Interindividual variation in the isomerization of 4- hydroxytamoxifen by human liver microsomes: involvement of cytochromes P450

Williams, Mary L.; Lennard, M. S.; Martin, Iain; Tucker, GT

doi:10.1093/carcin/15.12.2733

Cited by 40 publications

(33 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3). Isomerization was time-and NADPH-dependent as shown in previous work (Williams et al, 1994). For certain forms (3A4, 1A1, 2D6), interpretation of the isomerization data were complicated by the finding that substrate at the low concentrations employed appeared to be metabolized to secondary metabolites.…”

Section: Tamoxifen Metabolism By Cytochrome P450mentioning

confidence: 57%

“…HPLC was performed according to a previously described method (Williams et al, 1994), using a Novapak (Waters Corp.) 4 M C 8 column. The mobile phase was 10 mM KH 2 PO 4 , pH 3.7, buffer/ methanol/acetonitrile (3:3:4 v/v) and containing 0.02% (v/v) triethylamine.…”

Section: Methodsmentioning

confidence: 99%

“…P450-catalyzed isomerization of trans-4-hydroxytamoxifen has been reported (Williams et al, 1994), resulting in the conversion of a potent antiestrogen (trans-4-hydroxytamoxifen) into a weak estrogen agonist (cis-4-hydroxytamoxifen). Clinical resistance to tamoxifen therapy has been associated with decreased tumor concentrations of tamoxifen and an increase in the cis/trans ratio of 4-hydroxytamoxifen (Osborne et al, 1992).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Metabolism of Tamoxifen by Recombinant Human Cytochrome P450 Enzymes: Formation of the 4-Hydroxy, 4′-Hydroxy andN-Desmethyl Metabolites and Isomerization oftrans-4-Hydroxytamoxifen

Crewe

Notley²,

Wunsch³

et al. 2002

Drug Metab Dispos

223

140

View full text Add to dashboard Cite

ABSTRACT:The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPHcytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 ؎ 20 pmol/40 min/0.2 nmol P450 using 18 M tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 ؎ 0.8 and 3.1 ؎ 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 M tamoxifen). These two forms also catalyzed 4-hydroxylation (13.0 ؎ 1.9 and 1.4 ؎ 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 M tamoxifen; 0.51 ؎ 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 M tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

show abstract

Section: Tamoxifen Metabolism By Cytochrome P450mentioning

confidence: 57%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Metabolism of Tamoxifen by Recombinant Human Cytochrome P450 Enzymes: Formation of the 4-Hydroxy, 4′-Hydroxy andN-Desmethyl Metabolites and Isomerization oftrans-4-Hydroxytamoxifen

Crewe

Notley²,

Wunsch³

et al. 2002

Drug Metab Dispos

223

140

View full text Add to dashboard Cite

show abstract

“…at ASPET Journals on May 12, 2018 dmd.aspetjournals.org Downloaded from vidual variation in the isomerization of 4OHtam by human liver microsomes has been shown by Williams et al (1994). Figure 3, B and C, shows the MS spectrum of the GSHEE adduct and the MS/MS fragmentation patterns, respectively.…”

Section: Formation Of the Tamoxifen Quinone Methide By Cyp2b6mentioning

confidence: 95%

Bioactivation of the Cancer Chemopreventive Agent Tamoxifen to Quinone Methides by Cytochrome P4502B6 and Identification of the Modified Residue on the Apoprotein

2012

View full text Add to dashboard Cite

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 188 FHYQDQE 194 with the site of adduct formation localized to Gln193 as the site modified by the reactive metabolite formed during tamoxifen metabolism.

show abstract

“…Our objective was not to undertake a quantitative analysis of which P450s metabolize tamoxifen in vivo or to predict which forms will be more or less important. Rather we sought only to show which forms potentially may be involved, building on and extending previous in vitro studies using liver microsomes and recombinant systems (Jacolot et al, 1991;Simon et al, 1993;Williams et al, 1994;Crewe et al, 1997;Dehal and Kupfer, 1997). Indeed, given the stated aim to identify forms that might participate in extrahepatic metabolism and the paucity of information regarding the content of different P450s in extrahepatic tissues such as breast and endometrium, it is impossible at this stage to perform such a scaling procedure as suggested.…”

