Chem. Res. Toxicol. 2005, 18, 1611-1618
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Characterization of the Human Cytochrome P450 Forms Involved in Metabolism of Tamoxifen to Its r-Hydroxy and r,4-Dihydroxy Derivatives L. M. Notley,† K. H. Crewe,‡ P. J. Taylor,§ M. S. Lennard,*,‡ and E. M. J. Gillam*,† School of Biomedical Sciences, University of Queensland, St. Lucia, Australia 4072, Academic Unit of Clinical Pharmacology, Division of Clinical Sciences South, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom, and Department of Medicine, University of Queensland and Department of Clinical Pharmacology, Princess Alexandra Hospital, Woolloongabba 4102, Australia Received May 30, 2005
Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include R-hydroxytamoxifen, R,4-dihydroxytamoxifen, and R-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing R-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250 µM) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as R-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No R,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100 µM) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of R,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.
Introduction Tamoxifen is a nonsteroidal antiestrogen that has been used to successfully treat breast cancer for many years. More recently, tamoxifen has been used as a prophylactic agent in women considered at significant risk of developing the disease (1). However, clinical data have shown a link between tamoxifen therapy and an increased incidence of endometrial cancer (2). Although the underlying mechanism has not been conclusively established, tamoxifen metabolism may lead to the generation of reactive metabolites capable of binding to DNA in the endometrium and resulting in genotoxicity. To date, a number of metabolites have been identified as associated with adduct formation in animal models and/or in vitro studies (Figure 1), including R-hydroxytamoxifen, R,4-dihydroxytamoxifen (3), and R-hydroxy-N-desmethyltamoxifen (4). Tamoxifen is well-established as a hepatic carcinogen in rats. A number of studies have used 32P postlabeling to examine DNA adducts in rat hepatocytes treated with * To whom correspondence should be addressed. (M.S.L.) Tel: +44114-271 2578. Fax: +44-114-272 0275. E-mail: M.S.Lennard@ sheffield.ac.uk. (E.M.J.) Tel: +61-7-3365 1410. Fax: +61-7-3365 1766. E-mail:
[email protected]. † University of Queensland. ‡ Royal Hallamshire Hospital. § Princess Alexandra Hospital.
the drug. Phillips et al. (5) found a similar pattern of adduct formation in R-hydroxytamoxifen-treated hepatocytes as compared to those treated with tamoxifen. Further evidence that R-hydroxytamoxifen is involved in adduct formation came from the finding that the D5-ethyl derivative of tamoxifen is less genotoxic and forms lower amounts of adducts than tamoxifen (6). Although adducts could be formed in the absence of further metabolism (5), a further finding was that sulfonation dramatically enhances adduct formation over that seen with R-hydroxytamoxifen alone (7, 8). R-Hydroxytamoxifen and its N-demethylated glucuronide metabolite have been detected in the plasma of patients (9), but detection of any sulfate metabolite may not be possible due to its lability (8, 10). The adducts formed following the sulfonation of R-hydroxytamoxifen by hydroxysteroid sulfotransferases have been shown to be miscoding and mutagenic, leading predominantly to G-T transversions (11, 12). Shibutani et al. (13, 14) were able to show the presence of DNA adducts derived from R-hydroxytamoxifen in the endometrium of eight out of 16 women who received tamoxifen therapy, with the majority identified as the trans and cis epimers of R-(N2-deoxyguanosinyl) tamoxifen. Adducts involving breast, myometrial, and endometrial DNA and breast protein have also been detected in small amounts after a single dose of tamoxifen (15).
10.1021/tx050140s CCC: $30.25 © 2005 American Chemical Society Published on Web 09/30/2005
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Figure 1. Pathways of tamoxifen metabolism examined in this study.
