Determination of DNA Damage in F344 Rats Induced by Geometric

There were high adduct levels in the liver, but no uterine DNA adducts were detected ... Genotoxic Mechanism of Tamoxifen in Developing Endometrial Ca...
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Chem. Res. Toxicol. 1998, 11, 527-534

527

Determination of DNA Damage in F344 Rats Induced by Geometric Isomers of Tamoxifen and Analogues Karen Brown,*,†,§ John E. Brown,† Elizabeth A. Martin,§ Lewis L. Smith,§ and Ian N. H. White§ Pharmaceutical Chemistry, University of Bradford, West Yorkshire BD7 1DP, U.K., and MRC Toxicology Unit, University of Leicester, Leicestershire LE1 9HN, U.K. Received December 22, 1997

To investigate the activation mechanisms involved in tamoxifen carcinogenicity, analogues of tamoxifen isomers modified at the ethyl group were synthesized and assessed for their ability to induce hepatic DNA damage following their administration to female F344 rats. The cis isomer was prepared by acid-catalyzed isomerization of tamoxifen and isolated by preparative HPLC. The active metabolite R-hydroxytamoxifen and geometric isomers of bromotamoxifen and C-desmethylenetamoxifen, analogues in which the ethyl group has been replaced by a bromine atom and methyl group, respectively, were synthesized according to published procedures. The levels of hepatic DNA adducts induced were determined by 32P-postlabeling. Bromotamoxifen and tamoxifen 1,2-epoxide caused no detectable DNA damage relative to controls. Trans isomers of tamoxifen, C-desmethylenetamoxifen, and R-hydroxytamoxifen all produced DNA adducts at a 5-90-fold higher level than the corresponding cis isomers. In contrast, both the cis and trans isomers of R-hydroxytamoxifen showed similar reactivity toward calf thymus DNA in vitro. Molecular models of R-hydroxytamoxifen isomers suggest this difference in DNA adduct-forming ability is due to steric hindrance of the enzymes involved in the activation of this metabolite. There were high adduct levels in the liver, but no uterine DNA adducts were detected in rats treated with R-hydroxytamoxifen. This suggests that in contrast to the liver, R-hydroxytamoxifen is not further activated in rat uterus. This may help to explain the absence of uterine tumors in rats following long-term tamoxifen treatment.

Introduction The antiestrogen tamoxifen [trans-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene] (Chart 1) is currently undergoing clinical evaluation as a chemopreventive agent in women at an increased risk of developing breast cancer (1). Tamoxifen has complex pharmacology, acting as an estrogen antagonist or agonist depending on the target tissue and species involved. The cis isomer [cis-(E)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene] which is referred to in this paper as cis-tamoxifen, is fully estrogenic (2) and is not used clinically. (For tamoxifen and related analogues, trans and cis are used to refer to the relative positions of the substituted phenyl and alkyl, hydroxyl, or bromo function attached to the ethene moiety.) Although inactive in standard short-term tests for genotoxicity, longterm administration of tamoxifen to rats produces hepatocellular carcinomas (3-5). This drug has also been shown to form DNA adducts in rat liver (5, 6), bind irreversibly to human and rat microsomal proteins (7), and induce micronucleus formation in MCL-5 cells (8). In women, tamoxifen is associated with an increased incidence of endometrial carcinoma (9), but it is not clear whether this is associated with a genotoxic or hormonal mechanism. * To whom correspondence should be addressed at the MRC Toxicology Unit. † University of Bradford. § University of Leicester.

Chart 1. Structures of Tamoxifen and Its Cis Isomer

Like many chemical carcinogens, tamoxifen must be metabolically activated to reactive electrophiles which bind to DNA. It has been proposed that R-hydroxytamoxifen, a metabolite observed in the plasma of patients on tamoxifen therapy and also formed by rat, mouse, and human hepatocytes in vitro, is an intermediate in this activation process (10, 11). Other suggested reactive intermediates include tamoxifen 3,4-epoxides (12), 3,4dihydroxytamoxifen (13), 4-hydroxytamoxifen quinone methide (14), or a radical species formed from 4-hydroxytamoxifen (15). An adduct of tamoxifen and deoxyguanosine linked via the exocyclic amino group has been identified as the major product arising from the reaction of model esters prepared chemically from R-hydroxytamoxifen, R-acetoxytamoxifen or R-sulfate tamoxifen with DNA (16-18) (Chart 2). This adduct is chromatographically identical to the major adduct in rat liver DNA, following the administration of tamoxifen.

