The objective of the proposed research is to elucidate the molecular basis for species differences in the metabolism of two model polychlorinated biphenyls (PCBs) by the major phenobarbital-inducible forms of cytochrome P450 from dogs, rabbits, and rats. PCBs have been widely used in industry due to their resistance to thermal, chemical and biological degradation. This high stability has led to the persistence of PCBs in the environment, and the toxicity associated with many PCBs poses a serious human health hazard. One of the key factors that determines the stability in the environment and in animal and human tissues of PCBs is their metabolism. In general, highly chlorinated PCBs lacking two adjacent unsubstituted sites on one of the phenyl rings, are metabolized slowly by micro- organisms and mammals. One such compound which is highly persistent in the environment and in human tissues and breast milk is 2,2',4,4',5,5'- hexachlorobiphenyl (245-HCB), a known tumor promoter in rats. Of all species tested to date, only the dog appears to be able to metabolize and eliminate 245-HCB efficiently. This metabolism is attributable to a cytochrome P450 that is a member of the P450IIB (phenobarbital- inducible) subfamily in dog liver. The dog P450IIB is also active in the metabolism of 2,2',3,3;,6,6'-hexachlorobiphenyl (236-HCB), which has adjacent unsubstituted sites on both phenyl rings. In contrast, the structurally related cytochromes P450IIB in the rabbit and rat metabolize 245-HCB poorly, although rat P450IIB1 can metabolize 236-HCB. Presumably, electronic and steric properties of the enzymes and of the PCBs dictate which cytochromes P450IIB can metabolize which hexachlorobiphenyls. The hypothesis to be tested is that amino acid residues required for the metabolism of 245-HCB and 236-HCB by cytochromes P450IIB can be identified. The basic approach will be to express in heterologous systems cDNAs encoding native, hybrid, and mutant forms of the enzymes. These will be assayed for their ability to metabolize not only the two hexachlorobiphenyls but also the two model substrates androstenedione and progesterone. The steroids will serve as positive control of the overall functional integrity of any hybrid or mutant enzymes since the native cytochromes P450 from all three species hydroxylate these compounds. These studies should provide the rational basis for the design of superior catalysts of PCB detoxification.
Showing the most recent 10 out of 16 publications
