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Resolution of r-Hydroxytamoxifen; R-Isomer Forms More DNA Adducts in Rat Liver Cells Martin R. Osborne,* Alan Hewer, and David H. Phillips Section of Molecular Carcinogenesis, Haddow Laboratories, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Received February 6, 2001
The genotoxic tamoxifen metabolite R-hydroxytamoxifen has been resolved into R- and S-enantiomers. This was achieved by preparing its ester with S-camphanic acid, chromatographic separation into two diastereoisomers, and hydrolysis to give (+)- and (-)-R-hydroxytamoxifen. The configuration of the (-)-isomer was shown to be S- by degradation of an ester to a derivative of (-)-2-hydroxy-1-phenyl-1-propanone, which has already been shown to have S-configuration. Metabolism of tamoxifen by rat liver microsomes gave equal amounts of the two enantiomers. They have the same chemical properties but, on treatment of rat hepatocytes in culture, R-(+)-R-hydroxytamoxifen gave at least eight times as many DNA adducts as the S-(-)-isomer.
Introduction The anti-estrogenic drug tamoxifen1 has been shown to form DNA adducts in rat liver and to induce liver cancer in rats (1-3). Its genotoxic effects have been ascribed to the formation of R-hydroxytamoxifen (Scheme 1), which has been identified as a metabolite of tamoxifen in rat, mouse and human hepatocytes (4, 5), in human liver (6) and in rat, mouse, and human liver microsomes (7). It has been detected in the blood plasma of women undergoing treatment with tamoxifen (6). R-Hydroxytamoxifen reacts to a small extent with DNA without activation (8) and to a much greater extent following esterification. Modified nucleosides have been prepared from DNA treated with R-acetoxytamoxifen (8-10), tamoxifen R-sulfate (10), or R-acetoxy-N-desmethyltamoxifen (11), and characterized as deoxyguanosine adducts in which guanine is alkylated at the aminogroup. R-Hydroxytamoxifen has a chiral carbon atom and, therefore, contains two enantiomers, as shown in Scheme 1. It has not hitherto been established which enantiomer is formed by metabolism, which becomes covalently bound to DNA and which is responsible for the biological effects. In this paper, we report the isolation of (+)- and (-)-isomers of R-hydroxytamoxifen, the assignment of their absolute configuration, their formation by metabolism of tamoxifen in a rat liver preparation, and an assessment of their relative abilities to form covalent adducts in DNA.
Materials and Methods Tamoxifen, 4-hydroxytamoxifen (containing 2% cis-isomer), salmon testis DNA, propiophenone (1-phenyl-1-propanone), 1-phenyl-1,2-propandione and [1S]-(-)-camphanyl chloride (cam* To whom correspondence should be addressed. E-mail: martino@ icr.ac.uk. 1 Abbreviations: Tamoxifen, (Z)-1-{4-[2-(dimethylamino)ethoxy]phenyl}-1,2-diphenyl-1-butene; R-hydroxytamoxifen, (E)-4-{4-[2-(dimethylamino)ethoxy]phenyl}-3,4-diphenyl-3-buten-2-ol.
