Abstract
The nonsteroidal antiestrogen tamoxifen was introduced as a treatment for breast cancer 3 decades ago. It has also been approved as a chemopreventive agent and is prescribed to women at high risk for this disease. However, several studies have shown that use of tamoxifen leads to increased risk of endometrial cancer in humans. One potential pathway of tamoxifen toxicity could involve metabolism via hydroxylation to give 4-hydroxytamoxifen (4OHtam), which may be further oxidized to form a quinone methide. CYP2B6 is a highly polymorphic drug-metabolizing enzyme, and it metabolizes a number of clinically important drugs. Earlier studies from our laboratory have shown that tamoxifen is a mechanism-based inactivator of CYP2B6. The aim of the current study was to investigate the possible formation of reactive intermediates through detection of protein covalent binding and glutathione ethyl ester adduct (GSHEE) formation. The incubation of tamoxifen with 2B6 gave rise to an adduct of 4OHtam with glutathione, which was characterized as the 4OHtam quinone methide + GSHEE with an m/z value of 719, and the structure was characterized by liquid chromatography-tandem mass spectrometry. The metabolic activation of tamoxifen in the CYP2B6 reconstituted system also resulted in the formation of an adduct to the P4502B6 apoprotein, which was identified using liquid chromatography mass spectrometry. The site responsible for the inactivation of CYP2B6 was determined by proteolytic digestion and identification of the labeled peptide. This revealed a tryptic peptide 188FHYQDQE194 with the site of adduct formation localized to Gln193 as the site modified by the reactive metabolite formed during tamoxifen metabolism.
Footnotes
This work was supported in part by the National Institutes of Health National Cancer Institute [Grant CA16954].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS:
- 4OHtam
- 4-hydroxytamoxifen
- LC-MS
- liquid chromatography-mass spectrometry
- GSHEE
- glutathione ethyl ester
- HPLC
- high-performance liquid chromatography
- ESI
- electrospray ionization
- MS/MS
- tandem mass spectrometry
- TFA
- trifluoroacetic acid
- P450
- cytochrome P450
- 7-EFC
- 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation
- OHtam
- hydroxytamoxifen.
- Received June 14, 2012.
- Accepted August 31, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|