S0893-228x(97)00228-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/23/1998

528 Chem. Res. Toxicol., Vol. 11, No. 5, 1998 Chart 2. Structures of Tamoxifen-Related Compounds

C

The aim of this study was to synthesize geometric isomers of tamoxifen analogues and the putative active metabolite R-hydroxytamoxifen. These were assessed for their ability to cause DNA damage in the liver and reproductive tract of rats following short-term dosing, using the 32P-postlabeling assay (19). The results have implications in the understanding of the genotoxic potential and possible mechanisms of carcinogenicity of tamoxifen.

Experimental Section Chemical Methods. General Procedures. 1H NMR spectra were determined at 270 MHz using a JEOL JNM-GX270 Fourier transform (FT-NMR) spectrometer. Chemical shifts are expressed using the δ system of units relative to the internal standard, Me4Si. Routine mass spectra (electron impact, 70 eV) were obtained with an AEI (Kratos) MS902 spectrometer and MSS consul and data system. Microanalytical data were provided by the microanalysis service at the University of Newcastle upon Tyne using a Carlo Erba 1106 elemental analyzer, and infrared spectra were obtained from KBr disks using a Perkin-Elmer 297 infrared spectrophotometer. Melting points were determined on an Electrothermal 1A9000 series digital melting point apparatus and are uncorrected. Column chromatography was performed on silica gel for flash chromatography (40-63 µm; BDH, Poole). Unless stated otherwise the column was 20 cm × 2.5 cm. Isomers of R-hydroxytamoxifen and R-acetoxytamoxifen were prepared according to the published procedures (16, 20). Chemical reagents were purchased from Aldrich Chemical Co. (Poole, Dorset, U.K.) unless otherwise stated. cis-(E)-1-[4-[2-(Dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene, cis-Tamoxifen. The cis isomer of tamoxifen

