Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal,
1 Division of Biochemical Toxicology and
2 Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA
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Abstract |
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Abbreviations: alkO, alkoxy; Ar, aryl; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Cquat, quaternary carbon; N-desmethyltamoxifen, (Z)-1-[4-(2-methylaminoethoxy)phenyl]-1,2-diphenylbut-1-ene; N,N-didesmethyltamoxifen, (Z)-1-[4-(2-aminoethoxy)phenyl]-1,2-diphenylbut-1-ene; EI, electron impact; ENU, N-ethyl-N-nitrosourea; FAB, fast atom bombardment; 3-hydroxytamoxifen, (E)-1-[4'-(2-dimethylaminoethoxy)phenyl]-1-(3-hydroxyphenyl)-2-phenyl-but-1-ene; 4-hydroxytamoxifen, (Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1-(4-hydroxyphenyl)-2-phenylbut-1-ene; -hydroxy-N-desmethyltamoxifen, (E)-4-[4-(2-methylaminoethoxy)phenyl]-3,4-diphenylbut-3-en-2-ol;
-hydroxytamoxifen, (E)-4-[4-(2-dimethylaminoethoxy)phenyl]-3,4-diphenylbut-3-en-2-ol; Hprt, hypoxanthine phosphoribosyl transferase; Ph, phenyl; PNK, T4 polynucleotide kinase; rHSTarat hydroxysteroid sulfotransferase a; tamoxifen, (Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1-ene.
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Introduction |
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In addition to causing endometrial cancer in women, tamoxifen is carcinogenic in rodents. In rats treated orally, tamoxifen induced a high incidence of hepatic tumors (35), and endometrial tumors have been observed in rats and mice following transplacental, neonatal and/or chronic exposure (610). There is substantial evidence that the hepatic tumors in rats are due to a genotoxic mechanism resulting from the formation of tamoxifenDNA adducts (4,1115); however, as with humans, the mechanism for the induction of endometrial tumors in rodents has not been established, although hormonal imprinting (10) and genotoxic (6,7) mechanisms have been proposed.
In rat liver, tamoxifen is activated to an electrophile by sequential -hydroxylation and esterification (1622; Figure 1
). The
-hydroxylation is catalyzed primarily by cytochrome P450 3A4 (23), while esterification appears to occur mainly through sulfation, which is catalyzed by sulfotransferases, in particular SULT2A (2022,24). The major DNA adduct resulting from this metabolism is (E)-
-(deoxyguanosin-N2-yl)tamoxifen, which is accompanied by minor amounts of the Z diastereomer and deoxyadenosine adducts (2527). Another major activation pathway for tamoxifen in rat liver is N-desmethylation followed by
-hydroxylation (or
-hydroxylation followed by N-desmethylation) to give
-hydroxy-N-desmethyltamoxifen, which is presumably then esterified (2831; Figure 1
). The major adduct resulting from this pathway has recently been characterized as (E)-
-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (32). Both this adduct and its Z diastereomer have subsequently been identified in the reaction of
-acetoxy-N-desmethyltamoxifen with deoxyguanosine (33).
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Although activation pathways of tamoxifen in rat liver have been established, the contribution of the -hydroxy metabolites of tamoxifen to the toxicities observed in non-hepatic tissues (e.g. endometrial tumors) is presently unknown. Accordingly, we have compared the extent of DNA adduct formation in the livers and selected non-hepatic tissues of female SpragueDawley rats treated by gavage with tamoxifen,
-hydroxytamoxifen, N-desmethyltamoxifen,
-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen. In addition, spleen lymphocytes from rats treated with tamoxifen or
-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene.
The route of drug administration may have an important bearing on the metabolic activation pathways of tamoxifen. For example, oral dosing with tamoxifen resulted in decreased uterine peroxidase activity (39), whereas intraperitoneal administration caused a 10-fold induction of peroxidase activity (37). Although uterine DNA adducts were not detected after oral dosing with tamoxifen (15,39), the induction of peroxidase activity by intraperitoneal treatment could result in an increased production of 4-hydroxytamoxifen and its quinone methide. To test this possibility, a second experiment was conducted in which hepatic and uterine DNA adducts were assessed in female SpragueDawley rats injected intraperitoneally with tamoxifen, -hydroxytamoxifen and 4-hydroxytamoxifen. For comparison, additional rats were treated with 3-hydroxytamoxifen (droloxifene). This regio-isomer of 4-hydroxytamoxifen has been proposed as a chemotherapeutic agent for the treatment of advanced breast cancer in postmenopausal women (42,43). Moreover, 3-hydroxytamoxifen has estrogenic activity in the bone tissue of rats (44), which suggests its potential for the treatment of osteoporosis.
