Major inter-species differences in the rates of O-sulphonation and O-glucuronylation of {alpha}-hydroxytamoxifen in vitro: a metabolic disparity protecting human liver from the formation of tamoxifen–DNA adducts

David J. Boocock1, James L. Maggs1, Karen Brown2,3, Ian N.H. White2 and B.Kevin Park1,4

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


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Tamoxifen is a hepatic genotoxin in rats and mice but a hepatocarcinogen only in rats. It is not associated with DNA adducts and liver tumours in patients. The proposed major pathway for its bioactivation in rats involves {alpha}-hydroxylation, O-sulphonation and generation of a carbocation that reacts with DNA. Rat liver microsomes catalyse {alpha}-hydroxylation at ~2- and 4-fold the rate achieved by human and murine liver microsomes, respectively. O-glucuronylation will deactivate {alpha}-hydroxytamoxifen and compete with sulphonation. Rates of O-sulphonation of {alpha}-hydroxytamoxifen in hepatic cytosol have been determined by a HPLC assay of substrate-dependent 3'-phosphoadenosine 5'-phosphate production. The rank order of O-glucuronylation in hepatic microsomes was estimated by HPLC–mass spectrometry. The rate of sulphonation of trans-{alpha}-hydroxytamoxifen (25 µM) in cytosol from adult female Sprague–Dawley rats and CD1 mice was 5.3 ± 0.8 and 3.9 ± 0.5 pmol/min/mg protein (mean ± SD, n = 3), respectively. In cytosol fractions from women aged 40–65 years, the rate was 1.1 ± 0.4 pmol/min/mg protein (mean ± SD, n = 6). The Km for trans-{alpha}-hydroxytamoxifen in rat, mouse and human cytosol was 84.6 ± 3.8, 81.4 ± 4.6 and 104.3 ± 5.6 µM (mean ± SD, n = 3), respectively; the corresponding Vmax values were 22.4 ± 3.4, 17.1 ± 3.1 and 6.3 ± 1.9 pmol/min/mg protein. These Km were similar to a value obtained by others using purified rat liver hydroxysteroid sulphotransferase a. Turnover of the cis epimer was too slow for accurate determination of rates. Sulphonation of trans-{alpha}-hydroxytamoxifen in human uterine cytosol was undetectable. The rank order of O-glucuronylation of trans-{alpha}-hydroxy- tamoxifen in liver microsomes was human > > mouse > rat. In combination, lower rates of {alpha}-hydroxylation and O-sulphonation and a higher rate of O-glucuronylation in human liver would protect patients from the formation of tamoxifen–DNA adducts.

Abbreviations: CV, coefficient of variation; LC-MS, liquid chromatography–mass spectrometry; PAP, 3'-phosphoadenosine 5'-phosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulphate; SULT, sulphotransferase; tamoxifen, trans-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene; UDPGA, uridine 5'-diphosphoglucuronic acid.


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 Abstract
 Introduction
 Materials and methods
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Trans-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene (tamoxifen; Figure 1Go) is an important adjunct therapeutic agent for breast cancer (1). It is also given prophylactically to healthy women at great risk of developing the disease (2,3). Both therapeutic and chemopreventive regimens are associated with an increased risk of endometrial cancer (2,4). Although uterine tumours are not found in the rat (5), tamoxifen is a hepatocarcinogen for this species (6,7). It is genotoxic in the rat liver and isolated hepatocytes, forming persistent DNA adducts in vivo (6,811). Tamoxifen forms similar adducts in murine hepatocytes (8,12) but they are produced at lower concentrations and are less persistent in vivo (12) and, perhaps as a consequence, the drug is not hepatocarcinogenic in mice (12). While DNA binding has not been detected in liver biopsies from patients (13), the occurrence of adducts in certain leukocyte (14) and endometrial (15) samples, though such adducts are not always present (16,17), suggests that tamoxifen can be genotoxic in humans.



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Fig. 1. Proposed pathways of formation, activation (O-sulphonation) and inactivation (O-glucuronylation) of {alpha}-hydroxytamoxifen in rat, mouse and human liver.

