n-desmethyltamoxifen and alpha-hydroxytamoxifen

Tamoxifen, an antiestrogen used in the prevention and treatment of breast cancer, is extensively metabolized by cytochrome P450 enzymes. Its biotransformation to α-hydroxytamoxifen (α-OHT), which may be genotoxic, and to N-desmethyltamoxifen (N-DMT), which is partially hydroxylated to 4-hydroxy-N-DMT (endoxifen), a potent antiestrogen, is mediated by CYP3A enzymes. However, the potential contribution of CYP3A5 and the impact of its low-expression variants on the formation of these metabolites are not clear. Therefore, we assessed the contributions of CYP3A4 and CYP3A5 and examined the impact of CYP3A5 genotypes on the formation of α-OHT and N-DMT, by using recombinant CYP3A4 and CYP3A5 and human liver microsomes (HLM) genotyped for CYP3A5 variants. We observed that the catalytic efficiency [intrinsic clearance (CL(int))] for α-OHT formation with recombinant CYP3A4 was 5-fold higher than that with recombinant CYP3A5 (0.81 versus 0.16 nl · min⁻¹ · pmol cytochrome P450⁻¹). There was no significant difference in CL(int) values between the three CYP3A5-genotyped HLM (*1/*1, *1/*3, and *3/*3). For N-DMT formation, the CL(int) with recombinant CYP3A4 was only 1.7-fold higher, relative to that with recombinant CYP3A5. In addition, the CL(int) for N-DMT formation by HLM with CYP3A5*3/*3 alleles was approximately 3-fold lower than that for HLM expressing CYP3A5*1/*1. Regression analyses of tamoxifen metabolism with respect to testosterone 6β-hydroxylation facilitated assessment of CYP3A5 contributions to the formation of the two metabolites. The CYP3A5 contributions to α-OHT formation were negligible, whereas the contributions to N-DMT formation ranged from 51 to 61%. Our findings suggest that polymorphic CYP3A5 expression may affect the formation of N-DMT but not that of α-OHT.

Topics: Alleles; Antineoplastic Agents, Hormonal; Cytochrome P-450 CYP3A; Humans; Hydroxylation; Kinetics; Microsomes, Liver; Polymorphism, Genetic; Recombinant Proteins; Selective Estrogen Receptor Modulators; Substrate Specificity; Tamoxifen

Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.

Topics: Biotransformation; Culture Techniques; Cytochrome P-450 Enzyme System; DNA Adducts; Endometrium; Female; Humans; Immunohistochemistry; Recombinant Proteins; Tamoxifen

This study investigates which CYP forms are responsible for the conversion of tamoxifen to its putative active metabolite alpha-hydroxytamoxifen and irreversible binding to DNA. We have used eight different baculovirus expressed recombinant human CYP forms and liquid chromatography-mass spectrometry to show that only CYP3A4 is responsible for the NADPH-dependent alpha-hydroxylation of tamoxifen. Surprisingly, this CYP did not catalyse the formation of 4-hydroxytamoxifen. We demonstrate for the first time, by means of accelerator mass spectrometry, that CYP3A4 also catalysed the activation of [(14)C]tamoxifen to intermediates that irreversibly bind to exogenous DNA. Incubation of [(14)C]tamoxifen (20.6 kBq, 100 micro M) with CYP3A4, in the presence of NADPH for 60 min led to levels of DNA binding of 39.0+/-9.0 adducts/10(8) nucleotides (mean +/- SE, n = 6). While CYP3A4 converted tamoxifen to N-desmethyltamoxifen (38.3 +/- 7.20 pmol/20 min/pmol CYP, n = 4), the polymorphic CYP2D6 showed the highest activity for producing this metabolite (48.6+/-1.52pmol/20 min/pmol CYP). CYP2D6 was also the most active in catalysing 4-hydroxylation of tamoxifen, although an order of magnitude lower level was also detected with CYP2C19. With tamoxifen as substrate, no 3,4-dihydroxytamoxifen could be detected with any CYP form. CYP2B6 did not catalyse the metabolism or the binding of tamoxifen to DNA. It is concluded that CYP3A4 is the only P450 of those tested that converts tamoxifen to alpha-hydroxytamoxifen and the only one that results in appreciable levels of irreversible binding of tamoxifen to DNA.

Topics: Biotransformation; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; DNA; Humans; Hydroxylation; Isoenzymes; NADP; Prodrugs; Tamoxifen