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Materials and methods |
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Instrumentation
Melting temperatures were measured with a Leica Galen III hot-stage apparatus and are uncorrected.
HPLC analyses were conducted with a µBondapak C18 column (0.39 cmx30 cm; Waters, Milford, MA), using either a Varian system consisting of a Star 9012 ternary gradient pump and a Polychrom 9065 diode array spectrophotometric detector (Varian, Palo Alto, CA), equipped with a Rheodyne Model 7125 injector (Rheodyne, Cotati, CA), or a Waters system consisting of two Model 510 pumps and a Model 660 automated gradient controller, equipped with a Rheodyne Model 7125 injector and a Hewlett-Packard 1050 diode array spectrophotometric detector (Hewlett-Packard, Wilmington, DE). UV absorbance was monitored at 254 or 280 nm.
HPLC analyses of 32P-post-labeled samples were conducted with a 5 µ Delta-Pak C18-100 column (0.39 cmx15 cm; Waters) using a Waters system, as described above, equipped with a Radiomatic Flo-One Model A-500 on-line radioactivity detector (Packard Instruments, Meriden, CT).
(E)--(Deoxyguanosin-N2-yl)tamoxifen was weighed to an accuracy of 0.1 µg with a Sartorius 4504 Mp8-1 ultra-micro balance.
UV spectra were recorded with either a Beckman DU-40 UV/vis or a Shimadzu 1202 UV/vis spectrophotometer.
1H NMR spectra were obtained either on a Varian Unity 300 spectrometer, operating at 300 MHz, or a Bruker AM500 spectrometer, operating at 500 MHz. 13C NMR spectra were recorded on the Varian Unity 300 instrument, operating at 75.4 MHz. Chemical shifts are reported in parts per million (p.p.m.) downfield from tetramethylsilane, and coupling constants are reported in Hertz (Hz).
Mass spectra were recorded on either a Finnigan TSQ-700 GS/MS system, operated in the electron impact (EI) mode, with the sample being introduced via a direct exposure probe, or a VG Trio-2000 instrument, operated in the fast atom bombardment (FAB) mode, with the sample dispersed in a matrix of 3-nitrobenzyl alcohol.
Elemental analyses were performed at the M-H-W Laboratories (Phoenix, AZ) or the Analytical Laboratory, Instituto Superior Técnico, Lisboa, Portugal.
Syntheses of tamoxifen derivatives
4-Hydroxytamoxifen was prepared as a mixture (ca. 1:1) of the E and Z isomers, as described in Marques and Beland (38). Synthesis and characterization of -hydroxy-N-desmethyltamoxifen are described in Gamboa da Costa et al. (32).
(E)-4-[4-(2-Dimethylaminoethoxy)phenyl]-3,4-diphenylbut-3-en-2-ol (-hydroxy-tamoxifen)
This metabolite was synthesized by the method of Foster et al. (46). Some of the following spectral data have not been previously reported. Mp: 122123°C (lit46 126128°C). UV (methanol): max 238 (
= 19 300 M1cm1), 272 (
= 10 700 M1cm1) nm. 1H NMR (CDCl3):
1.20 (3H, d, J 6.6, CH3CH), 2.28 (6H, s, CH3N), 2.63 (2H, t, J 5.7, CH2N), 3.91 (2H, t, J 5.7, CH2O), 4.84 (1H, q, J 6.6, CH3CH), 6.55 (2H, d, J 8.4, alkOPhH), 6.81 (2H, d, J 8.4, alkOPhH), 7.207.37 (10H, m, PhH). 13C NMR (CDCl3):
22.43 (CH3CH), 45.81 (CH3N), 58.19 (CH2N), 65.59 (CH2O), 68.06 (CH3CH), 113.34 (ArCH), 126.52 (ArCH), 126.96 (ArCH), 127.72 (ArCH), 128.24 (ArCH), 129.50 (ArCH), 131.06 (ArCH), 131.35 (ArCH), 134.48 (Cquat), 138.37 (Cquat), 140.58 (Cquat), 141.50 (Cquat), 141.94 (Cquat), 156.98 (Cquat). MS (FAB): m/z388 (MH+), 370 (MH+-H2O), 72 [CH2CH2N(CH3)2+], 58 [CH2N(CH3)2+]. Anal. Calcd. for C26H29NO2: C, 80.59%; H, 7.54%; N, 3.61%. Found: C, 80.50%; H, 7.59%; N, 3.47%.
