Developmental exposure to multiple-ortho substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB-), dexamethasone (DEX-) and corn oil (VEH-)treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzymes. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer didn't affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes, but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.

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This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is a chiral and highly neurotoxic PCB congener of environmental relevance. (+)-PCB 136 was previously shown to be enriched in tissues from mice treated with racemic PCB 136. We investigated the spectral interactions of (+)-, (-)-, and (+/-)-PCB 136 with mouse and rat hepatic microsomal cytochrome P450 (P450) enzymes to test the hypothesis that enantioselective binding to specific P450 enzymes causes the enrichment of (+)-PCB 136 in vivo. Hepatic microsomes prepared from C57BL/6 mice or Long Evans rats treated with beta-naphthoflavone or 3-methylcholanthrene, phenobarbital, and dexamethasone (prototypical inducers of CYP1A, CYP2B, and CYP3A, respectively) were used to determine first, whether the (+)-PCB 136 atropisomer binds to hepatic microsomal P450 enzymes to a greater extent than does the (-)-PCB 136 atropisomer and second, whether P450 enzymes of one subfamily bind the two PCB 136 atropisomers more efficiently than do P450 enzymes of other subfamilies. Increasing concentrations of (+)-, (-)-, or (+/-)-PCB 136 were added to hepatic microsomes, and the difference spectrum and maximal absorbance change, a measure of PCB binding to P450 enzymes, were measured. A significantly larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four hepatic microsomal preparations in mice and rats, indicating that (+)-PCB 136 interacted with microsomal P450 enzymes to a greater degree than did (-)-PCB 136. In addition, binding of the PCB 136 atropisomers was greatest in microsomes from PB-treated mice and rats and was inhibited by CYP2B antibodies, indicating the involvement of CYP2B enzymes. Together, these results suggest preferential binding of (+)-PCB 136 to P450 enzymes (such as CYP2B and CYP3A) in hepatic microsomes, an observation that may explain the enantioselective enrichment of the (+)-PCB 136 atropisomer in tissues of mice.