Abstract
Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.
Highlights
Several studies have shown that reactive oxygen species in cellular systems can activate PLA2 activity, measured as increased release of radioactive arachidonic acid (AA) in prelabeled cells (4 –11)
PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA؉Fe was higher in the CYP2E1 expressing HepG2 cells
These results suggest that release of stored calcium by AA؉Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2؉, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that produces the rapid toxicity of AA؉Fe in CYP2E1-expressing HepG2 cells
Summary
Several studies have shown that reactive oxygen species in cellular systems can activate PLA2 activity, measured as increased release of radioactive AA in prelabeled cells (4 –11). Analogous to PLA2 activity, Ca2؉ depletion but [14], all conditions associated with increased oxidant producnot removal of extracellular calcium alone prevented the activation of calpain activity by AA؉Fe. Analogous to PLA2 activity, Ca2؉ depletion but [14], all conditions associated with increased oxidant producnot removal of extracellular calcium alone prevented the activation of calpain activity by AA؉Fe These results suggest that release of stored calcium by AA؉Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2؉, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that produces the rapid toxicity of AA؉Fe in CYP2E1-expressing HepG2 cells. PLA2 activation results in the degradation of membrane phospholipids and in the accumulation of unsaturated free fatty acids and Phospholipase A2 (PLA2) comprises a set of extracellular and intracellular enzymes that catalyze the hydrolysis of the sn-2 lysophospholipids, which by themselves can be injurious [17]. There is mounting evidence that membrane lipid peroxidation stimulates phospholipid hydrolysis via Ca2ϩ-dependent PLA2 activity. Amido-4-methylcoumarin; E47 cells, HepG2 cell line derived after AMC, 7-amido-4-methylcoumarin; BPB, 4-bromophenacyl bromide; transfection with pCI-neo vector containing the human CYP2E1 cDNA; BEL, bromoenol lactone; PTK, palmityl trifluoromethyl ketone; A.U., C34 cells, HepG2 cell line derived after transfection with pCI-neo vec- arbitrary units; MOPS, 4-morpholinepropanesulfonic acid; ANOVA, tor; CYP2E1, cytochrome P450 2E1; PBS, phosphate-buffered saline; analysis of variance
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