Metabolic activation of carbon tetrachloride: induction of cytochrome P-450 with phenobarbital or 3-methylcholanthrene and its effect on covalent binding

Abstract

Anaerobic incubation of [ 14C]carbon tetrachloride with normal rat liver microsomes and microsomes from rats treated with the inducers phenobartital (PB) and 3-methylcholanthrene (MC) reveals distinct differences in metabolic activation. While the increase in CO-binding pigment is comparable for both inducers, metabolism of CCl 4 is enhanced only by PB-induction; MC-induced microsomes are equivalent to microsomes from untreated animals. Sodium dodecyl sulphate (SDS)-electrophoresis of microsomal proteins confirms the expected increase at 52 000 daltons (cytochrome P-450 PB) on PB-induction, at 56 000 daltons (cytochrome P-450 MC) on MC-induction; after anaerobic incubation with [ 14C]CCl 4 the electrophoretic pattern is largely unchanged. The highly reactive radical intermediates of CCl 4-metabolism should attack the closest possible partner. Most of protein-bound radioactivity is located in the mass range between 47 000 and 54 000 daltons, indicating that cytochrome P-450 PB is the isoenzyme mainly responsible for CCl 4-activation; cytochrome P-450 MC plays virtually no role in metabolic activation. The direct participation of NADPH-cytochrome P-450 reductase appears unlikely, since the specific binding to proteins in the corresponding mass range is not elevated. A significant percentage of label is attached to proteins at 120 000 daltons and above, presumably oligomers of cytochrome P-450 apoprotein.