 
Metabolic activation is a prerequisite for the production of DNA adducts from tamoxifen (18). The pathway generally considered to predominate in vivo involves {alpha}-hydroxylation (8,1921) and O-sulphonation to {alpha}-sulphate tamoxifen (22,23), leading ultimately to the generation of an electrophilic carbocation (19,24) capable of reacting with DNA (Figure 1Go). Desmethyltamoxifen, the principal metabolite of the drug in humans (21), appears to undergo an analogous bioactivation in rat liver (11). While trans-{alpha}-hydroxytamoxifen does react directly with isolated DNA, the synthetic O-sulphonate gives a 180-fold greater yield of adducts (22). When {alpha}-sulphate trans-tamoxifen is incubated with DNA, it produces mutagenic {alpha}-(N2-deoxyguanosinyl)tamoxifen adducts identical to those found in the hepatic DNA of rats treated with tamoxifen (22,25).

The involvement of sulphonation in the bioactivation of tamoxifen was established by Davis et al. (23), who found that formation of DNA adducts from tamoxifen and {alpha}- hydroxytamoxifen in isolated hepatocytes was directly proportional to the concentration of inorganic sulphate in the medium. Evidence that {alpha}-hydroxytamoxifen is a substrate of rat and human hepatic sulphotransferase (SULT) was obtained by demonstrating an enhancement of adduct formation catalysed by purified and recombinant enzymes (2628). Rat hydroxysteroid SULTa was approximately three times more active than human SULT2A1 (28). Using the same DNA-binding assay, trans-{alpha}-hydroxytamoxifen was a better substrate for both rat and human enzymes than its epimer (27,28). Bacteria and a mammalian cell line expressing SULTa but none of those expressing a human SULT metabolized trans-{alpha}-hydroxytamoxifen to a mutagenic and DNA adduct-forming species (26,29).

The rate of sulphonation has been considered to be the principal determinant of DNA adduct formation from {alpha}-hydroxytamoxifen in the liver (30). However, it may not be the primary cause of the rat's particular vulnerability to DNA modification by tamoxifen because there are marked differences between the rates of {alpha}-hydroxylation in rodent and human hepatic microsomes: in the rank order rat > > mouse > human (31). A similar rank order (rat > mouse > > human) is obtained with hepatocytes; such that only the rodent hepatocytes generate adducts from tamoxifen itself (8).

The activation of {alpha}-hydroxytamoxifen to a DNA-binding intermediate by rat and human SULT has been characterized in some detail (2628). However, the implied inter-species difference in the rate of the sulphonation reaction has yet to be confirmed by direct, enzymological measurements using subcellular fractions. Nor has the intermediate level of binding in mouse hepatocytes been associated with hepatic SULT activity. During the present investigations, we have determined the kinetic parameters of the O-sulphonation of {alpha}-hydroxytamoxifen in hepatic cytosol. We have also confirmed an earlier observation (32) that the disparity in genotoxicity could be regulated additionally by the rate of {alpha}-hydroxytamoxifen deactivation via O-glucuronylation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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Chemicals
The sodium salts of estrone ß-glucuronide, estriol 3-glucuronide, estriol 17ß-glucuronide and estriol 16{alpha}-glucuronide and trisodium uridine 5'-diphosphoglucuronic acid (UDPGA), sodium 3'-phosphoadenosine 5'-phosphate (PAP), lithium 3'-phosphoadenosine 5'-phosphosulphate (PAPS) (manufacturer's assay, 83%), saccharolactone (D-saccharic acid 1,4-lactone), 20 cetyl ether (Brij 58) surfactant and Glucurase (beef liver ß-glucuronidase, 5x103 U/ml) were purchased from Sigma Chemical Co. (Poole, UK). [Glucuronyl-14C(U)]UDPGA (272.5 mCi/mmol) was obtained from NEN Life Science Products (Boston, MA). The cis and trans isomers of {alpha}-hydroxytamoxifen were prepared by Dr J.E.Brown (Pharmaceutical Chemistry, University of Bradford, Bradford, UK) according to a published method (33). They were chromatographically homogeneous: eluted at 0.9 ml/min from a Columbus 5-µm C8 column (250x0.46 mm i.d.; Phenomenex, Macclesfield, UK) with a gradient of acetonitrile (20–40% over 10 min and 40–85% over 3 min) in 0.1 M ammonium acetate, pH 6.9, they had Rt of 11.9 and 12.4 min, respectively. HPLC grade solvents were obtained from Fisher Scientific (Loughborough, UK). All other chemicals were purchased from BDH Chemicals (Poole, UK).