Section: Response To Letter To the Editor Evaluation Of Recombinant Cmentioning

confidence: 99%

Evaluation of Recombinant Cytochromes P450 Activity in Metabolic Pathways

Rochat¹

2003

Drug Metab Dispos

View full text Add to dashboard Cite

In the August issue of Drug Metabolism and Disposition, an article on the metabolism of the anticancer agent tamoxifen by Crewe et al. (2002) appeared. This article investigates the implication of cytochrome P450 (P450) isozymes involved in four different metabolic pathways.For more than a decade, tamoxifen biotransformation was extensively studied since its 4-hydroxy metabolite showed much more potent anti-estrogen efficacy than its parent drug and because protein and DNA adducts were discovered in animals as well as in treated women (Phillips, 2001). Therefore, correlations between tamoxifen resistance or toxicity and drug metabolism were investigated but unfortunately until now without real success.Recently, an interesting article reviewing tamoxifen resistance at the molecular level (Clarke et al., 2001) described mechanisms of resistance depending on the cell clones. In other words, one cell clone could show a resistance related to its metabolism whereas another resistant clone would not. Such heterogeneity of drug resistance underlines the need to investigate drug metabolism as a possible mechanism of resistance even if some data suggest the contrary in a specific cell clone. Thus, identifying P450s involved in tamoxifen metabolism remains a challenge in order to investigate a resistance to tamoxifen.It is clear that the interest is to identify P450s implication in vivo in the tumor cells or in the liver cells. In Crewe et al. (2002), various cDNA-expressed P450s (recombinant systems) were used to identify the specific isozyme(s) involved in four different metabolic pathways. For such investigation, the use of recombinant P450 isozymes seems one of the best tools.In this article, results are given at best by metabolic velocities (v) at two different tamoxifen concentrations. Such data are unfortunately poorly predictive of the real biotransformation in cancer or liver cells, and all discussion on tamoxifen biotransformation from such partial data may be quite unreliable.Indeed, implication in cells of a specific P450 isozyme depends on the drug-enzyme affinity, K m , and the maximal velocity of the metabolite production, V max . From these two Michaelis-Menten constants, an intrinsic clearance can be calculated by V max /K m . With recombinant P450s, intrinsic clearances can be easily determined for each isozyme. Unfortunately, no V max and K m values are available in this article. Moreover, it was described by Venkatakrishnan et al. (2000) that metabolic velocities in recombinant system preparation may be strongly different from microsome preparation. Indeed, the cellular amounts of P450 coenzymes (NADPH P450 oxidoreductase and, for some isozymes, cytochrome b 5 ) as well as lipidic membrane composition of recombinant systems (bacteria, yeast, etc.) can strongly modify P450 activity. As stated by these authors, "Use of metabolic rates ( When I studied the demethylation of the antidepressant, citalopram (Rochat et al., 1997), human recombinant CYP2D6 isozyme showed by far the highest production of demethy...

show abstract

Interindividual variation in the isomerization of 4- hydroxytamoxifen by human liver microsomes: involvement of cytochromes P450

Cited by 40 publications

References 0 publications

Metabolism of Tamoxifen by Recombinant Human Cytochrome P450 Enzymes: Formation of the 4-Hydroxy, 4′-Hydroxy andN-Desmethyl Metabolites and Isomerization oftrans-4-Hydroxytamoxifen

Metabolism of Tamoxifen by Recombinant Human Cytochrome P450 Enzymes: Formation of the 4-Hydroxy, 4′-Hydroxy andN-Desmethyl Metabolites and Isomerization oftrans-4-Hydroxytamoxifen

Bioactivation of the Cancer Chemopreventive Agent Tamoxifen to Quinone Methides by Cytochrome P4502B6 and Identification of the Modified Residue on the Apoprotein

Evaluation of Recombinant Cytochromes P450 Activity in Metabolic Pathways

Contact Info

Product

Resources

About