{alpha}-Hydroxytamoxifen, a genotoxic metabolite of tamoxifen in the rat: identification and quantification in vivo and in vitro

David J. Boocock, James L. Maggs, Ian N.H. White1 and B. Kevin Park2

Department of Pharmacology and Therapeutics, University of Liverpool, Ashton Street, Liverpool L69 3GE and
1 MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The metabolic formation of {alpha}-hydroxytamoxifen, a reactive metabolite of tamoxifen in rat liver, was characterized and quantified in vitro (hepatic microsomal incubations) and in vivo (bile-duct cannulated animals). This minor metabolite was identified by chromatographic and mass spectral comparisons with the authentic compound. The rates of formation of {alpha}-hydroxytamoxifen in incubations (30 min) of tamoxifen (25 µM) with liver microsomal preparations from women (pool of six), female CD1 mice or female Sprague–Dawley rats, as quantified by liquid chromatography–mass spectrometry (LC–MS), were 1.15 ± 0.03, 0.30 ± 0.05 and 2.70 ± 0.35 pmol/min/mg protein, respectively. Selective inhibition of microsomal P450 indicated that {alpha}-hydroxylation was catalysed predominantly by CYP3A in humans. Bile-duct cannulated and anaesthetized female rats and mice given [14C]tamoxifen (43 µmol/kg, i.v.) excreted, respectively, 24 and 21% of the administered radioactivity in bile over 5 and 3.5 h. The major radiolabelled biliary metabolite in rats, characterized by LC–MS after enzymic hydrolysis of conjugates, was the glucuronide of 4-hydroxytamoxifen (10% of dose) and only 0.1% of the dose was recovered as {alpha}-hydroxytamoxifen. After administration of {alpha}-hydroxytamoxifen (43 µmol/kg, i.v.) to rats, only 1.19% of the administered compound was recovered from a glucuronide metabolite in bile, indicating a possible 0.84% {alpha}-hydroxylation of tamoxifen in vivo. There was, however, no indication of the presence in bile of either O-sulphonate or glutathione conjugates derived from {alpha}-hydroxytamoxifen. This study shows for the first time that {alpha}-hydroxytamoxifen can be glucuronylated in rat liver. Whereas sulphonation results in electrophilic genotoxic intermediates, glucuronidation may represent a means of detoxifying {alpha}-hydroxytamoxifen.

Abbreviations: CV, coefficient of variation; DMSO, dimethyl sulphoxide; LC–MS, liquid chromatography–mass spectrometry; SIM, selected ion monitoring; tamoxifen, (Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene (tamoxifen) is the most widely used drug for the treatment of breast cancer (1) and is under investigation as a chemopreventive in healthy women considered to be at high risk of developing the disease (2). It has a low acute toxicity in humans (1) but has been associated with uterine cancer in patients (3,4) and in mice exposed to the compound neonatally (5). As a matter of particular concern, tamoxifen is a potent hepatocarcinogen in rats (6), though not in mice (6), subjected to either short-term or extended dosing regimens (7,8).

Tamoxifen acts as a genotoxin in rat liver (9). Numerous and relatively persistent hepatic DNA adducts are detected following repeated administration of the drug (10,11), and an identical pattern of damage is found in cultured rat hepatocytes (12). Adducts are also formed in mouse liver though they are present at lower concentrations and are less persistent (10,13). The major adduct isolated from rat liver is {alpha}-(N2-deoxyguanosinyl)tamoxifen, which has a strong miscoding potential (14). Studies employing human and rat hepatic microsomes (15,16) and hepatocytes (17) have demonstrated that metabolic activation is a prerequisite for the generation of tamoxifen–DNA adducts. The reactive intermediate generated during the oxidative metabolism of tamoxifen by microsomal P450 binds irreversibly to protein and DNA (16,18). Tamoxifen is metabolized variously via primary N-demethylation, 4-hydroxylation, {alpha}-hydroxylation and N-oxidation (Figure 1Go) by hepatic microsomes and cell lines (1923), and additionally via secondary O-glucuronidation in hepatocytes (24) and in vivo (2529), N-demethylation being the principal biotransformation in humans and human liver microsomes (20,23,28). Both aromatic hydroxylation and {alpha}-hydroxylation have been consistently implicated in the formation of reactive tamoxifen metabolites in vitro and in experimental animals, and they appear to represent the initial steps of distinct bioactivation pathways in vivo (16,30). Although {alpha}-hydroxytamoxifen reacts directly, if slowly, with isolated DNA (31), both it and the chemically unreactive 4-hydroxytamoxifen are regarded as precursors of DNA-binding derivatives that have yet to be identified as metabolites, namely {alpha}-esters such as the highly reactive {alpha}-sulphate tamoxifen (31,32) and 4-hydroxytamoxifen quinone methide (33), respectively. The balance of the published evidence suggests that {alpha}-hydroxytamoxifen is the precursor of the principal DNA adducts formed in rat liver (30,31,34). This would explain why toremifene (ß-chlorotamoxifen) does not give rise to such DNA adducts (11,35). The proposed {alpha}-hydroxylation–sulphonation pathway of bioactivation (30) is represented in Figure 2Go (transfer of a sulphonyl group to a hydroxyl function is termed O-sulphonation in this paper).