Scheme 1. Structure of r-Hydroxytamoxifen, R and S Stereoisomers
phanoyl chloride; 3-oxo-4,7,7-trimethyl-2-oxabicyclo[2.2.1]heptane1-carbonyl chloride), were from Sigma-Aldrich, Poole and Gillingham, U.K. R-Hydroxytamoxifen (12), N-desmethyltamoxifen, tamoxifen-N-oxide, and R-hydroxytamoxifen-N-oxide were synthesized and provided by I. R. Hardcastle (now at the University of Newcastle, U.K.). R-Acetoxytamoxifen (8) was prepared as described before. Ring-labeled 14C-tamoxifen (55 Ci/mol) was a gift from E. A. Martin, University of Leicester, U.K. Optical rotations were measured on a Perkin-Elmer 141 polarimeter with a sodium lamp. Mass spectroscopy was carried out using a Finnigan TSQ 700 triple-quadrupole mass spectrometer fitted with an electrospray ion source. Proton magnetic resonance spectra were obtained on a Bruker AC250 spectrometer. Liquid chromatography was carried out on Waters (Watford, U.K.) apparatus. The following systems were used. (a) Reversedphase: a “Jupiter” C18 or “Max RP” C12 column (4.6 × 250 mm; Phenomenex, Macclesfield, U.K.) eluted at 0.8 mL/min with a water-acetonitrile mixture, containing 0.05 M ammonium formate throughout, and varying acetonitrile concentrations as stated. (b) Normal phase: two Nucleosil silica columns (250 × 4.6 mm; Fisher, Loughborough, U.K.) connected in series and eluted with hexane-dichloromethane-methanol-triethylamine in the stated proportions. In each case, tamoxifen derivatives were detected in the eluate by their ultra-violet absorbance at 254 nm. 14C-Labeled compounds were detected by liquid scintillation counting of samples of the eluate in a Packard scintillation counter. Preparation of 2-Camphanyloxy-1-phenyl-1-propanone (C, Scheme 2). 2-Hydroxy-1-phenyl-1-propanone (E, Scheme 2) was prepared by incubation of 1-phenylpropan-1,2-dione with yeast and glucose as described (13). The product was extracted with ethyl acetate, and purified by chromatography on silica.
10.1021/tx010027b CCC: $20.00 © 2001 American Chemical Society Published on Web 06/20/2001
Isomers of R-Hydroxytamoxifen Scheme 2. Structures of (A) r-Hydroxytamoxifen; (B) Tamoxifen r-Camphanate; (C) 2-Camphanyloxy-1-phenyl-1-propanone; (D) 1-Phenylpropan-1,2-dione; (E) 2-Hydroxy-1-phenyl-1-propanone, S-Isomer Showna
Chem. Res. Toxicol., Vol. 14, No. 7, 2001 889 eluted with 40-70% MeCN in 0.05M ammonium formate in 30 min at 0.8 mL/min. Treatment of Rat Hepatocytes with r-Hydroxytamoxifen. Hepatocytes were isolated from female Fischer F-344 rats and maintained in culture as described before (4). R-Hydroxytamoxifen was dissolved in dimethyl sulfoxide (0.06 mL) and added to three flasks, each containing hepatocytes in 20 mL of medium, to a final concentration 1 µM. After 18 h at 37 °C, the cells from each flask were harvested, and their DNA extracted and analyzed for adducts as described before (8,15). Each sample was analyzed twice, giving six values for each compound.
Results
a (i), Camphanic acid chloride/pyridine; (ii), NaOEt/EtOH, water; (iii), KMnO4; (iv), yeast; (v), camphanic acid chloride/pyridine.
The specific rotation was estimated at -289° (RD; 3 g/L chloroform), of the same sign as found by Cheˆnevert and Thiboutot (-85°, ref 13) but of greater magnitude. 2-Hydroxy-1-phenyl-1-propanone (10 mg) was treated with 30 mg camphanyl chloride in 0.4 mL of dimethylformamide + 0.1 mL of pyridine at 24 °C overnight. Water was added and the mixture extracted with hexane. The extract (4 mL) was passed through a silica cartridge (Sep-Pak, Waters), followed by 20, 40, and 60% dichloromethane in hexane (2 mL each) whereupon the ester (ca. 11 mg) was eluted. Its UV absorption spectrum (λmax 245 nm) was like that of propiophenone. The positive ion mass spectrum showed peaks at m/z 133 (PhCOCHMe), 331 (MH), 348 (MNH4), 353 (MNa), and 683 (M2Na), consistent with the expected molecular weight 330. The proton magnetic resonance spectrum showed peaks at δ 8.0 (2, d, o-H), 7.7 (1, t, p-H), 7.6 (2, m, m-H), 6.3 (1, m, R-CH), 2.4 (2, m, CH2), 2.0 (2, m, CH2), 1.5 (3, d, β-CH3), 1.1 (3, s), and 1.0 (5 or 6, s) (camphor methyls; obscured by a contaminant). Reaction of r-Hydroxytamoxifen