It has been shown in DNA binding studies that 4-hydroxytamoxifen may be another intermediate in the binding of tamoxifen to DNA in vivo in rats, suggesting that a secondary metabolite of 4-hydroxytamoxifen is involved in adduct formation (16). Chemically synthesized R,4-dihydroxytamoxifen was found to produce 12fold greater amounts of adducts than R-hydroxytamoxifen (3) under acidic conditions. It is thought that some of the minor adducts detected in tamoxifen-treated rats may be formed from this metabolite (16), although other results have led to this conclusion being questioned (17, 18). Although the quinone methide was initially proposed as the proximal reactive metabolite formed from R,4dihydroxytamoxifen (19), studies by Bolton and colleagues have demonstrated that this compound is unexpectedly stable (20). Alternatively, a resonance form of the quinone methide may be responsible for the low extent of adduct formation (17). Interestingly, adducts formed in quinone methide-treated DNA appeared to be more mutagenic than those from R-acetoxytamoxifentreated DNA in human cell lines (21), despite producing less overall DNA damage. N-Demethylation is the major route of metabolism of tamoxifen in rats and humans, and a number of secondary metabolites of this compound have been detected in patients, including R-hydroxy-N-desmethyltamoxifen. When rats are treated with tamoxifen or R-hydroxytamoxifen, two major DNA adducts are formed. However, only one of these adducts is detected after treatment with N-desmethyltamoxifen (4). Attempts have been made to detect adducts arising from this metabolite in human endometrial samples. However, refinements to the current chromatographic techniques will be required in order to separate the adduct derived from the N-demethylated metabolite from one of the trans epimers of dG-N2tamoxifen (22, 23). Cytochrome P450 enzymes (P450s)1 are the principal catalysts of oxidative drug metabolism. The liver is
Notley et al.
considered to be the major site of activity of most P450 forms, but a number of forms have been found to be expressed to a significant extent in tissues such as the breast and endometrium. Whereas it is possible that a nonreactive metabolite could be produced in the liver and then be transported to an extrahepatic site where further activation (such as sulfonation) results in a genotoxic compound, the proximal reactive metabolite could be generated in the tissue of interest by P450-mediated metabolism. A number of P450s have been shown to be capable of metabolizing tamoxifen (24-29). Using bacterially expressed human P450 enzymes, it was shown that the primary metabolites, 4-hydroxytamoxifen and N-desmethyltamoxifen, could be produced by P450 2D6, P450 3A4, P450 3A5, P450 2C9, P450 2C19, P450 1A1, and P450 1A2, with P450 2B6 generating 4-hydroxytamoxifen only and P450 1B1 producing N-desmethyltamoxifen only (27). Other studies with recombinant enzymes (29, 30) and those using selective inhibitors of microsomal P450s from human livers (HLs) (10, 29) suggested that P450 3A4 is predominantly responsible for the production of R-hydroxytamoxifen, but the involvement of other forms has also been proposed (29, 31). Moreover, the ability of minor and/or extrahepatic P450s to catalyze tamoxifen R-hydroxylation remains to be assessed fully. Such forms may be of considerable importance in the bioactivation of tamoxifen in patients where the site of genotoxic events appears to be the endometrium. Additionally, the catalysts of R,4-dihydroxytamoxifen production remain to be identified. Thus, the aim of the present study was to determine which P450 forms are capable of producing the reactive metabolites, R-hydroxytamoxifen and R,4-dihydroxytamoxifen.
Experimental Procedures Caution: Tamoxifen has been identified as a human endometrial carcinogen following long-term use and should be handled with care. Chemicals and Drugs. The metabolite standards for HPLC analyses were kindly provided by Dr. I. N. H. White (MRC Toxicology Unit, University Of Leicester, United Kingdom; racemic 4-hydroxytamoxifen, N-desmethyltamoxifen, N,N-didesmethyltamoxifen, and tamoxifen N-oxide), Dr. P. Jank (Klinge Pharma Munich, Germany; N-desmethyldroloxifene, trans-4hydroxytamoxifen, racemic 4-hydroxytamoxifen, 4′-hydroxytamoxifen, N-desmethyltamoxifen, N,N-didesmethyltamoxifen, and tamoxifen N-oxide), Dr. J. L. Bolton (University of Illinois at Chicago; metabolite E), Dr. D. H. Phillips (Institute of the Cancer Research, Surrey, United Kingdom; R-hydroxytamoxifen and R,4-dihydroxytamoxifen), and Dr. D. A. Flockhart [Indiana University School of Medicine, Indianapolis; 4-hydroxy-N-desmethyltamoxifen (endoxifen)]. N,N-Didesmethyltoremifene was the generous gift of Orion Farmos Corporation (Turku, Finland). Tamoxifen citrate, trans-4-hydroxytamoxifen (>98% trans isomer), racemic 4-hydroxytamoxifen (∼70% trans; ∼30% cis isomer), and nafoxidine were purchased from the Sigma Chemical Co. (St. Louis, MO). All other chemicals were obtained from local suppliers at the highest quality commercially available. HL Microsomes. Samples of HL were obtained from organ donors according to procedures approved by University of Queensland ethics committees and frozen in liquid nitrogen for storage at -70 °C prior to use. Microsomes were prepared according to the method of Guengerich (32), with the addition 1 Abbreviations: HL, human liver; hNPR, human NADPH-cytochrome P450 reductase; P450, cytochrome P450.
