Role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells.
The current study was designed to test the role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity, and to determine whether it may serve as a source of catalytic iron. Hydrogen peroxide led to iron release (as measured by iron/bathophenanthroline complex) from the microsomes prepared from LLC-PK1 cells. Cimetidine, which inhibits cytochrome P-450 by interacting with the heme iron, significantly blocked iron release, whereas ranitidine, which has a similar structure as cimetidine but is a weak inhibitor of P-450, did not have an effect (H2O2, 0.42 +/- 0.04; H2O2 + cimetidine, 0.23 +/- 0.02 nmol/mg protein; N = 4, P < 0.01). Exposure of LLC-PK1 cells to hydrogen peroxide (2.5 mM) resulted in a significant increase in the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) content that was prevented by cimetidine, but not ranitidine. We then examined the effect of the inhibitors of cytochrome P-450 on cell death (as measured by Trypan blue exclusion) after exposure of LLC-PK1 cells to 2.5 mM hydrogen peroxide for 120 minutes. Inhibition of cytochrome P-450 by cimetidine significantly reduced the cell death; the effect was observed with 0.05 mM and was concentration dependent with 1 mM affording almost complete protection (H2O2, 59 +/- 1.3% vs. H2O2 + cimetidine, 11 +/- 0.7%; N = 5, P < 0.01). In contrast, ranitidine did not show any protection. We confirmed that the protective effect of cimetidine was not related to scavenging hydrogen peroxide or hydroxyl radicals or chelating iron. A second inhibitor of cytochrome P-450, piperonyl butoxide, had a similar dose-dependent beneficial effect against hydrogen peroxide-induced cell injury. Our data thus indicate an important role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells and suggest that cytochrome P-450 may serve as a source of catalytic iron.
Baliga
R
R
Department of Pediatrics, University of Mississippi Medical Center, Jackson, USA.
Zhang
Z
Z
Shah
S V
SV
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002927:Cimetidine / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000633:toxicity*; D017665:Hydroxyl Radical / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism; D010882:Piperonyl Butoxide / Q000494:pharmacology; D011899:Ranitidine / Q000494:pharmacology; D013552:Swine; D014343:Trypan Blue / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D017665:Hydroxyl Radical; D002927:Cimetidine; D011899:Ranitidine; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D007501:Iron; D014343:Trypan Blue; D010882:Piperonyl Butoxide