| Polybrominated diphenyl ethers and tetrabromobisphenol A are brominated flame retardants, which are widely used in building materials, cable insulation, electronic products, furniture and other materials. Polybrominated diphenyl ethers are persistent, lipophilic, bioaccumulative and toxic to animals.4,4’-dibromodiphenyl ether (BDE-15) and2,2’,4,4’-tetrabromodiphenyl ether (BDE-47) were detected in many biota samples and human blood and tissue samples, and BDE-47was detected as the dominant congener. Although the half-life of TBBPA in the environment is short, its ecological effects can not be ignored because of the huge amount of usage. Characterization of PBDEs and TBBPA metabolism in vitro can provide a better assessment of the role of metabolism in their bioaccumulation, persistence and toxicity.The studies on metabolism of brominated flame retardants in vitro mainly focus on model animals (rats, mice, etc.), and studies on human tissues are fewer. However, the CYP450enzymes of rats and mice are quite different from those of humans. Although the pig seems to be a good model of human metabolism, there is few data directly dealing with the metabolism of brominated flame retardants in the tissues of pigs. In this study, BDE-15, BDE-47and TBBPA were selected as the typical brominated flame retardants to study their biotransformation in pig liver subcellular fractions (microsomes and S9fractions) in vitro. The structures of metabolites of BDE-15, BDE-47and TBBPA formed in pig liver subcellular fractions were identified. The cytochrome P450(CYP450) inhibition studies and the effects of different concentrations of substrates on CYP450enzymes were determined so as to investigate the key enzymes involved in the metabolism of BDE-15, BDE-47and TBBPA in pig liver subcellular fractions. The results are summarized as follows:(1) BDE-47could be metabolized in pig liver microsomes significantly. Three metabolites were observed in BDE-47exposed microsomes, which were2,4-dibromophenol and two mono-OH-tetrabromodiphenyl ethers. There seem to be two metabolic pathways:hydroxylation and cleavage of the diphenyl ether bond. By comparing with the reference materials of OH-BDE-47, it could be concluded that the two hydroxylated metabolites of BDE-47may be formed through the1,2-shift mechanism. BDE-47at various concentrations (0.02,0.1,0.2,1,2,10and20μM) could protect the activity of7-ethoxycoumarin O-deethylase (ECOD) of pig liver microsomes, while BDE-47at the concentrations of1,2and10μM could inhibit the activity of7-ethoxyresorufin O-deethylase (EROD) significantly, and no significant effect were observed on the activity of aniline p-hydroxylase (ANH).(2) BDE-15could also be metabolized in pig liver subcellular fractions significantly. Three metabolites observed in BDE-15exposed microsomes or S9fractions were4-bromophenol and two mono-OH-dibromodiphenyl ethers. The metabolic pathways of BDE-15in pig liver subcellular fractions were hydroxylation and cleavage of the diphenyl ether bond, which were the same as those of BDE-47. Inhibition studies showed that CYP1A and CYP3A were involved in the BDE-15metabolism in pig liver microsomes.(3) TBBPA could be metabolized in pig liver subcellular fractions significantly. Km value of TBBPA metabolized by pig liver microsomes was14.42μM, and Vmax value was0.0975μM·min-1·mg-1protein. Six metabolites were observed in TBBPA exposed microsomes or S9fractions. Five metabolites were detected in GC-MS and one was found in LC-MS. Structures of two metabolites were proposed as4-isopropyl-2,6-dibromophenol and2,6-dibromo-4-(2’,6’-dibromo-1’-hydroxycumyl)-phenoxy-3",5"-dibromo-4"-hydroxybenzene. Other four metabolites contained two bromine atoms, and the cleavage of the bond adjacent to the central carbon atom may occur. The main metabolic ways of TBBPA may be through the cleavage of the bond adjacent to the central carbon atom and molecular polymerization. Inhibition studies showed that CYP1A and CYP3A were involved in the TBBPA metabolism in pig liver microsomes or S9fractions, and CYP3A played the major role. |
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