The R-Isomer Forms More DNA Adducts in Rat Liver Cells - American

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Stereoselective Metabolic Activation of r-Hydroxy-N-desmethyltamoxifen: The R-Isomer Forms More DNA Adducts in Rat Liver Cells Martin R. Osborne,* Alan Hewer, and David H. Phillips Section of Molecular Carcinogenesis, Brookes Lawley Building, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom Received January 26, 2004

The antiestrogenic drug tamoxifen forms DNA adducts in rat liver through two genotoxic metabolites, R-hydroxytamoxifen and R-hydroxy-N-desmethyltamoxifen. These have now each been resolved into R- and S-enantiomers. The work with R-hydroxytamoxifen was published earlier [Osborne, et al. (2001) Chem. Res. Toxicol. 14, 888-893]. Here, we publish results with R-hydroxy-N-desmethyltamoxifen. We prepared the derivative N-ethoxycarbonyl-N-desmethyltamoxifen-R-S-camphanate, separated it into two diastereoisomers, and hydrolyzed them to give (+)- and (-)-R-hydroxy-N-desmethyltamoxifen. The configuration of the (-)-isomer was shown to be S- by degradation of the above ester to a derivative of (-)-2-hydroxy-1-phenyl-1propanone, which has already been shown to have S-configuration. The two enantiomers have the same chemical properties and were equally reactive toward DNA in vitro at pH 6. However, on treatment of rat hepatocytes in culture, R-(+)-R-hydroxy-N-desmethyltamoxifen gave 10 times as many DNA adducts as the S-(-)-isomer. This suggests that the R-isomer more readily undergoes sulfate conjugation to generate a reactive carbocation that attacks DNA.

Introduction The antiestrogenic drug tamoxifen1 (Scheme 1, 1) has been shown to form DNA adducts in rat liver and to induce liver cancer in rats (reviewed in refs 1-3). Adduct formation takes place chiefly through the following mechanism. Metabolism by cytochrome P450 enzymes (4, 5) produces R-hydroxytamoxifen (2) and R-hydroxy-Ndesmethyltamoxifen (3, 4). Sulfotransferases convert these metabolites to sulfate esters (6-8), which are alkylating agents that attack DNA, chiefly at the amino group of guanine. The resulting adducts cause mutation and tumor induction in rat liver. It is unclear whether the same process occurs in humans (reviewed in refs 1 and 9). No tamoxifen-DNA adducts have been found in the livers of women receiving tamoxifen (10), although they have been detected in the livers of monkeys treated with much higher doses of tamoxifen (11, 12). Women given tamoxifen have an increased risk of endometrial cancer, despite the level of DNA adducts in human endometrium being low or zero (13-16); the cancer here may be due to hormonal effects rather than reaction with DNA (9, 17). While doubt remains about the mechanism of tamoxifen carcinogenicity in humans, shedding further light on the mechanism of genotoxicity in rat liver will assist in the process of making interspecies comparisons and extrapolations. The chief metabolites giving rise to bonding to DNA in rat liver, R-hydroxytamoxifen and R-hydroxy-N-des* To whom correspondence should be addressed. E-mail: [email protected]. 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; R-hydroxy-Ndesmethyltamoxifen, (E)-4-{4-[2-(methylamino)ethoxy]phenyl}-3,4diphenyl-3-buten-2-ol.

Scheme 1. Structures of 1-4

methyltamoxifen (18-21), each have a chiral carbon atom and therefore exist as two enantiomers. In our previous paper (22), we described the isolation of the R-(+)- and S-(-)-isomers of R-hydroxytamoxifen and showed that metabolism of tamoxifen by rat liver microsomes produces both isomers. When they were separately administered to rat hepatocytes in culture, the R-(+)-isomer induced eight times as many DNA adducts as the S(-)-isomer, and the racemate induced five times as many. In this paper, we report the isolation of (+)- and (-)isomers of R-hydroxy-N-desmethyltamoxifen (3 and 4), the assignment of their absolute configuration, and an assessment of their relative abilities to form covalent adducts in DNA by chemical means and in primary cultures of rat hepatocytes.