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Fig. 1. The primary routes of metabolism of tamoxifen in vivo (derived from refs 25–28).

 


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Fig. 2. Hypothetical scheme for the metabolic activation of tamoxifen to a genotoxic intermediate via {alpha}-hydroxylation and O-sulphonation.

 
{alpha}-Hydroxytamoxifen is a metabolite of tamoxifen in human plasma (22), in rat (12), mouse (12) and human (12,22) hepatocytes, in human liver homogenate (22) and in rat liver microsomes (36). Only the Z-isomer of tamoxifen undergoes {alpha}-hydroxylation by rat liver microsomes (36). {alpha}-Hydroxytamoxifen N-oxide is a metabolite of the N-oxide in rat liver microsomes (19,21) and N-desmethyl-{alpha}-hydroxytamoxifen has been identified in human plasma (22), as an O-glucuronide in human urine (28) and in incubations of rat liver microsomes (21), but their contributions to adduct formation are unknown. The production of {alpha}-hydroxytamoxifen by hepatocytes was found to be in the rank order rat > mouse >> human, i.e. the rank order of tamoxifen–DNA adduct formation in the cells (12) and in vivo (10,37). However, in none of the other in vitro studies was the metabolite quantified and in none of the studies was the rate of {alpha}-hydroxylation assayed. None of the {alpha}-hydroxylated metabolites formed in vivo have been quantified.

The objectives of the present study were, firstly, to determine the rate of tamoxifen {alpha}-hydroxylation in human, mouse and rat hepatic microsomes and to identify, at least preliminarily, the isoform(s) of P450 responsible for this reaction in human microsomes. Secondly, to identify and quantify {alpha}-hydroxytamoxifen as a biliary metabolite of tamoxifen in rats; the biliary route of excretion being predominant for tamoxifen in rats (25) and humans (26). Thirdly, to determine the biliary recovery in rats of exogenous {alpha}-hydroxytamoxifen, which is known to undergo activation to a DNA-binding product in vivo (34).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Chemicals
Tamoxifen, quinidine hydrochloride, NADPH, bovine serum albumin, Glucurase (beef liver ß-glucuronidase in acetate buffer; 5000 U/ml) and sulphohydrolase (Type V; ß-glucuronidase activity < 2 U/mg) were purchased from Sigma (Poole, UK). Ketoconazole was obtained from Janssen (Beerse, Belgium), furafylline from Parke Davis (Ann Arbor, MI), and sulphaphenazole from Ciba-Geigy (Basel, Switzerland). Ring-labelled [14C]tamoxifen (2.03 GBq/mmol; radiochemical purity of re-purified Z-isomer, 98%), [ring-14C]toremifene (4.44 GBq/mmol; radiochemical purity, 98%), E/Z-4-hydroxytamoxifen, unlabelled toremifene, N-desmethyltamoxifen and tamoxifen N-oxide were provided by Dr L.L.Smith (MRC Toxicology Unit, Leicester, UK). Z-{alpha}-hydroxytamoxifen was provided by Dr J.E.Brown (Pharmaceutical Chemistry, University of Bradford, UK). HPLC-grade solvents were products of Fisher Scientific (Loughborough, UK). All other chemicals were purchased from BDH Chemicals (Poole, UK).

Human liver samples
Histologically normal livers were obtained from female Caucasian organ donors aged 40–50 years. Livers were removed and transferred to the laboratory within 30 min of death. Approval was granted by the relevant ethical committees, and prior consent was obtained from the donors' relatives. The livers were cut into 10–20 g portions, frozen in liquid nitrogen, and stored at –80°C until used.

