The FIRCA application is an extension of grant proposal number 1 R01E504597-O1A3. This proposal derives from our recent finding that DFP, an organophosphate inhibitor of acetylcholinesterase (AChE), causes a significant increase in prostaglandin F2-like compounds (now named F2- isoprostanes) in skeletal muscle coinciding with the appearance of fasciculations and electron microscopic lesions. These effects are potentiated when endogenous glutathione levels are reduced. Because F2- isoprostanes are produced by a noncyclooxygenase mechanism involving free radical-catalyzed peroxidation of arachidonic acid, it is postulated that there is a link between lipid peroxidation, F2-isoprostane production and cell injury. Our goal is to examine this hypothesis and explore whether F2-isoprostanes serve as indicators of lipid peroxidation and are one of the mechanisms leading to cell injury. The similarity of electroencephalographic patterns, repetitive clonic convulsions and neuroanatomical changes in the brain found after status epilepticus (SE) induced by organophosphorus agent soman and excitotoxic kainic acid inspires the idea of common excitotoxic mechanisms of initiation and propagation of the lesions. The excitotoxic cell death is a consequence of a series of extra- and intracellular events terminating in generation of oxygen--:derived free radicals and of accumulation of calcium ions in the cells, with activation of proteolytic enzymes leading finally to irreversible destruction of cellular components such as plasmalemma, mitochondria, other intracellular membranes and cytoskeleton. We can detect F2-isoprostanes, following TLC purification, by gas chromatography/negative-ion chemical ionization. Quantification of these compounds provides a useful approach to determine whether oxygen free radicals initiate the (SE) induced neuronal necrosis by: (1) establishing the time course relationship between agent treatment, F2-isoprostanes production, and neuronal injury, using light and electronmicroscope techniques, (2) determining whether the agent induced SE is causally related to F2-isoprostanes increase and injury by preventing seizures, (3) determining whether antioxidants and free radical scavengers such as the endogenous glutathione, inhibitors of xanthine oxidase such as allopurinol and inhibitors of lipid peroxidation such as the lazaroids prevent increases of F2-isoprostanes and neuronal injury. Our preliminary data (1 R01ESO4597-01A3) support the hypothesis that oxygen free radicals play a causative role in organophosphate induced muscle necrosis. These proposed studies should determine whether status epilepticus induced by agents such as organophosphates and kainic acid induce neuronal necrosis via lipid peroxidation and generation of F2-isoprostanes. As such, these studies will provide fundamental new insights into the molecular events underlying SE induced injury as well as offer a therapeutic strategy for protection, namely treatment with antioxidants.
