Identification of human CYP forms involved in the activation of tamoxifen and irreversible binding to DNA
David J. Boocock,
Karen Brown1,
Anthony H. Gibbs,
Esther Sanchez2,
Kenneth W. Turteltaub2 and
Ian N.H. White3
MRC Molecular Endocrinology Group, Department of Obstetrics and Gynaecology, Robert Kilpatrick Building, University of Leicester, Leicester LE2 7LX, UK,
1 Cancer Biomarkers and Prevention Group, University of Leicester LE1 7RH, UK and
2 Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA
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Abstract
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This study investigates which CYP forms are responsible for the conversion of tamoxifen to its putative active metabolite
-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
-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 [14C]tamoxifen to intermediates that irreversibly bind to exogenous DNA. Incubation of [14C]tamoxifen (20.6 kBq, 100 µM) with CYP3A4, in the presence of NADPH for 60 min led to levels of DNA binding of 39.0±9.0 adducts/108 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
-hydroxytamoxifen and the only one that results in appreciable levels of irreversible binding of tamoxifen to DNA.
Abbreviations: AMS, accelerator mass spectrometry; LC-ESIMS, liquid chromatography-electrospray secondary ion mass spectrometry.
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Introduction
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Tamoxifen is a synthetic antioestrogen used for many years as an important adjunct therapeutic agent for breast cancer (1). It has also recently been through clinical trials to examine its efficacy in preventing the disease when given to healthy at risk women. However, both therapeutic and chemopreventative regimens are associated with an increased risk of endometrial cancer (2,3). Although no uterine tumours have been found in rats (4), tamoxifen is a hepatocarcinogen in this species (5,6) and results in persistent DNA adducts in vivo that have been detected by 32P-post-labelling (68). However, the presence or absence of DNA adducts derived from tamoxifen binding in the human endometrium is still a matter of debate.
Tamoxifen is metabolized by CYP enzymes to form several metabolites, primarily 4-hydroxytamoxifen and N-desmethyltamoxifen and to a lesser extent,
-hydroxytamoxifen (Figure 1
)(911). Additionally, flavin-containing monooxygenases produce tamoxifen-N-oxide (12). Activation of tamoxifen in vivo involves
-hydroxylation (1315) followed by phase II sulfonation (16,17) leading to the ultimate generation of an electrophilic carbocation that is capable of reacting with DNA (14,18).
-Hydroxytamoxifen itself can react with DNA, but esterified forms of the alcohol, such as the acetate or sulfate are in the order of 2001600-fold more reactive, respectively (17,19). This phase II activation via sulfonation can be balanced with phase II inactivation via the glucuronylation of
-hydroxytamoxifen (11) and this balance may go some way to explaining why rats seem more susceptible to hepatic DNA damage and tumour formation than humans (5,20).
As metabolic activation is a prerequisite for the formation of tamoxifen DNA adducts, the objective of this study was to examine the ability of different baculovirus expressed human CYP forms to activate tamoxifen to its putative reactive intermediate,
-hydroxytamoxifen. We expected the level of DNA adducts generated by this in vitro system to be close to, or below the limits of detection achievable with the 32P-post-labelling assay, therefore we used the ultra sensitive technique of accelerator mass spectrometry (AMS) to quantify DNA binding (21). For this purpose, AMS has proven to be at least 100-fold more sensitive than standard 32P-post-labelling, capable of detecting 110 adducts/1012 nucleotides. By these means we demonstrate directly for the first time, that CYP3A4 is the major form responsible for irreversible binding of tamoxifen to exogenous DNA.
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Materials and methods
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Chemicals
Tamoxifen and its metabolites were gifts from AstraZeneca (Macclesfield, UK). [1-Phenyl-U-14C]tamoxifen (sp. act. 2.03 GBq/mmol) of >98% radiochemical purity, as determined by HPLC, was from Cambridge Research Biochemicals (Cleveland, UK). HEPES, NADPH and salmon sperm DNA were from Sigma Chemical Co. (Poole, UK). CYP1A2, CYP1B1, CYP2B6, 2CYP2D6, CYP2E1, CYP2C9, CYP2C19 and CYP3A4 (SupersomesTM), from baculovirus-insect cell expressed protein with supplemental cDNA expressed reductase, were from BD Gentest (Woburn, MA, USA). In CYP2B6, CYP2C9, CYP2E1 and CYP3A4 preparations, cytochrome b5 was also co-expressed.