with DNA in Vitro. Reaction was carried out as described before (8). To DNA (1 mg) in 1 mL 0.12 M sodium cacodylate buffer, pH 6, was added 0.1 mg R-hydroxytamoxifen in 0.2 mL of ethanol. After 23 h at 37 °C, 0.1 mL of 2.5 M sodium acetate was added, and the mixture extracted five times with 0.6 mL of ether. The DNA was precipitated with ethanol, dried, and analyzed for adducts by the 32P-postlabeling procedure (8). Each sample was analyzed twice. Reaction of Tamoxifen r-Camphanate with DNA. Reaction was carried out as for R-acetoxytamoxifen (8). To DNA (5 mg) in 4 mL 0.01 M phosphate buffer, pH 7, was added 0.13 mg tamoxifen R-camphanate in 1 mL of ethanol. After 1 h at 37 °C, the mixture was extracted 10 times with 2 mL of ether and the DNA precipitated and analyzed as before. Metabolism of Tamoxifen by Rat Liver Microsomes. Microsomes were prepared from the livers (10 g) of three female Fischer F344 rats, by centrifugation (14). The resulting suspension (10 mL) contained 28 mg of protein/mL according to Lowry’s assay. It was stored at -70 °C in 1 mL aliquots. One microcurie [14C]tamoxifen in 5 µL of ethanol was added to 1 mL of suspension containing 0.1 mL of rat liver microsomal fraction, 20 mM KH2PO4 (pH 7.4), 5 mM MgSO4, and 2 mM NADPH, and kept at 37 °C for 2 h. The metabolites were extracted out with 3 × 0.7 mL of ethyl acetate, evaporated, dissolved in acetonitrile-water and analyzed on a C12 reverse phase column,
Preparation and Resolution of Tamoxifen r-Camphanate (B, Scheme 2). A mixture of 5 mg of R-hydroxytamoxifen, 30 mg of 1S-camphanyl chloride, 0.4 mL of dry dimethylformamide, and 0.1 mL of pyridine were mixed and incubated at 37 °C for 2 h. The ester was isolated by chromatography in multiple runs on a C18 column in 57% acetonitrile (retention time about 16 min). The product peaks were pooled, added to ether, dried over calcium chloride in three stages, and evaporated to dryness. This gave tamoxifen camphanate (mixed isomers) in 70% yield. This ester is unstable to hydrolysis, but the half-life (about 30 min in 3:2 water-ethanol, pH 7, 37 °C) was sufficient to allow easy handling. We saw some separation of tamoxifen camphanate by hplc on a single silica or C18 column, but not enough for preparative purposes. Better separation was achieved by two silica columns connected in series. The ester was dissolved in 4 mL of hexane-dichloromethane (2:1), injected in 0.1 mL aliquots onto the double silica column, and eluted with hexane-dichloromethane-methanol-triethylamine (100:100:10:0.05). The principal peaks eluted were at about 19 and 20 min: “B1” and “B2”. This gave 40 absorbance units (mL × absorbance at λmax, 278 nm; ca. 2 mg) of each isomer from 100 units of mixed ester. Each was at least 90% pure as shown by rechromatography. B1 and B2 gave identical positive ion mass spectra: peaks were obtained at m/z 325 (tam-2H-Me2N, 24%), 370 (tam-H, 100%), 371 (26%), 568 (MH, 12%) and 590 (MNa, 9%), consistent with the formula C36H41NO5 ) 567. Hydrolysis to Enantiomers of r-Hydroxytamoxifen. Hydrolysis of tamoxifen R-camphanate by usual methods failed to give optically active R-hydroxytamoxifen. In water at neutral pH, or in aqueous or aqueousethanolic sodium hydroxide, the ester gave a mixture of trans- and cis-hydroxytamoxifen (3:1), presumably with racemization at the R-carbon. This was unexpected, as R-acetoxytamoxifen gives only the trans-isomer on hydrolysis in alkali. Apparently, the camphanate anion is so good a leaving group that hydrolysis in a polar medium proceeds largely by dissociation to the carbocation, even in molar sodium hydroxide. (+)- and (-)- R-hydroxytamoxifen were obtained by hydrolysis in nonaqueous conditions. The ester (B1 or B2; 40 absorbance units) was dissolved in 0.8 mL of absolute ethanol, and treated with 0.2 mL of 0.5 M sodium ethoxide in ethanol for 2 h at 37 °C. The mixture was neutralized with ammonium formate solution, and separated on the C18 column eluted with 53% acetonitrile. This gave 20 absorbance units of each isomer of R-hydroxytamoxifen, about 0.8 mg based on the molar extinction coefficient of tamoxifen (about 10 000 at 270 nm).
