Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions.
Effects of oxidative stress on isolated rat ventricular myocytes were studied. Myocyte viability was determined by the ability of these cells to retain rod-shaped morphology and to exclude trypan blue. The mean life time of myocytes was quantitated using the Weibull distribution function. Superfusion with 200 microM tert-butyl hydroperoxide (t-BHP) led to a time-dependent loss of cell viability, generation of the products of lipid peroxidation, oxidation of protein and non-protein thiols, a decrease in [ATP]i and in the cellular energy charge. Dithiothreitol (DTT, 5 mM) prolonged survival of myocytes exposed to t-BHP, attenuated oxidation of protein and non-protein thiols, and preserved the energy charge. Exposure to DTT did not affect the concentration of t-BHP-generated lipid peroxidation products. Promethazine (1 microM) prevented t-BHP-induced increase in the concentration of lipid peroxidation products, but did not prevent either loss of thiols or loss of cell viability. Superfusion with N-ethylmaleimide (NEM, 5 microM) also led to loss of cell viability, with accompanying decreases in protein and non-protein thiols, ATP and energy charge without the accumulation of the products of lipid peroxidation. Superfusion with FeSO4 (400 microM) and ascorbate (1 mM), (Fe-Asc) did not result in loss of cell viability or a decrease protein thiols or the energy charge. Superfusion with Fe-Asc, did, however, lead to a slight decrease in the concentration of non-protein thiols and ATP and a large increase in the concentration of lipid peroxidation products. Accumulation of lipid peroxidation products induced by Fe-Asc was prevented by promethazine.(ABSTRACT TRUNCATED AT 250 WORDS)
Bhatnagar
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Department of Physiology and Biophysics and Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555.
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002470:Cell Survival; D005033:Ethylmaleimide / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D005609:Free Radicals; D015227:Lipid Peroxidation; D008297:Male; D009206:Myocardium / Q000378:metabolism / Q000473:pathology*; D018384:Oxidative Stress; D010545:Peroxides / Q000378:metabolism; D011398:Promethazine / Q000494:pharmacology; D011506:Proteins / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D020122:tert-Butylhydroperoxide
D005296:Ferrous Compounds; D005609:Free Radicals; D010545:Peroxides; D011506:Proteins; D017392:Thiobarbituric Acid Reactive Substances; C020748:ferrous sulfate; D020122:tert-Butylhydroperoxide; D011398:Promethazine; D005033:Ethylmaleimide; D001205:Ascorbic Acid