In vitro metabolism of BDE-47, BDE-99, and α-, β-, γ-HBCD isomers by chicken liver microsomes

The in vitro oxidative metabolism of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99), and the individual α-, β- and γ-hexabromocyclododecane (HBCD) isomers catalyzed by cytochrome P450 (CYP) enzymes was studied using chicken liver microsomes (CLMs). Metabolites were identified using a liquid chromatography-tandem mass spectrometry method and authentic standards for the oxidative metabolites of BDE-47 and BDE-99. Six hydroxylated tetra-BDEs, namely 4-hydroxy-2,2′,3,4′-tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (3-OH-BDE-47), 5-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE-47), 4′-hydroxy-2,2′,4,5′- tetrabromodiphenyl ether (4′-OH-BDE-49), and 2′-hydroxy-2,3′,4,4′-tetrabromodiphenyl ether (2′-OH-BDE-66), were identified and quantified after incubation of BDE-47 with CLMs. 4′-OH-BDE-49 was the major metabolite formed. Three hydroxylated penta-BDEs (5′-hydroxy-2,2′,4,4′,5-pentabromodiphenyl ether (5′-OH-BDE-99), 6′-hydroxy-2,2′,4,4′,5- pentabromodiphenyl ether (6′-OH-BDE-99), and 4′-hydroxy-2,2′,4,5,5′-pentabromodiphenyl ether, 4′-OH-BDE-101, were formed incubating BDE-99 with CLMs. Concentrations of BDE-99 metabolites were lower than those of BDE-47. More than four mono-hydroxylated HBCD (OH-HBCD), more than four di-hydroxylated HBCD (di-OH-HBCD), more than five mono-hydroxylated pentabromocyclododecenes (OH-PBCD), and more than five di-hydroxylated pentabromocyclododecenes (di-OH-PBCD) were detected when α-, β-, or γ-HBCD were individually incubated with CLMs. Response values (the ratio between the peak areas of the target compound and its internal standard) for OH-HBCD were 1-3 orders of magnitude higher than those for OH-PBCD, di-OH-HBCD, and di-OH-PBCD, suggesting that OH-HBCD might be the major metabolites of α-, β- and γ-HBCD produced by CLMs. No diastereoisomeric or enantiomeric bioisomerisation was observed incubating α-, β- or γ-HBCD with CLMs. Collectively, our data suggest that (i) BDE-47 is metabolized at a faster rate than BDE-99 by CLMs, (ii) OH-HBCD are the major hydroxylated metabolites of α-, β- and γ-HBCD produced by CLMs and (iii) the diastereoisomeric or enantiomeric bioisomerisation of α-, β- and γ-HBCD is not mediated by chicken CYP enzymes.