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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Effect of 17-α-Ethynylestradiol on Activities of Cytochrome P450 2B (P450 2B) Enzymes: Characterization of Inactivation of P450s 2B1 and 2B6 and Identification of Metabolites

Ute M. Kent, Danielle E. Mills, Rajendram V. Rajnarayanan, William L. Alworth and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics February 2002, 300 (2) 549-558; DOI: https://doi.org/10.1124/jpet.300.2.549
Ute M. Kent
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Danielle E. Mills
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Rajendram V. Rajnarayanan
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William L. Alworth
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Paul F. Hollenberg
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Abstract

17-α-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s 2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed pseudo-first order kinetics. The maximal rate constants for the inactivation of P450s 2B1 and 2B6 at 30°C were 0.2 and 0.03 min−1, respectively. For P450s 2B1 and 2B6 the apparentKI was 11 and 0.8 μM, respectively. Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 20 to 25% loss of the enzyme's ability to form a reduced CO complex. With P450 2B6, an 83% loss in enzymatic activity and a 5 to 10% loss in the CO reduced spectrum were observed. The extrapolated partition ratios for 17EE with P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation of an alternate substrate together with 17EE protected both enzymes from inactivation. A 1.3:1 stoichiometry of labeling for binding of the radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metabolites that were not produced by P450s 2B2 or 2B4.

Footnotes

  • This study was supported by National Institutes of Health Grant CA 16954 from the National Cancer Institute. Portions of this work were presented at the Ninth Annual Meeting of the International Society for the Study of Xenobiotics in Nashville, TN, Oct. 24–28, 1999.

  • Abbreviations:
    P450
    cytochrome P450
    17EE
    17-α-ethynylestradiol
    HPLC
    high-performance liquid chromatography
    DLPC, dilauroyl-l-α-phosphatidylcholine
    7-EFC
    7-ethoxy-4-(trifluoromethyl)coumarin
    HFC
    7-hydroxy-4-(trifluoromethyl)coumarin
    GC-MS
    gas chromatography-mass spectrometry
    GSH
    glutathione
    BSTFA
    N,O-bis(trimethylsilyl)trifluoroacetamide
    TMCS
    trimethylchlorosilane
    LC-MS
    liquid chromatography-gas chromatography
    TIC
    total ion chromatogram
    TMS
    trimethylsilane. RP73401, 3-cyclopentyloxy-N-(3,5-clichloro-4-pyridyl)-4-methoxybenzamide
    • Received August 27, 2001.
    • Accepted November 6, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 300 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 300, Issue 2
1 Feb 2002
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Effect of 17-α-Ethynylestradiol on Activities of Cytochrome P450 2B (P450 2B) Enzymes: Characterization of Inactivation of P450s 2B1 and 2B6 and Identification of Metabolites

Ute M. Kent, Danielle E. Mills, Rajendram V. Rajnarayanan, William L. Alworth and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics February 1, 2002, 300 (2) 549-558; DOI: https://doi.org/10.1124/jpet.300.2.549

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Effect of 17-α-Ethynylestradiol on Activities of Cytochrome P450 2B (P450 2B) Enzymes: Characterization of Inactivation of P450s 2B1 and 2B6 and Identification of Metabolites

Ute M. Kent, Danielle E. Mills, Rajendram V. Rajnarayanan, William L. Alworth and Paul F. Hollenberg
Journal of Pharmacology and Experimental Therapeutics February 1, 2002, 300 (2) 549-558; DOI: https://doi.org/10.1124/jpet.300.2.549
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