Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: Role of cytochrome P-450

Abstract

In order to define the site of bioactivation of CCl 4, CHCl 3 and CBrCl 3 in the NADPH cytochrome c reductase-cytochrome P-450 coupled systems of liver microsomes, the 14C-labeled hepatotoxins were incubated in vitro with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O 2 (8:2) and addition of a cytochrome c reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome c reductase activity was unchanged, the covalent binding of CCl 4, CHCl 3, and CBrCl 3 was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N 2 enhanced the binding of CCl 4, inhibited the binding of CHCl 3 and did not influence the binding of CBrCl 3. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome c reductase and that CCl 4 bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl 3 activation proceeds by cytochrome P-450 dependent oxidative pathways.