Determination of tamoxifen activation and metabolism
Reaction mixtures of 0.5ml volume in 0.05M HEPES buffer, pH7.4, contained 1 mM NADPH, 50 pmol CYP (0.10.4 mg protein) and where indicated, 1 mg salmon sperm DNA. Following equilibration to 37°C, reactions were started by addition of tamoxifen or [14C]tamoxifen (20.6 kBq, 100 µM) dissolved in methanol (5 µl). Following incubation for 20 or 60 min, reactions were extracted (x3) with 0.4 ml ice-cold 2% ethanol in n-heptane (22). The combined organic extracts were dried under N2 at 37°C and the residue dissolved in 50 µl methanol for HPLC or LC-ESIMS as described below. For the determination of DNA binding, reactions were stopped with chloroform at 20°C (0.5 ml) and the aqueous phase exhaustively extracted with organic solvents to remove the majority of unbound-radiolabelled tamoxifen and its metabolites as described (23). Further clean up of the DNA was achieved by treating the solution first with Nucleon II resin (Amersham Biosciences, Buckingham, UK) to remove proteins and secondly by Qiagen column chromatography (Qiagen, Crawley, UK), according to the manufacturers instructions.
HPLC and liquid chromatography-electrospray secondary ion mass spectrometry
HPLC was carried out using a Waters (Watford, UK) Alliance liquid chromatograph with a 996 photodiode array detector. Separations were performed on a 25x0.4 cm Phenomenex Luna column (Phenomenex, Macclesfield, UK) with an isocratic mobile phase of methanol/0.5 M ammonium acetate 75:25 (v/v) at a flow rate of 1 ml/min. Metabolites were detected by monitoring UV absorption at 243 nm. For liquid chromatography-electrospray secondary ion mass spectrometry (LC-ESIMS), the column outlet was connected to a VG-Platform electrospray mass spectrometer (VG Instruments, Manchester, UK) operating in the positive ion mode. The flow rate of the effluent from the column was reduced by splitting, such that 15% entered the electrospray. Samples were scanned over the mass range 300500 at the rate of one scan every 2 s. The ESI capillary and HV electrode potentials were 3.78 and 0.38 kV, respectively. The source temperature was 90°C and the cone voltage was set at 48 V. The residual flow was passed into a UV detector set at 243 nm.
AMS analysis
DNA samples from the incubations containing [14C]tamoxifen, in the presence or absence of NADPH, were analysed by AMS according to standard procedures (24). From the AMS measurement, the amount of non-extractable radioactivity could be determined, as a measure of [14C]tamoxifen DNA adduct formation. An aliquot of tributyrin (2 mg in methanol) was added to each DNA sample (2550 mg), to provide sufficient carbon for efficient graphitisation (25). The AMS measurement gives an isotope ratio, the amount of 14C relative to the amount of 13C present in each sample. This value was converted to DNA adduct levels after subtraction of the 14C contribution from tributyrin and also DNA controls (21). These controls were DNA samples incubated and purified in the same way as the other samples, but without [14C]tamoxifen added, and therefore take into account the amount of 14C naturally present in the salmon sperm DNA and potentially contributed through sample processing. The average value for all control samples (n = 4) was subtracted from each microsomal incubation containing [14C]tamoxifen.
Statistical analysis
Results are given as the mean ± SE and were tested using analysis of variance (ANOVA) with Dunnetts test for significance at the 5% level.
