Metabolism Of Typical Brominated Flame Retardants By Crucian Carp In Vivo And In Vitro

Brominated flame retardants (BFRs) are frequently applied to a number of different consumer products such as plastics, electronics, polyurethane foam, textiles, and so on. Polybrominated diphenyl ethers (PBDEs) are additive brominated flame retardants mixed into the materials and therefore may release from the surface of the products into the environment. Others, such as tetrabromobisphenol A (TBBPA), are reactive and are bound to the material chemically. However, some of the reactive flame retardants may still be released to the environment. Due to their high lipophilicity and environmental stability, PBDEs and TBBPA can be transported through environmental media and bioaccumulated in biotic samples, especially in the aquatic organisms. In exposed organisms, metabolism is an important factor determining the bioaccumulation, fate, and toxicity of contaminants. Development of an in vitro system to study the metabolism of BFRs would facilitate the elucidation of their metabolic pathways and the identification of key metabolism enzymes.As PBDE derivatives, OH-PBDEs have been detected in some fish species. While, it is not clear that these derivatives are resulted from in vivo metabolism or uptaken from the environment and accumulated to measurable levels. Up to date, most of the studies on the metabolism of PBDE congeners in fish tissues focused on the reductive debromination pathway. Therefore, the focus of this thesis was on the oxidative biotransformation of PBDEs and the identification of OH-PBDE metabolites in fish.Tetrabromobisphenol A (TBBPA) has also been detected in various animal species, including mammals, aquatic organisms and even human body. Despite its widespread use, there is limited knowledge about the metabolic fate of TBBPA in animals, and no studies directly dealing with the metabolism of TBBPA in fish have been published so far.In this thesis, TBBPA,4,4’-dibromodiphenyl ether (BDE15) and2,2’,4,4’-tetrabromodiphenyl ether (BDE47) were selected as representative study objects. The experiments in vivo and in vitro were both conducted to clarify the metabolic mechanism of these BFRs. The first work was to simulate the exposure of BDE47to assess the in vivo distribution and biotransformation behavior of lower brominated BDEs in freshwater fish. The second part of the work was to determine if reductive and/or oxidative metabolites of BDE15,47and TBBPA would be produced by fish liver subcellular fractions in vitro. Specific enzymes responsible for their biotransformation were also identified. The third purpose was to evaluate potential toxicity of polybrominated diphenyl ethers on freshwater fish. Main contents and results of the study are as follows:(1) The present study demonstrates that BDE47is bioaccumulative and biotransformative in crucian carp via intraperitoneal injection exposure. As a bioavailable substance, BDE47was well taken up, and rapidly accumulated in fish tissues, particularly the liver and gut. Relatively high levels of BDE47found in gut content suggested that excretion is one of the major elimination routes. Significantly, one oxidative product mono-OH-tetraBDE was detected, which indicated that oxidation metabolism was also a metabolic pathway of BDE47in crucian carp.(2) The subacute toxicity study on response of hepatic enzymes on the exposure of BDE47showed that BDE47had a significant effect on the induction of ECOD activity. The glutathione transferase (GST) activity was increased at lower concentrations while decreased at higher concentrations, which indicated that GST might be involved in the BDE47metabolism. Reductions of thyroxine (T4) and triiodothyronine (T3) were both noted after BDE47exposure, which might be caused by binding site competition of BDE47and its metabolite mono-OH-tetraBDE with T4plasma transport proteins and subsequently resulted in the lower of T4level. Histopathological changes of fish liver have also been observed after exposure to BDE47.(3) Optimization extraction method of crucian carp liver microsomes and S9was established for the experiment accuracy. Methods for determination of ethoxyresorufin-O-deethylase (EROD)、pentyloxyresorufin-O-deethylase (PROD)、 ECOD、hydroxylase (AH)、GST activity were established in order to evaluate the integrity and stability of the liver microsomes and S9.(4) An in vitro system to study the biotransformation of BDE15, BDE47and TBBPA in fish liver subcellular fractions has been developed. The GC/MS and HPLC/MS were used to identify the metabolites of the BFRs. Exposure of liver subcellular fractions to BDE15resulted in the formation of bromophenol and two monohydroxylated dibromodiphenyl ether metabolites. Neither in microsomes nor in S9studies has revealed the presence of hydroxylated metabolites with BDE47exposure, which indicated that the oxidation reactions in vitro were hindered by the increased number of bromine substituents on the PBDEs. TBBPA underwent an oxidative cleavage near the central carbon of the molecule, which led to the production of2,6-dibromo-4-isopropyl-phenol and three unidentified metabolites. Another metabolite of TBBPA characterized as a hexa-brominated compound with three aromatic rings was also found in the liver subcellular fractions. These results suggest that the biotransformation of BDE15and TBBPA in fish liver is mediated by cytochrome P450(CYP450) enzymes, as revealed by the formation of hydroxylated metabolites and oxidative bond cleavage products.(5) The inhibition study demonstrates that CYP1A is the major enzyme responsible for the biotransformation of BDE15and TBBPA in fish liver subcellular fractions and that CYP3A4also have a major role in metabolism of TBBPA. BDE15and BDE47inhibited crucian carp and pig hepatic CYPlA-catalyzed EROD activity in vitro, while TBBPA didn’t have inhibition effect on EROD activity. BDE15and BDE47were determined to be the competitive type inhibitor of EROD activity. It is indicated that BDE15and BDE47could combine with CYP1A active site, and be catalyzed by CYP1A. This competitive inhibition also may lead to unexpected toxicological interactions.