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Research ArticleToxicology

A Cytochrome P450–Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes

Kazuhisa Miyakawa, Ryan Albee, Lynda G. Letzig, Andreas F. Lehner, Michael A. Scott, John P. Buchweitz, Laura P. James, Patricia E. Ganey and Robert A. Roth
Journal of Pharmacology and Experimental Therapeutics August 2015, 354 (2) 230-237; DOI: https://doi.org/10.1124/jpet.115.223537
Kazuhisa Miyakawa
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.)
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Ryan Albee
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.)
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Lynda G. Letzig
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.)
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Andreas F. Lehner
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.)
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Michael A. Scott
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.)
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John P. Buchweitz
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.)
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Laura P. James
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.)
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Patricia E. Ganey
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.)
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Robert A. Roth
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.)
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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.

Footnotes

    • Received February 19, 2015.
    • Accepted May 29, 2015.
  • This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK087886].

  • L.P.J. is part owner of Acetaminophen Toxicity Diagnostics, LLC, and has a patent pending for the development of a commercial assay for measurement of acetaminophen protein adducts. The remaining authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or conflict with the subject matter or materials discussed in the manuscript.

  • dx.doi.org/10.1124/jpet.115.223537.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

  • Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 354 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 354, Issue 2
1 Aug 2015
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Research ArticleToxicology

P450-Independent APAP Hepatocellular Injury In Vitro

Kazuhisa Miyakawa, Ryan Albee, Lynda G. Letzig, Andreas F. Lehner, Michael A. Scott, John P. Buchweitz, Laura P. James, Patricia E. Ganey and Robert A. Roth
Journal of Pharmacology and Experimental Therapeutics August 1, 2015, 354 (2) 230-237; DOI: https://doi.org/10.1124/jpet.115.223537

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Research ArticleToxicology

P450-Independent APAP Hepatocellular Injury In Vitro

Kazuhisa Miyakawa, Ryan Albee, Lynda G. Letzig, Andreas F. Lehner, Michael A. Scott, John P. Buchweitz, Laura P. James, Patricia E. Ganey and Robert A. Roth
Journal of Pharmacology and Experimental Therapeutics August 1, 2015, 354 (2) 230-237; DOI: https://doi.org/10.1124/jpet.115.223537
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