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The optical rotation (RD; 0.5 g/L ethanol) of the alcohol derived from B1 was -330° and that from B2 +293°. Oxidation of Tamoxifen. To determine the absolute configuration of the above ester, we needed to split the molecule into simpler components. We first established a way in which this could be done, with the parent substance as a model. Tamoxifen (2 mg) in 0.5 mL of acetone was treated with 4 mg of potassium permanganate in 1 mL of 0.12 M sodium cacodylate, pH 6. After 20 min at 24 °C, the mixture was decolorized by adding sodium sulfite, and analyzed by HPLC on the C18 column. The most conspicuous oxidation product (retention time 14 min in 50% acetonitrile; 21 min on gradient, 30-60% acetonitrile in 30 min) co-chromatographed with propiophenone. The identification was confirmed by the UV spectrum (λmax 243 and 280 nm, absorbance ratio 100: 7; propiophenone has λmax 242 and 278 nm, ratio 100:8). The same product could also be obtained by treatment of tamoxifen successively with osmium tetroxide, mannitol, and sodium periodate. Oxidation of Tamoxifen Camphanate. Oxidation of tamoxifen esters at pH 6, as described for tamoxifen, did not result in appreciable splitting of the molecule, but this could be achieved in dilute acetic acid. One-tenth of a milligram of tamoxifen R-camphanate (mixed isomers; B, Scheme 2) was dissolved in 0.01 mL of acetic acid, and treated with 0.3 mg of potassium permanganate in 0.05 mL of water for 45 min at room temperature. The mixture was decolorized with 0.05 mL of 0.1 M sodium sulfite and neutralized with 0.1 mL of M sodium hydroxide and 0.2 mL of 0.2 M ammonium formate, then analyzed by HPLC on the C18 column, with a gradient of 30-60% acetonitrile over 30 min. The most conspicuous product appeared as a double peak at retention time about 34.0 and 34.6 min. This was identified as 2-camphanyloxy-1-phenyl-1-propanone (C, Scheme 2) by the similarity of its UV absorbance spectrum (λmax 245 nm, sh 280 nm, absorbance ratio 100:8) to that of propiophenone (above), and by co-chromatography with authentic ester prepared from 2-hydroxy-1-phenylpropanone. This identity was established in both hplc systems: on silica, eluted with hexane-dichloromethane-methanol-triethylamine (60:30:3:0.025; results shown in Figure 1) and on C18 (not shown). Isomer B1, derived from (-)-R-hydroxytamoxifen, gave the same isomer of 2-camphanyloxy-1phenyl-1-propanone (C) as prepared from [S]-(-)-2hydroxy-1-phenyl-1-propanone (E). Isomer B1 has therefore S-configuration at the R-carbon, and B2 R-configuration. Metabolism of Tamoxifen by Rat Liver Microsomes. [14C]Tamoxifen was incubated with rat liver microsomal fraction, and the products analyzed by reversed-phase chromatography as described in the Materials and Methods. The elution of 14C radioactivity is shown in Figure 2. Some of the peaks were tentatively identified by co-chromatography of a sample with unlabeled known metabolites, as shown in the legend to the figure. To determine the enantiomeric composition of R-hydroxytamoxifen produced by metabolism, the material of peak B (Figure 2) was dried and acylated with 5 mg camphanyl chloride in 5 µL of pyridine + 45 µL of dimethylformamide. The ester was purified by reversedphase chromatography and resolved on the double silica column as before, adding unlabeled racemic tamoxifen
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Figure 1. Assignment of configuration of 2-camphanyloxy-1phenyl-1-propanone by chromatography on silica; part of chromatogram shown. (A) Derived from (-)-R-hydroxytamoxifen; (B) derived from (+)-R-hydroxytamoxifen; (C) derived from racemic R-hydroxytamoxifen; Panels D, E, and F as A, B, and C, respectively, but spiked with 2-camphanyloxy-1-phenyl-1-propanone prepared from (S)-2-hydroxy-1-phenyl-1-propanone. This coeluted with the peaks numbered 2; peaks 1 were therefore derived from the (R)-isomer.
