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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Mechanism-Based Inactivation of Cytochrome P450 3A4 by 17α-Ethynylestradiol: Evidence for Heme Destruction and Covalent Binding to Protein

Hsia-lien Lin, Ute M. Kent and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics April 2002, 301 (1) 160-167; DOI: https://doi.org/10.1124/jpet.301.1.160
Hsia-lien Lin
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Ute M. Kent
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Paul F. Hollenberg
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Abstract

17α-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6β-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanism-based manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the KIand kinact were 18 μM and 0.04 min−1, respectively, and thet1/2 was 16 min. Incubation of 50 μM EE with P450 3A4 at 37°C for 30 min resulted in a 67% loss of testosterone 6β-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EE-dependent inactivation. The partition ratio was ∼50. The stoichiometry of binding was approximately 1.3 nmol of an EE metabolite bound per nmol of P450 3A4 inactivated. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [3H]EE was irreversibly bound to the P450 3A4 apoprotein. After extensive dialysis of the [3H]EE inactivated samples, high-pressure liquid chromatography (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M − H)− of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site.

Footnotes

  • This work was supported in part by National Institutes of Health Grant CA-16954 (P.F.H.) and by a grant (DRR-00480) from the Biotechnology Research Technology Program, National Center for Research Resources, National Institutes of Health (Michigan State University).

  • Abbreviations:
    P450
    cytochrome P450
    EE
    17α-ethynylestradiol
    3A4
    cytochrome P450 3A4
    reductase
    NADPH-cytochrome P450 reductase
    HPLC
    high-pressure liquid chromatography
    PAGE
    polyacrylamide gel electrophoresis
    2-OH-EE
    2-hydroxyethynylestradiol
    TIC
    total ion chromatogram
    GC-MS
    gas chromatography-mass spectrometry
    • Received October 18, 2001.
    • Accepted December 26, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 301 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 301, Issue 1
1 Apr 2002
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Mechanism-Based Inactivation of Cytochrome P450 3A4 by 17α-Ethynylestradiol: Evidence for Heme Destruction and Covalent Binding to Protein

Hsia-lien Lin, Ute M. Kent and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics April 1, 2002, 301 (1) 160-167; DOI: https://doi.org/10.1124/jpet.301.1.160

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Mechanism-Based Inactivation of Cytochrome P450 3A4 by 17α-Ethynylestradiol: Evidence for Heme Destruction and Covalent Binding to Protein

Hsia-lien Lin, Ute M. Kent and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics April 1, 2002, 301 (1) 160-167; DOI: https://doi.org/10.1124/jpet.301.1.160
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