Abstract
Earlier study suggested that 3,4-dihydroxytamoxifen (tam catechol), a tamoxifen metabolite, is proximate to the reactive intermediate that binds covalently to proteins and possibly to DNA (Dehal and Kupfer, 1996). The current study demonstrates that rat and human hepatic cytochrome P-450s (CYPs) catalyze tam catechol formation from tamoxifen (tam), 3-hydroxy-tam (Droloxifene), and 4-hydroxy-tam (4-OH-tam). Higher levels of catechol were formed from 4-OH-tam and 3-hydroxy-tam than from tam. Evidence that human hepatic CYP3A4 and 2D6 catalyze the formation of tam catechol from 4-OH-tam and supportive data that the catechol is proximate to the reactive intermediate, was obtained: 1) There was a good correlation (r = 0.82; p ≤ .0004) between steroidal 6β-hydroxylase (CYP3A activity) andortho hydroxylation of 4-OH-tam in human liver microsomes; 2) monospecific antibodies against CYP3A4 strongly inhibited catechol formation from 4-OH-tam and its covalent binding to proteins in human liver microsomes; 3) low levels of ketoconazole inhibited catechol tam accumulation and covalent binding of 4-OH-tam to human liver proteins; 4) among human P-450s expressed in insect cells (supersomes), only CYP3A4 and 2D6 noticeably catalyzed catechol formation, and cytochrome b5 markedly stimulated the CYP3A4 catalysis; and 5) human livers with high CYP3A and low or high CYP2D6 activity exhibited high catechol formation and those with low 3A and 2D6 activities formed only little catechol. These findings demonstrate that CYP3A4 and to a lesser extent 2D6 catalyze tam catechol formation and support the participation of tam catechol in covalent binding to proteins.
Footnotes
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Send reprint requests to: Dr. David Kupfer, Ph.D., Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, 55 Lake Ave. N., Worcester, MA 01655. E-mail: david.kupfer{at}ummed.edu
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This study was made possible by United States Public Health Service Grant ES00834 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. A preliminary account of a portion of this study was presented at the American Association for Cancer Research meeting in San Diego, CA. [ (1997) Proc Am Assoc Cancer Res38:3997.]
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↵2 Because 3,4-di-OH-tam catechol is not an end product of tam metabolism, but is further transformed and thus cannot be accurately assessed by chemico/physical means, we used a method that permits methylation of the formed catechol in situ, using endogenous or exogenous COMT and 3H-SAM, and the resulting radiolabeled catechol is quantified by scintillation spectroscopy. The value for catechol formation may represent an underestimation, because of competing oxidative reaction resulting in formation of quinones. However, if the rate of catechol oxidation is much slower than the COMT-mediated methylation, then catechol underestimation would be insignificant.
- Abbreviations used are::
- tam
- tamoxifen [Z-1-(4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenyl-1-butene]
- 4-OH-tam
- 4-hydroxy-tamoxifen
- 3,4-di-OH-tam
- 3,4-dihydroxy-tamoxifen (tam catechol)
- 3-OH-tam
- 3-hydroxy-tamoxifen (Droloxifene)
- CYP
- cytochrome P-450
- TPE
- triphenylethylene
- SAM
- S-adenosyl-l-methionine iodide salt
- COMT
- catechol-O-methyltransferase
- PB
- phenobarbital
- TAO
- troleandomycin
- Dex
- dexamethasone
- KZ
- ketoconazole
- RI
- reactive intermediate
- DTT
- dithiothreitol
- [3H]-SAM
- [3H-methyl]-S-adenosyl-l-methionine
- TLC
- thin-layer chromatography
- IIAM
- International Institute for the Advancement of Medicine
- Received December 16, 1998.
- Accepted February 24, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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