Carbon tetrachloride-induced cell death in perfused livers from phenobarbital-pretreated rats under hypoxic conditions and various ionic milieu: Further evidence for calcium-dependent irreversible changes

Abstract

The role of Ca 2+ in the initiation of carbon tetrachloride (CCl 4) hepatotoxicity was studied using perfused livers isolated from phenobarbital-pretreated rats in a single-pass system. Krebs-Henseleit bicarbonate buffer containing 1.3 mM CaCl 2 (KHB) was the regular ionic milieu. In the liver perfused with fructose-supplemented regular KHB equilibrated with 95% N 2−5% CO 2, infusion of 0.5 mM CCl 4 caused an early uptake of Ca 2+ coupled with K + leakage and Na + uptake within the infusion time of 30 min, which was followed by a marked lactic dehydrogenase (LDH) leakage into the effluent perfusate and further Ca 2+ uptake by the liver. With Ca 2+-free medium, the prenecrotic K + leakage and the successive LDH leakage were suppressed markedly. However, a perfusate exchange from regular to Ca 2+-free KHB at the end of the prenecrotic stage did not protect against the LDH leakage, and the perfusate exchange conversely did not produce LDH leakage. Perfusion of the liver with high K +(Cl −) medium under 20% O 2 markedly suppressed CCl 4-induced LDH leakage even in the presence of Ca 2+, whereas once CCl 4 had acted under regular KHB perfusion, changing the medium to high K + did not further prevent the LDH leakage. High K +-lactobionic acid medium containing Ca 2+ and supplemented with fructose also suppressed LDH leakage under 95% N 2 without the accompanying prenecrotic Ca 2+ uptake. However, a change of the medium after CCl 4 infusion to regular KHB containing Ca 2+ caused LDH leakage and K + leakage, with Ca 2+ uptake. The prevention of LDH leakage in a different ionic milieu may not be due to suppression of CCl 4 bioactivation, since the liver cytochrome P450 content decreased to a similar extent. These findings suggest that entry of extracellular Ca 2+ into hepatocytes coupled with K + leakage and Na + entry is a prerequisite for CCl 4-induced hepatocyte death and that association of Ca 2+ with a CCl 4-derived radical-mediated process may be necessary for early and irreversible plasma membrane damage.