Figure 2. Elution of tamoxifen and its metabolites from a reversed-phase column. Most peaks corresponded to known metabolites: (A) R-hydroxytamoxifen N-oxide; (B) R-hydroxytamoxifen; (D) 4-hydroxytamoxifen; (E) 4-hydroxy-cis-tamoxifen; (F) tamoxifen-N-oxide and N-desmethyltamoxifen. The last peak was unchanged tamoxifen. Peaks C and G were not identified.
R-camphanate as a marker. The result is shown in Figure 3. There were similar amounts of tamoxifen R-camphanate A and B, derived from (-)- and (+)-R-hydroxytamoxifen, respectively; the proportion of A was estimated at 50 ( 1%. To ensure that the extraction and acylation process does not cause racemization, the whole process of metabolism, extraction, separation, acylation and resolution on silica was carried out in a parallel experiment with
Isomers of R-Hydroxytamoxifen
Chem. Res. Toxicol., Vol. 14, No. 7, 2001 891 Table 1. Level of Adducts in DNA Exposed to r-Hydroxytamoxifena R (+)
S (-)
racemic
at pH 6, by chemical reaction 0.24 0.35 0.32 (200 µM) in rat hepatocytes 5.9 ( 0.3 0.7 (0.2 3.7 ( 0.4 (1 µM) a The results are given as adducts per million DNA bases. Those for chemical reaction are the result of single incubations; those for hepatocytes from three incubations, with results expressed as mean ( standard deviation.
Figure 3. Elution from silica of tamoxifen R-camphanate, prepared from R-hydroxytamoxifen formed by metabolism. The ester is resolved into isomers A and B, derived respectively from (-)- and (+)-R-hydroxytamoxifen.
(-)-R-hydroxytamoxifen. Here, the recovered tamoxifen camphanate was at least 90% A-isomer. Bonding to DNA. When R-hydroxytamoxifen is incubated with DNA in aqueous solution, a small amount becomes covalently bound, and tamoxifen-deoxyguanosine adducts have been isolated from the DNA. The reaction is acid-catalyzed (8). (+), (-), and racemic R-hydroxytamoxifen were now incubated separately with DNA at pH 6, and the levels of reaction determined by the 32P-postlabeling procedure. The results are shown in Figure 4 (panels A, B, and C) and in Table 1. The separated enantiomers and racemic mixture each became bonded to DNA to approximately equal extents, and gave the same pattern of adducts. This was to be expected, in
view of the mechanism by which reaction is thought to occur. Protonation of the hydroxyl group is followed by loss of water to give a carbocation, which reacts with DNA. Each isomer gives the same amount of the same carbocation, and hence the same level of reaction. Bonding of R-hydroxytamoxifen to DNA in tissues takes place mainly through the sulfate ester. As a model for this ester, we treated DNA with the camphanate esters of R-(+)- and S-(-)-R-hydroxytamoxifen. The level of adducts induced by the R-isomer (22 mg/L) was 0.153 per thousand bases, and by the S-isomer (28 mg/L) 0.153/ thousand bases, again showing comparable reactivity of the two isomers. We then treated rat hepatocytes with the resolved enantiomers of R-hydroxytamoxifen (1 µM) and determined the level of adducts in their DNA by 32P-postlabeling as before. The pattern of adducts (Figure 4, panels D and E) was the same in each case and identical to that previously obtained with the racemic substance (Figure 4F) and shown to correspond mainly to deox-
Figure 4. Autoradiographs of polyethyleneimine-cellulose thin layer plates after chromatography of 32P-labeled tamoxifen-nucleoside bisphosphates. The origins are at the bottom left corner, and the directions of development: D1 downward, D2 upward, and D3 to the right. The adducts were derived from salmon sperm DNA treated with (A) (+), (B) (-), or (C) (() R-hydroxytamoxifen at pH 6; or from DNA from rat hepatocytes treated with (D) (+), (E) (-), or (F) (() R-hydroxytamoxifen.