(Z)-1-[4-(2-Methylaminoethoxy)phenyl]-1,2-diphenylbut-1-ene (N-desmethyltamoxifen)
The synthetic methodology was based on an N-dealkylation procedure described by Olofson et al. (47). Briefly, tamoxifen (500 mg, 1.35 mmol) was dissolved in 1,2-dichloroethane (25 ml) and the solution was cooled in an ice-bath. A solution of 1-chloroethyl chloroformate (730 µl, 5 molar equivalents) in 1,2-dichloroethane (7.5 ml) was added dropwise over a period of 10 min and the mixture was refluxed overnight. Following evaporation of the solvent, the residue was redissolved in methanol (20 ml) and refluxed for 45 min. The solvent then was evaporated, and the crude mixture was washed three times with n-hexane and dried under vacuum. N-Desmethyltamoxifen was recovered as the corresponding hydrochloride salt (516 mg, 97%). Mp: 226227°C. UV (methanol): max 235 (
= 18 800 M1cm1), 276 (
= 11 800 M1cm1) nm. 1H NMR (DMSO-d6):
0.84 (3H, t, J 7.2, CH3CH2), 2.37 (2H, q, J 7.2, CH3CH2), 2.54 (3H, s, CH3NH), 3.21 (2H, t, J 5.1, CH2N), 4.09 (2H, t, J 5.1, CH2O), 6.65 (2H, d, J 9.0, alkOPhH), 6.76 (2H, d, J 9.0, alkOPhH), 7.107.30 (8H, m, PhH), 7.38 (2H, t, J 7.2, PhH), 9.02 (2H, bs, NH2+). 13C NMR (DMSO-d6):
13.31 (CH3CH2), 28.53 (CH3CH2), 32.68 (CH3N), 47.20 (CH2N), 62.99 (CH2O), 113.62 (ArCH), 126.23 (ArCH), 126.71 (ArCH), 127.96 (ArCH), 128.31 (ArCH), 128.94 (ArCH), 129.35 (ArCH), 131.38 (ArCH), 135.64 (Cquat), 137.78 (Cquat), 140.96 (Cquat), 141.73 (Cquat), 143.17 (Cquat), 155.65 (Cquat). MS (EI): m/z 357 (M+-HCl), 300 (M+-HCl-CH2CH2NHCH3+1), 285 (M+-HCl-CH2CH2NHCH3-CH3+1), 58 [(CH2CH2NHCH3)+], 44 [(CH2NHCH3)+]. Anal. Calcd. for C25H27NO.HCl: C, 76.22%; H, 7.16%; N, 3.56%. Found: C, 76.12%; H, 7.13%; N, 3.50%.
(Z)-1-[4-(2-Aminoethoxy)phenyl]-1,2-diphenylbut-1-ene (N,N-didesmethyltamoxifen)
N,N-Didesmethyltamoxifen was synthesized from (E,Z)-1-(4-hydroxyphenyl)-1,2-diphenylbut-1-ene, which was prepared from 4-hydroxybenzophenone using super-base metalated propylbenzene (48).
