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.

Thesis (M.S.)--Wisconsin. / Includes bibliographical references (leaves 70-79).

Polychlorinated biphenyls (PCBs) are ubiquitious environmental pollutants that cause a number of diverse toxicities. The chemical stability of PCBs is responsible for their persistence in the environment, while their lipid solubility and resistance to biotransformation results in their accumulation in a number of animal species. The rate of PCB elimination is dependent on the ability of each animal species to metabolize a particular PCB congener. The goal of this project was to determine if in vitro liver microsomal metabolism studies could predict in vivo metabolism and to examine the reasons for the species variation in PCB metabolism. Kinetic constants were developed from in vitro metabolism studies using 4,4'-dichlorobiphenyl (4-DCB), 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB) and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) and liver microsomes from the human, dog, monkey and rat. An excellent correlation between the in vitro Vmax values and the in vivo hepatic clearance values was obtained. Human microsomal PCB metabolism was most similar to the rat. The in vitro human results were consistent with available in vivo data. All species produced the same major metabolites. The major metabolite of 4-DCB was 4,4'-dichloro-3-biphenylol and the two major metabolites of 236-HCB were 2,2',3,3',6,6'-hexachloro-4-biphenylol and 2,2',3,3',6,6'-hexachloro-5-biphenylol. The dog was the only species found to metabolize 245-HCB in vitro. Metabolites of 245-HCB were not identified. Studies of metabolism, covalent binding of PCB-equivalents to microsomal protein and metabolites demonstrated that the dog can metabolize PCBs more readily than other species because the dog has an alternate pathway of PCB metabolism. This pathway is either not found in other species or only found to a limited extent. Furthermore, an arene oxide does not seem to be involved in this alternative pathway. In summary, for certain classes of compounds in vitro to in vivo extrapolation is possible and may prove to be very useful in predicting the appropriate animal model for humans. Secondly, the dog appears to be quite different in its metabolism of PCBs in that it may have an alternate route of metabolism not involving an arene oxide.

The metabolism and distribution of three commonly occurring PCB congeners, 4,4'-dichlorobiphenyl (4-DCB), 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB) and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), each displaying different structural features, were investigated at their principal metabolic site, the hepatocyte. Hepatocytes, isolated from male Sprague-Dawley rats (200-250 g) by collagenase perfusion, were suspended in medium 199 and maintained at 37°C in a gyratory shaker. The radiolabeled ¹⁴C-PCB congeners were added to the hepatocyte suspensions as a DMSO-albumin mixture. Each congener was rapidly taken up by the cells with less than 10% of the congener remaining in the medium. The congeners accumulated within the hepatocytes without being fully metabolized. Metabolism followed first order Michaelis-Menten kinetics for 20 min and plateaued by 90 min at which point only 32% of 4-DCB (0.01-100 uM) and 60% of 236-HCB (0.01-100 uM) was metabolized, while 245-HCB (0.1-200 uM) was not metabolized. Readdition of congener once metabolism had plateaued resulted in a reinitiation of metabolism with the same proportion of metabolites produced indicating that product inhibition was not the cause for the plateau. A partitioning of the PCB congeners within subcellular compartments and binding to cytosolic proteins influenced the extent of metabolism by decreasing the availability of congener for the drug metabolizing enzymes, cytochrome P-450. Spectral binding studies further revealed that the ability of a PCB congener to bind to the cytochrome P-450 system correlated with the extent of metabolism observed, with 236-HCB 4-DCB 245-HCB. The metabolic potential of the PCB congeners was influenced by both the affinity of the congener for cytochrome P-450 and the partitioning of congener within the hepatocyte, and not by product inhibition.