Purification of PAPS
PAP, a contaminant of commercial PAPS, is an inhibitor of SULTs (34). The PAP content of the product on receipt, as determined by a spectrophotometric HPLC assay (35), was 23%. The PAPS was purified by adapting the analytical HPLC method of Pennings and van Kempen (36), which employs a neutral eluent and thereby minimizes hydrolysis of PAPS subsequent to elution. Aliquots (100 µl) of an aqueous solution (2 ml, 5 mM) were eluted from an Ultracarb 5-µm C8 column (250x0.46 mm i.d.; Phenomenex) at room temperature with water:methanol (80:20 v/v) containing 30 mM potassium dihydrogen phosphate and 3 mM tetrabutylammomium perchlorate, pH 7.0. The flow rate was 1.0 ml/min. Volumes of eluate containing PAPS (detected at 260 nm) were collected in a vessel cooled with solid carbon dioxide and concentrated ~5-fold at 40°C under a stream of nitrogen. This solution was assayed for PAPS and PAP by HPLC (36) and diluted slightly with distilled water to obtain concentrations of 250 and 2.25 µM (0.9% PAPS), respectively. It was concentrated a further 2-fold when required.

Human livers and uterine samples
Histologically normal livers were obtained from six female Caucasian transplant donors (aged 40–65 years). The certified cause of death was in every case severe injuries arising from a road traffic accident. The livers were removed and transferred to the laboratory within 30 min of death. They were portioned, frozen in liquid nitrogen and stored at –80°C. Approval was granted by the relevant ethical committees and prior consent was obtained from the donors' relatives. Normal uterine tissue was obtained from a 42-year-old Caucasian subject undergoing hysterectomy for dismenorrhoea. It was frozen in liquid nitrogen and stored at –80°C.

Preparation of cytosol and microsomes
Samples (10 g) of the stored human livers were homogenized individually in 2 vol ice-cold 67 mM potassium phosphate buffer (pH 7.4) containing 0.15 M potassium chloride. Equal samples of the six human livers were homogenized together to obtain the preparation used for determination of enzymatic kinetic constants. The livers removed from either four female Sprague–Dawley rats (250 g body wt; Charles River UK, Margate, UK) or four female CD1 mice (40 g; Biomedical Services Unit, University of Liverpool, Liverpool, UK) immediately after the animals had been killed by cervical dislocation were combined and homogenized. The homogenates were centrifuged at 10 000 g for 20 min and the resulting supernatants at 105 000 g for 60 min. The supernatant was decanted and retained as the cytosol fraction. The microsomal pellets were resuspended in phosphate buffer, sedimented (105 000 g for 60 min) and resuspended in chloride-free phosphate buffer. Cytosol was prepared from a sample (3 g) of human uterine tissue by the method used to obtain hepatic cytosol. All of the subcellular fractions were stored at –80°C for no more than 12 weeks before use. Protein in the fractions was assayed by the method of Lowry et al. (37).

Sulphotransferase assay
Enzymatic sulphonation of {alpha}-hydroxytamoxifen was determined by an assay of substrate-dependent PAP formation, which is suitable for reactions yielding chemically unstable sulphates (35). The HPLC method of Pennings and van Kempen (36) was used. Substrate (cis- or trans-{alpha}-hydroxytamoxifen, final concentration 2.5–400 µM, in the standard assay 25 µM) in acetone (5 µl) was incubated in triplicate with either hepatic or uterine cytosol (final protein concentration 3 mg/ml), PAPS (60 µl, final concentration 1.0–200 µM, in the standard assay 100 µM) and 2-mercaptoethanol (8.3 mM) in a final volume of 150 µl made up with 0.25 M potassium phosphate buffer (pH 7.0). Reactions were performed in capped 1.5 ml microcentrifuge tubes (Eppendorf, Cambridge, UK). All preparations were carried out on ice. Reactions were initiated by addition of purified PAPS (60 µl of a 250 µM aqueous solution), incubated for 20 min at 37°C and terminated with 150 µl of ice-cold methanol. Substrate was omitted from control incubations. The precipitated protein was immediately sedimented by centrifugation at 2000 g for 5 min at 4°C. The supernatant was transferred to a glass vial in ice. Aliquots (50 µl) were injected onto the HPLC column within 6 h. Authentic PAPS was stable under these conditions: a 250 µM aqueous solution underwent <0.8% conversion to PAP over 18 h at 0°C. Chromatographic analyses were conducted at ambient temperature using a Kontron 325 system (Kontron Instruments, Milan, Italy) linked to an Ultracarb 5-µm C18 column (250x4.6 mm i.d.) protected by a C18 guard column. The eluent was water:methanol (80:20 v/v) containing 30 mM potassium dihydrogen phosphate and 3 mM tetrabutylammonium perchlorate (pH 7.0). The flow rate was 1.0 ml/min. PAP (Rt 8.9 min; PAPS Rt 15.0 min) was detected at 260 nm with a Kontron Model 332 detector. Chromatographic peak areas were determined using a Kontron Data System 450-PCIP operating v.4.0 software. A linear calibration graph (r2 >= 0.99) was constructed for each group of analytical samples using dilutions of a solution of PAP in distilled water (final concentration 0–50 µM). The limit of determination was taken to be 0.08 µM at a signal-to-noise ratio of 3:1. At the typical assay concentration of 1 µM, the within-assay coefficient of variation (CV) was 8.6 ± 4.2% (n = 6). The substrate-dependent formation of PAP was calculated by subtracting the amount found in incubations from which the substrate had been omitted.