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Results
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Tamoxifen metabolism by different CYP forms
Figure 2a
shows the elution of metabolite standards of tamoxifen, detected by UV absorption at 243 nm. Chromatograms b and c of Figure 2
show that for CYP3A4, only two major metabolites were detected by LC-MS, one with a protonated molecular ion at m/z 358 and a retention time of 19.3 min, the other at m/z 388 with a retention time of 6.7 min. These characteristics are consistent with N-desmethyltamoxifen and
-hydroxytamoxifen respectively. There was a minor product observed in several of the chromatograms (retention time 24.7 min), which may represent tamoxifen N-oxide formed non-enzymically during the extraction processes. There was no evidence that CYP3A4 catalysed the formation of 4-hydroxytamoxifen. In contrast, Figure 2d and e
shows that with CYP2D6, as well as formation of N-desmethyltamoxifen (m/z 358), there was a component that gave a protonated molecular ion with m/z 388 but had a retention time of 11.3 min, consistent with 4-hydroxytamoxifen formation. Only one major metabolic product was observed with CYP2C19, which had a retention time of 19.3 min, and m/z 358, consistent with the formation of N-desmethyltamoxifen. However, the presence of a small peak with m/z 388 indicated CYP2C19 also generates trace levels of 4-hydroxytamoxifen. Of the remaining five CYPs tested, CYP1A2, CYP1B1, CYP2B6, CYP2C9 and CYP2E1 gave no detectable metabolism in this system. Analysis included a search for a protonated metabolite, at m/z 402, which might have been indicative of 3,4-dihydroxytamoxifen formation but none was found. All of the reaction mixtures contained the same concentration of cytochrome P450. Reaction time courses (not shown) revealed rates of metabolism were linear up to 20 min. Table 1
shows the quantification of these metabolites by HPLC, based on their UV absorption. CYP3A4 was the only one catalysing
-hydroxylation. Somewhat surprisingly, in this study, CYP2D6 had the highest specific activity towards N-demethylation of tamoxifen, in addition to 4-hydroxylation.

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Fig. 2. On line LC-ESIMS analysis of the incubation products of tamoxifen with individual CYPs. Trace a, UV absorbance of standards at 243 nm. These were: (1) -hydroxytamoxifen; (2) 4-hydroxytamoxifen; (3) N-desmethyltamoxifen; (4) tamoxifen N-oxide; (5) tamoxifen. Traces b, d and f, reaction products with [M + H]+ at m/z 358. Traces c, e and g, reaction products with [M + H]+ at m/z 388. Metabolites generated by CYP3A4 (b and c), CYP2D6 (d and e) and CYP2C19 (f and g) are shown. None of the other CYPs formed any detectable tamoxifen metabolites.
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Table I. Comparison of the conversion of tamoxifen toN-desmethyltamoxifen or 4-hydroxytamoxifen by different CYP forms
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Tamoxifen activation to intermediates involved in DNA binding by different CYP forms
The NADPH-dependent irreversible binding of activated tamoxifen to exogenous DNA is shown in Figure 3
. It is clear that CYP3A4 is the major form involved in this reaction, as it resulted in a level of DNA binding over 12 times greater than that measured in incubations containing this enzyme without NADPH (39.0 ± 9.0 and 3.0 ± 1.73 adducts/108 nucleotides respectively, n = 6). The mean level of DNA binding without NADPH for all incubations was 1.74 ± 0.26 adducts/108 nucleotide (n = 14). Small non-significant NADPH-dependent binding of tamoxifen to DNA was also detected with CYP2D6 and CYP2C19 (Figure 3
). None of the other CYP forms tested gave adduct levels significantly different than the controls, indicating that they were not able to catalyse the NADPH-dependent binding of tamoxifen to DNA. In a separate study, using CYP3A4, an incubation time of 20 min gave levels of DNA binding of 78 ± 4% (mean ± SE, n = 3) those of 60 min incubations.

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Fig. 3. Irreversible binding of [14C]tamoxifen to DNA catalysed by CYPs. Following incubation of individual CYPS with [14C]tamoxifen (100 µM) and salmon sperm DNA, the amount of covalently bound radiolabel was determined by AMS. For the +NADPH samples, results represent the mean (±SE) of three to six separate incubations. For the NADPH samples, results were obtained from one (CYPs 1B1, 2B1, 2E1), two (1A2, 2D6, 2C9, 2C19) or three (3A4) separate incubations. *Significantly higher level of radiolabel in the presence of NADPH than in its absence.