10.1021/tx049957w CCC: $27.50 © 2004 American Chemical Society Published on Web 04/24/2004

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Chem. Res. Toxicol., Vol. 17, No. 5, 2004 Scheme 2. Structures of 4-6a

a R-S-isomers shown. Conversions by (i) (1) NaOEt/EtOH, (2) N2H4/KOH; (ii) KMnO4.

Experimental Procedures Tamoxifen, salmon testis DNA, and [1S]-(-)-camphanyl chloride (camphanoyl chloride; 3-oxo-4,7,7-trimethyl-2-oxabicyclo[2.2.1]heptane-1-carbonyl chloride) were from Sigma-Aldrich (Poole and Gillingham, U.K.). R-Hydroxytamoxifen was synthesized and provided by I. R. Hardcastle. 2-Camphanyloxy-1phenyl-1-propanone (S,S-isomer; 6, Scheme 2) was prepared as described before (22). Racemic R-hydroxy-N-desmethyltamoxifen and R-hydroxy-N-ethoxycarbonyl-N-desmethyltamoxifen were prepared by the method of Kitagawa et al. (23). Optical rotations were measured on a Perkin-Elmer 141 polarimeter with a sodium lamp. Mass spectrometry 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) Reverse phase: a “Jupiter” C18 column (4.6 mm × 250 mm; Phenomenex, Macclesfield, U.K.) eluted at 0.8 mL/min with a wateracetonitrile mixture, containing 0.05 M ammonium formate throughout, and varying acetonitrile concentrations as stated. (b) Normal phase: a Nucleosil silica column (4.6 mm × 250 mm; Fisher, Loughborough, U.K.) eluted with hexane-dichloromethane-methanol-triethylamine (80:20:0.5:0.025 or 80:20:0.8:0.025). Tamoxifen derivatives were detected in the eluate by their ultraviolet absorbance at 254 nm. Preparation and Resolution of N-Ethoxycarbonyl-Ndesmethyltamoxifen r-Camphanate (5, Scheme 2). A mixture of 15 mg of R-hydroxy-N-ethoxycarbonyl-N-desmethyltamoxifen (23), 30 mg of 1S-camphanyl chloride, 0.4 mL of dry dimethylformamide, and 0.1 mL of pyridine was incubated at 37 °C for 2 h. The ester was isolated by chromatography in multiple runs on a C18 column in 76 or 80% 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 N-ethoxycarbonyl-N-desmethyltamoxifen R-camphanate (5) in about 90% yield. This ester is unstable to hydrolysis; its half-life in 3:2 water-ethanol, pH 7, at 37 °C is about 12 min. It is therefore slightly more reactive than tamoxifen-R-camphanate (half-life, 30 min). The ester was dissolved in 4 mL of hexane-dichloromethane (2:1), injected in 0.1 mL aliquots onto a silica column, and eluted with hexane-dichloromethane-methanol-triethylamine (0.8%