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yguanylic acid adducts of tamoxifen and N-desmethyltamoxifen (8-10, 16-18). However, as shown in Table 1, (+)-R-hydroxytamoxifen (R-isomer) gave a much higher level of adducts than the (-), while the racemic mixture gave a level close to the mean value of the two isomers. There may have been up to 10% cross-contamination between the isomers in our preparations, in which case the true difference between the isomers would be greater than that shown in the Table.
Discussion Resolution of R-hydroxytamoxifen has been achieved by separation on a chiral HPLC column (A. Seidel, personal communication) but only with difficulty, and not with readily available columns. We therefore sought to resolve the substance by conversion to a diastereomeric ester, which can be separated by readily available nonchiral columns. We chose to work with esters of camphanic acid, since this acid and its chloride can be derived from natural sources as the pure 1S-enantiomers and are readily available. Separation of tamoxifen R-camphanate into its two isomers was straightforward. Hydrolysis to give the chiral alcohols was complicated by their tendency to dissociate in polar media with consequent racemization, but was eventually achieved by cleavage with sodium ethoxide. This afforded (+)- and (-)-R-hydroxytamoxifen in good yield. Several methods have been used for the assignment of absolute configuration of chiral alcohols, including NMR spectroscopy of esters with a chiral acid, degradation to simpler compounds of known configuration, X-ray crystallography, and comparison of the circular dichroism spectrum with that of similar compounds (19). The popular NMR (Mosher’s) method calls for preparation of the R-methoxy-R-trifluoromethylphenylacetate; we found this ester of R-hydroxytamoxifen so labile as to make the method impractical. We then tried degradation; the obvious approach was to split the molecule at the central double bond. The method is shown in Scheme 2. The hydroxy-group was first protected by esterification; we used the camphanate ester (B), since it was already available from our method of resolution. Oxidative splitting of this ester gave two ketones, of which one (C) still contained the chiral carbon. This is an ester of the hydroxyketone (E), which could be readily prepared by reduction of the commercially available diketone (D) with yeast. This yields the (-)isomer of E. The absolute stereochemistry of (-)-E was already known; it has been shown to have (S)-configuration by its preparation from (S)-lactic acid (19). As described above, we thus showed that the (+)- and (-)isomers of R-hydroxytamoxifen have (R) and (S) configuration, respectively (Scheme 1). None of the steps in this scheme involved the R-carbon atom, and so its stereochemistry should have remained constant throughout. Incubation of tamoxifen with the microsomal fraction of rat liver gave R-hydroxytamoxifen as a minor metabolite. This contained equal amounts of the (+)- and (-)enantiomers. This was somewhat unexpected, as metabolism is usually a stereoselective process, and raises the possibility that formation of R-hydroxytamoxifen in the liver proceeds through a planar intermediate such as the carbocation, resulting in the racemic alcohol. This carbocation must have remained bound to the enzyme and not released into solution, for such release would
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have resulted in trans-cis isomerization, and we could not detect any R-hydroxy-cis-tamoxifen among the metabolites. Although the enantiomers are chemically identical, they gave different levels of adducts in rat hepatocytes. Since the formation of adducts occurs mainly through conversion to a sulfate ester (21-25), the obvious explanations for the difference in adduct levels are (a) that the R-(+)-isomer is a better substrate for the sulfotransferases, or (b) that the R-sulfate reacts with DNA to a greater extent than the S-isomer. However, the reaction with DNA is almost certainly an SN1 process, in which the sulfate is lost to give tamoxifen R-carbocation which then reacts with the DNA. The same carbocation would be generated in equal amounts from either stereoisomer, so it is probable that the R- and S-sulfates alkylate DNA to equal extents. The reaction is stereoselective in that unequal amounts of R-R and R-S deoxyguanosine adducts are formed (10, 22), but this is determined by the chirality of DNA, not by the chirality of the R-hydroxytamoxifen, which is lost when the sulfate group leaves. It would therefore appear that in rat liver at least, the R-(+)-isomer is a better substrate for the sulfotransferases than the S-(-). Since the (+)-isomer forms more adducts, it should also be more potent than the (-)isomer in terms of the biological properties that depend on reaction with DNA, e.g. mutagenesis, chromosome damage, cell transformation, and carcinogenesis. Experiments now in progress will establish whether this is the case.