R-Hydroxylation of Tamoxifen by Human P450s of a final wash in resuspension buffer (10 mM Tris-acetate and 1 mM EDTA containing 20% glycerol) to remove residual drugs. P450 concentrations were determined as previously described (32). Preparation of Recombinant P450s. Human P450 enzymes were expressed in bacteria in bicistronic format with NADPH-cytochrome P450 reductase (hNPR) as described previously (33-36). DH5R strain Escherichia coli was transformed with bicistronic expression constructs each of which contained cDNAs encoding hNPR and one of the following recombinant P450s: P450 1A1, P450 1A2, P450 1B1 [four variants, designated RAVN, RALN, GSVN, and GSLN expressing the following amino acids at positions 48 (Arg or Gly), 119 (Ala or Ser), and 432 (Val or Leu); all four variants contained Asn at position 453 (35, 37)], P450 2A6, P450 2B6, P450 2C9*1 (wild type), P450 2C19, P450 2D6 [full length variant designated DB4 (38) but with wild type (Ala) at position 374], P450 2E1, P450 3A4, P450 3A5, and P450 3A7. Cells were also transformed with the monocistronic expression vector containing the cDNA for hNPR alone and with the empty vector, pCW. Bacteria were cultured and harvested as described previously (39) except that the expression of P450 1A1, P450 2A6, P450 2B6, P450 2D6, and P450 3A7 was augmented by coexpression of bacterial chaperones. Briefly, E. coli strain DH5R was cotransformed with the relevant bicistronic expression vector and pGro7 (40) using the method of Inoue et al. (41). Colonies harboring both plasmids were selected by growth on LB agar containing 100 µg/mL ampicillin and 20 µg/mL chloramphenicol. Isolated colonies were precultured at 37 °C overnight in LB media containing both antibiotics and then used to inoculate terrific broth containing 1 mM thiamine, trace elements, 100 µg/mL ampicillin, and 20 µg/mL chloramphenicol. Cultures were incubated for 5 h at 25 °C, 160-180 rpm shaking speed before initiation of induction by the addition of arabinose (4 mg/mL), IPTG (1 mM), and δ-aminolevulinic acid (0.5 mM). Flasks were then incubated for a further 43 h at 25 °C and 160-180 rpm before harvest. Membranes were prepared and characterized for P450 hemoprotein expression and hNPR activity. Bacterial membranes were used directly in enzyme assays. Enzyme Assays. Incubations contained 0.05-0.2 µM P450 from microsomes or bacterial membranes, substrate, an NADPHgenerating system (consisting of 1 mM NADPH, 2.5 mM glucose6-phosphate, and 0.5 U/mL glucose-6-phosphate dehydrogenase) in either 80 mM potassium phosphate, pH 7.4, with 0.46% (w/ v) KCl (experiments with 18 µM tamoxifen) or 100 mM potassium phosphate, pH 7.4, with 2 mM ascorbic acid. Incubations using P450 3A and P450 2C forms were supplemented with membranes obtained from cells expressing hNPR alone except where otherwise indicated. In the high concentration substrate reactions, P450 3A4, P450 3A5, P450 2C9, P450 2C19, P450 2E1, P450 2A6, and P450 2B6 were supplemented with a 1:1 molar ratio of purified rabbit b5. Control incubations were included in each assay run and contained membranes from cells expressing hNPR alone (hNPR) or from cells transformed with the empty expression vector (pCW). Other negative controls lacked either the NADPH-generating system or the substrate or were stopped immediately after initiation of the reaction (t ) 0). All procedures were performed under reduced light and using amber vessels to minimize photodecomposition of tamoxifen. The R-hydroxylation of tamoxifen was examined at two concentrations: 18 µM, based on previous studies showing satisfactory sensitivity for metabolite detection at this concentration (24), and 250 µM, to reflect metabolism at concentrations approaching saturation. Formation of secondary metabolites from 4-hydroxytamoxifen or N-desmethyltamoxifen was investigated using substrate concentrations of 100 and 22 µM, respectively. Substrate was added from a stock solution in methanol except that 1:1 acetone:ethanol was used as the vehicle for the experiments with 18 µM tamoxifen. Final concentrations of solvent were maintained below 1% v/v in all cases. Reaction mixtures were incubated at 37 °C with gentle