(E,Z)-1-(4-Hydroxyphenyl)-1,2-diphenylbut-1-ene
To a stirred suspension of 95% potassium t-butoxide (2.6 g, 22 mmol) in n-hexane (13 ml), kept under nitrogen at room temperature, were added sequentially n-propylbenzene (3 ml, 21.5 mmol), n-butyllithium (1.6 M in hexane, 12 ml, 19.2 mmol) and N,N,N',N'-tetramethylethylenediamine (6.5 ml, 43 mmol). The ensuing red suspension was stirred at room temperature for an additional period of 30 min and then cooled to 70°C. A solution of 4-hydroxybenzophenone (2.5 g, 12.6 mmol) in tetrahydrofuran (20 ml) was subsequently added over ~45 min, and the mixture was allowed to reach room temperature. After 2 h, the reaction was quenched by addition of a saturated ammonium chloride solution (350 ml) and the organic materials were extracted with methylene chloride. The crude mixture of diastereomeric carbinols was dehydrated by treatment with a 1:1 solution of methanol and 25% H2SO4. Following flash chromatography on silica gel H (Type 60; E. Merck, Darmstadt, Germany), using methylene chloride as the eluent, 1-(4-hydroxyphenyl)-1,2-diphenylbut-1-ene (2.28g, 60%) was isolated as a 1:1 mixture of the E and Z isomers. 1H NMR (acetone-d6): 0.89 (3H, t, J 7.5, CH3CH2, Z), 0.91 (3H, t, J 7.5, CH3CH2, E), 2.43 (2H, q, J 7.5, CH3CH2, Z), 2.51 (2H, q, J 7.5, CH3CH2, E), 6.49 (2H, d, J 8.4, HOPhH, Z), 6.71 (2H, d, J 8.4, HOPhH, Z), 6.836.91 (m, ArH), 6.957.00 (m, ArH), 7.077.16 (m, ArH), 7.187.38 (m, ArH), 8.15 (1H, s, OH, Z), 8.39 (1H, s, OH, E). 13C NMR (acetone-d6):
13.74 (CH3CH2), 29.52 (CH3CH2), 115.03 (ArCH), 115.74 (ArCH), 126.37 (ArCH), 126.79 (ArCH), 127.32 (ArCH), 128.05 (ArCH), 128.54 (ArCH), 128.58 (ArCH), 128.91 (ArCH), 130.03 (ArCH), 130.46 (ArCH), 131.21 (ArCH), 131.38 (ArCH), 132.52 (ArCH), 135.05 (Cquat), 135.47 (Cquat), 139.60 (Cquat), 139.72 (Cquat), 141.53 (Cquat), 142.27 (Cquat), 143.23 (Cquat), 143.29 (Cquat), 144.45 (Cquat), 144.76 (Cquat), 156.21 (Cquat), 157.07 (Cquat). Anal. Calcd. for C22H20O: C, 87.96%; H, 6.71%. Found: C, 87.61%; H, 6.91%.
N,N-Didesmethyltamoxifen
An excess of potassium hydroxide (20 g, 357 mmol) was added to a stirred solution of (E,Z)-1-(4-hydroxyphenyl)-1,2-diphenylbut-1-ene (1 g, 2.54 mmol) in dry dioxane:toluene (1:3, 20 ml) and the mixture was refluxed. 2-Chloroethylamine hydrochloride (1.5 g, 12.9 mmol) was then added portionwise during 3 h and the mixture was further refluxed for 3 h and then poured into ice water. Following extraction with methylene chloride, the organic layer was washed with 1 M sodium hydroxide and dried over anhydrous sodium sulfate. The product was separated from the E isomer by TLC on silica gel (Merck) by eluting twice with 15% triethylamine in methylene chloride. N,N-Didesmethyltamoxifen was recrystallized from ethanolic hydrogen chloride as the corresponding hydrochloride salt (453 mg, 47%). The elemental composition was determined for the picrate salt, which was prepared by standard precipitation from a mixture of the free base and picric acid in toluene. Mp (picrate): 185186.5°C. UV (hydrochloride, methanol): max 236 (
= 15 900 M1cm1), 275 (
= 10 200 M1cm1) nm. 1H NMR (hydrochloride, DMSO-d6):
0.84 (3H, t, J 7.2, CH3CH2), 2.36 (2H, q, J 7.2, CH3CH2), 3.11 (2H, bs, CH2N), 4.01 (2H, bs, CH2O), 6.64 (2H, d, J 7.8, alkOPhH), 6.76 (2H, d, J 7.8, alkOPhH), 7.137.30 (8H, m, PhH), 7.37 (2H, t, J 7.1, PhH), 8.09 (3H, bs, NH3+). 13C NMR (hydrochloride, DMSO-d6):
13.31 (CH3CH2), 28.52 (CH3CH2), 38.25 (CH2N), 63.97 (CH2O), 113.61 (ArCH), 126.22 (ArCH), 126.72 (ArCH), 127.97 (ArCH), 128.31 (ArCH), 128.94 (ArCH), 129.35 (ArCH), 131.39 (ArCH), 135.59 (Cquat), 137.78 (Cquat), 140.95 (Cquat), 141.74 (Cquat), 143.16 (Cquat), 155.76 (Cquat). MS (hydrochloride, EI): m/z 343 (M+-HCl), 300 (M+-HCl-CH2CH2NH2+1), 285 (M+-HCl-CH2CH2NH2-CH3+1), 44 [(CH2CH2NH2)+]. Anal. (picrate) Calcd. for C30H28N4O8.0.25 toluene: C, 64.03%; H, 5.08%; N, 9.41%. Found: C, 63.94%; H, 4.71%; N, 9.27%.