Acknowledgment. We thank Kathy Cole and Clive Lebozer for technical assistance, Ian Hardcastle for the R-hydroxytamoxifen, Elizabeth Martin for [14C]tamoxifen, and Amin Mirza for the mass and NMR spectra. The work was funded by the Cancer Research Campaign, U.K.
References (1) IARC Monographs on the Evaluation of the Carcinogenic Risks of Chemicals to Humans: Some Pharmaceutical Drugs (1996) Vol. 66, pp 253-365, International Agency for Research on Cancer, Lyon. (2) Osborne, M. R. (1999) Genotoxicity of tamoxifen and other antiestrogens. Recent Res. Devel. Cancer 1, 69-81. (3) Phillips, D. H. (2001) Understanding the genotoxicity of tamoxifen? Carcinogenesis 22, 839-849. (4) Phillips, D. H., Carmichael, P. L., Hewer, A., Cole, K. J., and Poon, G. K. (1994) R-Hydroxytamoxifen, a metabolite of tamoxifen with exceptionally high DNA-binding activity in rat hepatocytes. Cancer Res. 54, 5518-5522. (5) Phillips, D. H., Carmichael, P. L., Hewer, A., Cole, K. J., Hardcastle, I. R., Poon, G. K., Keogh, A., and Strain, A. J. (1996) Activation of tamoxifen and its metabolite R-hydroxytamoxifen to DNA-binding products: comparisons between human, rat and mouse hepatocytes. Carcinogenesis 17, 89-94. (6) Poon, G. K., Walter, B., Lønning, P. E., Horton, M. N., and McCague, R. (1995) Identification of tamoxifen metabolites in human Hep G2 cell line, human liver homogenate, and in patients on long-term therapy for breast cancer. Drug Metab. Dispos. 23, 377-382. (7) Boocock, D. J., Maggs, J. L., White, I. N. H., and Park, B. K. (1999). R-Hydroxytamoxifen, a genotoxic metabolite of tamoxifen in the rat: identification and quantification in vivo and in vitro. Carcinogenesis 20, 153-160. (8) Osborne, M. R., Hewer, A., Hardcastle, I. R., Carmichael, P. L., and Phillips, D. H. (1996) Identification of the major tamoxifendeoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res. 56, 66-71. (9) Osborne, M. R., Hardcastle, I. R., and Phillips, D. H. (1997) Minor products of reaction of DNA with R-acetoxytamoxifen. Carcinogenesis 18, 539-543.