Chem. Res. Toxicol., Vol. 18, No. 10, 2005 1613 agitation for 40 (18 µM experiments) or 120 min (all other experiments). For incubations carried out with 18 µM tamoxifen, incubations were terminated by addition of 1.8 mL of methyl-tert-butyl ether and 50 µL of internal standard (100 µg/mL nafoxidine). Aqueous and organic phases were mixed vigorously, and the top 1.5 mL was removed and evaporated to dryness under air. Extracts were resuspended in 250 µL of a mixture of mobile phase and water (2:1 v/v) prior to analysis by HPLC (isocratic method). For incubations carried out with 250 µM tamoxifen or containing either of the primary metabolites as substrates, incubations (1 mL) were allowed to proceed for 120 min before being terminated by addition of 2 mL of helium-purged ethyl acetate, followed by addition of 100 µL of 15 µM didesmethyl toremifene HCl (internal standard). The phases were mixed vigorously, and the top 1.4 mL was removed. A second extraction was then performed with a further 1.2 mL of ethyl acetate. The combined extracts were evaporated to dryness under argon and resuspended in 100 µL of helium-purged acetonitrile prior to analysis by HPLC (gradient method). HPLC Analysis of Metabolites. Two HPLC methods were used as follows: an isocratic method for analysis of metabolites from incubations carried out using 18 µM tamoxifen as substrate and a gradient method for all other incubations. Isocratic Conditions. HPLC was performed according to a previously described method (24) and using a 5 µm Spherisorb ODS-1 Reverse phase column (250 mm × 4.6 mm) maintained at 40 °C. The mobile phase consisted of 10 mM KH2PO4, pH 3.7, buffer:methanol:acetonitrile (2.5:3:4.5 v/v), and the flow rate was 1.3 mL/min. The column eluant was subjected to UV irradiation, and metabolites were subsequently detected by fluorescence at excitation and emission wavelengths of 260 and 375 nm, respectively. The retention times of tamoxifen, Ndesmethyltamoxifen, 4-hydroxytamoxifen, 4′-hydroxytamoxifen, tamoxifen N-oxide, and nafoxidine were 27.5, 21.8, 14.6, 16.2, 24.4, and 31.5 min, respectively. Gradient Conditions. HPLC was performed using a Shimadzu HPLC system fitted with an autoinjector and a 3.9 mm × 150 mm Waters Symmetry C8 reverse phase column. The mobile phase was 20 mM ammonium acetate:acetonitrile run on a gradient derived from that described in Poon et al. (9) and modified by extension of the second gradient step to optimize peak separation. Initial conditions were 95:5 20 mM ammonium acetate:acetonitrile. Steps were programmed as follows: 0-4 min linear gradient to 80:20 ammonium acetate:acetonitrile; 4-24 min linear gradient to 60:40 ammonium acetate:acetonitrile; 24-60 min linear gradient to 35:65 ammonium acetate: acetonitrile; 60-70 min constant at 35:65 ammonium acetate: acetonitrile; 70-80 min linear gradient to 95:5 ammonium acetate:acetonitrile; and a final reequilibration at 95:5 ammonium acetate:acetonitrile for 10 min. The flow rate was 0.75 mL/min. Metabolites were detected by absorbance at 280 nm. Quantification of metabolites was performed with reference to standard curves prepared using authentic metabolites after correction for recovery of the internal standard (N,N-didesmethyl toremifene hydrochloride).
Results The current study was designed to examine the formation in vitro of potentially reactive tamoxifen metabolites, some of which had not been previously examined. To achieve this end, the HPLC method reported by Poon et al. (9) was modified to resolve the R,4-dihydroxytamoxifen peak from a confounding peak found in the recombinant membranes used in these experiments. Using the modified gradient conditions, the approximate relative retention times of tamoxifen and its main metabolites were as follows: R,4-dihydroxytamoxifen, 28.4 min; R-hydroxytamoxifen, 36.3 min; trans-4-hydroxytamoxifen, 44.4 min; cis-4-hydroxytamoxifen, 46.1 min; 4′-hydroxytamox-
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Figure 2. Chromatogram showing separation of metabolites achieved with the gradient method described in the Experimental Procedures. Authentic metabolites (0.4 nmol each) were injected. Table 1. Effect of Ascorbate Supplementation on r,4-Dihydroxytamoxifen Production from 4-Hydroxytamoxifen by Recombinant P450 3A4 and Liver Microsomes R,4-dihydroxytamoxifen production (µM)b enzyme preparationa
control
+ ascorbate
P450 3A4/hNPR P450 3A4/hNPR + hNPR supplementation HL microsomes rat liver microsomes
0.38 ( 0.03 0.52 ( 0.01#
0.71 ( 0.02** 0.55 ( 0.02#
0.14 ( 0.01 0.10 ( 0.01
0.16 ( 0.01 0.22 ( 0.02*
a Incubations were undertaken as described in the Experimental Procedures for 120 min using 100 µM racemic 4-hydroxytamoxifen and 0.2 µM P450 from microsomes or membranes isolated from bacteria coexpressing P450 3A4 and hNPR. Where indicated, incubations were supplemented with additional hNPR (in bacterial membranes) to a hNPR:P450 ratio of 2:1. b Data represent means ( SD and were analyzed using the Student’s t-test. Asterisks denote data significantly different from the ascorbate deficient control: *p < 0.05 and **p < 0.005. Hatch symbols denote data significantly different from the nonsupplemented control: #p < 0.05.
