Ca2+-dependent and independent mitochondrial damage in HepG2 cells that overexpress CYP2E1.
CYP2E1-dependent mitochondrial damage, in the presence or absence of extracellular calcium, was investigated. HepG2 cells expressing CYP2E1 (E47 cells) were preloaded with arachidonic acid (AA), washed, and incubated with iron-nitrilotriacetate 1:3 complex (Fe-NTA) in minimum essential medium (MEM) (1.8mM Ca(2+)) or Ca(2+)-free MEM (SMEM). Toxicity in SMEM was CYP2E1-dependent, necrotic, and lipid peroxidation-dependent. Intracellular calcium did not significantly change during the incubation in SMEM. Mitochondrial damage preceded the loss of plasma membrane integrity and was significant at 12h of incubation, in coincidence with the toxicity. E47 cells treated with AA+Fe in MEM also showed a decline of mitochondrial membrane potential (Delta(Psi)(m)) that preceded the loss of plasma membrane integrity, but starting at earlier times, e.g., 3h than in SMEM. The decline in Delta(Psi)(m) and the toxicity in both MEM and SMEM were inhibited by alpha-tocopherol and cyclosporin A, while the calpain inhibitor calpeptin was only effective in MEM. In conclusion, oxidative damage to mitochondria and the permeability transition plays a role in the CYP2E1-dependent toxicity of Fe+AA in HepG2 cells, both in MEM and SMEM. Ca(2+) mobilization and activation of calpain contributes to the more rapid onset of mitochondrial damage in MEM, while oxidative damage and lipid peroxidation are involved in the Ca(2+)-independent later onset of mitochondrial damage.
Caro
Andres A
AA
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA.
Cederbaum
Arthur I
AI
D016718:Arachidonic Acid / Q000633:toxicity; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D003470:Culture Media; D016572:Cyclosporine / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects; D007501:Iron / Q000633:toxicity; D015227:Lipid Peroxidation / Q000187:drug effects; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D018384:Oxidative Stress; D010539:Permeability; D014407:Tumor Cells, Cultured; D024502:alpha-Tocopherol / Q000494:pharmacology
D003470:Culture Media; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; C071482:calpeptin; D016718:Arachidonic Acid; D016572:Cyclosporine; D007501:Iron; D019392:Cytochrome P-450 CYP2E1; D002154:Calpain; D024502:alpha-Tocopherol; D002118:Calcium