Glucuronyltransferase assay
Substrate (cis- or trans-{alpha}-hydroxytamoxifen, final concentration 0–750 µM, in standard assay trans-{alpha}-hydroxytamoxifen at 66 µM) in acetone (5 µl) was incubated in triplicate with hepatic microsomal preparation (final protein concentration 2.0 mg/ml), UDPGA (2.4 mM), MgCl2 (20 mM) and 67 mM sodium phosphate buffer (pH 7.4) in a final volume of 250 µl in capped 1.5 ml microcentrifuge tubes in a shaking water bath at 37°C for 45 min. Some of the incubations also contained either Brij 58 surfactant (0.24 mg/ml) or saccharo-lactone (5 mM). Either the substrate or UDPGA was omitted from control incubations. Reactions were initiated by addition of UDPGA (20 µl of 30 mM aqueous solution). They were terminated by adding ice-cold methanol (150 µl). Estrone ß-glucuronide (2 µl of a 10 mM aqueous solution) was added immediately afterwards as an internal standard for estimation of the {alpha}-hydroxytamoxifen glucuronide. The final mixture was kept at 4°C for 2 h. Precipitated protein was sedimented by centrifugation and the supernatant analysed by liquid chromatography–mass spectrometry (LC-MS) within 2 h. Aliquots (100 µl) were eluted from a Columbus 5-µm C8 column at room temperature with a gradient of acetonitrile (20–40% over 10 min and 40–85% over 3 min) in 0.1 M ammonium acetate (pH 6.9). The flow rate was 0.9 ml/min. The {alpha}-hydroxytamoxifen glucuronide (Rt 11.7 min) was monitored at m/z 564 ([M+H]+) and the estrone ß-glucuronide (Rt 10.1 min) at m/z 464 ([M+NH4]+). Selected ion chromatogram peak areas were determined using MassLynx 2.0 software (Micromass UK, Manchester, UK). Linear calibration graphs (r2 > 0.98) for the estimation of trans-{alpha}-hydroxytamoxifen glucuronide were constructed on each day of analysis using dilutions of an aqueous solution of estrone ß-glucuronide (final concentration 0–100 µM). Thereby, the amount of substrate glucuronide was expressed as nmol equivalents. The limit of determination of estrone ß-glucuronide was taken to be 0.1 µM (CV 8.7%). At 1 µM, the within-assay CV was 5.8% (n = 6). At a typical assay concentration of 10 µM equivalents of trans-{alpha}-hydroxytamoxifen glucuronide the CV was 6.4%.

Determination of kinetic constants
The sulphonation (in rat liver cytosol) and glucuronylation (in human liver microsomes) reactions were optimized for linear turnover of trans-{alpha}-hydroxytamoxifen (25 and 66 µM, respectively) in respect of incubation time and protein concentration. PAP formation was linear (r2 > 0.98) for at least 30 min at a protein concentration of 3 mg/ml and up to a protein concentration of 4 mg/ml over 30 min (r2 > 0.98). Glucuronide formation was linear (r2 > 0.98) for at least 60 min at a protein concentration of 2 mg/ml and up to a protein concentration of 4 mg/ml over 45 min (r2 > 0.98).

