This proposal is focused on the mechanism of the tissue injury caused by exposure to the herbicide, paraquat. Because of its inherent chemical stability, paraquat may pose a serious problem in the environment, as a consequence of its accumulation in soil and crops. Subsequently, it may enter into the water supply and food chain and eventually into the human body. Paraquat in vivo undergoes an enzyme-catalyzed cyclic one-electron reduction and reoxidation which results in the formation of superoxide radicals. Tissue injury caused by paraquat is believed to be associated with molecular oxygen production. The molecular oxygen-induced tissue injury had been often observed to be greatly enhanced by traces of transition metals. An obligatory role for traces of Cu and Fe in the mediation of tissue injury caused by paraquat has been recently demonstrated in a bacterial model and in mice. The experiments proposed in this application are designed to a) determine the degree of correlation between the dose of paraquat, the cellular level of transition metals, and the observed intensity of in vivo production of free radicals, and b) determine the degree of correlation between the dose of paraquat, the cellular level of transition metals, and the observed severity of injury to the organism. An evaluation of the results of studies described in a) and b) should provide insights into the relationship between the level of in vivo free radical production and the degree of biological damage. The dose of paraquat will be varied and the cellular level of transition metals will be manipulated in order to determine how these parameter affect both free radical production and cellular injury. Two biological models will be used: E. coli and mice. In studying each model we will employ three recently developed methods for monitoring the in vivo production of free radicals: 1) HPLC combined with electrochemical detection (HPLC-ECD) of 8-OH-2-deoxyguanosine in DNA, as a measure of .OH attack on chromosomes and subsequent damage to DNA structure, 2) HPLC-EDC analyses of the conversion of salicylate into dihydroxybenzoate as a reporter of the production of .OH radicals in the cytoplasm of cells, and 3) electron paramagnetic resonance (EPR) determination of spin adducts formed from a variety of oxygen- and carbon-centered free radicals as a measure of free radical production in cell membranes. This proposal offers a unique approach for critical evaluation of the role of free radicals in tissue injury in vivo.
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