DNA adduct standards
-Acetoxytamoxifen was prepared from
-hydroxytamoxifen and reacted with DNA using the method of Osborne et al. (25), as modified by Beland et al. (39). Following sequential extraction of unbound materials with diethyl ether and n-butanol, both of which had been presaturated with 5 mM Bis-Tris, 0.1 mM EDTA (pH 7.1), the modified DNA was precipitated with NaCl and ethanol, and redissolved in 5 mM Bis-Tris, 0.1 mM EDTA (pH 7.1) at a concentration of ~1 mg/ml. The DNA was hydrolyzed to nucleosides by treatment with DNase I, followed by alkaline phosphatase and phosphodiesterase I (49). The adducts were then partitioned into n-butanol, which had been presaturated with 5 mM Bis-Tris, 0.1 mM EDTA (pH 7.1) and the n-butanol was evaporated. The residue was redissolved in methanol and purified by HPLC, at a flow rate of 2 ml/min, using a 17 min linear gradient of 060% acetonitrile in 100 mM ammonium acetate (pH 5.7), followed by a 3 min linear gradient to 100% acetonitrile and a 5 min isocratic elution with acetonitrile. The major adduct, which has been characterized as (E)-
-(deoxyguanosin-N2-yl)tamoxifen (25), was collected and the sample was thoroughly evaporated under vacuum and weighed in a microbalance. Additional confirmation of this quantification was obtained by 1H NMR in methanol-d4, using nitromethane as an internal standard. Based upon the UV absorbance of a 1.83x105 M methanolic solution, the molar extinction coefficients of the adduct were determined to be 16 800 and 13 200 M1cm1 at 250 and 275 nm, respectively.
To obtain DNA samples with different extents of modification, a series of reactions was conducted in which the amount of -acetoxytamoxifen was varied from 1 mg/mg DNA to 0.01 µg/mg DNA. After purification, aliquots of the DNA samples from modifications conducted with 1, 0.1 and 0.01 mg
-acetoxytamoxifen/mg DNA were enzymatically hydrolyzed, as described above, and analyzed directly by HPLC without prior n-butanol extraction of the adducts. Based upon the molar extinction coefficient for (E)-
-(deoxyguanosin-N2-yl)tamoxifen, the extent of modification was 207, 58 and 3.9 adducts/104 nucleotides, respectively. Modifications conducted at 1, 0.1 and 0.01 µg
-acetoxytamoxifen/mg DNA were hydrolyzed in a similar manner and analyzed by mass spectrometry (50), which indicated binding levels of 48, 5.2 and 0.59 adducts/106 nucleotides, respectively.
-Sulfoxy-N-desmethyltamoxifen was prepared from
-hydroxy-N-desmethyltamoxifen and reacted with DNA as detailed in Gamboa da Costa et al. (32). Following extraction of unbound materials and precipitation, the modified DNA was hydrolyzed to nucleosides as described above. HPLC analysis indicated one major adduct, which has been characterized as (E)-
-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (32).
4-Hydroxytamoxifen quinone methide was prepared from (E,Z)-4-hydroxytamoxifen and reacted with DNA as described in Marques and Beland (38). Two major adducts, which have been identified as (E)- and (Z)--(deoxyguanosin-N2-yl)-4-hydroxytamoxifen (38), were detected by HPLC.
