title: 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.
abstract: 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 (∼ 4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min 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 90 min. 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.
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Racine
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: Travis
authors.initials: T
authors.lastname: Ferguson
authors.affiliation: Department of Pediatrics, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: Debbie
authors.initials: D
authors.lastname: Preston
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: Dakota
authors.initials: D
authors.lastname: Ward
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: John
authors.initials: J
authors.lastname: Ball
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: Dianne
authors.initials: D
authors.lastname: Anestis
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: Valentovic
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA. Electronic address: rankin@marshall.edu.
authors.forename: Gary O
authors.initials: GO
authors.lastname: Rankin
mesh_terms: D000814:Aniline Compounds / Q000493:pharmacokinetics / Q000633:toxicity*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001711:Biotransformation; D002469:Cell Separation; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism / Q000473:pathology*; D007674:Kidney Diseases / Q000139:chemically induced* / Q000378:metabolism / Q000517:prevention & control; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors; D018384:Oxidative Stress / Q000187:drug effects*; D050050:Protein Carbonylation / Q000187:drug effects; D051381:Rats; D011916:Rats, Inbred F344
chemical_list: D000814:Aniline Compounds; D000975:Antioxidants; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006899:Mixed Function Oxygenases; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; C037267:3,5-dichloroaniline
keywords: 3,5-Dichloroaniline; In vitro; Nephrotoxicity; Oxidative stress; Rat