Hepatic microsomal incubations
Thawed human liver samples (10 g), and livers removed from adult female rats and mice (eight mouse livers pooled for homogenization) immediately after the animals were killed by cervical dislocation, were minced in 2 vol ice-cold 67 mM potassium phosphate buffer (pH 7.4) containing 0.15 M KCl, and homogenized using a motor-driven homogenizer. The homogenates were centrifuged at 10 000 g for 20 min, and the resulting supernatants at 105 000 g for 60 min. The microsomal pellets were resuspended in phosphate buffer and sedimented (60 min at 105 000 g). All centrifugations were performed at 4°C. The washed pellets were resuspended in chloride-free phosphate buffer and stored at –80°C for no more than 5 weeks before use. Microsomes from equal samples of six human livers were pooled to obtain the preparation used in the incubations. Protein was assayed by the method of Lowry et al. (38) using bovine serum albumin as the standard.

Tamoxifen (final concentration, 25 µM) or [14C]tamoxifen (25 µM, 4 µCi) as a methanol solution (10 µl) was incubated with microsomal preparation (1.5 mg protein), NADPH (0.1 mM), MgCl2 (20 µM) and sodium phosphate buffer (67 mM, pH 7.4; final volume, 1.5 ml) in 12 ml glass tubes within a shaking water bath at 37°C for 30 min. Substrate or NADPH was omitted from the control incubations. Inhibitors of P450 were dissolved in methanol. Incubations were performed in triplicate. All of the inhibition studies and the incubations performed for quantification of {alpha}-hydroxytamoxifen by liquid chromatography–mass spectrometry (LC–MS) made use of the unlabelled tamoxifen. Reactions were initiated by addition of the NADPH (150 µl of 1 mM solution) except that the mechanism-based inhibitor, furafylline, was pre-incubated with microsomes and NADPH for 10 min before addition of the substrate (39). Reactions were terminated by adding methyl tert-butyl ether (4 ml). Toremifene (10 µl of 40 µM methanol solution) was added as an internal standard for quantification of {alpha}-hydroxytamoxifen immediately after addition of the ether. The final mixture was subject to rotation-mixing for 15 min. The ether of two combined extracts was evaporated to dryness under nitrogen at 40°C, and the residue reconstituted in methanol (300 µl) for immediate analysis by either radiometric HPLC with LC–MS or quantitative LC–MS. The recovery of radioactivity from rat microsomal incubations extracted immediately and after 30 min was 81.2 ± 4.2% (mean ± SD, n = 4) and 78.3 ± 5.4%, respectively. The recovery of [14C]toremifene (0.2 µM, 0.5 µCi) from incubation mixtures on immediate extraction was 79.6 ± 3.2%.

Metabolism inhibition studies in vitro
Furafylline (final concentration, 10 µM), ketoconazole (2 µM), quinidine (10 µM) and sulphaphenazole (100 µM) were used as selective inhibitors of human microsomal CYP1A2, CYP3A4, CYP2D6 and CYP2C9, respectively (23,39). The inhibitors were also incubated with human liver microsomes and NADPH in the absence of tamoxifen to establish that they neither contained contaminants nor yielded during the incubation any compounds that co-eluted with {alpha}-hydroxytamoxifen and gave an isobaric ion in the LC–MS assay.

Animal experiments
Female Sprague–Dawley rats (250 g) and female CD1 mice (40 g), obtained from breeding colonies maintained by the Biomedical Services Unit, University of Liverpool, UK, were anaesthetized with urethane (1.0 ml/kg, 15.7 M isotonic solution, i.p.) and sodium pentobarbitone (70 mg/kg, i.p.), respectively. Cannulae were inserted into the jugular vein and common bile duct. Tamoxifen (43 µmol/kg) or [14C]tamoxifen (43 µmol/kg, 2 µCi), or {alpha}-hydroxytamoxifen (rats only; 43 µmol/kg), in dimethyl sulphoxide (DMSO; 100 µl) was given by i.v. injection over 2 min. Bile was collected for 30 min before the administration of the drug to obtain a `drug blank' sample for mass spectral comparisons, and thereafter either as a single 3.5 h fraction (mice) or as hourly fractions for 5 h (rats). It was stored at –80°C if not analysed immediately. After 5 h, urine was aspirated from the bladder. Radioactivity in the bile, urine and tissues was determined by liquid scintillation counting as described previously (40).

Enzymic hydrolysis of biliary conjugates
Bile (200 µl) from animals given either [14C]tamoxifen or unlabelled tamoxifen or {alpha}-hydroxytamoxifen was incubated with Glucurase (500 µl) in capped glass test tubes at 37°C for 16 h. Additional aliquots of bile were co-incubated with either Glucurase and the ß-glucuronidase inhibitor, saccharic acid 1,4-lactone (saccharolactone; 4 mM), or with type V sulphatase (20 U/ml). Toremifene (5 µl of 40 µM methanol solution) was added as internal standard when required for quantification of {alpha}-hydroxytamoxifen in the Glucurase incubations, and each incubation was extracted twice with methyl tert-butyl ether (4 ml). The combined ether extracts were evaporated to dryness under nitrogen, and reconstituted in methanol (150 µl) for either radiochromatographic analysis (100 µl aliquots) or quantification of {alpha}-hydroxytamoxifen by LC–MS (100 µl aliquots).

