Correlation between cytosolic Ca²⁺ concentration and cytotoxicity in hepatocytes exposed to oxidative stress

To investigate the relationship between alterations of cytosolic Ca²⁺ concentration and development of cytotoxicity, isolated rat hepatocytes were loaded with the fluorescent indicator Quin-2 AM and then incubated with non-toxic or toxic levels of menadione (2-methyl-1,4-napthoquinone) or tert-butyl hydroperoxide (t-BH). The resulting changes in cytosolic Ca²⁺ concentration were compared to those seen upon exposure of the hepatocytes to an α₁-adrenergic agonist, phenylephrine, as well as to those induced by menadione and (t-BH) in hepatocytes pretreated with agents that modify their toxicity. Exposure of hepatocytes to phenylephrine or non-toxic levels of menadione caused a moderate and transient increase in cytosolic Ca²⁺({slanted equal to or less-than}0.7 μM), whereas a toxic concentration of menadione produced a marked, sustained increase in Ca²⁺ which fully saturated the binding capacity of Quin-2 (>1.5 μM). Treatment of the hepatocytes with the protective agent, dithiothreitol, prevented both the increase in cytosolic Ca²⁺ and the cytotoxicity induced by menadione. On the other hand, pretreatment of cells with diethylmaleate to deplete intracellular glutathione made otherwise non-toxic concentrations of menadione cause both a sustained increase in cytosolic Ca²⁺ and cytotoxicity. Similarly, toxic concentrations of t-BH also caused a sustained increase in cytosolic Ca²⁺. The iron chelator, desferrioxamine, and dithiothreitol (DTT), which protected the cells from t-BH toxicity, also prevented the sustained elevation of cytosolic Ca²⁺. Our findings provide further support for the hypothesis that a perturbation of intracellular Ca²⁺ homeostasis is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress.