CCl₄-Induced Cytochrome P-450 Loss and Lipid Peroxidation in Rat Liver Slices

Abstract

Previous studies from this laboratory have shown a concentration-time dependent, site specific cytotoxicity of CCl₄ in slices. The present study examined radical formation and lipid peroxidation as a potential mechanism of toxicity. Liver slices from male Sprague-Dawley rats (220– 250 g) were exposed to 0.57 mM CCl₄ by vaporization using a roller incubation system. Cytochrome P-450 is responsible for the bioactivation of CCl₄ generating the CCl₃ radical, which also acts as a suicide inhibitor. In slices exposed to CCl₄, cytochrome P-450 loss occurred in a timedependent manner relative to controls (↓ 58% at 9 hr). A 48 hr fast prior to sacrifice both enhanced and accelerated both cytochrome P-450 loss as well as CCl₄ toxicity in slices. Unlike cytochrome P-450, glutathione levels were not altered over the course of the experiment. These studies suggest that centrilobular hepatocytes are more susceptible to CCl₄ induced injury. Covalent binding studies using ¹⁴CC1₄ confirmed CCl₃ radical formation by cytochrome P-450. Binding to slice proteins plateaued as early as 30 min following CCl₄ administration whereas lipid binding was saturated by 60 min. Covalent binding of the CCl₃ radical was increased two-fold 60 min following phenobarbital pretreatment whereas allylisopropylacetamide caused the converse (50% at 60 min). Conjugated diene formation, an index of lipid peroxidation as detected by 15 min following