Brown et al. was prepared by acid-catalyzed isomerization of tamoxifen (21). A solution of tamoxifen (4.0 g, 10.78 mmol) in absolute ethanol (100 mL) and concentrated HCl (60 mL) was refluxed for 4 h. The reaction mixture was cooled and partitioned between aqueous NaOH (3 M, 200 mL) and ether (200 mL). The ether layer was washed (2 × 100 mL of water), concentrated, and dissolved in methanol (5 mL) to give a 1:1 mixture of cis and trans isomers as determined by HPLC analysis (22). Reversephase preparative HPLC was then used to isolate the pure cis isomer. The system comprised a Shandon 5-µm Hypersil BDS C18 (25 cm × 2.5 cm i.d.) column, a Waters 510 pump operating at a flow rate of 8.0 mL/min, and a Waters 440 UV detector monitoring absorbance at 280 nm. A mobile phase of methanol/ 5% ammonium acetate (75:25) produced baseline separation of the tamoxifen isomers. Aliquots (25 µL, approximately 10 mg of cis-tamoxifen) of the methanol mixture were injected on to the column, the cis isomer eluting first. Fractions (10 mL), were pooled, diluted with water (100 mL), and extracted with ether (3 × 100 mL); then the organic phase was dried (Na2SO4), filtered, and concentrated. The residue was dissolved in methanol (1 mL) and isomeric purity confirmed using a BDS Hypersil column (250 × 4.6 mm i.d.) and the HPLC system previously described (22), monitoring the peak absorbance at 243 nm. The concentration was determined using a calibration curve, and the volume containing the required mass of cistamoxifen was concentrated to dryness and redissolved in an appropriate vehicle for subsequent experiments. A total of 24 injections produced approximately 132 mg of cis-tamoxifen: HPLC retention time, 17.5 min (tamoxifen retention time, 18.7 min); MS m/z 371 (M+, 2%), 72 (Me2NCH2CH2+, 24%), 58 (Me2NdCH2+, 100%). cis-2-Ethyl-2,3-diphenyl-3-[4-[2-(dimethylamino)ethoxy]phenyl]oxirane, Tamoxifen 1,2-Epoxide. Tamoxifen 1,2epoxide was previously synthesized from a chloroethoxy derivative and by N-deoxygenation of tamoxifen N-oxide 1,2-epoxide (23, 24). When tamoxifen N-oxide 1,2-epoxide was reduced using sulfur dioxide, the product was reported to form DNA adducts following in vitro reaction with DNA (24). In this study the DNA reactivity of tamoxifen 1,2-epoxide prepared using triphenylphosphine as the reducing agent (23) is discussed. trans-(E)- and cis-(Z)-1-Bromo-2-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenylethene, Bromotamoxifen and cis-Bromotamoxifen (20). Geometric isomers of bromotamoxifen were synthesized from either cis/trans-1-bromo-1,2diphenyl-2-(4-hydroxyphenyl)ethene or the pure cis isomer (25). The products were assigned cis or trans configuration by proton NMR spectroscopy. In tamoxifen derivatives where the 1- and 2-phenyl groups are unsubstituted, the isomers can be identified according to the chemical shifts of the AB quartet for the protons in the remaining aromatic ring (20), since only in the trans isomers is the AB quartet upfield of the signals for the protons of the phenyl groups. To a stirred solution of cis/trans-1-bromo1,2-diphenyl-2-(4-hydroxyphenyl)ethene (933 mg, 2.76 mmol) in anhydrous DMF under N2 at room temperature was added NaH (500 mg, 20.8 mmol), and the mixture was heated to 60 °C. (Dimethylamino)ethyl chloride hydrochloride (1.0 g, 7.0 mmol) was then added portionwise over a 20-min period and the vessel maintained at this temperature for 15 min. After cooling to room temperature, the mixture was poured into water at 0 °C (500 mL) and extracted with ether (3 × 300 mL); the combined organic extracts were then washed with water (300 mL), dried (Na2SO4), and concentrated. The residue was crystallized from petroleum ether (bp 40-60 °C) to give a 2:1 trans:cis isomeric mixture of bromotamoxifen (826 mg, 73% yield). Further recrystallization of this mixture (640 mg) from petroleum ether (bp 80-100 °C) gave the pure trans isomer (360 mg): mp 116118 °C (lit. mp 117-118 °C); MS m/z 423 (M+, 1%), 421 (M+, 1%), 253 (M+ - C6H5CBr, 1%), 72 (Me2NCH2CH2+, 16%), 58 (Me2NdCH2+, base peak, 100%); 1H NMR (CDCl3) δ 2.29 (s, 6H, N(CH3)2), 2.65 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 3.94 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 6.60 (d, J ) 9.0 Hz, 2H, H-3,5 of C-C6H4O), 6.83 (d, J ) 8.8 Hz, 2H, H-2,6 of C-C6H4-O), 7.0-7.4 (m,