LC–MS
Positive-ion electrospray mass spectra and selected ion monitoring (SIM) data were acquired with a Quattro II instrument (Micromass, Manchester, UK). Eluate was delivered by two Jasco PU-980 pumps via an HG-980-30 solvent mixing module (Jasco UK, Great Dunmow, UK). Biliary metabolites and methanol solutions of standards and other analytes were eluted from an Ultracarb 5-µm C8 column (250x46 mm; Phenomenex, Macclesfield, UK) at room temperature with gradients of either acetonitrile or methanol in 0.1 M ammonium acetate, pH 6.9, as follows: bile, with acetonitrile at 10–30% over 20 min, 30% for 10 min, 30–40% over 10 min and 40–50% over 5 min; and methanol solutions of metabolites extracted from microsomal incubations and biliary hydrolysates, with methanol at 60–85% over 15 min (system I). For chromatographic identification of the {alpha}-hydroxytamoxifen recovered from enzymic hydrolysates of rat bile, two additional columns were employed with the latter gradient: system II, a Hichrom 5-µm CN column (250x4.6 mm; Hichrom, Reading, UK); and system III, a Radial Pak 4-µm phenyl cartridge column (100x8 mm; Waters Associates, Milford, MA) housed in a Z module. UV-absorbing analytes were monitored at 254 nm with a Jasco UV-975 spectrophotometer. The flow rate was 0.9 ml/min; the split-flow of effluent to the LC–MS interface was ~50 µl/min. The configuration of the LC–MS system and parallel radioactivity detector (Radiomatic A-250; Canberra-Packard, Pangbourne, UK) has been described previously (40). The temperature of the LC–MS interface was 60°C, the capillary voltage was 3.9x103 V, and the HV lens voltage and RF lens voltage were 0.5x103 and 0.1 V, respectively. Spectra were acquired between m/z 100–1050 over a scan time of 5 s. Analyte fragmentation was enhanced by increasing the cone voltage from 30 V. Daughter spectra (MS–MS) were acquired from m/z 50 at 1 scan/5 s using argon (~7x10–4 mBar) as collision gas. Data were processed via MassLynx software (Micromass). Column effluent was mixed (1:1, v/v) with Flo-Scint A (Canberra-Packard) for detection of radioactivity.

Quantification of {alpha}-hydroxytamoxifen by LC–MS
Calibration graphs for {alpha}-hydroxytamoxifen were constructed on each day of analysis using dilutions of a freshly prepared methanol solution (0.5–40 µM) containing toremifene (40 µM) as internal standard. Aliquots (100 µl) were eluted from an Ultracarb 5-µm C8 column with methanol (60–85% over 15 min) in 0.1 M ammonium acetate, pH 6.9, at 0.9 ml/min. The mass spectrometer monitored five channels for protonated analytes ([M + 1]+), viz. m/z 358 (desmethyltamoxifen), m/z 372 (tamoxifen), m/z 388 ({alpha}-hydroxytamoxifen; Rt 18 min), m/z 404 (dihydroxytamoxifen) and m/z 406 (toremifene; 21 min), with a dwell time of 0.2 s and an inter-channel delay of 0.02 s. Several control microsomal incubations (Figure 3Go) and biliary hydrolysates were analysed for any interfering (isobaric) endogenous material. Peak areas for {alpha}-hydroxytamoxifen and toremifene were computed with MassLynx integration software. Calibration graphs were linear (r2 > 0.98) over the range 0.5–40 µM. At 0.5 µM and 40 µM, the intra-assay coefficients of variation (CV) were 9.2% (n = 8) and 8.3% (n = 8), respectively. The limit of determination (CV >= 15%) was taken to be 0.2 µM (CV = 15.3%). Analytical samples contained {alpha}-hydroxytamoxifen at concentrations between 0.52 and 34.6 µM. The recovery of authentic {alpha}-hydroxytamoxifen (final concentration, 25 µM) added to rat liver microsomes (2 mg protein) in the presence of NADPH and extracted with methyl tert-butyl ether immediately was 81.2 ± 4.8% (mean, n = 6). After 30 min at 37°C, the recovery was 78.6 ± 3.2%; the corresponding figure for the pooled human liver microsomes was 79.3 ± 6.2% (mean, n = 6). The immediate recovery from rat liver microsomes of a lower concentration of {alpha}-hydroxytamoxifen (0.25 µM) in the absence of NADPH was 76.9 ± 4.7% (n = 6). The recovery of {alpha}-hydroxytamoxifen (final concentration, 25 µM) added to a mixture of Glucurase (500 µl) and `drug blank' rat bile (200 µl) was 76.9 ± 5.4% (n = 6) on immediate extraction and 73.8 ± 6.2% after incubation at 37°C for 16 h.