Osborne et al. methanol). Resolution was easier than with tamoxifen-R-camphanate. The principal peaks “E1” and “E2” were eluted at about 19 and 20 min, respectively. This gave about 5 mg of each isomer, at least 90% pure as shown by rechromatography. Isomers E1 and E2 had identical UV absorption spectra, with maxima at 239 and 277 nm (absorbance ratio 1.66:1). The mass spectrum of isomer E2 gave positive ions at m/z 648 (34%, [M + Na]+), 512 (19%, unknown), 450 (57%, [M - camphanate H + Na]+), 428 (28%, [M - camphanate]+), and 382 (100%, CH2: CH‚CPh:CPh‚C6H4‚O(CH2)2NMe+:CO), consistent with the formula C38H43NO7 (M ) 625). 1H NMR [isomer E1, (CD3)2SO]: δ 7.3 (m, 10H, phenyl), 6.8 and 6.5 [d, 2H + 2H, J ) 8.8 Hz, 3,5 and 2,6-phenylene; compare spectrum of R-hydroxytamoxifen, (27)], 5.9 (q, 1H, J ) 6.6 Hz, R), 4.1 (q, 2H, J ) 7.1 Hz, ethyl-R), 3.7 and 3.5 (m, 2H + 2H, OCH2CH2N), 3.0 (s, 3H, MeN), 1.8 and 1.6 (m, 4H, camphanyl CH2), 1.3 (d, 3H, β), 1.2 (t, 3H, ethylβ), 1.0 (s, 3H, CMe), 0.9 and 0.8 (s, 3H+3H, CMe2), with some contaminant peaks. Hydrolysis to Enantiomers of r-Hydroxy-N-desmethyltamoxifen. N-Ethoxycarbonyl-N-desmethyltamoxifen R-camphanate (isomer E1 or E2; about 4 mg) was treated with 0.2 mL of sodium ethoxide in ethanol (0.6 M, 37 °C, 1.5 h) and then with 0.06 mL of hydrazine hydrate + 0.1 g of KOH in 0.6 mL of 1,2-ethanediol (140 °C, 2 h). The mixture was extracted between ether and water, and the ether phase was dried and evaporated to give about 2 mg of (+)- or (-)-R-hydroxydesmethyltamoxifen. It may have been unnecessary to use sodium ethoxide to hydrolyze the camphanate esters; the hydrazine/KOH in the second stage will also do this, but we have not determined whether they cause racemization. The two isomers had identical UV absorbance spectra, with maxima at about 235 nm and inflection at 272 nm (absorbance ratio 1.7:1). This is close to the spectrum of the racemic substance, which had absorbance maxima at 235 and 267 nm (21). The specific optical rotation of each isomer was measured in ethanol solution at 20 °C. The isomer derived from E1 showed a specific rotation estimated at -290° and that from E2 at +270°. These values are comparable with those of R-hydroxytamoxifen, which are about -300 and +300° (22). The mass spectrum of the (-)-isomer showed a positive ion at m/z 374 (100%, [M + H]+) consistent with the formula C25H27NO2. Determination of Absolute Stereochemistry. Racemic N-ethoxycarbonyl-N-desmethyltamoxifen R-camphanate (5, 0.2 mg) was treated with 10 µL of acetic acid + 0.3 mg of KMnO4 in 50 µL of water for an hour at room temperature. The mixture was decolorized with sodium sulfite (50 µL, 0.1 M) and neutralized with sodium hydroxide and ammonium formate. It was analyzed on two Jupiter ODS columns connected in series, run in 55% acetonitrile. The least polar components were eluted in twin peaks at about 36 and 37 min. The second of these corresponded to phenylpropanone camphanate prepared from S-(-)-2-hydroxy-1-phenylpropanone (6, Scheme 2) as established by cochromatography. Oxidation of isomer E1 produced a single peak, identical to the second peak, thus identifying E1 as the R-S-isomer. This was confirmed by extracting out the oxidation product with hexane-dichloromethane and separating by chromatography on silica (0.5% methanol). In this system, the product from isomer E1 cochromatographed with R-S-2-camphanyloxy-1-phenyl-1-propanone (retention time about 13 min) and that from E2 was eluted 0.5 min later. Reaction of r-Hydroxy-N-desmethyltamoxifen with DNA in Vitro. Reaction was carried out as described for R-hydroxytamoxifen (24). To DNA (1 mg) in 1 mL of 0.12 M sodium cacodylate buffer, pH 6, was added 0.1 mg of R-hydroxy-Ndesmethyltamoxifen 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 was 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 (below). Each sample was analyzed twice. Treatment of Rat Hepatocytes with r-Hydroxy-N-desmethyltamoxifen. Hepatocytes were isolated from female

