Mechanisms of Biotransformation of Methoxylated and Hydroxylated Polybrominated Diphenyl Ethers
Polybrominated diphenyl ethers (PBDEs) were the most commonly used non-chemically bound brominated flame retardants (BFRs). The PBDEs as well as their methoxylated- (MeO-) and hydroxylated- (OH-) analogs are distributed in environmental matrices worldwide and have been detected in several organisms. There is concern over the occurrence of the OH-BDEs because they have greater toxicities relative to the PBDEs, including neurotoxicity, immunotoxicity, disruption of energy metabolism, and reproductive and endocrine disruption. Relationships among PBDEs, OH-BDEs and MeO-BDEs are unclear. While MeO-BDEs and OH-BDEs are naturally occurring in the marine environment, it has been reported that OH-BDEs are formed by biotransformation of PBDEs. However, in these studies, the OH-BDEs occurred at trace levels, from <0.01-1% of exposed PBDEs levels. A recent study by Wan et al. (2009) suggested that instead of PBDEs, in vitro, naturally occurring MeO-BDEs are precursors of OH-BDEs. However, there is no evidence from in vivo studies that the MeO-BDEs are transformed to OH-BDEs. Further, the enzyme(s) that catalyze the transformation of MeO-BDEs to OH-BDEs were unknown. To further demonstrate in vivo, biotransformation relationships among these structurally related compounds dietary accumulation, maternal transfer, and tissue distribution of PBDEs, MeO-BDEs and OH-BDEs and their transformation products were investigated in sexually mature Japanese medaka (Oryzias latipes). Medaka were exposed to BDE-47, 6-OH-BDE-47, and 6-MeO-BDE-47 through their diet for 14 days. Concentrations of all compounds were quantified in livers and whole carcass (minus livers) of female medaka as well as in eggs. Significant concentrations of 6-OH-BDE-47 were quantified in medaka exposed to 6-MeO-BDE-47. Significant concentrations of 6-MeO-BDE-47 were also detected in fish exposed to 6-OH-BDE-47. However, 6-MeO-BDE-47 was not observed when microsomes from livers of medaka were exposed to 6-OH-BDE-47. OH-PBDEs and MeO-PBDEs were not detected in medaka exposed to BDE-47. Similar results were demonstrated in eggs from female medaka. Furthermore, as hypothesized, concentrations of BDE-47, 6-MeO-BDE-47 and 6-OH-BDE-47 in medaka eggs increased during exposure. Therefore, this study demonstrated in vivo biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47 as a primary pathway, while conversion from BDE-47 to 6-OH-BDE-47 did not occur. The enzyme(s) that catalyze transformation of MeO-BDEs to OH-BDEs was characterized in liver of rainbow trout (Oncorhynchus mykiss). Significantly greater concentrations of 6-OH-BDE-47 were detected in microsomes than were observed in S9 fractions exposed to 6-MeO-BDE-47, which suggests that biotransformation of 6-MeO-BDE-47 to 6-OH-BDE-47 is localized in microsomes. The requirement for the co-factor NADPH further confirmed the catalysis by phase Ι enzymes in this biotransformation reaction. Non-significant transformation of 6-MeO-BDE-47 to 6-OH-BDE-47 in microsomes isolated from livers of rainbow trout exposed to the aryl-hydrocarbon receptor (AhR) agonist β-napthoflavone (βNF) compared to unexposed rainbow trout indicated that members of the CYP 1 family enzymes were not involved in this transformation. Inhibitors of CYP enzymes, clotrimazole (CL), 1-benzylimidazole (BI) and gestodene (GE) and an anti-CYP 3A antibody were used to further investigate the enzymes involved. Transformation of 6-MeO-BDE-47 to 6-OH-BDE-47 was significantly inhibited by the broad spectrum inhibitors of CYP enzymes, CL and BI. However, neither the CYP 3A inhibitor GE nor the anti-CYP 3A antibody significantly altered the rate of transformation in microsomes exposed to 6-MeO-BDE-47. Therefore, transformation of 6-MeO-BDE-47 to 6-OH-BDE-47 is more likely to be catalyzed by enzymes of the CYP 2 family. Because activities of CYP enzymes can be different among fishes, it was hypothesized that transformation of 6-MeO-BDE-47 to 6-OH-BDE-47 would also differ among fishes. Differences of transformation from 6-MeO-BDE-47 to 6-OH-BDE-47 among rainbow trout, white sturgeon (Acipenser transmontanus) and goldfish (Carassius auratus) were investigated. Microsomes isolated from these three species were incubated with 6-MeO-BDE-47 for 0.5, 1, 2, 6 and 24 h. The greatest concentrations of 6-OH-BDE-47 were detected in microsomes isolated from livers of rainbow trout, followed by goldfish and white sturgeon. The initial rate of transformation for rainbow trout was significantly greater than that of goldfish and white sturgeon, while goldfish also had a significantly greater initial rate than white sturgeon. The final concentration of transformation product after 24 h exposure for rainbow trout was greater than that of goldfish and white sturgeon. Similarly, the final concentration of OH-BDE-47 in goldfish was significantly greater than white sturgeon. In addition, differences in the concentrations of OH-BDEs determined in different species of fish could be due to differences in CYP-catalyzed transformation of MeO-BDEs to OH-BDEs. Taken together, the results are consistent with the conclusion that naturally occurring MeO-BDEs, and not the synthetic PBDEs, are the primary source of the biologically potent OH-BDEs. An enzyme of the CYP 2 family was suggested to transform 6-MeO-BDE-47 to 6-OH-BDE-47. It was demonstrated that the degree of biotransformation of MeO-BDEs to OH-BDEs is dependent on species of fish. Therefore, since the OH-BDEs have greater toxicity than PBDEs, different species exposed to the same concentration of MeO-BDEs may be differentially impacted by this exposure due to differences in the capacity to convert MeO-BDEs to OH-BDEs.
DegreeMaster of Science (M.Sc.)
DepartmentGraduate Studies and Research
SupervisorGiesy, John P.
CommitteePietrock, Michael; Hecker, Markus
Copyright DateDecember 2011