DNA Damage Induced by Tamoxifen and Analogues 10H, ArH). Anal. Calcd for C24H24NOBr: C, 68.25; H, 5.69; N, 3.32. Found: C, 68.60; H, 5.62; N, 3.20. When the pure cis isomer of 1-bromo-1,2-diphenyl-2-(4hydroxyphenyl)ethene was reacted in the same way, recrystallization from petroleum ether (bp 80-100 °C) gave cisbromotamoxifen: mp 108.5-110 °C; 1H NMR (CDCl3) δ 2.35 (s, 6H, N(CH3)2), 2.75 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 4.10 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 6.90-7.40 (m, 14H, ArH). Anal. Calcd for C24H24NOBr: C, 68.25; H, 5.69; N, 3.32. Found: C, 68.49; H, 5.59; N, 3.22. trans-(Z)- and cis-(E)-1-[4-[(Dimethylamino)ethoxy]phenyl]-1,2-diphenylprop-1-ene, C-Desmethylenetamoxifen and cis-C-Desmethylenetamoxifen. C-Desmethylenetamoxifen was synthesized in an organometallic coupling reaction, where the bromine was substituted for a methyl group (26). The stereoselective mechanism enables the production of pure cis or trans isomers from the corresponding bromotamoxifen. A refluxing solution of bromotamoxifen (710 mg, 1.68 mmol) in anhydrous THF under N2 was treated with methylzinc chloride under catalysis by trans-benzyl(chloro)bis(triphenylphosphine)palladium(II) (10 mg). The methylzinc chloride was prepared by adding methyllithium (9.45 mmol of a 1.0 M solution in THF/ cumene) to zinc chloride (9.45 mmol of a 0.5 M solution in THF) at -78 °C. After cooling, the reaction was terminated with water (100 mL) and extracted with ether (3 × 100 mL). The organic phase was dried (Na2SO4), concentrated, and subjected to column chromatography using an initial mobile phase of petroleum ether (bp 60-80 °C)/ether (80:20) and then ether/ petroleum ether (bp 60-80 °C)/triethylamine (80:20:1) to elute the product. Crystallization from petroleum ether (bp 60-80 °C) gave white crystals of C-desmethylenetamoxifen (430 mg, 72% yield): mp 86-87.5 °C; 1H NMR (CDCl3) δ 2.11 (s, 3H, CH3), 2.29 (s, 6H, N(CH3)2), 2.64 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 3.93 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 6.58 (d, J ) 8.8 Hz, 2H, H-3,5 of C-C6H4-O), 6.76 (d, J ) 8.8 Hz, 2H, H-2,6 of C-C6H4O), 7.10-7.40 (m, 10H, ArH); MS m/z 357 (M+, 4%), 253 (M+ C6H5 - C - CH3, 1%), 72 (Me2NCH2CH2+, 33%), 58 (Me2NdCH2+, base peak, 100%). Anal. Calcd for C25H27O: C, 84.03; H, 7.56; N, 3.92. Found: C, 83.95; H, 7.53; N, 3.81. When cis-bromotamoxifen (360 mg) was treated in the same manner, the product was the cis isomer of C-desmethylenetamoxifen (150 mg, 49% yield): mp 88-89 °C; 1H NMR (CDCl3) δ 2.15 (s, 3H, CH3), 2.34 (s, 6H, N(CH3)2), 2.74 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 4.08 (t, J ) 5.9 Hz, 2H, OCH2CH2N), 6.86-7.26 (m, 14H, ArH). Anal. Calcd for C25H27NO: C, 84.03; H, 7.56; N, 3.92. Found: C, 84.08; H, 7.48; N, 3.72. Direct Reaction of Tamoxifen Analogues with DNA. To calf thymus DNA (500 µg) in 500 µL of 1:100 SSC-EDTA (0.15 M NaCl and 0.015 M sodium citrate-10 mM EDTA) buffer was added 50 µg of R-hydroxytamoxifen or 55 µg of R-acetoxytamoxifen dissolved in 150 µL of ethanol and incubated at 37 °C overnight. Ethanol (150 µL) was added to DNA to provide a control sample. Unreacted compound was removed by ether extraction (4 × 700 µL), and DNA precipitated from the aqueous phase by addition of 50 µL of 5 M NaCl and 1 mL of ice-cold ethanol. After centrifugation and removal of the supernatant, the DNA was washed with 1 mL of 70% and then 100% ethanol before being redissolved in 500 µL of 1:100 SSC-EDTA buffer. Microsomal Activation of Tamoxifen Isomers. Washed microsomal preparations were prepared by differential centrifugation (27) from the livers of 6-week-old female F344 rats (Harlan Olac Ltd.). Microsomal protein concentrations were determined by the method of Lowry (28) using a bovine serum albumin standard. Reaction mixtures of 400-µL volume, in 0.1 M HEPES buffer, pH 7.5, contained EDTA (2 mM), MgCl2 (5 mM), NADPH (1 mM), and 0.16 mM tamoxifen or cis-tamoxifen (added in 4 µL of methanol). Following equilibration to 37 °C, reactions were started by the addition of microsomal protein (250 µg). After 1 h, the reactions were stopped by the addition of ice-cold ethanol (800 µL). Following centrifugation (14000g for 10 min at 4 °C), the supernatant was removed for chromatographic analysis.

