Renal
reperfusion injury results from
oxygen radical generation. During reoxygenation of hypoxic kidney cells,
xanthine oxidase produces
superoxide radical, which eventuates in
hydroxyl radical formation by the Fenton reaction. This reaction, catalyzed by transition metals such as
iron, is particularly important because
hydroxyl radical is highly reactive with a wide variety of biomolecules. We tested the hypothesis that this catalytic function is fostered by
iron released from the
heme moiety of
cytochrome P-450. Primary cultures of rat proximal tubule epithelial cells studied in a subconfluent stage were subjected to 60 min of
hypoxia and 30 min of reoxygenation. When cells were pretreated with one of three
cytochrome P-450 inhibitors (
piperonyl butoxide,
cimetidine, or
ketoconazole), lethal cell injury was attenuated. There was the expected increase in O2-. production during
hypoxia/reoxygenation that
cytochrome P-450 inhibitors did not prevent; on the other hand, inhibitors did prevent reoxygenation-induced
hydroxyl radical formation. Analogously, the increase in catalytic
iron (bleomycin-detectable
iron) that accompanies
hypoxia/reoxygenation did not occur in the presence of
cytochrome P-450 inhibitors. In vivo studies confirmed a protective effect of
cytochrome P-450 inhibition because glomerular filtration rate was better preserved in rats pretreated with
cimetidine and then subjected to renal artery occlusion. In summary, several chemically distinct
cytochrome P-450 inhibitors reduced
iron release, and thereby,
hydroxyl radical formation and reoxygenation-induced lethal cell injury, without inhibiting
superoxide radical formation. We conclude that highly labile P-450 may act as an Fe-donating catalyst for Fenton reaction production of HO.-mediated
reperfusion injury.