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

Andres A Caro,Arthur I Cederbaum

doi:10.1074/jbc.m107864200

Abstract

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 microm) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 microm) 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 alpha-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40-fold). The toxicity in E47 cells mirrored 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.

Highlights

According to the calcium hypothesis of cell injury, a perturbation of intracellular Ca2ϩ homeostasis leading to a sustained increase in cytosolic Ca2ϩ is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress, causing the ultimate loss of cell viability through activation of various Ca2ϩ-dependent processes [1, 2]
CYP2E1-dependent Toxicity in HepG2 Cells Exposed to Iron plus Arachidonic Acid—Fig. 1A shows that the combination of 25 ␮M Fe-NTA and 5 ␮M arachidonic acid was highly toxic in
The toxicity in E47 cells was evident starting from early incubation times (i.e. 3 h, 62% viability), while no significant toxicity could be detected in C34 cells exposed to iron plus arachidonic acid at early times (Fig. 1A)

Summary

Introduction

According to the calcium hypothesis of cell injury, a perturbation of intracellular Ca2ϩ homeostasis leading to a sustained increase in cytosolic Ca2ϩ is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress, causing the ultimate loss of cell viability through activation of various Ca2ϩ-dependent processes [1, 2]. The initial source of Ca2ϩ depends on the specific oxidant and cell type employed, since certain toxins cause calcium release from intracellular stores [5, 7], whereas others cause Ca2ϩ influx from the extracellular space [6, 8]. Some reports suggest that both calcium-dependent and calcium-independent events contribute to the cell toxicity [15]; an influx of extracellular Ca2ϩ ions may aggravate the mechanism of iron-mediated cell injury [16].

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Conclusion