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 529 Animals and Treatment. Geometric isomers of tamoxifen, bromotamoxifen, C-desmethylenetamoxifen, R-hydroxytamoxifen, and tamoxifen 1,2-epoxide were assessed for their ability to induce DNA damage as detected by 32P-postlabeling following in vivo activation. Female F344 rats, aged 6 weeks, were from Harlan Olac Ltd. Animals received 0.11 mmol/kg compound (equivalent to 40 mg/kg tamoxifen) by ip injection for 4 days. Dosing solutions were made up in tricaprylin (Sigma) (0.11 mmol/mL). The animals were culled 24 h after the last dose. Liver and uterine tissues were removed, immediately frozen in liquid nitrogen, and stored at -80 °C. DNA Isolation and 32P-Postlabeling. Liver DNA was extracted by the method of Gupta (29) using proteinase K digestion, phenol/chloroform extraction, and digestion with RNase A and T1. Uterus DNA was isolated using Qiagen Genomic Tips according to the manufacturer’s protocol (30). DNA purity and concentration were established spectrophotometrically. Only DNA with a A260 nm/A280 nm ratio of 1.7 to 1.9 was used. 32P-Postlabeling using nuclease P1 enhancement was carried out as previously described (19). Adducts were visualized by autoradiography using OMAT-AR film with intensifying screens for between 5 min and 24 h at room temperature or -80 °C as indicated. For each plate, adduct spots were excised along with a similarly sized background region from an area of the plate without visible radioactivity. Radioactivity was then quantified by scintillation counting. Adduct levels were determined by subtracting radioactivity in background regions from radioactivity in adduct spots. In samples where no adducts were detected, including vehicle-dosed animals, a similarly sized area was excised from the region where the major tamoxifen adducts would be expected to elute, along with a background area, to provide an indication of the levels of radioactivity in this region. Mean levels of adducts ( SD are expressed as relative adduct labeling, RAL × 108. Adduct levels in DNA from vehicle control animals were not subtracted from any of the dosed animal data. HPLC Determination of Tamoxifen and Metabolites in Rat Liver. Liver (100 mg) from female F344 rats was homogenized in ice-cold methanol/DMSO (95:5, v/v, 900 µL). Samples were centrifuged (14000g for 10 min at 4 °C) and the supernatant (100 µL) analyzed by an isocratic reversed-phase HPLC system previously described (22). LC/ESI-MS Analysis. For on-line LC/ESI-MS analysis (31) the column outlet was connected to a VG-Quattro BIOQ electrospray mass spectrometer (Fisons Instruments Ltd.), operating in the positive ion mode. Capillary and high-voltage electrode potentials were 0.38 and 3.78 kV, respectively, with a source temperature of 150 °C. A Varian 9012 solvent delivery system was used, and sample injections were via a 200-µL Rheodyne loop injector 7125 (Cotati). The metabolites were separated isocratically on a Varian Res Elute-BD column (5 µm, 250 × 4.6 mm i.d.) using a mobile phase of methanol/0.5 M ammonium acetate (70:30, v/v) at a flow rate of 1 mL/min. The flow leaving the column was split in the ratio 1:6 with one part entering the electrospray. Typically 200 µL of sample was loaded onto the column, and ion spectra were acquired over the mass range of 300-800 atomic mass units at a rate of 1 scan every 2 s. Molecular Modeling of r-Hydroxytamoxifen Isomers. The crystal structure of tamoxifen citrate was obtained from the Cambridge database (32) and read into Insight II, Biopolymer: Molecular modeling system (MSI, San Diego). The structure of R-hydroxytamoxifen was built by substitution of a hydroxyl group for a hydrogen atom at the R-carbon of the ethyl side chain. The aim was to determine whether the protonated nitrogen of the (dimethylamino)ethoxy side chain could interact with the R-hydroxyl function in the cis or trans position. This was achieved by manually adjusting the torsion angles within the side chain, keeping within (20° of the minimum (180 ( 60°) and allowing no steric clashes greater than 20% of the sum of the van der Waals radii of the respective atoms.

