The objective of the proposed research is to characterize and quantitate the production of superoxide and H2O2 by rat lung microsomes and purified components of the lung microsomal electron transport system. Previous work with rat liver microsomes has revealed that purified NADPH-cytochrome P450 reductase (reductase) generates very low amounts of superoxide with respect to its potential for reduction of other electron acceptors. The addition of certain purified cytochrome P450 isozymes and dilaurylphosphatidylcholine stimulates superoxide and H2O2 production by the reductase. Critical to these studies is the elimination of iron from this system as particular ferric chelates appear to act as artificial electron acceptors of the reductase, artifactually enhancing the NADPH-oxidase activity of the reductase and masking its stimulation by purified cytochromes P450. Also critical to these studies is the removal of contaminants such as catalase, ferritin, ans superoxide dismutase from microsomes. We propose to isolate lung microsomes from rats and quantitate NADH and NADPH-dependent superoxide and H2O2 production under normoxic and hyperoxic conditions. We will also purify NADPH-cytochrome P450 reductase and the two major forms of cytochrome P450, as well as cytochrome b5 from rat lung microsomes and examine the generation of superoxide and H2O2 in the reconstituted monooxygenase system.
