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J. Biol. Chem., Vol. 277, Issue 1, 104-113, January 4, 2002

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Role of Calcium and Calcium-activated Proteases in CYP2E1-dependent Toxicity in HEPG2 Cells^*

Andres A. Caro and Arthur I. Cederbaum

From the Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029

The objective of this work was to investigate whether CYP2E1- and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca²⁺ and activation of Ca²⁺-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 µM) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 µM) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca²⁺ were inhibited by omission of Ca²⁺ from the extracellular medium, and toxicity was restored by reincorporation of Ca²⁺. An inhibitor of Ca²⁺ release from intracellular stores did not prevent the toxicity or the increase in Ca²⁺, reflecting a role for the influx of extracellular Ca²⁺ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca²⁺ in E47 cells exposed to iron-AA were inhibited by -tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrorred calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca²⁺ and activation of Ca²⁺-dependent proteases.

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