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Figure 1. Autoradiographs of PEI-cellulose TLC maps of 32P-postlabeled DNA adducts from rat liver. Female F344 rats were treated with tamoxifen or analogues (0.11 mmol/kg daily) by ip injection for 4 days: (A) positive control rat administered dietary tamoxifen (420 ppm) for six months (1 h); (B) tamoxifen 1,2-epoxide (16 h); (C) bromotamoxifen (16 h); (D) cis-bromotamoxifen (16 h); (E) tamoxifen (16 h); (F) cis-tamoxifen (16 h); (G) C-desmethylenetamoxifen, (6 h); (H) cis-C-desmethylenetamoxifen (4 h); (I) R-hydroxytamoxifen (30 min); (J) cis-R-hydroxytamoxifen (2 h); (K) vehicle control (tricaprylin) (6 h). Autoradiography was carried out at either room temperature (A, I) or -80 °C (B-H, J, K). Exposure times are indicated in parentheses. Table 1. Levels of Hepatic

32P-Postlabeled

DNA Adducts in Female F344 Rats Treated with Tamoxifen or Analogues (0.11 mmol/kg daily) by ip Injection for 4 Days

compound tamoxifen 1,2-epoxide bromotamoxifen cis-bromotamoxifen tamoxifen cis-tamoxifen C-desmethylenetamoxifen cis-C-desmethylenetamoxifen R-hydroxytamoxifen cis-R-hydroxytamoxifen vehicle controls

no. of animals treated

RALa × 108 (meanb ( SD)

4 2 2 4 4 4 2 4 4 5

( 2.3 3.6d,e 4.2d,e 118f ( 39.6 12.0 ( 2.3 60.2 ( 27.8 4.3e 1857f ( 870 35.5 ( 22.3 5.1d ( 2.1 3.1d

significancec NS *** ** ** NS ** *

a RAL, relative adduct labeling. b Each value represents the mean ( SD of at least three determinations. c Probability of significance of difference in adduct accumulation relative to vehicle-dosed controls: ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant. d Since no adduct spots were visibly detectable in these sample, values correspond to background levels of radioactivity in the region of the plate where the major tamoxifen adducts would be expected to elute. e Two animals were treated; therefore only the mean level of adducts is given. f Probability of significance of difference in adduct accumulation between trans and cis isomers; P < 0.01.

Results and Discussion It has been proposed that R-hydroxytamoxifen or an activated ester may be the major DNA-reactive metabolite of tamoxifen (10, 16, 17). In this study, the cis isomer of tamoxifen was synthesized together with analogues in which the ethyl function was modified. These included bromotamoxifen isomers and the C-desmethylene analogues. In addition, tamoxifen 1,2-epoxide, previously suggested to be a putative reactive species (24), was prepared and reassessed for its ability to form DNA adducts. Hepatic DNA Damage in Rats as Assessed by 32PPostlabeling. Following the administration of equimolar doses of tamoxifen 1,2-epoxide or either isomer of bromotamoxifen to rats, no 32P-postlabeled DNA adducts could be detected above the background level of radio-

activity and 32P-postlabeled TLC plates were indistinguishable from vehicle-dosed controls (Figure 1). The absence of DNA adducts in the liver of rats treated with the epoxide is consistent with more recent results which indicate that the pure compound is devoid of DNAbinding ability (16) and is therefore unlikely to be involved in the activation pathway. The inactivity of the bromotamoxifen isomers supports the view that R-hydroxylation is a necessary first step in the activation of tamoxifen. However, the present results show that an ethyl function per se is not required since C-desmethylenetamoxifen was metabolically activated in rat liver in vivo, producing a distinct pattern of hepatic DNA adducts (Figure 1). This shows that a methyl function can also undergo activation, presumably via R-hydroxylation, although the extent to which 32P-postlabeled adducts

DNA Damage Induced by Tamoxifen and Analogues Table 2. Levels of

a

32P-Postlabeled

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 531

DNA Adducts following in Vitro Reaction of Tamoxifen Analogues with Calf Thymus DNA

compound

no. of determinations

RAL × 108 (mean ( SD)

R-hydroxytamoxifen cis-R-hydroxytamoxifen R-acetoxytamoxifen cis-R-acetoxytamoxifen

3 3 3 4

233 ( 52 166 ( 37 61066 ( 30288 47775 ( 19575

significancea NS NS

Probability of significance of difference between trans and cis isomers; NS, not significant.

