The effects of x-irradiation and hyperthermia on oxygen uptake into intact cells and isolated mitochondria and on membrane fluidity of intact Chinese hamster ovary and HeLa cells and isolated mitochondria and lysosomes under oxic and hypoxic conditions will be studied using electron spin resonance spectroscopy and related methods. The oxygen uptake will be determined using a spin probe which quantitatively changes its spectrum in response to different concentrations of oxygen in solution. Both the environmental concentration and cellular uptake rates will be determined. New methods will be employed to measure oxygen uptake at concentrations producing maximum radioresistance and to measure O2 diffusion in mitochondrial membranes in addition to uptake across the membrane. The study will attempt to differentiate acutely hypoxic from chronically hypoxic cells based on biochemical parameters such as the rate of O2 uptake. The O2 uptake rates following different periods of hypoxia will be perturbed by hyperthermia and x-rays during the reoxygenation processes. Measurement of membrane fluidity will be based on ESR spin-probe methods which label the membranes of cultured mammalian cells with fatty acid spin probes. Conventional ESR (both 9 and 35 GHz) and saturation-transfer ESR will be used for measuring rotational diffusion of lipids and electron electron double resonance (ELDOR) techniques will be used for measuring lateral diffusion of lipids in membranes of intact cells and of isolated mitochondria and lysosomes. Sensitivity of these parameters to hyperthermia and radiation will be altered by agents known to modify cell survival. The determination of the effects of x-rays and heat on O2 uptake of previously hypoxic cells may be important in determining the mechanism of reoxygenation processes in solid tumors. Damage to membrane structure may provide insight into the mechanism of cell killing. The possible correlation between membrane fluidity and O2 uptake will be investigated in detail.
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