Treatment of animals
Rats were treated according to the protocol of White et al. (12). Specifically, 12 female SpragueDawley rats [Crl:COBS CD (SD) BR outbred, 8 weeks old, 199 ± 20 g, obtained from the breeding colony at the National Center for Toxicological Research] were treated by gavage with seven daily doses of tamoxifen (20 mg/kg, 54 µmol/kg, dissolved in 200 µl trioctanoin). Additional animals were treated in the same manner with equimolar doses of -hydroxytamoxifen (12 rats; 20.9 mg/kg), N-desmethyltamoxifen hydrochloride (12 rats; 21.2 mg/kg),
-hydroxy-N-desmethyltamoxifen (4 rats; 20.1 mg/kg), N,N-didesmethyltamoxifen hydrochloride (4 rats; 20.5 mg/kg) or the solvent alone (12 rats; 200 µl trioctanoin). Two additional rats, which served as positive controls for the mutation analyses, were given a single intraperitoneal injection of 150 mg/kg N-ethyl-N-nitrosourea (ENU; Sigma), administered in 2 ml phosphate-buffered saline.
Twenty-four hours after the last treatment, four animals from each of the groups treated with tamoxifen and its derivatives were killed by exposure to carbon dioxide. The livers, spleens, thymuses and uteri were quickly excised and bone marrow was aspirated from the femurs and humeri. Hepatic nuclei were isolated by the method of Basler et al. (51) and DNA was prepared from liver nuclei, spleen, thymus, uterus and bone marrow by slight modifications of the method described in Beland et al. (52) to conduct DNA adduct analyses. Four additional rats from the tamoxifen, -hydroxytamoxifen, N-desmethyltamoxifen and control groups were killed 1 and 3 months after the last treatment. DNA adduct analyses were conducted on liver DNA from these rats and the Hprt mutant frequency was assessed in spleen lymphocytes. One ENU-treated rat was killed at each of these time points for Hprt mutant frequency analysis.
In a separate experiment, eight female SpragueDawley rats (8 weeks old, 220 ± 19 g, obtained from the breeding colony at the National Center for Toxicological Research) were treated by intraperitoneal injection, according to the protocol of Pathak et al. (37), with seven daily doses of tamoxifen (20 mg/kg; 54 µmol/kg; dissolved in 200 µl trioctanoin). Eight additional rats were treated daily for 7 days with equimolar doses of -hydroxytamoxifen (20.9 mg/kg), 3-hydroxytamoxifen citrate (31.1 mg/kg), (E, Z)-4-hydroxytamoxifen (20.9 mg/kg) or the solvent (200 µl trioctanoin) alone. Twenty-four hours following the last treatment, the rats were killed. Four animals from each group were used to determine the type and extent of DNA adduct formation in the liver and uterus. The remaining rats were used to assess the induction of hepatic cytochrome P-450 and uterine peroxidase activities.
Hepatic microsomes and uterine extracts were prepared and hepatic microsomal and uterine peroxidase activities were measured as described previously (39).
32P-Post-labeling analyses
32P-Post-labeling analyses were conducted by the nuclease P1 enrichment procedure of Reddy and Randerath (53), essentially as described in Gamboa da Costa et al. (32). Briefly, 10 µg DNA was hydrolyzed with microccocal endonuclease and spleen phosphodiesterase for 3 h at 37°C and then treated for 1 h with nuclease P1. After evaporation, each sample was resuspended in water and labeled with 20 µCi carrier-free [-32P]ATP in the presence of PNK. Analysis of each labeled mixture was conducted by HPLC. Elution conditions were as follows: from 0 to 10 min, isocratic elution with 58% solvent A [1.2 M ammonium formate and 10 mM ammonium phosphate (pH 4.5)] and 42% solvent B [24% acetonitrile in 1.2 M ammonium formate and 10 mM ammonium phosphate (pH 4.5)]; from 10 to 20 min, a linear gradient to 83% solvent B; from 20 to 40 min, isocratic elution with 83% solvent B and from 40 to 60 min, a linear gradient to 100% solvent B. The eluent from the first 15 min was diverted from the detector to avoid interference from the high amounts of radioactivity associated with free 32P, unreacted [
-32P]ATP and 32P-post-labeled normal nucleotides. The adducts formed in vivo were characterized by comparison with the DNA adduct standards modified in vitro with
-acetoxytamoxifen,
-sulfoxy-N-desmethyltamoxifen and 4-hydroxytamoxifen quinone methide. The adduct levels were quantified through comparison to a 32P-post-labeled DNA adduct standard containing (E)-
-(deoxyguanosin-N2-yl)tamoxifen at a level of 5.2 adducts/106 nucleotides.
