There is accelerating interest in the possibility that oxidative events may play a central role in cancer chemotherapy. Understanding has been hampered in the past by the lack of sensitive and reliable techniques for the measurement of seminal peroxidative events. Many free radicals, especially lipid-derived free radicals, could be measured only in extracts of killed cells; this limited kinetic and intervention studies. We have developed several new electron paramagnetic resonance (EPR) techniques to measure free radicals generated from live cells undergoing oxidative stress in real time. Using these methods, anticancer drugs and agents that modulate oxidative events can be added to the cell suspension during EPR monitoring. In addition we have adapted a method for measuring peroxidation using the generation of hydrocarbons, and this sensitive method can be used for tissue-cultured cells and for measuring in vivo oxidation in small animals. We will utilize these techniques and others to determine the importance of oxidative events in the cytotoxicity of anticancer drugs with different cellular targets. Membrane-active and DNA-active drugs will be compared. Sensitive/resistant cell line pairs will be used to explore the relationship of peroxidation and cytotoxicity. The kinetic and concentration relationship of each peroxidative event to immediate cytotoxicity and clonogenic survival will be determined. Manipulation of oxidative susceptibility will be done using superoxide dismutase transfection, glutathione depletion, antioxidants and oxidative metabolic inhibitors. The sequence of ascorbate, lipid-derived and tocopheroxyl free radical generation will be explored to attempt to explain some interesting preliminary antioxidant observations on the effect of vitamin E concentration on lipid-derived radical generation. The subcellular site of free radical and oxidative events will be determined. A membrane modification model by which cells can be enriched with polyunsaturated fatty acids of various types, thereby increasing their susceptibility to peroxidative events, will be used in some studies to amplify events not otherwise detectable. Central to the project are experimental therapeutic objectives in which tumor-bearing mice fed diets to increase the polyunsaturation and bis-allylic positions of their tumors will be treated using drugs with differing cellular targets. These studies will build upon the preliminary in vitro experiments. Clinical contexts will be pursued, but not forced. Studies with normal tissues will allow an estimate of selectivity of events and role in toxic side effects. The results could lead to the design of innovative ways to experimentally manipulate free radical events to increase selectivity of agents for neoplastic cells or overcome resistance.
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