Thiol oxidation and cytochrome P450-dependent metabolism of CCl4 triggers Ca2+ release from liver microsomes

Elevation of cytosolic calcium levels has been shown to occur after exposure to hepatotoxins such as CCl4. This has been associated with inhibition of the Ca2+, Mg(2+)-ATPase which pumps calcium into the endoplasmic reticulum. Elevated cytosolic Ca2+ may also result from activation of calcium releasing channels. In the presence of NADPH, CCl4 produced a concentration-dependent release of calcium from liver microsomes after a lag period. The lag period was shorter with microsomes from pyrazole-treated rats in which CYP2E1 is induced, as compared to saline microsomes. The calcium releasing process appears to be very sensitive to activation by CCl4 as effective concentrations (e.g., 50 microM) did not affect the Ca2+, Mg(2+)-ATPase or produce lipid peroxidation. Inhibition of the CCl4-induced release of calcium by 4-methylpyrazole and by anti-CYP2E1 IgG, and the requirement for NADPH, indicates that CCl4 metabolism is required for the activation of calcium release. The lag period for CCl4-induced release of calcium was associated with the time required to deplete alpha-tocopherol from the microsomal membranes; however, lipid peroxidation was not observed at these levels of CCl4, and the lag period for CCl4-induced release of calcium was shorter under anaerobic than aerobic conditions, suggesting a possible role for CCl3 in the mechanism of activation. Production of CCl3 was observed by ESR spin-trapping experiments with PBN; PBN prevented the CCl4-induced calcium release, presumably by interacting with CCl3 and other reactive species. Calcium release was produced by thiol oxidants such as 2,2'-dithiodipyridine. Lipophilic thiols such as mercaptoethanol or cysteamine could partially reverse the CCl4-induced calcium release, whereas GSH was ineffective. While the IP3 receptor system is considered as the main regulator of calcium release, liver also contains ryanodine-sensitive calcium releasing stores. The CCl4-induced calcium release was blocked by ruthenium red, a specific inhibitor of the ryanodine receptor; ruthenium red did not block CCl4 metabolism to CCl3. CCl4 increased the binding of ryanodine, a specific ligand for the ryanodine-sensitive calcium channel. These results suggest that metabolism of CCl4 to reactive species by cytochrome P450 results in an activation of a ryanodine-sensitive calcium channel, perhaps due to oxidation of lipophilic thiols of the channel. Activation of calcium releasing channels may play a role in the elevated cytosolic calcium levels found in the liver after treatment with hepatotoxins.