Cytochromes P450 with Bisallylic Hydroxylation Activity on Arachidonic and Linoleic Acids Studied with Human Recombinant Enzymes and with Human and Rat Liver Microsomes1

  1. Johan Bylund,
  2. Tina Kunz,
  3. Karin Valmsen and
  4. Ernst H. Oliw
  1. Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden

    Abstract

    Bisallylic carbons of polyunsaturated fatty acids can be hydroxylated in NADPH-dependent reactions in liver microsomes. Human recombinant cytochromes P450 and human and rat liver microsomes were assayed for bisallylic hydroxylation activity. CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 converted [14C]linoleic acid to14C-labeled 11-hydroxyoctadecadienoic acid (11-HODE), whereas [14C]arachidonic acid was oxygenated by CYP1A2 and CYP3A4 to 14C-labeled 13-hydroxyeicosatrienoic acid (13-HETE), 10-HETE and 7-HETE as determined by HPLC. Both substrates were also converted to many other metabolites. CYP2C9 appeared to form 12R-HETE and 13-HETE, whereas CYP2C8 formed 13-HETE, 11-HETE and 15-HETE as main monohydroxy metabolites. Fetal human liver microsomes metabolized linoleic acid to 11-HODE as a major hydroxy metabolite, whereas arachidonic acid appeared to be hydroxylated at C13, C20 and, to some extent, at C10, C19 and C7. Fetal liver microsomes mainly formed 13R-HETE, whereas adult human liver microsomes and CYP1A2 mainly formed 13S-HETE. 7,8-Benzoflavone (5 μM) and furafylline (20 μM), two inhibitors of CYP1A2, reduced the bisallylic hydroxylation activity of adult human liver microsomes. Treatment of rats with erythromycin or dexamethasone induced bisallylic hydroxylation of linoleic acid to 11-HODE in liver microsomes by 2- and 10-fold, respectively. The biosynthesis of 11-HODE by microsomes of dexamethasone-treated rats was inhibited by troleandomycin (ED50 = 1 μM) and by polyclonal antibodies against CYP3A1, suggesting that CYP3A1 could catalyze bisallylic hydroxylations in the dexamethasone-treated rat. We conclude from steric analysis of 13-HETE and the effects of CYP inhibitors on adult human liver microsomes that CYP1A2 might contribute to its bisallylic hydroxylation activity.

    Footnotes

    • Send reprint requests to: Dr. Ernst H Oliw, Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, P.O. Box 591, S-751 24 Uppsala, Sweden.

    • ↵1 This work was supported by the Swedish Medical Research Council (Grant 06523). K.V. is supported by the Swedish Institute.

    • ↵2 This was identified by liquid chromatography-mass spectrometry (J. Ericsson, J. Bylund, C. Su and E. H. Oliw, unpublished observation).

    • Abbreviations:
      CYP
      cytochrome P450
      DEX
      dexamethasone
      ERY
      erythromycin
      GC-MS
      gas chromatography-mass spectrometry
      HETE
      hydroxyeicosatetraenoic acid
      HODE
      hydroxyoctadecadienoic acid
      HPLC
      high-performance liquid chromatography
      P450
      cytochrome P450
      RP
      reverse phase
      SP
      straight phase
      TAO
      troleandomycin
      TLC
      thin-layer chromatography
      • Received April 22, 1997.
      • Accepted September 8, 1997.
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    1. JPET January 1, 1998 vol. 284 no. 1 51-60
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