title: Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1.
abstract: Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.
authors.affiliation: Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, Box 1603, One Gustave L. Levy Place, New York, NY 10029, USA. Andres.Caro@mssm.edu
authors.forename: Andres A
authors.initials: AA
authors.lastname: Caro
authors.forename: Arthur I
authors.initials: AI
authors.lastname: Cederbaum
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000502:physiology* / Q000633:toxicity; D002118:Calcium / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis*; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004789:Enzyme Activation; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D015227:Lipid Peroxidation; D053078:Membrane Potential, Mitochondrial; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000502:physiology*; D054467:Phospholipases A2; D051381:Rats; D024502:alpha-Tocopherol / Q000494:pharmacology
chemical_list: D065691:Cytochrome P-450 CYP2E1 Inhibitors; D016718:Arachidonic Acid; D019392:Cytochrome P-450 CYP2E1; D010741:Phospholipases A; D054467:Phospholipases A2; D024502:alpha-Tocopherol; D002118:Calcium