afimoxifene and alpha-hydroxytamoxifen

An increased risk of developing endometrial cancer is observed in breast cancer patients treated with tamoxifen (TAM) and in healthy women undergoing TAM chemoprevention therapy. TAM-DNA adducts were detected in the endometrium of women taking TAM (Shibutani, S., et al. (2000) Carcinogenesis 21, 1461-1467) and are formed primarily through O-sulfonation of alpha-hydroxytamoxifen (alpha-OHTAM). To explore the genotoxicic mechanisms of TAM, TAM was incubated with one of multiple human cytochrome P450 enzymes, i.e., P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3A, or 4F3B, in a NADPH regenerating system, and the metabolites were identified using HPLC/UV analysis with authentic standards. Among the 18 human P450 enzymes, P450 3A4 generated a significant amount of alpha-OHTAM. When some rat P450 enzymes were examined, P450 3A2 also catalyzed alpha-hydroxylation of TAM. Similarly, human P450 3A4 and rat P450 3A1 and 3A2 converted toremifene (TOR, a chlorinated TAM analogue) to alpha-hydroxytoremifene (alpha-OHTOR). The formation of alpha-OHTAM and alpha-OHTOR by these P450 enzymes was confirmed by tandem mass spectroscopy. Only the P450 3A subfamily enzymes are able to alpha-hydroxylate TAM and TOR. Although the formation of alpha-OHTOR by these enzymes was much higher than that of alpha-OHTAM, TOR is known to be much less genotoxic than TAM. The results support our proposed mechanism that the lower genotoxicity of TOR is due to limited O-sulfonation of alpha-OHTOR by hydroxysteroid sulfotransferases, resulting in the poor formation of DNA adducts (Shibutani, S., et al. (2001) Cancer Res. 61, 3925-3931).

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Deoxyguanine Nucleotides; Female; Humans; Hydroxylation; Mass Spectrometry; NADPH Oxidases; Rats; Tamoxifen; Toremifene

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