Mechanistic aspects of 4-amino-2,6-dichlorophenol-induced in vitro nephrotoxicity

• Published in: Toxicology (Amsterdam) 2008-03, Vol.245 (1), p.123-129
• Main Authors: Rankin, Gary O, Hong, Suk-kil, Anestis, Dianne K, Ball, John G, Valentovic, Monica A
• Format: Article
• Language: English
• Subjects: 4-Amino-2,6-dichlorophenol; Acetylcysteine - pharmacology; Animals; Antioxidants - pharmacology; Biological and medical sciences; Chlorophenols - toxicity; Co-oxidation; Cytochrome P-450 Enzyme Inhibitors; Cytosol - drug effects; Cytosol - enzymology; Cytosol - pathology; Emergency; Enzyme Inhibitors - pharmacology; In vitro; In Vitro Techniques; Kidney Cortex - drug effects; Kidney Cortex - enzymology; Kidney Cortex - pathology; L-Lactate Dehydrogenase - metabolism; Male; Medical sciences; Nephrotoxicity; Organic Anion Transporters - antagonists & inhibitors; Rats; Rats, Inbred F344; Toxicology
• ISSN: 0300-483X; 1879-3185
• DOI: 10.1016/j.tox.2007.12.014

Abstract 4-Amino-2,6-dichlorophenol (ADCP) is a potent acute nephrotoxicant in vivo inducing prominent renal corticomedullary necrosis. In vitro, ADCP exposure increases lactate dehydrogenase (LDH) release from rat renal cortical slices at 0.05 mM or greater. The purpose of this study was to examine the ability of antioxidants, cytochrome P450 (CYP) and flavin adenine dinucleotide monooxygenase (FMO) activity modulators, indomethacin, glutathione and inhibitors of glutathione conjugate metabolism to attenuate ADCP cytotoxicity in vitro. Renal cortical slices prepared from untreated male Fischer 344 rats ( N = 4/group) were preincubated at 37 °C under a 100% oxygen atmosphere with an inhibitor or vehicle for 5–30 min. ADCP (0.05–0.5 mM) or vehicle was added and incubations continued for 120 min. At the end of the incubation period, LDH release was measured as an index of nephrotoxicity. ADCP cytotoxicity was partially attenuated by ascorbate (1.0 or 2.0 mM), but not by N , N ′-diphenyl- p -phenylenediamine (DPPD), α-tocopherol or deferoxamine. Inhibitors of CYP (metyrapone, piperonyl butoxide and isoniazid) and FMO activity modulators (methimazole, N -octylamine) had no effect on ADCP cytotoxicity. Indomethacin or glutathione 1.0 mM completely and partially blocked ADCP 0.1 and 0.5 mM cytotoxicity, respectively. N -acetylcysteine, AOAA (an inhibitor of cysteine conjugate β-lyase) and probenecid (an organic anion transport inhibitor), but not AT-125 (an inhibitor of γ-glutamyl transferase), partially attenuated ADCP 0.1 mM cytotoxicity. Overall, these results suggest that reactive metabolites may be produced from ADCP primarily via a co-oxidation-mediated mechanism. The difference in the ability of ascorbate and glutathione to attenuate ADCP-induced cytotoxicity in vitro in kidney cells could indicate that alkylation via the reactive benzoquinoneimine metabolite might be responsible for cytotoxicity rather than a free radical-mediated mechanism.