Isomers of R-Hydroxy-N-desmethyltamoxifen Fischer F-344 rats and maintained in culture as described before (25). R-Hydroxy-N-desmethyltamoxifen 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 of 1 µM. After 18 h at 37 °C, the cells from each flask were harvested, and their DNA was extracted and analyzed for adducts as described below. Each sample was analyzed twice, giving six values for each compound. 32P-Postlabeling. DNA was isolated from cells by the phenol-chloroform procedure as described before (26) and subjected to the nuclease P1 enrichment method of postlabeling analysis, as follows. Aliquots of DNA (4 µg) were taken and evaporated to dryness using a Savant Speedvac SVC100 vacuum centrifuge. The DNA was digested overnight at 37 °C with micrococcal nuclease (0.14 U) and spleen phosphodiesterase (20 milliunits) in 5 µL of buffer (pH 6) containing sodium succinate (4 mM) and calcium chloride (2 mM). Nuclease P1 (0.15 U, 0.96 µL), sodium acetate (62.5 mM, 2.4 µL, pH 5), and zinc chloride (0.3 mM, 1.44 µL) were added, and digestion continued for a further hour at 37 °C. The reaction was stopped by adding 1.92 µL of 0.5 M Tris base. 32P-Postlabeling of each sample was carried out by incubation at 37 °C for 30 min with [γ-32P]ATP (50 µCi, specific activity approximately 4000 Ci mmol-1) and polynucleotide kinase (6 units, 0.6 µL) in bicine (14 mM, pH 9.0)/magnesium chloride (7 mM)/dithiothreitol (7 mM)/spermidine (0.7 mM) (1 µL). TLC of DNA Adducts. Radiolabeled digests were applied to the origins of 10 cm × 10 cm polyethyleneimine-cellulose TLC plates (Macherey-Nagel, Duren, Germany). Multidirectional chromatography was carried out using the following solutions: D1, sodium phosphate (2.3 M, pH 5.8) overnight onto a paper wick outside the tank; D2, lithium formate (2.275 M)/ urea (5.525 M), pH 3.5; D3, lithium chloride (0.52 M)/Tris-HCl (0.325 M)/urea (5.525 M), pH 8.0. DNA adducts were detected as radioactive spots on the TLC plates, visualized using a Packard Instant Imager (Canberra Packard, Pangbourne, Berks, U.K.). Quantification was carried out using the measured specific activity of the [γ-32P]ATP with background subtraction.

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Figure 1. Autoradiographs of polyethyleneimine-cellulose thin layer plates after chromatography of 32P-labeled tamoxifennucleoside bisphosphates. The origins are at the bottom left corner, and the directions of development are as follows: D1, downward; D2, upward; and D3, to the right. The adducts were derived from salmon sperm DNA treated with (a) (+)-, (b) (-)-, or (c) (()-R-hydroxy-N-desmethyltamoxifen at pH 6 or from DNA from rat hepatocytes treated with (d) (+)-, (e) (-)-, or (f) (()-Rhydroxy-N-desmethyltamoxifen. Table 1. Level of Adducts in DNA Exposed to r-Hydroxy-N-desmethyltamoxifena

Results Chemical Bonding to DNA. When R-hydroxytamoxifen is incubated with DNA in aqueous solution, a small amount becomes covalently bound, and tamoxifendeoxyguanosine adducts have been isolated from the DNA. The reaction is acid-catalyzed (24). This also occurs with R-hydroxy-N-desmethyltamoxifen. The R-(+), S(-), and racemic substances were incubated separately with DNA at pH 6, and the levels of reaction were determined by the 32P-postlabeling procedure. The results are shown in Figure 1 and in Table 1. The separated enantiomers and racemic mixture each became bonded to DNA to give the same pattern of adducts (Figure 1ac). The mobility of the principal adduct was similar to that of the principal tamoxifen-deoxyguanosine bisphosphate adduct isolated from the liver of tamoxifentreated rats or from DNA treated with R-hydroxytamoxifen or R-acetoxytamoxifen (24). The extent of bonding to DNA was approximately the same for each isomer. 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. Adducts in Rat Hepatocytes. Each isomer of R-hydroxy-N-desmethyltamoxifen was dissolved in DMSO and administered to rat hepatocytes at 1 µM concentration. The adduct levels in the DNA were determined by the

