Identification of human CYP forms involved in the activation of tamoxifen and irreversible binding to DNA

Abstract

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.

Tamoxifen Forms DNA Adducts in Human Colon after Administration of a Single [14C]-Labeled Therapeutic Dose

“…Tamoxifen is metabolically activated via a-hydroxylation of the parent compound and other phase I metabolites, a reaction catalyzed by CYP3A4 in human tissues (13). Although in rat liver the proximate reactive species is considered to arise as a consequence of further sulfation, the alcohol itself shows intrinsic activity toward DNA albeit f1,600-fold weaker than the sulfate ester (16).…”

“…Interestingly, the urinary metabolite profile of the dietary carcinogen 2-amino-1-methyl-6-phenyimidazo [4,5-b ]pyridine (PhIP) has recently been described as a possible predictor of DNA adduct levels in colon tissue of humans administered a dietary relevant dose of [2][3][4][5][6][7][8][9][10][11][12][13][14] C]-PhIP (45). PhIP is bioactivated by CYP1A2 to 2-N-hydroxy-PhIP, which is subsequently esterified, producing the ultimate DNA-reactive species, O-sulfonyl or Oacetyl esters.…”

“…These liver tumors are considered to arise through a genotoxic mechanism. Tamoxifen activation involves conversion to a-hydroxytamoxifen, catalyzed primarily by CYP3A4 in humans (12,13). This metabolite can then be further conjugated, generating the more reactive a-sulfate ester, but whether this step actually occurs in human tissues has not yet been confirmed (14).…”

Efficacy of tamoxifen based on cytochrome P450 2D6, CYP2C19 and SULT1A1 genotype in the Italian Tamoxifen Prevention Trial

“…CYP2D6 is one of the most significant enzymes that modulates plasma endoxifen concentration. 14 The relationship of CYP2D6 activity, the level of endoxifen and the antiestrogenic activity has been recently clarified by Wu et al 16 However, many other enzymes are involved in the pathway, including CYP3A4, CYP2C9, CYP2B6 and CYP2C19 17 and may support the metabolic pathway in case of CYP2D6 PM.…”

Digital PCR for determination of cytochrome P450 2D6 and sulfotransferase 1A1 gene copy number variations

“…Cytochrome P450 2D6 (CYP2D6) metabolizes several different drugs, including timolol, propranolol, amitriptyline, propafenone, flecainide, and tamoxifen (1)(2)(3)(4)(5)(6). The metabolic ratios of the probe drugs vary, thus patients can be classified into 4 different genotypes; poor metabolizer (PM), intermediate metabolizer (IM), extensive metabolizer (EM), and ultra-rapid metabolizer (UM) (7).…”