IN VITRO METABOLISM OF POLYCHLORINATED BIPHENYLS BY DOG HEPATIC CYTOCHROMES P-450

Abstract

The biochemical basis for the unique ability of Beagle dog liver microsomes to metabolize 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) was investigated. The major phenobarbital (PB)-inducible cytochrome P-450 isozyme, called PBD-2, was purified to 95% homogeneity from liver microsomes of both control and PB-treated dogs, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a reconstituted system containing cytochrome b₅, PBD-2 metabolized 245-HCB at a rate greater than three-fold that seen in liver microsomes from PB-treated dogs. Immunoblot analysis revealed that, upon PB treatment, the increase in the level of PBD-2 in dog liver microsomes correlated well with the increase in the rate of hepatic microsomal metabolism of 245-HCB by dogs. Anti-PBD-2 IgG caused a > 90% inhibition of 245-HCB metabolism by microsomes obtained from control and PB-treated dogs. Studies were also conducted to assess the ability of PBD-2 to metabolize 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB) and 4,4'-dichlorobiphenyl (4-DCB). Dog liver microsomes readily metabolized 236-HCB, and PB treatment led to a dramatic increase in this rate of metabolism, suggesting a role for PBD-2 in the metabolism of 236-HCB. In a reconstituted system containing cytochrome b₅, PBD-2 metabolized 236-HCB at a rate greater than two-fold that observed in microsomes from PB-treated dogs. Pretreatment of microsomes obtained from PB-treated dogs with chloramphenicol (a highly selective inactivator of PBD-2) caused a nearly 70% decrease in the microsomal metabolism of 236-HCB. Anti-PBD-2 IgG inhibited by > 90% 236-HCB metabolism by microsomes from both control and PB-treated dogs. In contrast, PB treatment caused no significant alteration in the metabolism of 4-DCB by dog liver microsomes, and PBD-2 metabolized this compound poorly, even in the presence of cytochrome b₅. Taken together, these data indicate that the dog hepatic cytochrome P-450 isozyme, PBD-2, is present in microsomes obtained from both control and PB-treated animals. PBD-2 is responsible for the microsomal metabolism of 245-HCB, and this isozyme likely accounts for the unique ability of Beagle dogs to readily metabolize and eliminate this compound in vivo. The data also strongly suggest that PBD-2 is responsible for the microsomal metabolism of 236-HCB in dogs. However, PBD-2 is not likely involved in the microsomal metabolism of 4-DCB by this species.