The proposed research will elucidate the roles of superoxide dismutase and catalase in preventing oxygen toxicity in Escherichia coli and determine the nature and the toxic effects of oxygen free radical species in vivo. In addition, a bacteriological plate assay will be developed to rapidly screen drugs, toxic compounds and naturally occurring plant and bacterial compounds for their abilities to generate oxygen free radicals in cells. The Carbon-Clarke clone bank will be screened for chimeric plasmids containing superoxide dismutase or catalase. Appropriate plasmids will be amplified, purified and the superoxide dismutase and catalase genes will be excised and subcloned into additional plasmid vehicles. These plasmids (PSC101, COL E1, PBR322) appear in different copy numbers and transformation of recipient cells will give strains that overproduce superoxide dismutase or catalase in variable amounts. The strains will be examined for their relative abilities to resist oxygen toxicity due to hyperoxic oxygen and to compounds that exacerbate oxygen toxicity. Damage will be assayed as a loss of viability and as the stimulation of membrane lipid perioxidation. Lipid peroxidation will serve as an indicator for the production of toxic oxygen free radicals within the bacteria. The degree of protection afforded the cells by either superoxide dismutase or catalase will indicate the nature of the oxygen radical species involved in damage. Overproducing strains will also be compared to control strains in a plate assay under anaerobic and aerobic conditions to examine their relative resistances to the toxic effects of redox active compounds. The chief goals of the work are to elucidate the molecular basis of oxygen toxicity in cells, to understand the enzymatic scavenging system that has evolved to detoxify oxygen, and to develop a method of identifiying compounds in the human environment that exacerbate oxygen toxicity. The proposal involves biochemistry, molecular biology, and microbiology and is directed to solving problems of drug and environmental toxicology. These studies should clarify the mechanisms by which oxygen radicals mediate: 1) red blood cell lysis in G6PDH deficient individuals by antimalarial drugs and by dietary substances, 2) the cardiotoxicity of antitumor antibiotics, 3) the destruction of the lung capillary beds following exposure to some herbicides.