Apparent kinetic constants (Km and Vmax) were determined from plots of the initial rates (1/V) against 1/[S] generated by non-linear least squares analysis using GraFit 3.01 software (Erithacus Software, Staines, UK).

LC-MS
Positive ion electrospray mass spectra and selected ion monitoring data were acquired by LC-MS using a Quattro II tandem quadrupole instrument, fitted with an in-line source, as described previously (31). Cone voltage fragmentation of analytes was achieved at 70 V when the capillary voltage was 3.9 kV and the source temperature 70°C. Daughter spectra of protonated trans-{alpha}-hydroxytamoxifen glucuronide were acquired by LC-MS-MS on a Q-Tof time-of-flight instrument fitted with a Z-spray orthogonal interface (Micromass UK). For daughter scans, the source and desolvation temperatures were 140 and 350°C, respectively, the cone voltage 35 V, the collision energy 20 eV, the collision gas argon at 0.5x10–3 mBar and the MS2 scanning range m/z 60–600.

Stability of {alpha}-hydroxytamoxifen glucuronide
The hydrolytic stability of trans-{alpha}-hydroxytamoxifen glucuronide formed by human liver microsomes was determined by incubating the conjugate with 67 mM potassium phosphate buffer (pH 7.4). Glucuronide in the supernatants of 10 0.5 ml microsomal incubations was concentrated on a Sep-Pak Plus C18 cartridge (Millipore Corp., Milford, MS) pre-conditioned with methanol and water and eluted with methanol. Effluent concentrated by evaporation under nitrogen was chromatographed on a Columbus column as described in respect of the glucuronyltransferase assay. Fractions containing the glucuronide were collected, combined, concentrated by evaporation of the acetonitrile under a stream of nitrogen and diluted to 5 ml with phosphate buffer. This solution was then incubated at 37°C. Aliquots (100 µl) were removed at intervals over 72 h and assayed for {alpha}-hydroxytamoxifen by LC-MS (31).

Aliquots (150 µl) of supernatant from the incubations containing human liver microsomes were concentrated under a stream of nitrogen to remove methanol and incubated with beef liver ß-glucuronidase (100 U) in 0.5 ml of 0.1 M sodium acetate buffer (pH 5.0) at 37°C for 6 h. Saccharolactone (4 mM) was included in some of the mixtures to inhibit the ß-glucuronidase. The enzymic incubations (100 µl) were analysed by LC-MS.


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Sulphonation of {alpha}-hydroxytamoxifen
Trans-{alpha}-hydroxytamoxifen was a substrate of the SULT(s) in rat, mouse and human hepatic cytosol (Table IGo). However, the rate of sulphonation of the cis form was invariably too slow to be measured accurately; at a substrate concentration of 25 µM the signal-to-noise ratio for PAP was 1:3. The initial rates of reaction could be fitted, with r2 > 0.98, to a plot of 1/V against 1/[S] (Figure 2Go) from which apparent kinetic constants were determined (Table IIGo). Rat and mouse cytosol catalysed much more rapid sulphonation than human but the apparent Km for trans-{alpha}-hydroxytamoxifen was essentially unaffected by the source of the enzyme. The reaction was saturable in respect of the PAPS concentration; 100 µM was used. Under standard assay conditions, the apparent Km for PAPS in rat liver cytosol was 79 µM.


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Table I. Sulphotransferase (cytosolic) and glucuronyltransferase (microsomal) activities of hepatic subcellular fractions toward {alpha}-hydroxytamoxifen
 


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Fig. 2. Determination of apparent kinetic constants from initial rates for the O-sulphonation of trans-{alpha}-hydroxytamoxifen by pooled liver cytosol from (a) female Sprague–Dawley rats (n = 4) and (b) female humans (n = 6). Product formation was assayed by measuring substrate-dependent generation of PAP. Incubations were performed at a protein concentration of 3 mg/ml and temperature of 37°C for 20 min. Each point is the mean for triplicate incubations. The lines of best fit (r2 > 0.98) were calculated by least squares regression.

 

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Table II. Apparent kinetic constants for the O-sulphonation of {alpha}-hydroxytamoxifen by human, rat and mouse hepatic cytosol
 
The cytosol prepared from a sample of human uterine tissue was devoid of measurable activity against trans-{alpha}-hydroxytamoxifen.