Lymphocyte Hprt mutant assay
The lymphocyte Hprt mutant assay was performed as described previously (54,55). Briefly, spleens were removed aseptically from rats, teased apart with 2526 gauge needles and washed with cold, supplemented phosphate-buffered saline to release the cells. Lymphocytes were isolated by Accu-Paque (Accurate Chemical, Westbury, NY) density-gradient centrifugation. For each sample, two sets of lymphocyte cultures were established in three 96-well microtiter plates. One set of plates was used for determining the cloning efficiency under non-selective conditions, and the other was supplemented with 2.5 µg/ml 6-thioguanine and used to select 6-thioguanine-resistant lymphocytes. Both sets of plates were incubated in a humidified atmosphere of 5% CO2 in air and after 11 days the plates were scored for clone formation (56). The cloning efficiency for each set of cultures and the frequency of 6-thioguanine-resistant lymphocytes were then calculated.
Statistical analyses
Statistical analyses were conducted by one-way ANOVA followed by Dunnett's test. Whenever necessary, the data were transformed before the analyses to maintain an equal variance, normal distribution, or both.
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Results |
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Discussion |
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Tamoxifen is also carcinogenic in experimental animals (310). In rats, tumors occur in the liver (35) and uterus (6,7,10), with the hepatic tumors being ascribed to a genotoxic mechanism resulting from the formation of tamoxifen DNA adducts (4,1115). Since the mechanism for the induction of uterine tumors in any species is not known, we have used the rat model to investigate if a genotoxic pathway could be responsible for the endometrial tumors. In order to maximize the possibility of adduct detection, the animals were also treated with tamoxifen derivatives that have been proposed to be proximate carcinogens in the metabolic activation of this anti-estrogen.
DNA adducts were readily detected in hepatic DNA after treating rats with tamoxifen by either gavage or intraperitoneal injection. The adduct levels did not differ between the two routes of administration but are ~10-fold higher than we previously reported after intraperitoneal dosing (39). In the present study, the adduct levels were quantified through comparison to a DNA standard that had been modified with tamoxifen at a known level, whereas previously (39) the levels were estimated based upon the extent of 32P incorporation and the specific activity of the [-32P]ATP used for 32P-post-labeling. These results indicate that the efficiency of labeling tamoxifen DNA adducts is ~10%, which emphasizes the importance of having well characterized DNA standards for DNA adduct quantification by 32P-post-labeling. A similar finding was reported by us when assessing DNA adduct levels from the carcinogen 4-aminobiphenyl (63). Other investigators, however, have reported a considerably higher 32P-post-labeling efficiency for tamoxifen DNA adducts when using relative adduct labeling as the method of quantitation (30).
Three DNA adducts were detected in liver DNA from rats treated with tamoxifen. As previously reported (25,28,31,32), two of these arise from -hydroxylation of tamoxifen (adduct c, Figure 2B
) and N-desmethyltamoxifen (adduct b, Figure 2B
). The identity of the third adduct (adduct a, Figure 2B
) is not known. It does not appear to result from N,N-didesmethyltamoxifen because this metabolite gave only very low binding to hepatic DNA (Table II
). A similar low level of hepatic DNA binding by N,N-didesmethyltamoxifen has been reported by Brown et al. (31). Considering the structural similarity of tamoxifen, N-desmethyltamoxifen and N,N-didesmethyltamoxifen, it is likely that the
-hydroxylation of N,N-didesmethyltamoxifen and subsequent esterification occurs in vivo. It is conceivable that the presence of a primary amino group in N,N-didesmethyltamoxifen may allow an efficient conjugation and excretion. However, adduct a could still arise from
-hydroxy-N,N-didesmethyltamoxifen, formed by an alternative pathway involving N-desmethylation of
-hydroxy-N-desmethyltamoxifen, as suggested by Phillips et al. (30). Further experiments will be necessary to test this possibility.
When tamoxifen and -hydroxytamoxifen were administered by intraperitoneal injection,
-hydroxytamoxifen bound to hepatic DNA to a 5-fold greater extent than was found for tamoxifen (Table II
). This difference is similar, although of a lower magnitude, to what has been reported previously in F344 rats treated intraperitoneally (18). When administered by gavage, the binding of
-hydroxytamoxifen to hepatic DNA was only 2-fold greater than that observed with tamoxifen (Table II
) and the binding of
-hydroxy-N-desmethyltamoxifen did not exceed that found with N-desmethyltamoxifen (Table II
). These results were unexpected because in F344 rats,
-hydroxytamoxifen gave 30-fold higher hepatic DNA adduct levels than tamoxifen when administered by gavage (30). The relatively small difference in binding of the
-hydroxy derivatives compared with tamoxifen after gavage dosing in the current experiments may be due to the use of SpragueDawley rats or the multiple dose sequence. In addition, a comparison of the results obtained after intraperitoneal versus gavage treatment indicates that decomposition of the
-hydroxy derivatives may occur when administered by gavage due to protonation of the hydroxyl functions in the acidic environment of the stomach.
