Date of Award


Degree Type


Degree Name

Master of Science in Pharmacology and Toxicology


Pharmacology and Toxicology

First Advisor

John R. Babson


The primary biochemical mechanisms involved in chemically-induced cell injury remain to be elucidated. Elevation of intracellular ea2+ is a common feature to cell death due to a wide array of toxic chemicals, and on this basis hypotheses have been put forth suggesting that the chemically-induced elevation of cytosolic ea2+ is responsible for the onset of cell death. The mechanism by which elevated ea2+ causes cell damage may involve activation of ea2+-dependent proteases, phospholipases and endonucleases (2). Previous evidence suggests that a link between intracellular thiol status and ea2+ homeostasis exists (1, 2). Based on these observations, it has been speculated that thiol depletion may lead to an elevation of intracellular ea2+ to cytotoxic levels (1, 2, 4). Glutathione, the major cellular thiol, is primarily a defense mechanism against cytotoxic reaction to oxidative stress or alkylating agents. Glutathione also plays an important role in maintaining protein thiols in a reduced state, which is required for their normal enzymatic activity (6, 7). Such enzymes include the sarcolemmal and sarcoplasmic reticular ea2+-A TPase's, which are involved in the maintenance of low levels of cytosolic ea2+. Therefore depletion of intracellular glutathione may limit the capacity of these enzymes with modified thiol groups to maintain low levels of cytosolic ea2+ (10, 11). The loss of GSH as an antioxidant may promote oxidative stress and the resultant peroxidative damage to plasma membrane may be an alternate cause of cell death by a ea2+-independent mechanism. The relative importance of elevated cytosolic free ea2+ or oxidative stress in cell death, in the face of a chemical challenge that alters intracellular thiol status is the subject of this thesis. Our approach towards this problem was to create a chemical model of oxidative stress in cardiomyocytes using ethacrynic acid. Ethacrynic acid depletes thiols by alkylation with a subsequent increase in cytosolic free ea2+, thereby permitting us to examine lethal cell injury due to thiol depletion, including the proposed link to ea2+ homeostasis. Exposure of primary rat myocardial cells to ethacrynic acid (150 μM) resulted in a rapid (within 7min) loss of glutathione and protein thiols that preceded an increase in cytosolic free ea2+ levels (within 45 min), as detected by the activation of phosphorylase a. The leakage of cytosolic lactate dehydrogenase due to loss of membrane integrity was used as a criterion of loss of cell viability. All of these biochemical events preceded the loss of cell viability, thus permitting us to examine whether thiol depletion or changes in cytosolic free ea2+ had the primary effect on the loss of cell viability. Pretreatment of cells with specific intracellular ea2+ chelators, Quin-2- acetoxymethylester and EGTA-acetoxymethylester, were used in an attempt to sequester ea2+, in order to prevent an ethacrynic acid-induced elevation of intracellular ea2+. Both intracellular chelators reduced lactate dehydrogenase leakage, protected against lipid peroxidation, but failed to reduce the marked elevation of intracellular ea2+. The latter observation required examination of the mechanism of protection afforded by the putative chelators. The antioxidant N,N'-Diphenyl-p-phenylenediamine was employed to investigate the importance of lipid peroxidation in ethacrynic acid-induced cell death. N,N'-Diphenyl-p-phenylenediamine reduced lipid peroxidation and lethal cell injury to control levels but had no effect on intracellular glutathione and ea2+ levels. Thus, it would appear that the antioxidant activity of the putative chelators might account for their protection. The possibility that cytotoxicity was due to an ethacrynic acid-induced alteration of cellular energy status was also examined. Ethacrynic acid had no significant effect on cellular ATP levels or mitochondrial membrane potential. In our model of myocardial cell injury the temporal relationship observed between the loss of intracellular thiol status and ea2+ homeostasis supports the hypothesis that thiol status is linked to ea2+. However elevated ea2+ levels alone, had no effect on cell viability over the time course we observed, further supporting that peroxidative damage is a requisite event for cell death in our model of myocardial cell injury.



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