Glucuronylation of {alpha}-hydroxytamoxifen
From LC-MS analyses, trans-{alpha}-hydroxytamoxifen was found to be metabolized to a single glucuronide (Rt 11.7 min) by human, mouse and rat liver microsomes in the presence of UDPGA (Figure 3Go). It co-chromatographed with the glucuronide excreted in bile by female rats administered trans-{alpha}-hydroxytamoxifen i.v. (31). The conjugate formed in human microsomes yielded unchanged substrate, identified by co-elution with the authentic trans epimer and resolution from the more polar cis epimer, when it was hydrolysed by ß-glucuronidase. Hydrolysis was inhibited by saccharolactone. At a cone voltage of 70 V the protonated glucuronide (m/z 564) fragmented principally by loss of the equivalent of glucuronic acid (194 a.m.u.) to give m/z 370; interpreted as the consequence of scission of the {alpha}-carbon–oxygen bond (Figure 4Go). The product of the more familiar loss of dehydroglucuronic acid (176 a.m.u.), represented by m/z 388, was always less abundant. Poon et al. (38) obtained this distinctive loss of 194 a.m.u. in the spectrum of a urinary metabolite of tamoxifen and, consequently, inferred the structure of {alpha}-hydroxydesmethyltamoxifen O-glucuronide. Ions derived from fragmentation of the aglycone dimethylaminoethyl side chain were also obtained, namely m/z 325 ([370-NH(CH3)2]+) and m/z 72 ([CH2CH2N(CH3)2]+). The collision-induced daughter spectrum of m/z 564 consisted of the fragments at m/z 370 (base peak) and m/z 325 (5% relative intensity).



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Fig. 3. Selected ion mass chromatograms (m/z 564, [M+H]+) representing the formation by human liver microsomes of an O-glucuronide from trans-{alpha}-hydroxytamoxifen: (a) complete incubation; (b) control incubation (– substrate). Peaks i and ii are of endogenous origin.

 


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Fig. 4. Electrospray mass spectrum of the {alpha}-hydroxytamoxifen glucuronide formed by human liver microsomes. Fragmentation was achieved at a cone voltage of 70 V.

 
In the absence of an authentic conjugate, as in the present case, glucuronylation can be assayed by the incorporation of radiolabelled glucuronic acid (39). However, not even the much more extensive turnover of trans-{alpha}-hydroxytamoxifen in human microsomes was sufficient to incorporate a detectable quantity of radiolabel from 2 µCi of [14C]UDPGA over 2 h when the substrate and cofactor concentrations were 400 and 600 µM, respectively. Moreover, rather than being enhanced by Brij 58 surfactant, an established activator of microsomal glucuronyltransferase (40), the glucuronylation of {alpha}-hydroxytamoxifen was less extensive at a surfactant:protein (w/w) ratio of 0.12 (data not shown). Therefore {alpha}-hydroxytamoxifen glucuronide formed metabolically was measured by LC-MS as the area of its peak in the mass chromatogram for m/z 564, which was expressed as molar equivalents of estrone ß-glucuronide using a calibration graph for the authentic conjugate's corresponding m/z 464 ion. This should be regarded only as a means of deriving a quantitative ranking of relative rather than absolute glucuronyltransferase activities in vitro. A unitary molar equivalence cannot be presumed because analyte response factors under LC-MS conditions can be highly sensitive to chemical structure (41). Thus the response factors (units of ion current peak area/nmol) of estriol 3-glucuronide, estriol 16{alpha}-glucuronide and estriol 17ß-glucuronide under the conditions used during the present work were 170 ± 34, 452 ± 51 and 478 ± 23 (mean ± SD, n = 6), respectively.

Human liver microsomes glucuronylated trans-{alpha}-hydroxytamoxifen much more rapidly than either rat or mouse microsomes (Table IGo). The rates achieved with the rat microsomes were too slow to enable accurate determinations but they were estimated to be >=50-fold lower. The initial rates of the reaction in human microsomes could be fitted, with r2 > 0.99, to a plot of 1/V against 1/[S] (Figure 5Go) from which apparent kinetic constants were calculated: Km and Vmax values were 75 µM and 1.3 nmol equivalents/min/mg protein, respectively. Co-incubation with the ß-glucuronidase inhibitor saccharolactone had no effect on apparent enzyme activity.



