Comparative In Vitro Nephrotoxicity of Three 3,5-Dihaloanilines in Isolated Renal Cortical Cells from Male Fischer 344 Rats

Halogenated anilines are widely used to manufacture dyes, pesticides, pharmaceuticals and industrial products. Toxicity induced by these compounds includes hepatotoxicity, hematotoxicity, splenotoxicity, immunotoxicity and nephrotoxicity. We have shown that among the mono‐ and dichloroanilines, 3,5‐dichloroaniline (DCA) is the most potent nephrotoxicant in vivo and in vitro. DCA nephrotoxicity in vitro is attenuated by primary antioxidants (α‐tocopherol, ascorbate) and antioxidant nucleophiles (glutathione, N‐acetyl‐L‐cysteine), suggesting a role for free radicals in DCA nephrotoxicity. The purpose of this study was to compare the nephrotoxic potential of DCA with the nephrotoxic potential of 3,5‐dibromo‐ and 3,5‐diiodoaniline (DBA and DIA, respectively) in isolated renal cortical cells (IRCC) from male Fischer 344 rats. The comparative effect of antioxidants on 3,5‐dihaloaniline (DHA) nephrotoxicity was also determined. IRCC (~4 million cells/ml; 3 ml) were incubated with shaking at 37°C under a 95% oxygen/5% carbon dioxide atmosphere with a DHA (0.25 – 1.0 mM) or vehicle (dimethyl sulfoxide) for 15–90 min with or without a pretreatment. General cytotoxicity was determined by assessing trypan blue exclusion of IRCC and measuring changes in lactate dehydrogenase (LDH) release. Among the three DHAs, DBA was the most potent nephrotoxicant, inducing LDH release at 0.25 mM by 30 min. DCA was the least potent nephrotoxicant, requiring 90 min at 0.5 mM exposure to induce cytotoxicity, while DIA was intermediate in potency by inducing cytotoxicity at 60 min with 1.0 mM exposure. Pretreatment with an antioxidant (α‐tocopherol, ascorbate) or antioxidant/nucleophile (glutathione, N‐acetyl‐L‐cysteine) attenuated cytotoxicity induced by the three DHAs (1.0 mM). These results indicate that the decreasing order of nephrotoxicity in IRCC was DBA > DIA > DCA, and that free radicals play a role in the DHA‐induced nephrotoxicity induced by the three compounds tested. Support or Funding Information Supported in part by NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence.