2-Amino-5-chlorophenol toxicity in renal cortical slices from Fischer 344 rats: effect of antioxidants and sulfhydryl agents.
2-Amino-5-chlorophenol is nephrotoxic through an unidentified mechanism. This study examined the in vitro toxicity of 2-amino-5-chlorophenol in renal cortical slices from Fischer 344 rats and specifically assessed induction of lipid peroxidation and depletion of renal glutathione. Renal cortical slices exposed to 0, 0.25, 0.5, and 1 mM 2-amino-5-chlorophenol exhibited a concentration- and time-dependent increase in lactate dehydrogenase (LDH) leakage. Pyruvate-directed gluconeogenesis was diminished in a concentration-dependent manner following a 90-min incubation with 0, 0.25, 0.5, and 1 mM 2-amino-5-chlorophenol. Lipid peroxidation was induced within 60 min by 1 mM 2-amino-5-chlorophenol in renal slices relative to control tissue. Total glutathione (GSH) levels were decreased below control values within 30 min of exposure to 0.5 and 1 mM 2-amino-5-chlorophenol. These results indicated that GSH levels were decreased prior to the appearance of increased LDH leakage and diminished membrane integrity. 2-Amino-5-chlorophenol toxicity was increased in renal slices isolated from animals pretreated with buthionine sulfoximine (BSO, 890 mg/kg ip). Pretreatment of renal slices with the phenolic antioxidant N,N'-diphenyl-1, 4-phenylenediamine (DPPD, 50 microM) or the iron chelator deferoxamine did not reduce 2-amino-5-chlorophenol cytotoxicity. These results suggest that 2-amino-5-chlorophenol toxicity was not mediated through an iron-dependent mechanism. 2-Amino-5-chlorophenol cytotoxicity was reduced by a 15-min pre-incubation with 2 mM ascorbate or a 30-min preincubation with the thiol-containing agents GSH (1 mM) or dithiothreitol (1 mM, DTT). Pretreatment with GSH, DTT, or ascorbate reduced LDH leakage and lipid peroxide generation induced by 2-amino-5-chlorophenol. These results suggest that 2-amino-5-chlorophenol cytotoxicity involved free radical generation through an iron-independent mechanism. Toxicity was reduced by the presence of the antioxidant ascorbate or by addition of glutathione.
Valentovic
M
M
Department of Pharmacology, Marshall University School of Medicine, 1542 Spring Valley Drive, Huntington, West Virginia, 25704-9388, USA. Valentov@Marshall.edu
Meadows
M K
MK
Harmon
R C
RC
Ball
J G
JG
Hong
S K
SK
Rankin
G O
GO
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002733:Chlorophenols / Q000037:antagonists & inhibitors / Q000633:toxicity*; D003676:Deferoxamine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005609:Free Radicals / Q000378:metabolism; D005943:Gluconeogenesis / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007501:Iron / Q000378:metabolism; D007502:Iron Chelating Agents / Q000494:pharmacology; D007672:Kidney Cortex / Q000187:drug effects* / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D010655:Phenylenediamines / Q000494:pharmacology; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D000975:Antioxidants; D002733:Chlorophenols; D005609:Free Radicals; D007502:Iron Chelating Agents; D010655:Phenylenediamines; D013439:Sulfhydryl Reagents; C099673:2-amino-5-chlorophenol; D019328:Buthionine Sulfoximine; D019289:Pyruvic Acid; C004845:N,N'-diphenyl-4-phenylenediamine; D007501:Iron; D005978:Glutathione; D003676:Deferoxamine; D001205:Ascorbic Acid; D004229:Dithiothreitol