ifen, 46.9 min; N,N-didesmethyltamoxifen, 53.1 min; N-desmethyltamoxifen, 56.1 min; metabolite E isomers, 61.4/64.2 min; tamoxifen N-oxide, 62.4 min; and tamoxifen, 62.3 min (Figure 2). Further improvements to the methodology were the addition of ascorbate to incubations, helium purging, and argon evaporation. These modifications were introduced to enhance the recovery of metabolites, in particular R,4dihydroxytamoxifen. When P450 3A4 was incubated with 4-hydroxytamoxifen, an 87% increase in R,4-dihydroxytamoxifen production was seen in incubations containing ascorbate (10 mM) (Table 1). At the low tamoxifen substrate concentration (18 µM), P450 2D6, P450 3A4, and P450 2C9 produced statistically significant amounts (as compared to control incubations) of a metabolite with the same retention time as R-hydroxytamoxifen (Figure 3A). Limiting amounts of authentic R-hydroxytamoxifen precluded precise quantification of product formed at the lower concentration. Thus, relative rather than absolute product formation is presented. Similarly, apparent R-hydroxytamoxifen production was seen when the high concentration (250 µM) of tamoxifen was incubated with P450 3A4, P450 2D6, P450 2B6, P450 2C19, and P450 3A5 (Figure 3B). Under these conditions, racemic 4-hydroxy-N-desmethyltamoxifen eluted as a pair of peaks approximately 5-6 min later than
Figure 3. R-Hydroxylation of tamoxifen by recombinant human P450 forms and liver microsomes. Bacterial membranes containing recombinant P450 [0.05 µM (A) and 0.2 µM (B)] coexpressed with reductase or liver microsomes were incubated with tamoxifen in the presence or absence of an NADPH generating system and metabolites were analyzed by HPLC. In all cases, incubations containing P450 3A and P450 2C forms were supplemented with membranes obtained from cells expressing hNPR alone. Control incubations were included in each assay and contained membranes from cells expressing hNPR alone (hNPR) and from cells transformed with the empty expression vector (pCW). Data represent the means ( SD of three determinations. Asterisks signify data significantly different to controls (Student’s t-test) at the following levels: *p < 0.05; **p < 0.01; and ***p < 0.005. (A) Incubations were carried out with 18 µM tamoxifen for 40 min, and metabolites were separated using the isocratic method. No apparent metabolite formation was noted in NADPH deficient controls. Results are expressed as peak height ratio relative to the internal standard. (B) Incubations were carried out with 250 µM tamoxifen for 120 min. Reactions with P450 3A4, P450 3A5, P450 2C9, P450 2C19, P450 2E1, P450 2A6, and P450 2B6 were supplemented with a 1:1 molar ratio of purified rabbit b5. Metabolites were separated using the gradient method. No apparent metabolite formation was noted in t ) 0 controls.
R-hydroxytamoxifen (Figure 4). A doublet of peaks that coeluted with authentic 4-hydroxy-N-desmethyltamoxifen was seen in incubations with P450 2D6 and to a lesser
R-Hydroxylation of Tamoxifen by Human P450s
Figure 4. Chromatographic separation of R-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen. Chromatogram showing the relative retention of R-hydroxytamoxifen (trace a), racemic 4-hydroxy-N-desmethyltamoxifen (trace b), and racemic-4-hydroxytamoxifen (trace c) achieved with the gradient method described in the Experimental Procedures.
