Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms

Abstract

Degradation of intrinsic hepatic [14C] heme was analyzed as 14CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16 h after pulse-labeling with 5-amino[5-14C]laevulinic acid, a precursor that labels bridge carbons of heme in non-erythroid tissues. NADPH-catalyzed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [14C] heme (largely cytochrome P-450 heme), but little 14CO formation. No additional 14CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [14C] heme. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl2 or phenyl-t-butylnitrone(a spin-trapping agent for radicals), carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide may promote loss of cytochrome P-450 heme through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic heme, catabolism of [14C]methemalbumin by microsomal heme oxygenase in vitro produced equimolar quantities of 14CO and bilirubin, although these catabolites reflected only 18% of the degraded [14C] heme. This value was increased to 100% by addition of MnCl2, which suggests that lipid peroxidation may be involved in degradation of exogenous heme to products other than CO. Phenyl-t-butylnitrone completely blocked heme oxygenase activity, which suggests that hydroxy free radicals may represent a species of active O2 used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [14C] heme was decreased and heme oxygenase activity was unchanged; however, 14CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [14C] heme and 14CO excretion, an important fraction of hepatic [14C] heme in normal rats is degraded by endogenous pathways not involving CO. Carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 heme through mechanisms that do not yield CO as an end product, and that are insenstive to cycloheximide and independent of heme oxygenase activity.