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Fig. 3. HPLC separation (methanol–ammonium acetate eluent) with electrospray SIM detection of {alpha}-hydroxytamoxifen ([M + H]+, m/z 388) and the internal standard, toremifene (m/z 406). (A) Authentic standards of (I) {alpha}-hydroxytamoxifen (25 µM) and (II) toremifene (50 µM). (B) {alpha}-Hydroxytamoxifen and endogenous isobaric material from (I) rat liver microsomes incubated with tamoxifen and NADPH, (II) rat liver microsomes incubated with NADPH in the absence of tamoxifen, (III) human liver microsomes incubated with tamoxifen and NADPH and (IV) human liver microsomes incubated with NADPH in the absence of tamoxifen. *, {alpha}-hydroxytamoxifen.

 

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[14C]tamoxifen metabolism by hepatic microsomes
Radiochromatographic analysis of the metabolites recovered from incubations of [14C]tamoxifen with rat and human liver microsomes (Figure 4Go) resolved two peaks, namely 4-hydroxytamoxifen (Rt 22.6 min) and N-desmethyltamoxifen (Rt 20.1 min), which were identified by co-elution with synthetic standards. The 4-hydroxytamoxifen represented 28.1 ± 2.3% (mean ± SD, n = 4) and 14.3 ± 1.9% of the chromatographed radioactivity, respectively. The N-desmethyltamoxifen represented 4.9 ± 1.4% (mean ± SD, n = 4) and 5.2 ± 0.7%, respectively.



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Fig. 4. HPLC separation, with parallel radiometric and electrospray SIM detection, of the metabolites recovered from incubations of [14C]tamoxifen with rat (A) and human (B) liver microsomes in the presence of NADPH. The SIM chromatograms represent [M + H]+ for unchanged tamoxifen (m/z 372) and monohydroxytamoxifen (m/z 388). *, {alpha}-hydroxytamoxifen; (1), N-desmethyltamoxifen; (2), 4-hydroxytamoxifen; (3), tamoxifen.

 
{alpha}-Hydroxylation of tamoxifen by hepatic microsomes
The rate of tamoxifen {alpha}-hydroxylation by human, mouse and rat hepatic microsomes was linear for at least 30 min of incubation at a substrate concentration of 25 µM and at microsomal protein concentrations between 0.2 and 2.4 mg/ml (data not shown). All subsequent incubations were performed at a protein concentration of 1 mg/ml. Authentic {alpha}-hydroxytamoxifen at 1% of the substrate concentration was efficiently (76.9 ± 4.7%, n = 6) recovered from the rat microsomal incubation mixture by solvent extraction. Conversion of tamoxifen to {alpha}-hydroxytamoxifen over a 30 min incubation period represented only 0.092 ± 0.010% (mean ± SD, n = 6), 0.024 ± 0.001% and 0.216 ± 0.011% of the substrate, respectively. The corresponding activities were 1.15 ± 0.03, 0.30 ± 0.05 and 2.70 ± 0.35 pmol/min/mg protein.

Inhibition of {alpha}-hydroxylation in human microsomes
Incubations with the various inhibitors of P450 showed that only ketoconazole at 2 µM gave any appreciable inhibition of {alpha}-hydroxylation (49.6 ± 6.3%, n = 6).

