| Polybrominated diphenyl ethers (PBDEs) are common flame retardants widely used in the manufacture of industrial and domestic equipments such as textiles, furniture, and electronic as well as electrical items to prevent fire and minimize fire damage. A number of rodent studies reported that PBDEs should have potential developmental neurotoxicity, developmental reproductive toxicity, and endocrine disruptive effects. During the past decades, large amounts of PBDEs have been produced and applied, which results in widespread contamination of the environment and accumulation of PBDEs in food webs. It is noteworthy that the concentrations of PBDEs in human and other body tissues have been increasing with doubling times being approximately 4~6 years. BDEs 47, 99, 100, 153, 154, 183 and 209 are the major BDE congeners found in most human and environmental biota. A number of studies on laboratory animal exposure of PBDE congeners reported that PBDEs could produce reductively debrominated metabolites and oxidative metabolites. Due to debrominated metabolites'high bioaccumulation and persistence [1] and increasing levels in the environment which therefore contribute to the endocrine-disrupting effects of PBDEs. Laboratory studies have confirmed that OH-BDE metabolites have greater adverse effects compared to the PBDEs. They have received increasing concern in recent years. Therefore, the information on the extent of formation of reductively debrominated metabolites and hydroxylated metabolites is critical to understand the complete risks associated with PBDE bioaccumulation and metabolism in humans and wildlife. However, the metabolic processes of PBDEs are not clear.Cytochrome P450 monooxgenases, composing 70~80% of all phase I xenobiotic-metabolizing enzymes, can metabolize a large number of endogenous compounds and are involved in many cellular functions. Usually when hydroxylation, dealkylation or oxidation occurs, ring-opening, and reduction can take place at the same time. Mainly the P450s families 1, 2, and 3 appear to be responsible for the metabolism of a variety of exdogenous and endogenous compounds. The liver is the predominant sites for P450-mediated compound elimination, while the other tissues contribute to a much smaller extent for compound elimination. Primary liver cells, containing abundant metabolizing enzymes such as cytochrome P450s, are considered to be a better model to study the metabolism of PBDEs. But, it is very limited because the primary human hepatocytes are difficult to obtain. Therefore, primary rat hepatocytes are used instead to establish a system for PBDEs metabolism. To date, there are several studies on the PBDE metabolism in human microsomes and hepatocytes. The up-regulation of the CYP genes and the formation of several oxidative metabolites of PBDEs suggested a key role of CYP-mediated metabolism. However, so far, little information on BDE congeners metabolism in human cytochrome P450s has been provided. In the present study, we established the in vitro model using primary rat hepatocytes and recombinant cytochrome P450s to detect whether reductively debrominated, OH and/or MeO metabolites of BDE-99 would be produced and which cytochrome P450 was involved in the BDE-47, BDE-99 and BDE-153 metabolism, and the compare metabolic activities of cytochrome P450s to BDE-47, BDE-99 and BDE-153. The study will also provide a better understanding on the pathways through which PBDEs are metabolized in humans.Part one Identification of the metabolites of PBDEs and initially determined its related cytochrome P450s in vitroObjectives We undertook this study to determine whether BDE-47, BDE-99 and BDE-153could be metabolized by rat liver cells in vitro and to identify what types of metabolites are formed. Methods First, the rat primary hepatocytes were isolated and treated with three environmentally relevant and prominent PBDE congeners—BDE-47, BDE-99 or BDE-153—for 24~72 hrs. Metabolites were then extracted from the hepatocytes and media, and detected by GC/MS. Several mRNA of metabolic enzymes were also extracted from the same cells and the gene expression level were determined using quantative real-time RT-PCR. Results Several CYP genes CYP1A2, CYP2B1/2, especially CYP3A23/3A1 were found to be the average about up-regulated to 15%, 20% and 120% in PBDEs exposed primary rat hepatocytes. After 72 hr treatment, about 12.2 %, 13.5% and 5.3% of BDE-47, BDE-99 and BDE-153 mass were uncovered, respect. Instead, two oxidative metabolites, 3-OH-BDE-47 and 5-OH-BDE-47 were identified from the BDE-47 treated rat hepatocytes; one reductively debrominated metabolite, BDE-47, and three oxidative metabolites, 2, 4, 5-tribromophenol, 5-OH-BDE-47 and 5'-OH-BDE-99, were identified from the BDE-99 treated rat hepatocytes. While no debrominated or hydroxylated metabolites were observed in the cells exposed to BDE-153.Part two Human CYP enzymes on metabolism of PBDEs in vitroObjectives Baculovirus/Sf9 system was utilized for expression of CYP450s and POR enzymes. Then explaining the bioactivities of PBDEs by CYP450s and identifying key enzymes. Methods Recombinant baculovirus were generated by CYP1A2, CYP2A6, CYP2A13, CYP2C9, CYP2E1, CYP3A4 and POR were expressed in Sf9 cells infected with the virus particles by addingδ-ALA and Fe3+, then the target proteins were detected by Western blotting analysis; CYP contents were measured by standard reduced CO-difference spectroscopy. In addition, selected recombinant cytochrome P450s were used to explore the metabolism of BDE-47, BDE-99 or BDE-153 in vitro. The parent depletion approach was used for screening the CYPs'ability to eliminate BDE-47, BDE-99 or BDE-153. Results Reconstructed Ac-Bacmid-CYPs(POR)were get subsequently, then transfected into Sf9 cells to obtain the recombinant baculovirus particles. Baculovirus/Sf9 system were used to express CYP1A1, CYP1A2, CYP2A6, CYP2A13, CYP2C9, CYP2E1, CYP3A4 and POR in vitro. A single protein band was detected by immunoblot analysis with the expected molecular weight in the microsomes prepared from the Sf9 cells that were infected with the expression vectors containing CYP450 cDNAs; All the microsome samples expressing the recombinant CYP450s proteins displayed the characteristic absorption peak at 450 nm. As determined by CO-difference spectroscopy analysis. CYP450 contents ranging from 0.08 to 0.35 nmol/mg microsome proteins. There were very large differences among the different CYPs in the metabolism of PBDEs, CYP1A2 and CYP3A4 showed the highest metabolic efficiency and the metabolic clearance rate to BDE-47 and BDE-99 approximate to 30.0%, to BDE-153 approximate to 20.0%, respectively.ConclusionsIn our available system of in vitro metabolism, two metabolites of BDE-47 and four metabolites of BDE-99 were identified. These metabolites have been shown to elicit greater toxicity than the parent BDE congeners in laboratory bioassays; Several CYP genes (CYP1A2, CYP2B1/2, especially CYP3A23/3A1) were found to be up-regulated in PBDEs exposed primary rat hepatocytes; Human CYP3A4 as well as CYP1A2 were demonstrated to be involved in the PBDEs metabolism. |
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