Hydrogen peroxide cytotoxicity in LLC-PK1 cells: a role for iron.
Reactive oxygen metabolites have been postulated to play an important role in both toxic and ischemic forms of acute renal tubular epithelial injury. In the present study, we examined the effect of enzymatically generated hydrogen peroxide on LLC-PK1 cells, a renal proximal tubule cell line. Exposure of LLC-PK1 cells to glucose and glucose oxidase (GO; which generates hydrogen peroxide) resulted in cytotoxicity (as measured by trypan blue exclusion) which was dose dependent and increased linearly over time to 81 +/- 5% at 180 minutes (8 +/- 1% at time 0; mean +/- SEM, N = 3 to 7). Catalase (which decomposes hydrogen peroxide) completely prevented the cytotoxicity, confirming that the toxicity was due to hydrogen peroxide production. To assess whether the hydrogen peroxide toxicity was a direct effect or mediated by other toxic oxygen metabolites, several scavengers of reactive oxygen metabolites and iron chelators were used. Superoxide dismutase (a scavenger of superoxide) had no effect. Deferoxamine (DFO), an iron chelator, provided marked protection (GO alone 45.9 +/- 4.4%; GO + DFO 13.0 +/- 2.0%; control 7.1 +/- 1.2%; N = 15 to 17, P less than 0.001). Pretreatment with DFO (1 hr, then 2 washes to remove DFO before GO addition) also markedly inhibited the cytotoxicity, suggesting that DFO's effect was due to iron chelation. Two other metal chelators (dihydroxybenzoic acid and 1,10-phenanthroline) also significantly decreased the GO-induced cytotoxicity. However, three of four hydroxyl radical scavengers used (mannitol, dimethyl sulfoxide, sodium benzoate) did not significantly decrease cell death. Only dimethylthiourea provided protection.(ABSTRACT TRUNCATED AT 250 WORDS)
Walker
P D
PD
Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana.
Shah
S V
SV
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003676:Deferoxamine / Q000494:pharmacology; D016166:Free Radical Scavengers; D005609:Free Radicals; D005947:Glucose / Q000494:pharmacology; D005949:Glucose Oxidase / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000633:toxicity*; D006878:Hydroxides / Q000378:metabolism; D017665:Hydroxyl Radical; D007501:Iron / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
D016166:Free Radical Scavengers; D005609:Free Radicals; D006878:Hydroxides; D017665:Hydroxyl Radical; C038983:1,3-dimethylthiourea; D006861:Hydrogen Peroxide; D007501:Iron; D005949:Glucose Oxidase; D013890:Thiourea; D005947:Glucose; D003676:Deferoxamine