Identification of {alpha}-hydroxytamoxifen in rat bile
Radiochromatograms of bile samples (24.0 ± 4.3%, n = 4, radioactive dose excreted over 5 h) from female Spague–Dawley rats treated with tamoxifen (43 µmol/kg, i.v.) showed several metabolites were being excreted. These metabolites were mainly in the form of glucuronides: analyses of the bile using LC–MS with SIM detection revealed the presence in most instances of corresponding peaks in the ion-current chromatograms ([M + 1]+) for glucuronides of either monohydroxytamoxifen (m/z 564), dihydroxytamoxifen (m/z 580) or methoxymonohydroxytamoxifen (m/z 594); the last was presumed to be the conjugate of a methylated catechol. LC–MS analysis of the radiolabelled aglycones recovered from enzymic hydrolysates of whole bile (Figure 5Go) resolved three peaks in the ion-current chromatogram ([M+1]+) for monohydroxytamoxifen (m/z 388). The major radiolabelled monohydroxylated metabolite co-eluted with authentic 4-hydroxytamoxifen (Rt, 22.6 min), and is assumed to be found in bile as the glucuronide of Rt 29.5 min. The monohydroxylated metabolite of Rt 23 min was not assigned. The smallest peak in the ion-current chromatogram co-eluted with authentic {alpha}-hydroxytamoxifen on three distinct HPLC systems: I (Ultracarb; Figure 5Go), Rt 18.3 min (Rt 4-hydroxytamoxifen, 22.6 min); II (CN), Rt 12.1 min (10.2 min); III (phenyl), Rt 16.1 min (19.3 min). LC–MS analyses of the material recovered from `drug blank' bile following incubation of the bile with ß-glucuronidase established that none of the endogenous biliary compounds, nor any compounds formed during the period of incubation, yielded an isobaric ion, i.e. a protonated molecule or ammonium adduct of m/z 388, which co-chromatographed with authentic {alpha}-hydroxytamoxifen. It was also established that detection of toremifene, the internal standard used for quantifying {alpha}-hydroxytamoxifen in bile, was not subject to interference from isobaric ions. The chromatographic identification of {alpha}-hydroxytamoxifen in bile was corroborated by daughter-scan analyses of the monohydroxylated aglycone metabolites resolved by HPLC: the metabolite which co-chromatographed with {alpha}-hydroxytamoxifen, in common with the standard compound and all tamoxifen metabolites possessing an intact dimethylaminoethyl side chain (28), yielded a daughter ion at m/z 72 corresponding to [Me2NCH2CH2]+. Attempts were made by enzymic hydrolysis and HPLC to isolate {alpha}-hydroxytamoxifen from 1.5 ml of bile for further (direct probe) mass spectral analysis, but the quantity of metabolite recovered was insufficient. Since no unconjugated {alpha}-hydroxytamoxifen was found in bile by either radiometric or mass spectral analysis, it was presumed that the metabolite found in the enzymic hydrolysate derived overwhelmingly from a glucuronide. However, it was not possible to assign the putative {alpha}-hydroxytamoxifen glucuronide to a peak in the biliary radiochromatogram. Incubation of rat bile with sulphatase in the presence of the glucuronidase inhibitor, saccharolactone, did not liberate any {alpha}-hydroxytamoxifen or 4-hydroxytamoxifen detectable by SIM LC–MS. No substantive peaks were located in the mass chromatograms ([M + 1]+) corresponding to glutathione conjugates of either tamoxifen (m/z 677), tamoxifen epoxide (m/z 693) or 4-hydroxytamoxifen quinone methide (m/z 693). In addition, no glucuronide of a dihydrodiol (m/z 582) derived from an epoxide intermediate was found in bile.



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Fig. 5. HPLC separation, with parallel radiometric and electroscopy MS detection, of the aglycone metabolites of [14C]tamoxifen recovered from female rat bile following enzymic hydrolysis of conjugates. *, {alpha}-hydroxytamoxifen.

 
Quantification of {alpha}-hydroxytamoxifen in the bile of rats administered tamoxifen
The metabolite was quantified by LC–MS after hydrolysis of biliary conjugates with ß-glucuronidase (Table IGo). No {alpha}-hydroxytamoxifen was seen in the absence of ß-glucuronidase, in incubations containing saccharolactone and after incubation with sulphohydrolase. It was not detected in whole bile by either radiometric or LC–MS analysis. The combined {alpha}-hydroxytamoxifen recovered from the five 1 h bile collections (10.75 ± 2.9 nmol, n = 4) represented 0.10 ± 0.03% of the dose.


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Table I. The recovery of {alpha}-hydroxytamoxifen in the enzymic hydrolysate of bile from female Sprague–Dawley rats administered tamoxifena
 
Quantification of {alpha}-hydroxytamoxifen in the bile of mice administered tamoxifen
Samples of bile (0–3.5 h collections containing 21 ± 3.2% of the administered radiolabel) from female CD1 mice dosed with tamoxifen (43 µmol/kg, i.v.) were incubated as before with ß-glucuronidase, and the {alpha}-hydroxytamoxifen quantified by LC–MS. The metabolite recovered from mouse bile (0.256 ± 0.040 nmol, n = 4) equalled only ~3% of the {alpha}-hydroxytamoxifen recovered from rat bile collected over the same period, and the amounts available for quantification were close to the limit of determination of the assay.

