of oxy-PAHs increase their mobility within the environment which increases the risk of exposure to fauna and flora compared to parent PAHs. Regioselective toxicity has been observed in several oxy-PAHs and the oxidation state of the oxygen on a specific PAH can have dramatic impacts on the toxicity. Previous studies have found that exposure to hydroxychrysenes at critical developmental time-points in fish models impairs red blood cell concentrations in a regioselective manner, with 2-hydroxychrysene (2-OHCHR) being more potent than 6-hydroxychrysene (6-OHCHR). The mechanisms of toxicity of oxy-PAHs are largely unknown and we aimed to characterize the pathways of toxicity of 2- and 6-OHCHR in fish embryos. Our first aim was to characterize the toxic effects in Japanese medaka embryos and to find a sensitive window of development to hydroxychrysenes. We found that 2-OHCHR caused anemia and morality in medaka embryos in contrast to in zebrafish embryos, where 2-OHCHR caused only anemia and 6-OHCHR only caused mortality. A sensitive window to 2-OHCHR toxicity was found between 52-100 hpf which closely coincided with liver development. This led us to our second aim, exploring the metabolism and toxicokinetics of the hydroxychrysenes. We found that although 6-OHCHR was taken up 97.2% ± 0.18 more rapidly than 2-OHCHR, it was also eliminated 57.7% ± 0.36 faster as a glucuronide conjugate. Pretreatment with the general cytochrome P450 inhibitor ketoconazole reduced anemia by 96.8% ± 3.19 and mortality by 95.2% ± 4.76 of 2-OHCHR treatments. In addition, formation of the 1,2-catechol was also reduced by 64.4% ± 2.14. However, while pretreatment with the UGT inhibitor nilotinib reduced glucuronidation of 2-OHCHR by 52.4% ± 2.55 and of 6-OHCHR by 63.7% ± 3.19, it did not alter toxicity for either compound. These results indicated that CYP mediated activation, potentially to the oxidatively active metabolite 1,2-catechol, may be driving the isomeric differences in toxicity. Previous studies have found 2-OHCHR to be a four-fold more potent aryl hydrocarbon receptor (AhR) agonist compared to 6-OHCHR. Therefore, in aim 3, we explored the role of the and oxidative stress in 2-OHCHR toxicity. While treatments with the AhR agonists PCB126 and 2-methoxychrysene (2-MeOCHR) did not cause significant anemia or mortality, pretreatments with AhR antagonist CH-223191 reduced anemia by 97.2% ± 0.84 and mortality by 96.6% ± 0.69. AhR inhibition was confirmed by a significant reduction (91.0% ± 9.94) in EROD activity. Thiobarbituric acid reactive substances (TBARS) concentrations were 32.9% ± 3.56 higher (p<0.05) in 2-OHCHR treatments at 100 hpf compared to controls, indicating oxidative stress. Staining with 2’,7’-Dichlorofluorescin diacetate (DCFDA) revealed 42.6% ± 2.69 of embryos exhibiting high concentrations of ROS in caudal tissues, which is a site for embryonic hematopoiesis. Both muscle and skeletal tissues were affected, as well as some caudal vasculature. Overall, our findings indicate that AhR may mediate 2-OHCHR toxicity, upregulating CYP and potentially forming the 1,2-catechol that generates ROS in the embryos within caudal tissues, potentially disrupting hematopoiesis leading to anemia and subsequent mortality. Further studies should investigate additional key events and construct adverse outcome pathways for oxy-PAHs.