Although extensive hepatic DNA adduct formation occurred with tamoxifen, N-desmethyltamoxifen and their -hydroxy metabolites, binding was not detected in other tissues. Two major pathways have been proposed for the metabolic activation of tamoxifen: sulfotransferase-catalyzed sulfation of
-hydroxytamoxifen and
-hydroxy-N-desmethyltamoxifen (2022,24) and peroxidase-catalyzed oxidation of 4-hydroxytamoxifen (3437). In the rat, the sulfation of the
-hydroxy metabolites is catalyzed by hydroxysteroid sulfotransferase a (rHSTa; 21). rHSTa is found at high levels in rat liver, but is expressed at very low levels in non-hepatic tissues (6466).
-Hydroxytamoxifen has been detected in the bile of rats administered tamoxifen (67) and the weight changes we observed in the uterus (Table I
) suggest that the administered compounds were distributed systemically. The failure to detect DNA adducts derived from
-hydroxytamoxifen or
-hydroxy-N-desmethyltamoxifen in the uterus or other non-hepatic tissues is consistent with the low levels of rHSTa in extrahepatic tissues. It also suggests that any
-sulfoxy derivatives formed in the liver are not sufficiently stable to be transported systemically.
Pathak et al. (37) reported that intraperitoneal administration of tamoxifen increased uterine peroxidase activity 10-fold, which could result in an increased oxidation of 4-hydroxytamoxifen to 4-hydroxytamoxifen quinone methide. We were unable to confirm this observation; instead of an increase in uterine peroxidase activity, we detected a 2-fold decrease after intraperitoneal administration of tamoxifen and a number of its derivatives (Table III), which is very similar to what we previously found after treating rats with tamoxifen by gavage (39). This decrease in peroxidase activity is consistent with the anti-estrogenicity of tamoxifen for the rat uterus (68). We also obtained no evidence for DNA adducts being formed from 4-hydroxytamoxifen quinone methide in either the liver or uterus. Fan et al. (69) have suggested that 4-hydroxytamoxifen quinone methide has unusual stability; nonetheless, even if this metabolite is formed in vivo, it either does not have sufficient reactivity or is trapped by other nucleophiles before reacting with DNA.
Although we did not detect DNA adducts indicative of -hydroxytamoxifen,
-hydroxy-N-desmethyltamoxifen or 4-hydroxytamoxifen in non-hepatic tissues, it is possible that DNA damage was occurring that was not detected by the 32P-post-labeling analyses. For example, tamoxifen and certain of its metabolites have been proposed to lead to oxidative DNA damage (e.g. 8-oxodeoxyguanosine; 70,71), a type of DNA adduct that would not be detected by our methodology. To evaluate the possibility of other types of DNA damage being formed extrahepatically, we assessed the mutant frequency in the Hprt gene of spleen lymphocytes from rats treated with tamoxifen and
-hydroxytamoxifen. When measured 1 and 3 months after the last dose, no increase in the mutant frequency was observed (Figure 4
), thus supporting the conclusion that tamoxifen and its metabolites are not genotoxic extrahepatically. As part of the mutagenesis experiment, hepatic DNA adduct levels were also determined and found to have decreased to 69% their initial levels 3 months after the last dose (Figure 3
). This decrease in adduct levels is consistent with what has been reported by Divi et al. (72) using an immunochemical approach.
In conclusion, these experiments confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through -hydroxylation. We found no evidence for DNA adduct formation through a quinone methide pathway and, furthermore, found no evidence for tamoxifen genotoxicity in non-hepatic tissues. Since tamoxifen is tumorigenic in endometrial tissue in rats, our results suggest that these tumors do not arise from the formation of tamoxifenDNA adducts. This implies that tamoxifen analogues designed to be less genotoxic than tamoxifen (e.g. toremifene) but which retain a similar estrogen/anti-estrogen profile may pose a risk for endometrial tissue.
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