extent with P450 3A4. This doublet was significantly increased in size in incubations carried out with P450 2D6 using either 4-hydroxytamoxifen or N-desmethyltamoxifen as a substrate (results not shown). No peaks were evident at the same retention times as either R-hydroxytamoxifen or 4-hydroxy-N-desmethyltamoxifen above background noise in extracts from incubations with tamoxifen lacking NADPH or substrate. The metabolism of tamoxifen (250 µM) by P450 1A1 produced a compound that had a retention time very close to that of R-hydroxytamoxifen. However, addition of authentic metabolite to a P450 1A1 incubation sample revealed that the unidentified compound was not trans(E)-R-hydroxytamoxifen. The retention time of the compound was slightly but reproducibly shorter than that of R-hydroxytamoxifen. No R,4-dihydroxytamoxifen formation was seen in the incubations using tamoxifen as substrate. At a concentration of 100 µM 4-hydroxytamoxifen, production of R,4-dihydroxytamoxifen by P450 2B6, P450 1B1 (all four allelic variants), P450 3A4, P450 1A1, P450 3A5, and P450 2D6 was observed (Figure 5). The val 432 variants of P450 1B1 showed significantly higher activity than the cognate leu 432 forms (RAVN > RALN, p < 0.0005; GSVN > GSLN, p < 0.00001; Students t-test). Incubations were performed with N-desmethyltamoxifen (22 µM) as the substrate. Incubations of N-desmethyltamoxifen with P450 2D6, P450 2B6, P450 1A1, P450 1B1, and P450 2C9 all produced a metabolite peak with a retention time within a few minutes prior to R-hydroxytamoxifen, which was absent from the controls.
Discussion The objective of the current investigation was to examine the enzymology of R-hydroxylation of tamoxifen and 4-hydroxytamoxifen in humans. These metabolites have been proposed to be intermediates along pathways
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Figure 5. Formation of R,4-dihydroxytamoxifen by recombinant human P450 forms. Incubations were carried out with 100 µM 4-hydroxytamoxifen for 120 min. Reactions with P450 3A4, P450 3A5, P450 2C9, P450 2C19, P450 2E1, P450 2A6, and P450 2B6 were supplemented with a 1:1 molar ratio of purified rabbit b5. Metabolites were analyzed using a gradient method for separation. Data are means ( SD for three independent determinations. No apparent metabolite formation was noted in t ) 0 controls. Data for the P450 1B1 GSLN variant were significantly different to t ) 0 controls (Student’s t-test) at the p < 0.01 level; all other P450s showing detectable activity were statistically different to t ) 0 controls at the p < 0.005 level or lower.
leading to DNA adducts that may be important in tamoxifen-induced carcinogenesis. Initially, an analytical method was refined to allow separation of multiple primary and secondary metabolites, and conditions were established to allow measurement of those metabolites sensitive to oxidation. Incorporation of ascorbate, helium purging, and evaporation under argon into the experimental workup markedly improved the recovery of the R,4-dihydroxy metabolite. This suggests that minimizing the presence of reactive oxygen species in the samples improves metabolite production or recovery. Ascorbate may afford particular protection against the effects of reactive oxygen species produced in P450 3A4 incubations by inefficient coupling of substrate, since it was less marked in incubations supplemented with additional hNPR (previously shown to enhance coupling in recombinant P450 3A4 incubations; Notley et al.2) and in liver microsomes. Previous studies (10, 28, 29) have shown that tamoxifen R-hydroxylation is catalyzed by P450 3A4. In the study by Kim et al. (28), R-hydroxytamoxifen could not be chromatographically resolved from 4-hydroxy-N-desmethyltamoxifen. HPLC-mass spectrometric analysis was used to show that, whereas P450 3A4 produced authentic R-hydroxytamoxifen, P450 2D6 generated 4-hydroxy-N-desmethyltamoxifen. Desta et al. (29) also showed that P450 3A4 was the principal catalyst of R-hydroxylation with some contribution from P450 3A5. By contrast, 4-hydroxy-N-desmethyltamoxifen was formed from N-desmethyltamoxifen by P450 2D6 and from 4-hydroxytamoxifen by P450 2D6 and P450 3A4. The results obtained in the present work suggested that several other P450s, specifically P450 2D6, P450 2
Unpublished results.
