In Vitro 2,3- and 3,4-Dibromophenol Nephrotoxicity in Isolated Kidney Cells

Abstract ID 128749 Poster Board 215 Brominated benzenes are frequently used chemical intermediates in the synthesis of a wide variety of industrial, pharmaceutical and agricultural products. Recent studies in our laboratory demonstrated that 1,2-dibromobenzene (1,2-DBB) is a nephrotoxicant in isolated kidney cells (IKC) from male Fischer 344 rats and that one or more metabolites may play a role in 1,2-DBB nephrotoxicity in vitro. The purpose of this study was to investigate the nephrotoxic potential of two 1,2-DBB metabolites, 2,3- and 3,4-dibromophenol (2,3- and 3,4-DBP) using IKC from male Fischer 344 rats as the model. We also investigated whether free radicals or further oxidative metabolism contributed to 2,3-DBP nephrotoxicity. IKC (∼4 million cells/ml; 3 ml) from male Fischer 344 rats were incubated with shaking at 37oC under a 95% oxygen/5% carbon dioxide atmosphere with a DBP (0.25, 0.50 or 1.0 mM) or vehicle (dimethyl sulfoxide) for 30 or 60 min. Cytotoxicity was measured by determining trypan blue exclusion by IKC and measuring changes in lactate dehydrogenase (LDH) release. In some experiments, IKC were pretreated with an antioxidant (glutathione, 1.0 mM, 30 min or ascorbate, 1.0 mM, 5 min) or a non-selective cytochrome P450 (CYP) inhibitor (piperonyl butoxide, 0.1 mM, 15 min) before addition of 2,3-DBP (1.0 mM) and a 60 min incubation period. At 30 min, only 2,3- or 3,4-DBP 1.0 mM induced cytotoxicity in IKC, while 2,3- or 3,4-DBP 0.5 mM or greater induced cytotoxicity with a 60 min incubation period. Between the two DBPs, 2,3-DBP induced slightly greater nephrotoxicity than 3,4-DBP at comparable concentrations and times. In the antioxidant pretreatment studies, 2,3-DBP-induced cytotoxicity was partially attenuated by ascorbate and fully attenuated by glutathione pretreatment. Pretreatment with the CYP inhibitor markedly reduced 2,3-DBP cytotoxicity. These results suggest that 2,3- and 3,4-DBP are potential nephrotoxic metabolites of 1,2-DBB, with 2,3-DBP being slightly more nephrotoxic. In addition, (1) further metabolism of 2,3-DBP to catechol and/or hydroquinone toxic metabolites and (2) the generation of free radicals contribute to 2,3-DBP nephrotoxicity in vitro. Supported in part by NIH grant P20GM103434.