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

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 Ca 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 Ca 2؉ 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 Ca 2؉

Role of Phospholipase A2 Activation and Calcium in CYP2E1-dependent Toxicity in HepG2 Cells

“…This increase occurs prior to the developing toxicity and is inhibited by ␣-tocopherol, but not by calpeptin (34). Fig.…”

“…Measurement of Calpain Activity in Situ-The activity of calpain in intact cells was assessed with the use of the cell permeable fluorogenic substrate SUCC-LLVY-AMC (45) as previously described (34). The tested compounds at the concentrations used did not interfere with the quantification of AMC fluorescence.…”

“…These cells when exposed to Fe-NTA showed an elevation of intracellular calcium and reactive oxygen species that occurs before the onset of cellular toxicity, events blocked by antioxidants. Control HepG2 cells not expressing P450 (C34 cells) showed very low toxicity and a small increase in intracellular calcium under the same conditions (34). Activation of calpain was observed together with cell death, both processes blocked by calpain inhibitors (34).…”

“…CYP2E1-dependent Activation of Calpain and Calcium-It has been previously shown that calpain is activated in the FeϩAA-treated cells expressing CYP2E1, and that toxicity is partially inhibited by 2 calpain inhibitors, calpeptin and E64d (34). Experiments were performed in order to evaluate if FeϩAA-activated calpain activity requires extracellular calcium, or if intracellular calcium stores can play a role in the activation analogous to what was observed for PLA2 activation.…”

“…Control HepG2 cells not expressing P450 (C34 cells) showed very low toxicity and a small increase in intracellular calcium under the same conditions (34). Activation of calpain was observed together with cell death, both processes blocked by calpain inhibitors (34). An increase in intracellular calcium seems to be a very early and critical event in the toxicity caused by CYP2E1-dependent oxidative stress, leading to the activation of Ca 2ϩ -dependent processes such as calpain activity.…”