were formed in the liver was slightly lower than with tamoxifen itself (Table 1). We show for the first time that with both tamoxifen and C-desmethylenetamoxifen there is a significant difference between the ability of the cis/ trans isomers to undergo metabolic activation and cause DNA adducts, the trans isomers forming around 10-14fold higher levels than the cis (Table 1). Treatment of female F344 rats for 4 days with R-hydroxytamoxifen produced adduct levels of approximately 2000 (RAL × 108) (Table 1), similar to that found when rats received tamoxifen for 6 months (5). Administration of the cis isomer, however, resulted in a 50-fold lower level of adducts. Quantitative analysis of the 32Ppostlabeled adduct levels formed in DNA following in vitro reaction with geometric isomers of R-hydroxytamoxifen and its ester R-acetoxytamoxifen showed that there was no difference in the intrinsic reactivity between isomers toward DNA (Table 2). As demonstrated previously, R-acetoxytamoxifen shows an approximate 200-fold increase in adduct formation relative to R-hydroxytamoxifen, supporting the prerequisite for conjugation of R-hydroxytamoxifen in the generation of the ultimate electrophilic species (16). It is proposed that the lower level of adduct formation observed with the cis isomers in vivo is due to steric hindrance of the enzymes involved in the activation step. Molecular modeling of R-hydroxytamoxifen isomers (Figure 2) has shown that in the trans isomer the shortest possible distance between the protonated amino group and the hydroxyl substituent is approximately 6.0 Å whereas in the cis form this distance is estimated at 2.5 Å. The latter is close enough to enable hydrogen bonding. In this conformation the methyl groups could protect the R-hydroxyl function from further activation. In contrast, there is no such interaction in the trans isomers which are readily conjugated, facilitating generation of the much more reactive carbocation. The PEI-cellulose thin layer chromatograms obtained from 32P-postlabeled uterus DNA from R-hydroxytamoxifen-treated rats were indistinguishable from those obtained from control rats (Figure 3). The absence of uterine DNA adducts in this species suggests that R-hydroxytamoxifen is not further activated in the rat uterus. Low levels of uterine DNA adducts have been reported following short-term administration of tamoxifen or 4-hydroxytamoxifen (33) to rats. DNA adducts have also been detected in the endometrium of tamoxifentreated breast cancer patients by Hemminki et al. using HPLC radioactivity detection (34), but a study by Carmichael et al. (35) found no evidence for tamoxifeninduced endometrial DNA adducts in women. Treatment of rats with R-hydroxytamoxifen produced high levels of hepatic DNA adducts, but none were detected in uterine tissues. In women, it is not clear if a genotoxic mechanism is associated with the increase in endometrial tumors seen during tamoxifen therapy (9). However, the

Figure 2. Molecular models of (A) R-hydroxytamoxifen and (B) cis-R-hydroxytamoxifen. The distance between the nitrogen of the (dimethylamino)ethoxy side chain and the R-hydroxyl function is indicated.

Figure 3. Representative autoradiographs of 32P-postlabeled uterine DNA digests. Female F344 rats received (A) tricaprylin or (B) R-hydroxytamoxifen (0.11 mmol/kg daily) by ip injection for 4 days. Autoradiography was for 8 h at -80 °C.

lack of DNA damage in the uterus of rats given R-hydroxytamoxifen may help to explain the absence of uterine tumors in this species following long-term tamoxifen treatment. Comparative Accumulation of Metabolites of Tamoxifen Isomers in Rat Liver. HPLC analysis of rat liver extracts (Figure 4) showed the major metabolites

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Table 3. Hepatic Concentrations of Tamoxifen and Metabolites in F344 Rats Treated with Tamoxifen Isomers (0.11 mmol/kg daily) by ip Injection for 4 Days metabolite concentration (µg/g of tissue, mean ( SD)a

a

compound

tamoxifen

N-desmethyltamoxifen

4-hydroxytamoxifen

tamoxifen cis-tamoxifen

14.7 ( 5.1 13.2 ( 3.8

13.7 ( 5.7 21.0 ( 7.6

3.8 ( 0.7 not detected

Values are the mean of three determinations from four rats.

Figure 5. Selected ion chromatograms of tamoxifen standards and metabolites with [M + H] at m/z 388 formed by the incubation of (b) tamoxifen or (c) cis-tamoxifen with rat liver microsomes; (a) standards of R-hydroxytamoxifen (1), cis-Rhydroxytamoxifen (2), cis/trans-4-hydroxytamoxifen (3), tamoxifen 1,2-epoxide (4), and tamoxifen N-oxide (5).