Isomers of R-Hydroxytamoxifen (10) Dasaradhi, L., and Shibutani, S. (1997) Identification of tamoxifen-DNA adducts formed by R-sulfate tamoxifen and R-acetoxytamoxifen. Chem. Res. Toxicol. 10, 189-196. (11) Kitagawa, M., Ravindernath, A., Suzuki, N., Rieger, R., Terashima, I., Umemoto, A., and Shibutani, S. (2000) Identification of tamoxifen-DNA adducts induced by R-acetoxy-N-desmethyltamoxifen. Chem. Res. Toxicol. 13, 761-769. (12) Foster, A. B., Jarman, M., Leung, O.-T., McCague, R., Leclercq, G., and Devleeschouwer, N. (1985) Hydroxy derivatives of tamoxifen. J. Med. Chem. 28, 1491-1497. (13) Cheˆnevert, R., and Thiboutot, S. (1988) Bakers’ yeast reduction of 1,2-diketones. Preparation of pure (S)-(-)-2-hydroxy-1-phenyl1-propanone. Chem. Lett. 1191-1192. (14) Boocock, D. J., Maggs, J. L., Brown, K., White, I. N. H., and Park, B. K. (2000) Major inter-species differences in the rates of O-sulfonation and O-glucuronylation of R-hydroxytamoxifen in vitro: a metabolic disparity protecting human liver from the formation of tamoxifen-DNA adducts. Carcinogenesis 21, 18511858. (15) Osborne, M. R., Davis, W., Hewer, A. J., Hardcastle, I. R., and Phillips, D. H. (1999) 4-Hydroxytamoxifen gives DNA adducts by chemical activation, but not in rat liver cells. Chem. Res. Toxicol. 12, 151-158. (16) Phillips, D. H., Hewer, A., Horton, M. N., Cole, K. J., Carmichael, P. L., Davis, W., and Osborne,M. R. (1999) N-Demethylation accompanies R-hydroxylation in the metabolic activation of tamoxifen in rat liver cells. Carcinogenesis 20, 2003-2009. (17) Gamboa da Costa, G., Hamilton, L. P., Beland, F. A., and Marques, M. M. (2000) Characterization of the major DNA adduct formed by R-hydroxy-N-desmethyltamoxifen in vitro and in vivo. Chem. Res. Toxicol. 13, 200-207. (18) Firozi, P. F., Vulimiri, S. V., Rajaniemi, H., Hemminki, K., Dragan, Y., Pitot, H. C., DiGiovanni, J., Zhu, Y. H., and Li, D.
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(2000) Characterization of the major DNA adducts in the liver of rats chronically exposed to tamoxifen for 18 months. Chem-Biol. Interact. 126, 33-43. Shapiro, S. (1996) Chemical and spectroscopic methods for determining absolute configurations of chiral alcohols. Enantiomer 1, 151-166. Enders, D., Lotter, H., Maigrot, N., Mazaleyrat, J. P., and Welvart, Z. (1984) Asymmetric nucleophilic acylation via metalated chiral aminonitriles. Enantioselective synthesis of R-hydroxyketones. Nouv. J. Chim. 8, 747-750. Davis, W., Venitt, S., and Phillips, D. H. (1998) The metabolic activation of tamoxifen and R-hydroxytamoxifen to DNA-binding species proceeds via sulphation. Carcinogenesis 19, 861-866. Shibutani, S., Shaw, P. M., Suzuki, N., Dasaradhi, L., Duffel, M. W., and Terashima, I. (1998) Sulfation of R-hydroxytamoxifen catalysed by human hydroxysteroid sulfotransferase results in tamoxifen-DNA adducts. Carcinogenesis 19, 2007-2011. Shibutani, S., Dasaradhi, L., Terashima, I., Banoglu, E., and Duffel, M. W. (1998) R-Hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) transferase, resulting in tamoxifen DNA adducts. Cancer Res. 58, 647-653. Glatt, H., Davis, W., Meinl, W., Hermersdo¨rfer, H., Venitt, S., and Phillips, D. H. (1998) Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture. Carcinogenesis 19, 1709-1713. Davis, W., Hewer, A., Rajkowski, K. M., Meinl, W., Glatt, H., and Phillips, D. H. (2000) Sex differences in the activation of tamoxifen to DNA binding species in rat liver in vivo and in rat hepatocytes in vitro: role of sulfotransferase induction. Cancer Res. 60, 2887-2891.
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