title: A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.
abstract: Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Miyakawa
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: Ryan
authors.initials: R
authors.lastname: Albee
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: Lynda G
authors.initials: LG
authors.lastname: Letzig
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: Andreas F
authors.initials: AF
authors.lastname: Lehner
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: Michael A
authors.initials: MA
authors.lastname: Scott
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: John P
authors.initials: JP
authors.lastname: Buchweitz
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: Laura P
authors.initials: LP
authors.lastname: James
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.).
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Ganey
authors.affiliation: Department of Pathobiology and Diagnostic Investigation (K.M., M.A.S., J.P.B.), Department of Pharmacology and Toxicology, Institute for Integrative Toxicology (R.A., P.E.G., R.A.R.), and Diagnostic Center for Population and Animal Health, Section of Toxicology (A.F.L.), Michigan State University, East Lansing, Michigan; and Department of Pediatrics, University of Arkansas for Medical Sciences and Clinical Pharmacology and Toxicology Section, Arkansas Children's Hospital, Little Rock, Arkansas (L.G.L., L.P.J.) rothr@msu.edu.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Roth
mesh_terms: D000082:Acetaminophen / Q000378:metabolism* / Q000633:toxicity*; D018712:Analgesics, Non-Narcotic / Q000378:metabolism / Q000633:toxicity; D000818:Animals; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D018712:Analgesics, Non-Narcotic; D000082:Acetaminophen; D003577:Cytochrome P-450 Enzyme System