title: The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.
abstract: 3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.
authors.affiliation: Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences Little Rock, AR 72205.
authors.forename: Sudip
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Department of Pediatrics, Arkansas Children's Hospital Research Institute, Little Rock, AR 72205.
authors.forename: Stepan B
authors.initials: SB
authors.lastname: Melnyk
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, 72205.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Krager
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, 72205.
authors.forename: Nukhet
authors.initials: N
authors.lastname: Aykin-Burns
authors.affiliation: Department of Pediatrics, Arkansas Children's Hospital Research Institute, Little Rock, AR 72205.
authors.forename: Lynda G
authors.initials: LG
authors.lastname: Letzig
authors.affiliation: Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences Little Rock, AR 72205; Department of Pediatrics, Arkansas Children's Hospital Research Institute, Little Rock, AR 72205.
authors.forename: Laura P
authors.initials: LP
authors.lastname: James
authors.affiliation: Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences Little Rock, AR 72205. Electronic address: HinsonJackA@uams.edu.
authors.forename: Jack A
authors.initials: JA
authors.lastname: Hinson
mesh_terms: D000082:Acetaminophen / Q000633:toxicity*; D018712:Analgesics, Non-Narcotic / Q000633:toxicity*; D000818:Animals; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006146:Guanidines / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D009243:NAD / Q000187:drug effects / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors; D018384:Oxidative Stress / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D018712:Analgesics, Non-Narcotic; D004791:Enzyme Inhibitors; D006146:Guanidines; C000611304:N-(4-amino-5-((2-aminoethyl)amino)pentyl)-N'-nitroguanidine; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D009243:NAD; D000082:Acetaminophen; D052248:Nitric Oxide Synthase Type I
keywords: 3-nitrotyrosine; Acetaminophen; Hepatotoxicity; Mitochondria; Nitric Oxide; Reactive nitrogen and oxygen species; S-nitrosoglutathione