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

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{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:O₂ (8:2) and addition of a cytochrome $\text{c}$ 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 $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.