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The role of biotransformation and oxidative stress in 3,5-dichloroaniline (3,5-DCA) induced nephrotoxicity in isolated renal cortical cells from male Fischer 344 rats

Racine, Christopher R., Ferguson, Travis, Preston, Debbie, Ward, Dakota, Ball, John, Anestis, Dianne, Valentovic, Monica, Rankin, Gary O.
Toxicology 2016 v.341-343 pp. 47-55
acetylcysteine, alpha-tocopherol, antioxidants, biotransformation, cytochrome P-450, cytotoxicity, free radicals, glutathione, indomethacin, kidneys, lactate dehydrogenase, males, metabolites, metyrapone, nephrotoxicity, oxidative stress, peroxidase, piperonyl butoxide, pyruvic acid, rats, unspecific monooxygenase
Among the mono- and dichloroanilines, 3,5-dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (∼4millioncells/ml) from male rats were exposed to 3,5-DCA (0–1.0mM) for up to 120min. In IRCC, 3,5-DCA was cytotoxic at 1.0mM by 60min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120min was required for 3,5-DCA 0.5mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0mM) for 90min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-l-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro.