We propose to study alterations in functions that occur in cells in the course of oxygen toxicity. The hypothesis to be tested is that production of free radicals and/or depletion of reductants in cells occurs first, followed by lipid peroxidation and a sequential loss of membrane functions. Rat macrophages will be exposed in vitro to air or O2 for 0 to 24 hours and assays made for: lipid peroxidation; reduced and oxidized glutathione, NADPH and ascorbate; ATP, ADP, and mitochondrial function; CA++ distribution; binding of respiratory burst stimuli; mitochondrial and plasma membrane potentials; and the respiratory burst (stimulated superoxide release). The respiratory burst of alveolar macrophages, a plasma membrane function, can be inhibited by in vivo or in vitro hyperoxic incubation. Several of the parameters we will measure are involved in the respiratory burst response. A comparison of the rates of alterations in cellular activities should therefore reveal the specific functional losses responsible for inhibition of the respiratory burst activity as well as the sequence of events in O2 toxicity. The net rates of superoxide, hydrogen peroxide, and lipid peroxide production by isolated organelles from macrophages will also be measured. Attention will be focused on the possible correlations of higher net rates of radical production with lipid peroxidation in the organelles. Spectrophotometric and fluorometric assays will be used for metabolites. Mitochondrial function in intact cells will be assessed with an oxygen electrode. Oligomycin and an uncoupler will be added to determine respiratory control. CA++ distribution will be measured by null point titration with the dye, arsenazo III. An uncoupler and a CA++-ionophore will be used to release CA++ from organelles. Membrane potentials will be measued using both the distribution of radio-labeled compounds and microelectrode impalement. Our long-term objective is an understanding of the molecular mechanisms in toxicologic processes. In the process of achieving that goal, we hope to gain insight into the interrelationship of metabolism and cellular functions involved both in oxygen toxicity and also in normal cellular processes, such as the respiratory burst. Since altered macrophage function may result in increased susceptibility of oxygen exposed individuals to pulmonary infection and increasing evidence suggests an important contributory role of the respiratory burst in the bactericidal activity of alveolar macrophages, this research may be relevant to practical aspects of O2 toxicity.
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