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2B6, P450 2C9, P450 2C19, as well as P450 3A4 and P450 3A5, can produce relatively small amounts of a metabolite with the same retention time as R-hydroxytamoxifen at one or both substrate concentrations. Although the structure of this metabolite was not confirmed, our results are consistent with it being R-hydroxytamoxifen, since under the chromatographic conditions used (gradient method), synthetic 4-hydroxy-N-desmethyltamoxifen eluted substantially more slowly than authentic R-hydroxytamoxifen (Figure 4). Peaks with the same retention time as one or other peak in the doublet seen with authentic 4-hydroxy-N-desmethyltamoxifen were present following analysis in incubations of tamoxifen with P450 2D6 and P450 3A4 (to a lesser extent). In the present study, the higher tamoxifen concentrations and long incubation times could have potentiated secondary metabolite formation from tamoxifen. This hypothesis is supported by the putative 4-hydroxy-N-desmethyltamoxifen peaks seen following analysis of incubations of P450 2D6 with both 4-hydroxytamoxifen and N-desmethyltamoxifen. These data are consistent with the results of Desta et al. (29). It is unclear why R-hydroxytamoxifen production with P450s other than P450 3A4 was detectable in the current study but not in previous work. However, differences in assay sensitivity, enzyme source, accessory enzyme concentrations, or incubation time may contribute to this discrepancy. In the present work at 250 µM tamoxifen, P450 3A4 produced more than twice the amount of apparent R-hydroxytamoxifen production (0.52 ( 0.04 µM after 120 min, mean ( SD) as that generated by P450 2D6 (0.23 ( 0.02 µM after 120 min) or any other form examined. Amounts of apparent R-hydroxytamoxifen produced are comparable to those seen in other studies using substrate concentrations in the same overall range (10, 28). Limiting amounts of authentic R-hydroxytamoxifen precluded accurate quantification of product formation at the lower substrate concentration. However, relative R-hydroxytamoxifen production by the different P450 forms could be determined (quantified relative to peak height of the internal standard). At 18 µM tamoxifen, no R-hydroxylase activity was seen with P450 2C19 or P450 3A5, which suggests that these forms are low affinity catalysts. Conversely, P450 2C9 showed very low but statistically significant activity at the low but not at the high substrate concentration. The maximal activity of this form may have produced insufficient metabolite to be detected by the HPLC-UV method used for measuring metabolite formation at the high substrate concentration. Furthermore, we cannot exclude the possibility that the presence of cytochrome b5 in incubations performed at high substrate concentrations may have led to the difference in catalytic selectivity between the two substrate concentrations. However, experiments carried out at the higher concentration in the absence of b5 showed qualitatively similar results to those obtained when b5 was added, and supplementation with b5 led to only relatively modest (e50%) increases in the metabolism of other probe substrates by the various recombinant P450s (Notley and Gillam, unpublished results). Additional studies to determine the kinetic parameters for the R-hydroxylation of tamoxifen by these enzymes are required to explore further the in vivo relevance of these enzyme activities. However, it is notable that P450
Notley et al.
2D6 was the best catalyst of R-hydroxylation at the lower substrate concentration, which is closer to expected therapeutically relevant values. Whereas to our knowledge P450 2D6 has not been detected in the endometrium (31, 42), its expression in liver and other tissues is influenced by a genetic polymorphism, with poor metabolizers (representing ∼8% of Caucasian populations) being predisposed to, or protected from, the effects of a drug or its metabolite. From the present results, it might be anticipated that poor metabolizers would be protected from the effects of tamoxifen that are mediated through R-hydroxylation. However, P450 2D6 was also shown to catalyze tamoxifen 4-hydroxylation, 4′-hydroxylation, and N-demethylation (27). Both 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen are more potent ligands at the estrogen receptor than tamoxifen (43). Thus, poor metabolizers may experience less therapeutic effects (29) but may also be protected from the proliferative effects of these metabolites on the endometrium. The net effect of P450 2D6 genotype may depend on the relative proportions of each metabolite formed at pharmacologically relevant concentrations. P450 1A1 produced trace amounts of a metabolite that coeluted with authentic R-hydroxytamoxifen at the lower substrate concentration, but these failed to reach statistical significance relative to the controls. Extracts from incubations with P450 1A1 at the high tamoxifen concentration contained a metabolite that chromatographically ran very close to but was clearly resolved from authentic trans-R-hydroxytamoxifen. Although the transR-hydroxytamoxifen standard used in this study elutes as one peak using the gradient method, it is known that R-hydroxytamoxifen exists as isomers (30). As the cis and trans isomers of 4-hydroxytamoxifen could be separated using our gradient HPLC method (Figure 2), it is feasible that the unidentified compound generated by P450 1A1 is the cis isomer of R-hydroxytamoxifen. Boocock et al. (30) reported that the cis isomer is more polar than