ROLE OF CYTOCHROME-P-450 IN THE STIMULATION OF MICROSOMAL PRODUCTION OF REACTIVE OXYGEN SPECIES BY FERRITIN

Microsomes can remove iron from ferritin by a superoxide-dependent rea ction. The released iron can then catalyze formation of a variety of r eactive oxygen species. Experiments were carried out to evaluate the r ole of cytochrome P-450 in the release of iron from ferritin, and whet her induction of certain P-450 isoforms alters ferritin-dependent reac tive oxygen radical production. Rats were treated with phenobarbital, 3-methylcholanthrene, 4-methylpyrazole, or saline to produce microsome s with varying P-450 content and composition. Oxidation of 2,7'-dichlo rofluorescein diacetate to a fluorescent product and chemiluminescence were used as indices of production of reactive oxygen species. The ex treme sensitivity of these reactions to trolox, a potent chain-breakin g oxidant, indicates the involvement of lipid peroxidation products in these reactions. In the absence of ferritin, formation of reactive ox ygen species was higher in microsomes from the treated rats compared t o saline controls when results were expressed on a per mg protein basi s but not per nmol P-450, suggesting that the increased content of tot al P-450 (2-fold increases) rather than the population of isoforms was responsible for the increase. Superoxide dismutase had no effect on t he non-ferritin catalyzed reactions. Ferritin increased production of reactive oxygen species with all the microsomal preparations; the incr ease by ferritin was completely prevented by superoxide dismutase. The net increase by ferritin was higher in microsomes from the treated ra ts compared to saline controls, but this, again, largely reflected the increased content, rather than the type of isoforms of P-450 present. Similar results were obtained with either NADPH or NADH as microsomal reductants, although NADPH was much more effective in supporting ferr itin-dependent reactive oxygen formation. In microsomes from phenobarb ital-treated rats, anti-CYP2B1/B2 IgG completely prevented the NADPH- and NADH-dependent increases in reactive oxygen formation produced by ferritin. Anti-cytochrome b(5) IgG produced partial inhibition of the ferritin-stimulation. These results indicate that P-450, and to a less er extent, cytochrome b(5), play a role in the ferritin-dependent incr ease in formation of reactive oxygen species with either NADPH or NADH , most likely reflecting the requirement of these enzymes for microsom al production of superoxide anion.