Figure 4. HPLC chromatograms of tamoxifen metabolite standards and liver extracts from F344 rats treated with tamoxifen isomers (0.11 mmol/kg daily) by ip injection for 4 days: (a) standards of R-hydroxytamoxifen (1), cis-R-hydroxytamoxifen (2), cis/trans-4-hydroxytamoxifen (3), tamoxifen 1,2epoxide (4), N-desmethyltamoxifen (5), tamoxifen N-oxide (6), and tamoxifen (7); (b) liver extract from rat treated with tamoxifen, peaks identified as (a) 4-hydroxytamoxifen, (b) N-desmethyltamoxifen, and (c) tamoxifen; (c) liver extracts from rat treated with the cis isomer, peaks identified as (a′) cis-Ndesmethyltamoxifen and (b′) cis-tamoxifen.

which accumulated in the tissue following administration of tamoxifen isomers. Quantitation of these metabolites showed similar levels of both tamoxifen and cis-tamoxifen and their corresponding N-demethylated metabolites in the liver (Table 3). In contrast, 4-hydroxytamoxifen, which was present at concentrations of approximately 4 µg/g in the tamoxifen-treated rat, was undetectable in livers of rats administered with the cis isomer. This suggests that accumulation of 4-hydroxytamoxifen in the livers of rats dosed with the cis isomer is less than in those animals given tamoxifen itself. HPLC analysis showed only the presence of detoxification products, N-desmethyltamoxifen, and 4-hydroxytamoxifen. We wished to establish whether the difference in levels of 4-hydroxylated metabolites was due to

a lower rate of hydroxylation of the cis isomer rather than to differences in tissue accumulation and if this also applied to the formation of R-hydroxylated metabolites. Since R-hydroxytamoxifen might be expected to either react with nucleophiles or undergo further activation in the liver, on-line LC/ESI-MS analysis was carried out using rat liver microsomal preparations in vitro (Figure 5). Following incubation of tamoxifen with microsomes in the presence of NADPH, single-ion monitoring of extracts of the incubation mixture at m/z 388, which corresponds formally to the addition of oxygen to tamoxifen, shows the formation of products which correspond in retention time to R-hydroxytamoxifen (peak a), 4-hydroxytamoxifen (peak b), 4′-hydroxytamoxifen (peak c) (12), and tamoxifen N-oxide (peak d). When the incubations were performed in the presence of the cis isomer, R-hydroxytamoxifen was not detected and 4-hydroxytamoxifen (peak a′) was formed at lower levels. The reasons for these differences in enzyme specificity toward cis/trans isomers are not clear, but the results offer a plausible explanation as to why the levels of DNA adducts produced following the administration of the cis isomer are lower than those seen following tamoxifen treatment.

Conclusions In this study we have shown that the cis isomers of tamoxifen, C-desmethylenetamoxifen, or the putative reactive species R-hydroxytamoxifen, when given to rats, result in a 10-50-fold lower level of hepatic DNA adducts, as assessed by 32P-postlabeling, than the respective trans isomers. When reacted with calf thymus DNA in vitro, there is little difference in the chemical reactivity

DNA Damage Induced by Tamoxifen and Analogues

between the cis/trans isomers of R-hydroxytamoxifen, suggesting steric selectivity, in either the activating enzyme or detoxification pathways. It is demonstrated that bromotamoxifen does not undergo metabolic activation in rat liver. However C-desmethylenetamoxifen is activated in this system indicating the ethyl function is not a necessary prerequisite. Previous studies strongly suggest there is a causal relationship between the presence of DNA damage, assessed by 32P-postlabeling, and the development of liver tumors (36). Results from microsomal enzyme metabolism studies in vitro show little or no detectable formation of R-hydroxytamoxifen when cis-tamoxifen is used as a substrate. This and the results from 32P-postlabeling studies suggest the cis isomer will be a much weaker rat liver carcinogen than tamoxifen if it acts by a similar mechanism, although to our knowledge no carcinogenicity studies have been carried out with cis-tamoxifen.

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 533

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Acknowledgment. The authors would like to thank M. Gaskell, R. Heydon, A. Davies, R. Smith, and N. Razvi for their expert technical assistance, M. Sutcliffe (Chemistry Department, Leicester University) for the molecular modeling, and J. L. Luo for performing the LC/ESI-MS analysis. K. Brown is funded by a University of Bradford studentship.

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