Effect of extracellular Ca++ omission on isolated hepatocytes. I. Induction of oxidative stress and cell injury.
The effect of varying extracellular Ca++ concentration and various antioxidants and metal ion chelators on the viability of isolated rat hepatocytes incubated under 95% O2-5% CO2 was evaluated. Decreasing the extracellular Ca++ concentration from 2 mM to 50 microM or less resulted in a progressive increase in cell injury as determined by lactate dehydrogenase (LDH) leakage. The generation of an oxidative stress, indicated by malondialdehyde formation, occurred before and concomitant with LDH leakage. The antioxidants vitamin E, N,N'-diphenyl-p-phenyl-enediamine, chlorpromazine and promethazine, as well as the iron chelators desferrioxamine and EDTA, all prevented both malondialdehyde production and LDH leakage induced by the absence of extracellular Ca++. A marked loss of cytosolic and mitochondrial glutathione was observed in cells incubated without Ca++, which could be significantly prevented by antioxidants and iron chelators. Similar effects on hepatocyte vitamin E levels also were noted. Addition of the Ca++ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid to the incubation medium totally prevented LDH leakage and malondialdehyde production and partially ameliorated glutathione and vitamin E loss. Conversely, cell injury resulting from disrupted cellular Ca++ homeostasis after treatment with the Ca++ ionophore A23187 plus 2 mM Ca++ was only slightly inhibited by antioxidants and iron chelators. However, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid also prevented A23187-induced cell injury. These results indicate that a decrease in extracellular Ca++ generates an oxidative stress in hepatocytes which leads ultimately to severe cell injury. This oxidative stress appears to be a Ca++-dependent phenomenon, precipitating all toxicity by a mechanism distinct from that induced by A23187 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D010655:Phenylenediamines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D000975:Antioxidants; D010655:Phenylenediamines; D008315:Malondialdehyde; D004533:Egtazic Acid; C004845:N,N'-diphenyl-4-phenylenediamine; D007770:L-Lactate Dehydrogenase; D003676:Deferoxamine; D002118:Calcium