Glucuronylation of {alpha}-hydroxytamoxifen in vivo
A comparison of bile from female Sprague–Dawley rats given {alpha}-hydroxytamoxifen (43 µmol/kg, i.v.) with `drug blank' bile revealed the presence of only one major peak in the ion-current chromatogram for hydroxytamoxifen glucuronide (Figure 6Go). At raised cone voltages (70 V), a fragment ion was obtained at m/z 370 (65%). This is attributed to [M + H – glucuronic acid]+ and is indicative of the alcohol glucuronide of {alpha}-hydroxytamoxifen (28).



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Fig. 6. HPLC separation, with electrospray MS detection, of (I) `drug blank' bile from female Sprague–Dawley rat (1–2 h collection) and (II) bile from rat administered {alpha}-hydroxytamoxifen; showing m/z 564, representing the glucuronide of {alpha}-hydroxytamoxifen.

 
Quantification of {alpha}-hydroxytamoxifen in the bile of rats administered {alpha}-hydroxytamoxifen
Bile samples from female Sprague–Dawley rats dosed with {alpha}-hydroxytamoxifen (43 µmol/kg, i.v.) were analysed by LC–MS for quantification of the unchanged compound. Only trace amounts were found in the five 1 h collections, collectively representing 0.05% of the dose. Hydrolysis of biliary conjugates with ß-glucuronidase before quantification resulted in the recovery of 2.14 ± 0.60% (mean ± SD, n = 4) of the dose (Table IIGo).


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Table II. The recovery of {alpha}-hydroxytamoxifen in the bile of female Sprague–Dawley rats administered {alpha}-hydroxytamoxifena
 

    Discussion
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 References
 
The major route of excretion of tamoxifen metabolites in the rat was found to be biliary, with 24% of the radiolabelled dose recovered over 5 h, while only negligible quantities of radiolabel were excreted in urine. This is in agreement with earlier studies that showed [14C]tamoxifen was eliminated largely in the faeces from i.p. administration (25). The metabolism of tamoxifen is complex, but the major hepatic metabolites, 4-hydroxytamoxifen and N-desmethyltamoxifen, are not reactive towards DNA. There is a substantial body of evidence that the bioactivation of tamoxifen responsible for DNA adduct formation in vitro and in rat liver is a result of oxidative metabolism by microsomal cytochrome P450 (8,15,41).

Several mechanisms have been proposed to explain the formation of DNA adducts from the metabolism of tamoxifen. {alpha}-Hydroxylation, epoxidation and generation of a quinone methide have all been presented as likely candidates (22,4244) but {alpha}-hydroxylation is considered to be the major pathway in rat liver (34). The key step in the mechanism of DNA adduct formation from {alpha}-hydroxytamoxifen is thought to involve the reversible O-sulphonation of the hydroxyl moiety generating a highly reactive carbocation (30,45). In support of this, experiments using 32P-post-labelling of DNA have shown that {alpha}-sulphate cis-tamoxifen is highly reactive to DNA, yielding up to 1600 times the number of DNA adducts obtained from {alpha}-hydroxytamoxifen (31). It has also been shown that the number of DNA adducts in the livers of rats treated with {alpha}-hydroxytamoxifen is >50 times that found in rats treated with the corresponding cis isomer (36).

The principal purpose of this study was to investigate the formation of {alpha}-hydroxytamoxifen in vivo by measurement of the excreted metabolite after administration of tamoxifen.

Recovery of a substantial fraction of the dose in bile enabled analysis of even minor metabolites by LC–MS. This showed that 4-hydroxytamoxifen glucuronide was the major metabolite (~10% of the dose) and only 0.1% of the dose was recovered as {alpha}-hydroxytamoxifen. Although the sulphonation of phenols is well documented (46,47), no evidence for a 4-sulphate of tamoxifen was obtained. It is important to note that we did not find either glutathione conjugates or dihydrodiols derived from the putative 1,2-epoxide of tamoxifen (44); nor did we find a conjugate of 4-hydroxytamoxifen, i.e. a product of the addition of glutathione to 4-hydroxytamoxifen's quinone methide (16,33). Furthermore, no glutathione conjugate of tamoxifen, such as might have been formed from the {alpha}-carbocation intermediate (43), was found in rat bile.

