The in vitro NADPH-dependent inhibition by CCl₄ of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl₃·radical: Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCL_3. radical

The hepatotoxicity of CCl₄ is mediated through its initial reduction by cytochrome P-450 to the CCl₃· radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca²⁺ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCi₄ (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca²⁺ uptake from 28% without CCl₄ to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H.H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca²⁺ uptake was due both to a decrease in the Ca²⁺-dependent ATPase and to an enhanced release of Ca²⁺ from the microsomes. The NADPH-dependent CCl₄ inhibition was greater under N₂ and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl₄ prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl₄, the CCl₄ metabolism was stopped, and then the Ca²⁺ uptake was determined; GSH reversed the CCl₄ inhibition of CA²⁺ uptake. This reversal showed saturation kinetics for GSH with two K(m) values of 0.315 and 93 μM when both the preincubation and the Ca²⁺ uptake were performed under air, and 0.512 and 31 μM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl₄ inhibition of Ca²⁺ uptake. CCl₄ increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove ¹⁴C bound to samples preincubated with the ¹⁴CCl₄. Although EDTA (100 μM) decreased the CCl₄ inhibition, the metal-complexing agents deferoxamine (100 μM) and diethyldithiocarbamate (100 μM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 μg/ml), superoxide dismutase (15 μg/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCI₄ for the Ca²⁺ pump results from the metabolism of CCl₄ to the CCl₃· radical. This radical then directly oxidizes the Ca²⁺ pump, leading to decreased Ca²⁺ uptake. Finally, GSH would appear to reduce protein disulfides enzymatically back to the sulfhydryls and to reactivate the pump.