R-(+)

S-(-)

racemic R-OH-tam

at pH 6, by chemical 4.1 5.5 5.5 reaction (200 µM) in rat hepatocytes 6.5 ( 0.4 0.65 ( 0.04 4.7 ( 0.2 4.8 ( 0.4 (1 µM) a The table gives the number of tamoxifen adducts per million nucleotides, found (a) in salmon testis DNA after treatment with the substance at pH 6 and (b) in DNA from rat hepatocytes treated with it. The figure for racemic R-hydroxytamoxifen in hepatocytes is included for comparison. Each value is the mean of two determinations.

32

P-postlabeling procedure. The pattern of adducts (Figure 1d-f) was the same in each case, with a major spot in the same position as the major adduct obtained by reaction in vitro (Figure 1a-c). However, as shown in Table 1, (+)-R-hydroxy-N-desmethyltamoxifen (R-isomer) gave a much higher level of adducts than the (-)-isomer, 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 Table 1. The same preparation of hepatocytes was also treated with racemic R-hydroxytamoxifen. This resulted in adducts at a level of 4.8 per million bases, comparable with that found in an earlier experiment (3.7 per million; ref 22) and with that now found for R-hydroxydesmethyltamoxifen.

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Discussion Following the resolution of R-hydroxytamoxifen by conversion to the diastereomeric camphanate and separation by liquid chromatography, we applied the same method to resolve another tamoxifen metabolite, R-hydroxy-N-desmethyltamoxifen. Here, the tertiary amine function of tamoxifen has become a secondary amine, which needs to be protected before esterification of the hydroxyl group. A suitable protected derivative became available when Kitagawa et al. (23) devised a new synthetic route to R-hydroxy-N-desmethyltamoxifen. Their intermediate, R-hydroxy-N-ethoxycarbonyl-N-desmethyltamoxifen, could be converted to a diastereomeric camphanate ester (Scheme 2, 5), and this was readily separated into two isomers, E1 and E2, on a silica HPLC column. Hydrolysis gave (-)- and (+)-R-hydroxy-N-desmethyltamoxifen in good yield. The absolute stereochemistry of the diastereoisomeric esters was determined by splitting the molecule at the central double bond. The method is shown in Scheme 2. Oxidative splitting of this ester gave two ketones, of which one (6, Scheme 2) still contained the chiral carbon. One of the isomers, E1, gave S,S-2-camphanyloxy-1phenyl-1-propanone, which had been previously prepared (22) and whose configuration has been determined. E1 is therefore the S,S-isomer, and because hydrolysis should take place without affecting the R-carbon atom, (-)-R-hydroxy-N-desmethyltamoxifen must also have the (S)-configuration (Scheme 2, 4). Although the enantiomers are chemically identical, they gave different levels of adducts in rat hepatocytes. This was to be expected, as the formation of adducts involves a further enzymatic step: conjugation of R-hydroxy-N-desmethyltamoxifen with sulfate ion, followed by loss of sulfate to give the carbocation (6), which is the same for each isomer. Thus, each isomer gives the same DNA adducts, but the level of adducts depends on the extent of sulfate conjugation. It would appear that in rat liver at least, the R-(+)-isomer is a better substrate for enzymatic activation than the S-(-)-isomer. The same result was obtained for R-hydroxytamoxifen (22). This would suggest that the R-isomers of these metabolites fit well and that the S-isomers fit poorly into the active site of the activating enzyme [one of the three isoforms of rat SULT2A1 (7)]. The reaction of R-acetoxy-N-desmethyltamoxifen with deoxyguanosine has been used as a model for the alkylation of DNA by the sulfate ester. This reaction gives four adducts, all of which contain N-desmethyltamoxifen linked through the R-carbon to the amino group of deoxyguanosine (23). They differ in their configuration at the chiral R-carbon and in that two of these adducts have undergone rotation of the central double bond to give cis-isomers. Both epimerization and trans-cisisomerization occur because the reacting species is a carbocation, which has no chiral carbon and no fixed double bond in the center. Therefore, determination of the absolute stereochemistry of tamoxifen adducts in DNA would not establish which metabolite isomer they were derived from. It is not yet clear whether R-hydroxy-N-desmethyltamoxifen is more or less potent a genotoxin than R-hydroxytamoxifen. It appears that R-acetoxy-N-desmethyltamoxifen is a more reactive and unstable substance than R-acetoxytamoxifen (23). However, R-hy-

