Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress.
To investigate the relationship between alterations of cytosolic Ca2+ concentration and development of cytotoxicity, isolated rat hepatocytes were loaded with the fluorescent indicator Quin-2 AM and then incubated with non-toxic or toxic levels of menadione (2-methyl-1,4-naphthoquinone) or tert-butyl hydroperoxide (t-BH). The resulting changes in cytosolic Ca2+ concentration were compared to those seen upon exposure of the hepatocytes to an alpha 1-adrenergic agonist, phenylephrine, as well as to those induced by menadione and t-BH in hepatocytes pretreated with agents that modify their toxicity. Exposure of hepatocytes to phenylephrine or non-toxic levels of menadione caused a moderate and transient increase in cytosolic Ca2+ (less than or equal to 0.7 microM), whereas a toxic concentration of menadione produced a marked, sustained increase in Ca2+ which fully saturated the binding capacity of Quin-2 (greater than 1.5 microM). Treatment of the hepatocytes with the protective agent, dithiothreitol, prevented both the increase in cytosolic Ca2+ and the cytotoxicity induced by menadione. On the other hand, pretreatment of cells with diethylmaleate to deplete intracellular glutathione made otherwise non-toxic concentrations of menadione cause both a sustained increase in cytosolic Ca2+ and cytotoxicity. Similarly, toxic concentrations of t-BH also caused a sustained increase in cytosolic Ca2+. The iron chelator, desferrioxamine, and dithiothreitol (DTT), which protected the cells from t-BH toxicity, also prevented the sustained elevation of cytosolic Ca2+. Our findings provide further support for the hypothesis that a perturbation of intracellular Ca2+ homeostasis is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress.
Nicotera
P
P
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
McConkey
D
D
Svensson
S A
SA
Bellomo
G
G
Orrenius
S
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism*; D004347:Drug Interactions; D006706:Homeostasis; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D010545:Peroxides / Q000633:toxicity*; D010656:Phenylephrine / Q000494:pharmacology; D010762:Phosphorylase a / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014812:Vitamin K / Q000633:toxicity*; D020122:tert-Butylhydroperoxide
D010545:Peroxides; D014812:Vitamin K; D010656:Phenylephrine; D020122:tert-Butylhydroperoxide; D010762:Phosphorylase a; D002118:Calcium