Cytochrome P-450 mediates tissue-damaging hydroxyl radical formation during reoxygenation of the kidney.
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.
Paller
M S
MS
Department of Medicine, University of Minnesota, Minneapolis 55455.
Jacob
H S
HS
D000818:Animals; D002384:Catalysis; D002478:Cells, Cultured; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005609:Free Radicals; D017665:Hydroxyl Radical / Q000378:metabolism*; D007511:Ischemia / Q000201:enzymology; D007687:Kidney Tubules, Proximal / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008297:Male; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D013481:Superoxides / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D005609:Free Radicals; D013481:Superoxides; D017665:Hydroxyl Radical; D003577:Cytochrome P-450 Enzyme System; D010100:Oxygen