Toxic drug reactions frequently result from highly reactive metabolites that initiate a lethal biological cascade by widespread alkylation. With descriptive aspects of this discovery well investigated, attention now focuses on prevention of this form of injury. For this to happen, we must understand much more about specific sites and/or functions that are target(s) of lethal alkylation. Growing evidence points to ion regulation, particularly calcium, as an early casualty of drug alkylation which may program later cell demise. Support for the Calcium Hypothesis comes from in vivo evidence that is indicative but not conclusive and from in vitro models that require substantial extrapolation. The Calcium Hypothesis, if correct, must ultimately be shown in vivo but this has been hampered by the lack of methods to measure ion activity in this challenging setting. We propose to work toward this goal by examining Ca2+- sensitive enzymes in cytosol (phosphorylase a, synthase- phosphorylase kinase) and mitochondria (pyruvate dehydrogenase/phosphatase, 2-oxoglutarate dehydrogenase) and by following Ca2+-endonuclease DNA cleavage in nuclei as biological indicators of Ca2+ activity in these important pools. We will trace how indicators change in vivo during rodent liver injury by a panel of hepatotoxins (ACETAMINOPHEN, DIMETHYLNITROSAMINE, ALLYL ALCOHOL, D- GALACTOSAMINE) designed to distinguish specific molecular consequences of alkylation and thiol depletion. We will test the proposed cause-effect sequence of alkylation-induced loss of Ca2+ regulation leading to hepatocellular death. Calcium Hypothesis testing will feature agents that up-regulate (alpha-adrenergic drugs, hormones, tolbutamide) and down-regulate Ca2+ (calcium chelators, channel blockers, antagonists), that inhibit specific Ca2+ effects (proteinase inhibitors), and that """"""""dissociate"""""""" alkylation from cell death (antioxidants, esterase inhibitors, and calcium blockers). Physical damage (alkylation, peroxidation) to the plasma membrane and impairment of associated Ca2+ regulators (Ca2+-ATPase of Lin, Ca2+/Mg2+-ATPase of Schanne and Moore, Na+/Ca2+ Exchanger, Calmodulin) will be evaluated as possible molecular loci of Ca2+ deregulation, and the nucleus as a possible site of lethal Ca2+ effects. Data inconsistent with the Calcium Hypothesis will accelerate studies to examine regulation of sodium and other essential electrolytes and to probe other possible mechanisms of Ca2+-independent injury such as the premature catabolism of proteins and ploynucleotides induced by alkylation.
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