Critical role of lipid peroxidation in carbon tetrachloride-induced loss of aminopyrine demethylase, cytochrome P-450 and glucose 6-phosphatase

Liver microsomal glucose 6-phosphatase. cytochrome P-450 and aminopyrine demethylase all decrease rapidly in the CCl 4-poisoned rat. It has been suggested that such enzyme loss may be due to direct attack on enzyme catalytic sites by free radical cleavage products of CCl 4 metabolism. An alternate view has favored the suggestion that peroxidative decomposition of lipids is an intermediate link between initial homolytic cleavage of the CCl 3-Cl bond and eventual loss of these enzymes. We have subjected these two ideas to a critical test. In an anaerobic system in vitro containing liver microsomes supplemented with an NADPH-generating system, and in the presence of EDTA, all added CCl 4 is metabolized in 90 min. About one-third appears as CHCl 3 amd most of the remainder is covalently bound to microsomal lipids and proteins. In this anaerobic system in vitro there is no evolution of malonic dialdehyde. During the period of conversion of CCl 4 to CHCl 3, when extensive binding of 14C from 14CCl 4 is also taking place, there was no detectable loss of either glucose 6-phosphatase or cytochrome P-450, and aminopyrine demethylase activity decreased minimally. In the same system, under aerobic conditions and without EDTA, there is vigorous lipid peroxidation and all three of these enzyme systems decrease markedly. These experiments demonstrate conclusively that CCl 3-Cl bond cleavage and covalent binding of products of CCl 4 metabolism do not constitute a mechanism for loss of microsomal glucose 6-phosphatase, cytochrome P-450 or aminopyrine demethylase for the particular anaerobic conditions employed in vitro. By extension they suggest, but do not prove, that covalent binding of CCl 4 carbon probably does not constitute a mechanism for CCl 4-induced loss of these enzymes in vivo. Rather, these experiments support the view that lipid peroxidation is an obligatory link between initial CCl 3-Cl bond cleavage and loss of these enzymes.