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Fig. 5. Determination of apparent kinetic constants from initial rates for the O-glucuronylation of trans-{alpha}-hydroxytamoxifen by pooled human liver microsomes (n = 6). The glucuronide was measured by LC-MS and selected ion peak areas expressed as molar equivalents with respect to authentic estrone ß-glucuronide. Incubations were performed at a protein concentration of 2 mg/ml and temperature of 37°C for 45 min. Each point is the mean for triplicate incubations. The line of best fit (r2 > 0.98) was calculated by least squares regression.

 
The glucuronide isolated from incubations of trans-{alpha}- hydroxytamoxifen with human liver microsomes was essentially stable in an aqueous solution, pH 7.4, for up to 72 h, i.e. it was resistant to spontaneous hydrolysis: after 2 and 72 h, respectively, only 3.7 ± 1.4% and 5.5 ± 1.9% (mean ± SD, n = 6) of the conjugate was hydrolysed to {alpha}-hydroxytamoxifen.


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The ultimate reactive metabolite of tamoxifen is believed to be a carbocation (19,20,24) derived from a microsomal metabolite (31,42), {alpha}-hydroxytamoxifen (Figure 1Go). Since the {alpha}-hydroxyl can be eliminated as water, with formation of the carbocation (19), {alpha}-hydroxytamoxifen reacts slowly with isolated DNA (22,43,44). Nevertheless, the greater binding in rat as against human isolated hepatocytes (8) indicates that at least one additional enzymatic reaction is required to produce most of the genotoxic metabolite generated by the former. The dependence of binding in rat hepatocytes on exogenous sulphate (23) and the greatly enhanced reactivity of synthetic {alpha}-sulphate tamoxifen, which produces adducts identical to those formed in vivo (22), implicates the sulphate as the principal source of this metabolite.

The potential of SULTs to regulate DNA binding of {alpha}-hydroxytamoxifen was confirmed by the greater activity of rat hydroxysteroid SULTa against an equivalent human enzyme (28). However, the assay employed depends upon SULT-mediated binding rather than measurement of a product of the enzymatic reaction and thereby will be influenced by competing reactions and especially the rapid hydrolysis of {alpha}-sulphate tamoxifen (22,24). Indeed, synthetic {alpha}-sulphate cis-tamoxifen is thought to give a (9-fold) greater yield of adducts than its trans isomer in vitro because it has a longer half-life in aqueous solution (22). Notwithstanding this consideration, the apparent Km values for SULT activity in rat, mouse and human cytosol, namely 84.6, 81.4 and 104.3 µM, respectively, were similar to a value (72 ± 29 µM) obtained using purified rat liver hydroxysteroid SULTa and the DNA-binding assay (27). Hypothetically, any assay is compromised by the fact that the relatively long-lived carbocation derived from {alpha}-sulphate tamoxifen undergoes isomerization to yield, after hydration, a mixture of cis- and trans-{alpha}-hydroxytamoxifen (24), which could result in a recycling of the alcohol (Figure 1Go). Conversion of trans- to cis-{alpha}-hydroxytamoxifen is catalysed by rat liver SULT (27). Formation of the cis epimer might effect DNA binding because it is a poorer substrate for rat and human liver SULT (27,28); binding is approximately one-tenth that achieved with trans-{alpha}-hydroxytamoxifen. The presently reported stereoselectivity of sulphonation in hepatic cytosol conforms with these earlier findings. It also provides an explanation for an estimate that trans-{alpha}-hydroxytamoxifen administered to female rats produces ~50-fold higher levels of hepatic DNA adducts than the cis isomer (42).

The {alpha}-hydroxytamoxifen SULT activities were determined with rigorously purified PAPS (0.9% PAPS as PAP) because the commercial product contains substantial, if variable, amounts of PAP, a competitive inhibitor (34). Nevertheless, the values of Km and Vmax were not significantly different when a relatively pure batch of this product, containing only 8% PAPS as PAP, was used as cofactor (data not shown). Matsui et al. (45), using PAPS which was from 80 to >98% pure, also found that hydroxysteroid SULT activities are not critically dependent upon the purity of the PAPS.