Osborne et al.

droxy-N-desmethyltamoxifen and R-hydroxytamoxifen gave equal amounts of DNA adducts in rat hepatocytes, and the liver of rats treated with tamoxifen contained comparable amounts of tamoxifen and N-desmethyltamoxifen adducts (18-21). When rats were treated with tamoxifen metabolites by gavage, R-hydroxy-N-desmethyltamoxifen actually gave fewer liver DNA adducts than R-hydroxytamoxifen (28). N-Desmethyltamoxifen is the major metabolite of tamoxifen found in the blood of women undergoing tamoxifen treatment (29, 30). One might therefore expect that adducts derived from R-hydroxy-N-desmethyltamoxifen should be found wherever adducts derived from R-hydroxytamoxifen are found. However, in reports of tamoxifen-related DNA adducts in human endometrium, only adducts from R-hydroxytamoxifen have been reported (15), and the interpretation of these results remains a matter of debate (9).

Acknowledgment. We thank Amin Mirza for the mass and NMR spectra, Kathy Cole for technical assistance, and Ian Hardcastle for the R-hydroxytamoxifen. The work was funded by Cancer Research UK.

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Isomers of R-Hydroxy-N-desmethyltamoxifen

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A. J., and Phillips, D. H. (1999) Lack of evidence from HPLC 32Ppost-labeling for tamoxifen-DNA adducts in the human endometrium. Carcinogenesis 20, 339-342. Shibutani, S., Ravindernath, A., Suzuki, N., Terashima, I., Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (2000) Identification of tamoxifen-DNA adducts in the endometrium of women treated with tamoxifen. Carcinogenesis 21, 1461-1467. Martin, E. A., Brown, K., Gaskell, M., Al-Azzawi, F., Garner, R. C., Boocock, D. J., Mattock, E., Pring, D. W., Dingley, K., Turteltaub, K. W., Smith, L. L., and White, I. N. (2003) Tamoxifen DNA damage detected in human endometrium using accelerator mass spectrometry. Cancer Res. 63, 8461-8465. White, I. N. (2001) Anti-oestrogenic drugs and endometrial cancers. Toxicol. Lett. 120, 21-29. Rajaniemi, H., Rasanen, I., Koivisto, P., Peltonen, K., and Hemminki, K. (1999) Identification of the major tamoxifen-DNA adducts in rat liver by mass spectroscopy. Carcinogenesis 20, 305309. 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. Brown, K., Heydon, R. T., Jukes, R., White, I. N. H., and Martin, E. A. (1999) Further characterisation of the DNA adducts formed in rat liver after the administration of tamoxifen, N-desmethyltamoxifen or N,N-didesmethyltamoxifen. Carcinogenesis 20, 20112016. Gamboa da Costa, G., Hamilton, L. P., Beland, F. A., and Marques, M. M. (2000) Characterization of the major DNA adduct formed by alpha-hydroxy-N-desmethyltamoxifen in vitro and in vivo. Chem. Res. Toxicol. 13, 200-207. Osborne, M. R., Hewer, A., and Phillips, D. H. (2001) Resolution of alpha-hydroxytamoxifen; R-isomer forms more DNA adducts in rat liver cells. Chem. Res. Toxicol. 14, 888-893.

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