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Discussion
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This study has shown, through the use of eight baculovirus expressed human cytochrome P450s (SupersomesTM), only CYP3A4 is responsible for the conversion of tamoxifen to the putative active metabolite,
-hydroxytamoxifen. For the first time, by using accelerator mass spectrometry, we have demonstrated directly that CYP3A4 also catalyses the activation of [14C]tamoxifen, resulting in irreversible binding to exogenous DNA. As CYP3A4 produced no detectable 4-hydroxytamoxifen, these results do not support the view that this metabolite is on the path of formation of the reactive intermediate that binds covalently to DNA (26).
There has been much circumstantial evidence as to the role of different cytochrome P450s in the activation of tamoxifen. The initial studies of Jacolot et al. (27) using human liver microsomes identified the involvement of the CYP3A family in the N-demethylation of this drug. Subsequently, a CYP3A4 construct in a human lymphoblastoma cell line was shown to be responsible for the clastogenic action of this drug (28). DNA adduct formation as a consequence of liver microsomal NADPH-dependent tamoxifen activation has been illustrated using 32P-post-labelling (23). However, all of the evidence in vitro as to the role that different CYP forms play in activating tamoxifen has come from studies using protein binding as a surrogate for DNA binding. Results using human liver microsomes, depending on the quantification of protein by western blotting and the use of specific CYP inhibitors, have suggested that in addition to CYP3A4, others such as CYP2B6 and CYP2C19 may be involved (23,29,30). Using recombinant CYP proteins isolated from Escherichia coli, Notley et al. (30) showed the highest level of covalently bound tamoxifen protein adducts were formed in the presence of CYP3A4 but there was also significant binding catalysed by CYP2B6 and CYP2C19. These approaches have their limitations, in that no tamoxifen-protein adducts have been structurally identified and therefore it is not known if the active species which binds to protein is the same as that which binds to DNA.
Although it is generally accepted that the route of activation of tamoxifen lies via formation of
-hydroxytamoxifen (Figure 1
), other studies have suggested that 3,4-dihydroxytamoxifen (tamoxifen catechol) may be proximate to the reactive intermediate that binds covalently to proteins and possibly to DNA (31). Using LC-ESIMS, we looked for, but failed to find, a protonated molecular ion at m/z 402 that would be consistent with 3,4-dihydroxytamoxifen formation. This may be because the overall level of activity of the individual CYP preparations are lower than that of liver microsomes so the metabolite, if formed, could be below the limit of detection. The present study shows that there is a clear difference in specificity between the different CYPs with respect to metabolite formation. 4-Hydroxytamoxifen was only formed by CYP2D6 and CYP2C19 but these did not catalyse any significant covalent binding of tamoxifen to DNA. We did not investigate if 4-hydroxtamoxifen could be converted into the catechol by CYP3A4, as has been suggested in a recent publication (30). While the half-life of the sulfate ester of
-hydroxytamoxifen is in the order of a few minutes in phosphate buffer at neutral pH values (17),
-hydroxytamoxifen itself is more stable and reacts with DNA relatively slowly. For this reason incubations looking for binding of active intermediates of tamoxifen were carried out for 1 h, a time used previously by other investigators (17,23,30). However, shorter incubations (20 min) were sufficient to detect significant levels of DNA binding in incubations of tamoxifen with CYP3A4.
CYP3A4 is the most abundant form of cytochrome P450 in the human liver and the evidence suggests that this is the major form of cytochrome P450 responsible for the metabolism of tamoxifen. However, the present results show in terms of activity per picomole CYP, CYP2D6 is the most active both with respect to N-demethylation and 4-hydroxylation. While quantitatively both CYP2D6 and CYP2C19 represent only a minor fraction of the total drug metabolising capacity of the liver, both are polymorphic and therefore, may alter the balance of metabolism of tamoxifen towards the activation pathways involving CYP3A4.
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Notes
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3 To whom correspondence should be addressed Email: iw6{at}le.ac.uk 
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Acknowledgments
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Portions of this work conducted under the auspices of the U.S. DOE by LLNL (W-7405-ENG-48) with support from RR13461.
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Received May 23, 2002;
revised July 26, 2002;
accepted July 29, 2002.