The proposition that the species-dependent formation of DNA adducts from {alpha}-hydroxytamoxifen might be determined principally by O-sulphonation (23,30) is supported by the 3-fold greater specific activity of rat hepatic hydroxysteroid SULTa versus human hydroxysteroid SULT2A1 (28). The data reported here establish for the first time that the rank order of formation of hepatic tamoxifen–DNA adducts in vivo, namely rat > mouse > > human (9,12,13), conforms with that of the total cytosolic SULT activity against {alpha}-hydroxytamoxifen (rat >= mouse > humans). In particular, we have shown that the intermediate, strain-dependent level of DNA binding in murine liver (approximately one-tenth of that in rats after 4 days) (12) can be related, by rank order if not proportionately, to SULT activity. Nevertheless, the fate of tamoxifen and its adducts in mice reveals that binding is not solely an expression of the rate of bioactivation via {alpha}-hydroxylation and O-sulphonation. A faster, non-activating clearance of tamoxifen from mouse liver and the rapid removal of the DNA adducts (12) will tend to counterbalance the relatively high SULT activity in murine liver.

The partial elimination of tamoxifen and {alpha}-hydroxy- tamoxifen as {alpha}-hydroxytamoxifen O-glucuronide in rat bile (31) introduces the possibility that DNA adduction might also be regulated by an alternative pathway for {alpha}-hydroxytamoxifen. Glucuronylation, as a detoxification reaction, can be regarded as one of three processes, namely the rendering of either a parent compound or a pro-reactive metabolite incapable of (further) activation (46) and deactivation of a reactive intermediate (47). Since {alpha}-hydroxytamoxifen combines spontaneously with DNA, glucuronylation would not only prevent further bioactivation through O-sulphonation but also deactivate the metabolite. The present findings indicate that the inter-species variation in DNA binding might arise partly from an opposite ranking of the extent to which {alpha}-hydroxytamoxifen is glucuronylated. This could be important if bioactivation in human liver is limited significantly by the low activity of hydroxysteroid SULT toward {alpha}-hydroxytamoxifen (26,28), in which case the intrinsic reactivity of the alcohol (22,43,44) may represent the principal pathway to the DNA-binding carbocation. A similar deduction applies to the human endometrium: measurable DNA binding occurs in vivo (15) notwithstanding that O-sulphonation of {alpha}-hydroxytamoxifen by uterine cytosol was below the limit of detection. Glucuronylation would also break the recycling of {alpha}-hydroxytamoxifen (Figure 1Go) via either hydrolysis of its sulphonate or hydration of its carbocation (19,24).

It remains the case that a higher rate of tamoxifen {alpha}-hydroxylation in rodent hepatocytes (8,31) might be the principal as well as the primary metabolic determinant of their greater DNA adduction: {alpha}-hydroxytamoxifen is metabolized to a reactive intermediate in rat, mouse and human hepatocytes in vitro but only the rodent hepatocytes form a DNA-binding product from tamoxifen (8). However, if the variance in O-glucuronylation operates in the intact cell, it will reinforce human hepatocyte protection from reactive tamoxifen metabolites afforded by an intrinsic deficiency of bioactivation and make the rat even more susceptible to adduct formation. It is important to note that prolonged exposure to tamoxifen might alter the balance of activation and deactivation: in female rats, tamoxifen increases the expression of certain CYP450 and at least one hepatic glucuronyltransferase without affecting the level of hydroxysteroid SULT mRNA (48).

The current model of tamoxifen hepatic genotoxicity (19,49) identifies two reactions, {alpha}-hydroxylation and O-sulphonation, as potentially crucial determinants of bioactivation. To these may be added O-glucuronylation. From this mechanistic understanding, the present and previous (31) studies predict low levels of DNA adduction in human liver and, by implication, a low risk of liver tumours in patients. When the standard techniques for measuring hepatic DNA damage in rodents were applied to a small sample of patients, no tamoxifen-specific adducts could be detected (13).


    Notes
 
3 Present address: Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94551-9900, USA Back

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


    Acknowledgments
 
We thank Dr A.G.Attey (Micromass UK) for LC-MS-MS analyses. The LC-MS system was purchased and maintained with grants from the Wellcome Trust. D.J.B. was supported by a studentship from the University of Liverpool and the MRC Toxicology Unit, Leicester. B.K.P. is a Wellcome Principal Fellow.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 1, 1999; revised June 26, 2000; accepted June 30, 2000.