Characterization of 2-amino-4,5-dichlorophenol (2A45CP) in vitro toxicity in renal cortical slices from male Fischer 344 rats

Abstract

2-Amino-4,5-dichlorophenol (2A45CP) is a major, aromatic ring hydroxylated metabolite of the renal toxicant, 3,4-dichloroaniline. 3,4-Dichloroaniline is nephrotoxic with primary damage located to the proximal tubules. The purpose of this study was to first characterize the in vitro toxicity of 2A45CP in renal cortical slices. Second, the effect of antioxidants and sulfhydryl containing agents on the severity of 2A45CP toxicity was explored since part of the mechanism of toxicity for aminophenols may involve redox cycling. Renal tissue was isolated from male Fischer 344 rats (190–220 g). Renal slices were rinsed three times for 3 min each in 5-ml Krebs buffer. Tissues were then incubated for 90–120 min with varying concentrations of 2A45CP between 0 and 0.5 mM. In a separate series of experiments, the slices (50–100 mg) were preincubated for 30 min with 1 mM dithiothreitol (DTT), 1 mM glutathione (GSH) or 2 mM ascorbic acid prior to exposure to 0, 0.05, 0.1 or 0.25 mM 2A45CP. 2A45CP produced a concentration and time dependent increase in LDH leakage from renal cortical slices. Total glutathione levels were diminished by 0.5 mM 2A45CP within 30 min. Renal slices incubated for 60 and 120 min with 0.05 and 0.1 mM 2A45CP had lower malondialdehyde levels than control. Pretreatment with DTT did not alter 2A45CP toxicity. Pretreatment of renal cortical slices with GSH or ascorbic acid reduced 2A45CP toxicity. These findings indicate that 2A45CP is directly toxic to renal cortical slices and that cytotoxicity is at least partially mediated by a reactive intermediate.