the trans isomer, which is consistent with the relative retention times observed. Under the isocratic HPLC conditions, where some isomerization would be expected to occur, authentic trans-R-hydroxytamoxifen eluted as a single peak, suggesting the results obtained at the low substrate conditions may reflect the sum of the two isomers. This is consistent with P450 1A1 preferentially forming the cis metabolite at both tamoxifen concentrations, with product formation at or below the limit of sensitivity at the lower concentration. Further studies are required to determine the stereospecificity of tamoxifen R-hydroxylation by P450 1A1 and other P450s, but the peak tentatively attributed to cis-R-hydroxytamoxifen was also seen following incubation of 250 µM tamoxifen with P450 3A4 and P450 3A5 as part of a doublet, the second peak of which coeluted with authentic trans-Rhydroxytamoxifen in the gradient method. The latter was clearly dominant in P450 3A4 extracts. Studies suggest that this metabolite is more readily sulfonated than the cis isomer (44) yet the cis-sulfate, once formed, may generate more adducts due to its longer half-life in water (8). Some isomerization is known to occur during sulfonation and adduct formation (45). Additionally, cis adducts may be more mutagenic (11, 12) and cis and trans epimers of the major DNA adduct differ in their susceptibility to excision repair (46). Thus, it will be of considerable interest to determine whether this unidentified compound is an isomer of R-OH tamoxifen,
R-Hydroxylation of Tamoxifen by Human P450s
as well as to establish which isomer is preferentially produced by the other P450 forms. P450 2B6, P450 1B1 (all four allelic variants), P450 3A4, P450 1A1, P450 3A5, and P450 2D6 were all found to generate R,4-dihydroxytamoxifen from 4-hydroxytamoxifen. Some of these P450s demonstrated pronounced differences in selectivity toward 4-hydroxy metabolite and parent drug. At high substrate concentrations, P450 3A4, P450 2D6, P450 2B6, and P450 3A5 were all capable of R-hydroxylating both tamoxifen and 4-hydroxytamoxifen. However, P450 2C19 and P450 2C9 failed to show activity toward R-hydroxylation of 4-hydroxytamoxifen. By contrast, P450 1B1 R-hydroxylated 4-hydroxytamoxifen but not tamoxifen. Moreover, significant differences were seen between allelic variants of P450 1B1, in that leu 432 forms showed significantly lower activity as compared to val 432 variants. This allelic variation has been associated with differences in activity toward other P450 1B1 substrates (37, 47). We attempted to demonstrate the formation of R-hydroxy-N-desmethyltamoxifen from N-desmethyltamoxifen, but because of a lack an authentic standard, the suspected peak on the chromatogram could not be positively assigned. This compound would be expected to elute a few minutes earlier than R-hydroxytamoxifen [as seen by Desta et al. (29)], and chromatograms from the analysis of incubations of N-desmethyltamoxifen with P450 2D6, P450 2B6, P450 1A1, P450 1B1, and P450 2C9 all had peaks in this region. By contrast, Desta et al. showed P450 3A4 to be a major catalyst of the R-hydroxylation of N-desmethyltamoxifen, with P450 2D6, P450 2C9, and P450 2C19 playing a minor role. Previous studies have shown that R-hydroxy-N-desmethyltamoxifen may be involved in DNA adduct formation (4). Adducts similar to those derived from adduct formation at the R-position of tamoxifen, but lacking a methyl group, have been characterized in rats and rat hepatocytes treated with N-desmethyltamoxifen (4, 22, 48). Further studies are required to determine the enzymology underlying the formation of R-hydroxy-N-desmethyltamoxifen. N-Desmethyl- and 4-hydroxytamoxifen have been detected in patient plasma at concentrations approximately twice and half, respectively, of those of tamoxifen (49). While metabolite concentrations within the uterus may differ from those in plasma, both metabolites may be delivered by the circulation at sufficient concentrations to serve as substrates for secondary metabolite production (50). Although the concentration of in vitro reactive metabolite formation was found to be relatively low, if the production occurs within the tissue of interest, little metabolite may be required to exert genotoxic effects. Of the P450 forms shown in the present work to be capable of producing R-hydroxytamoxifen and R,4-dihydroxytamoxifen, mRNA transcripts of P450 3A4, P450 3A5, P450 2B6, P450 2C9, P450 1A1, and P450 1B1 have all been detected in uterine tissue (31, 42, 51), although the concentrations of expressed protein are yet to be determined. Thus, it is plausible that production of these metabolites takes place in the endometrium where DNA adduct formation may then occur, but the quantitative importance of any such metabolism remains to be established. The significance of P450-mediated bioactivation of tamoxifen or its primary metabolites in the endometrium will depend on both the concentrations and the kinetic properties of the enzymes concerned. Further
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studies using human tissue will be required to confirm whether bioactivation occurs to a significant extent and at pharmacologically relevant concentrations.
Acknowledgment. Grateful thanks are extended to Drs. I. N. H. White, P. Jank, J. L. Bolton, D. H. Phillips, and D. A. Flockhart for donating metabolic standards used in this study and Drs. J. J. DeVoss, D. H. Phillips, and B. K. Park for helpful discussions.
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