Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

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Abstract

Acetaminophen (APAP), a widely used over-the-counter analgesic, is known to cause hepatotoxicity when ingested in large quantities in both animals and man, especially when administered after chronic ethanol consumption. Hepatotoxicity stems from APAP activation by microsomal P450 monooxygenases to a reactive metabolite that binds to tissue macromolecules, thereby initiating cellular necrosis. Alcohol consumption also causes the induction of P450IIE1, a liver microsomal enzyme that in reconstitution studies has proven to be an effective catalyst of APAP oxidation. Thus, elevated microsomal P450IIE1 levels could explain not only the known increase in APAP bioactivating activity of liver microsomes after prolonged ethanol ingestion but also the enhanced susceptibility to APAP toxicity. We therefore examined the role of P450IIE1 in human liver microsomal APAP activation. Liver microsomes from seven non-alcoholic subjects were found to convert 1 mm APAP to a reactive intermediate (detected as an APAP-cysteine conjugate by high-pressure liquid chromatography) at a rate of 0.25 ± 0.1 nmol conjugate formed/min/nmol microsomal P450 (mean ± SD), whereas at 10 mm, this rate increased to 0.73 ± 0.2 nmol product/min/nmol P450. In a reconstituted system, purified human liver P450IIE1 catalyzed APAP activation at rates threefold higher than those obtained with microsomes whereas two other human P450s, P450IIC8 and P450IIC9, exhibited negligible APAP-oxidizing activity. Monospecific antibodies (IgG) directed against human P450IIE1 inhibited APAP activation in each of the human samples, with anti-P450IIE1 IgG-mediated inhibition averaging 52% (range = 30–78%) of the rates determined in the presence of control IgG. The ability of anti-P450IIE1 IgG to inhibit only one-half of the total APAP activation by microsomes suggests, however, that other P450 isozymes besides P450IIE1 contribute to bioactivation of this compound in human liver. Of the other purified P450 isozymes examined, a β-naphthoflavone (BNF)-inducible hamster liver P450 promoted APAP activation at rates even higher than those obtained with human P450IIE1. The extensive APAP-oxidizing capacity of this hamster P450, designated P450IA2 based upon its similarity to rat P450d and rabbit form 4 in terms of NH2-terminal amino acid sequence, spectral characteristics, immunochemical properties, and inducibility by BNF, agrees with previous reports concerning the APAP substrate specificity of the rat and rabbit P450IA2 proteins. Liver microsomes from BNF-treated hamsters activated APAP at enhanced rates when compared to microsomes from control animals whereas, surprisingly, ethanol treatment had a negligible effect on rates of microsomal APAP activation. On immunoblots, anti-hamster P450IA2 IgG cross-reacted primarily with a single protein (Mr 54,000) in human liver microsomes that was distinct from P450IIE1. Furthermore, anti-P450IA2 IgG also inhibited APAP activation in all seven human microsomal samples, with inhibition ranging from 30 to 56% of the total activity. With each of the human samples, antibody-inhibited rates of APAP activation determined in the presence of anti-P450IA2 IgG, when added to those obtained in the presence of anti-P450IIE1 IgG, were equal to APAP activation rates determined in the presence of control IgG. Our results indicate that two P450 isozymes, namely P450IIE1 and P450IA2, catalyze nearly all of the APAP activation in human liver microsomes. Since bioactivation of APAP is presumed to mediate its toxicity, then those factors affecting the hepatic levels of P450IIE1 (e.g., chronic alcohol consumption) and/or P450IA2 may markedly influence the susceptibility of certain individuals to APAP-promoted liver damage.

References (52)

  • P. Moldeus

    Biochem. Pharmacol

    (1978)
  • D.W. Potter et al.

    J. Biol. Chem

    (1987)
  • T. Nouchi et al.

    Toxicol. Lett

    (1986)
  • C. Sato et al.

    Gastroenterology

    (1981)
  • P. Moldeus et al.

    Biochem. Pharmacol

    (1980)
  • E.T. Morgan et al.

    Biochem. Biophys. Ren. Commun

    (1983)
  • P.E. Thomas et al.

    J. Biol. Chem

    (1983)
  • T.A. Van der Hoeven et al.

    J. Biol. Chem

    (1974)
  • A. Bensadoun et al.

    Anal. Biochem

    (1976)
  • T. Omura et al.

    J. Biol. Chem

    (1964)
  • C.M. Ardies et al.

    Biochem. Pharmacol

    (1987)
  • J.M. Lasker et al.

    Biochem. Biophys. Res. Commun

    (1987)
  • M.M. McKinney et al.

    J. Immunol. Methods

    (1987)
  • D.A. Johnson et al.

    Gene Anal. Tech

    (1984)
  • P.E. Thomas et al.

    Arch. Biochem. Biophys

    (1984)
  • L.B.G. Tee et al.

    Biochem. Pharmacol

    (1987)
  • C. Ioannides et al.

    Toxicol. Lett

    (1983)
  • D.E. Ryan et al.

    J. Biol. Chem

    (1980)
  • D.A. Haugen et al.

    J. Biol. Chem

    (1976)
  • D. Ochs

    Anal. Biochem

    (1983)
  • J.R. Mitchell et al.

    J. Pharmacol. Exp. Ther

    (1973)
  • W.Z. Potter et al.

    J. Pharmacol. Exp. Ther

    (1973)
  • G.G. Corcoran et al.

    Mol. Pharmacol

    (1980)
  • E. Albano et al.

    Mol. Pharmacol

    (1985)
  • D.J. Jollow et al.

    J. Pharmacol. Exp. Ther

    (1973)
  • D.G.D. Davidson et al.

    Brit. Med. J

    (1966)
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    This research was supported by Smith Kline & French Labs, by DHHS Grants AA-05934, AA-03508, and AA-08139, by American Heart Association Grant 880925, and by the Veterans Administration.

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