The ratio of {alpha}-hydroxytamoxifen:4-hydroxytamoxifen in microsomal incubations was much greater than in vivo, which suggested that the clearance of {alpha}-hydroxytamoxifen via conjugation and biliary excretion might be low. To examine this further, {alpha}-hydroxytamoxifen was given at the same high dose (43 µmol/kg) as tamoxifen. On analysis of the deconjugated biliary metabolites by LC–MS it was found that only ~2% of the {alpha}-hydroxytamoxifen was excreted compared with ~24% for tamoxifen in the form of its metabolites. Direct analysis of bile by LC–MS revealed a major peak of m/z 564, not present in the `drug blank' bile, indicating an intact glucuronide of a mono-hydroxylated tamoxifen derivative. The fragmentation pattern at higher cone voltages was consistent with the metabolite being the {alpha}-O-glucuronide. We were unable to detect any phase I metabolites of {alpha}-hydroxytamoxifen (e.g. hydroxy or desmethyl) in either free or conjugated form by means of SIM.

The low recovery of {alpha}-hydroxytamoxifen in vivo could be partly due to sulphonation followed by formation of DNA adducts (42). Interestingly, there was no evidence for a glutathione conjugate formed from either {alpha}-hydroxytamoxifen sulphate or the corresponding carbocation. This would be consistent with the ultimate reactive species being a hard electrophile, which would react preferentially with a nitrogen centre in DNA or protein rather than a sulphur centre such as the sulphydryl group of glutathione; notwithstanding, the hepatocellular concentration of glutathione is 10 mM (48,49).

The rate of {alpha}-hydroxylation, as measured using hepatic microsomes from humans, rats and mice, would appear to be at least a partial determinant of the marked species variation in tamoxifen's hepatocarcinogenicity: tamoxifen is carcinogenic in the rat but not in the mouse, and is not known to be hepatocarcinogenic in humans (6). In these studies the rate of formation of {alpha}-hydroxytamoxifen was in the rank order rat > human > mouse, with mouse microsomes catalysing an ~10-fold lower rate of {alpha}-hydroxylation and human an ~3-fold lower rate. However, the overall production of {alpha}-hydroxytamoxifen by isolated hepatocytes, in the rank order rat > mouse >> human, is at first sight closer to the apparent ranking of tamoxifen–DNA adduct formation in vivo (11,37), all be it on the basis of meagre data relating to adduct formation in human liver (37).

In addition to the rate of {alpha}-hydroxylation, a possible determinant of the risk of genotoxicity from tamoxifen therapy is the rate of sulphonation of {alpha}-hydroxytamoxifen. Indeed this may be the major metabolic factor that determines both species and individual (human) risk. Evidence to support this comes from studies using recombinant indicator cells (50): {alpha}-hydroxytamoxifen showed a pronounced mutagenic and bacteriotoxic effect on cells expressing rat hydroxysteroid sulphotransferase a, whilst only very weak or no mutagenic activity was observed when the human sulphotransferases were expressed in the bacteria. The pattern of DNA adducts observed in sulphotransferase proficient cells was indistinguishable from that formed by tamoxifen in the livers of rodents. It is therefore possible that the {alpha}-hydroxylation–sulphonation pathway, which can lead to the formation of hepatocarcinogenic metabolites in rats, is of minor relevance to humans. However, direct quantitative analysis of the relative rates of sulphonation and glucuronidation of {alpha}-hydroxytamoxifen by rat and human transferases is necessary to confirm this, and thus define precisely the metabolic risk factors that not only display species differences but may be subject to inter-individual variation in the target patient population.

Epidemiological studies suggest that in breast cancer patients taking tamoxifen there is an increase in the incidence of endometrial but not liver tumours (3,51). In the reproductive tract of these patients, the extent of DNA damage is either very low or absent (4). Thus although {alpha}-hydroxytamoxifen has been detected in the blood of women taking tamoxifen (22), it is presently not clear if this metabolite is able to interact with the DNA of cells in the reproductive tract. The balance between the glucuronylation (detoxication) and sulphonation (activation) pathways may play an important role in determining the extent of DNA lesions in target tissues.


    Notes
 
2 To whom correspondence should be addressed Email: bkpark{at}liv.ac.uk Back


    Acknowledgments
 
We are indebted to Dr L.L.Smith, MRC Toxicology Unit, University of Leicester, for his generous assistance and advice. We thank Miss S.Newby for her assistance with the animal experiments. We thank Drs J.E.Brown and K.Brown, Pharmaceutical Chemistry, University of Bradford, for the synthesis of {alpha}-hydroxytamoxifen. D.J.B. is in receipt of a studentship from the University of Liverpool and the MRC Toxicology Unit, Leicester. B.K.P. is a Wellcome Senior Fellow. The LC–MS system was purchased and maintained by means of grants from the Wellcome Trust.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 13, 1998; revised July 13, 1998; accepted September 29, 1998.