THIOL OXIDATION AND CYTOCHROME P450-DEPENDENT METABOLISM OF CCL4 TRIGGERS CA2-MICROSOMES( RELEASE FROM LIVER)

Elevation of cytosolic calcium levels has been shown to occur after ex posure to hepatotoxins such as CCl4. This has been associated with inh ibition of the Ca2+, Mg2+-ATPase which pumps calcium into the endoplas mic reticulum. Elevated cytosolic Ca2+ may also result from activation of calcium releasing channels. In the presence of NADPH, CCl4 produce d a concentration-dependent release of calcium from liver microsomes a fter a lag period. The lag period was shorter with microsomes from pyr azole-treated rats in which CYP2E1 is induced, as compared to saline m icrosomes. The calcium releasing process appears to be very sensitive to activation by CCl4 as effective concentrations (e.g., 50 mu M) did not affect the Ca2+,Mg2+-ATPase or produce lipid peroxidation. Inhibit ion of the CCl4-induced release of calcium by 4-methylpyrazole and by anti-CYP2E1 IgG, and the requirement for NADPH, indicates that CCl4 me tabolism is required for the activation of calcium release. The lag pe riod for CCl4-induced release of calcium was associated with the time required to deplete cr-tocopherol from the microsomal membranes; howev er, lipid peroxidation was not observed at these levels of CCl4, and t he lag period for CCl4-induced release of calcium was shorter under an aerobic than aerobic conditions, suggesting a possible role for (CCl4) -C-. in the mechanism of activation. Production of (.)CC1(3) was obser ved by ESR spin-trapping experiments with PEN; PEN prevented the CCl4- induced calcium release, presumably by interacting with (CCl3)-C-. and other reactive species. Calcium release was produced by thiol oxidant s such as 2,2'-dithiodipyridine. Lipophilic thiols such as mercaptoeth anol or cysteamine could partially reverse the CCl4-induced calcium re lease, whereas GSH was ineffective. While the IP3 receptor system is c onsidered as the main regulator of calcium release, liver also contain s ryanodine-sensitive calcium releasing stores. The CCl4-induced calci um release was blocked by ruthenium red, a specific inhibitor of the r yanodine receptor; ruthenium red did not block CCl4 metabolism to (CCl 3)-C-.. CCl4 increased the binding of ryanodine, a specific ligand for the ryanodine-sensitive calcium channel. These results suggest that m etabolism of CCl4 to reactive species by cytochrome P450 results in an activation of a ryanodine-sensitive calcium channel, perhaps due to o xidation of lipophilic thiols of the channel. Activation of calcium re leasing channels may play a role in the elevated cytosolic calcium lev els found in the liver after treatment with hepatotoxins.