Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

Frederick A. Beland2, L. Patrice McDaniel1 and M. Matilde Marques1

Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA and
1 Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Avenida Rovisco Pais, P-1049-001 Lisboa, Portugal


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study, we compared the extent of DNA adduct formation in female Sprague–Dawley rats treated by gavage with seven daily doses of 54 µmol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of {alpha}-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague– Dawley rats.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Tamoxifen is an important adjunct chemotherapeutic agent for treating women with breast cancer (1). This non-steroidal antiestrogen has also been shown to decrease breast cancer incidence in healthy women at high risk for the disease (2). In spite of these benefits, tamoxifen is known to be a liver carcinogen in rats (35) and has been found to increase the risk of endometrial cancer in women (6–12; reviewed in refs 1,13,14). In addition, the occurrence of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggests that it may be genotoxic to humans (1517).

One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide (18–22; Figure 1Go, pathway B). We have recently shown that 4-hydroxytamoxifen quinone methide reacts with DNA to form covalent adducts (23). The major products, which resulted from 1,8-addition of the exocyclic nitrogen of deoxyguanosine to the conjugated system of 4-hydroxytamoxifen quinone methide, were characterized as (E)- and (Z)-{alpha}-(deoxyguanosin-N2-yl)-4-hydroxytamoxifen (23). We have now compared the extent of DNA adduct formation in female Sprague–Dawley rats treated by gavage with tamoxifen and 4-hydroxytamoxifen. We have also assessed the effect of tamoxifen and 4-hydroxytamoxifen upon liver and uterine weights and microsomal enzyme activities.



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Fig. 1. Proposed metabolic activation pathways for tamoxifen. Only the major DNA adducts resulting from {alpha}-hydroxytamoxifen and 4-hydroxytamoxifen quinone methide are shown.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Instrumentation
Reversed phase HPLC analyses were conducted using a µBondapak C18 column (0.39x30 cm; Waters Associates, Milford, MA) on an HPLC system consisting of two Waters Model 510 pumps, a Rheodyne Model 7125 injector (Rheodyne, Cotati, CA) and a Waters Model 660 automated gradient controller. The peaks were monitored at 280 nm with a Hewlett-Packard 1050 diode array spectrophotometric detector (Hewlett-Packard, Wilmington, DE). When conducting 32P-post-labeling analyses by HPLC, 32P was monitored with a Radiomatic Flo-One Model A-500 on-line radioactivity detector (Packard Instruments, Meriden, CT).

Chemicals
Tamoxifen, salmon testes DNA, bis[2-hydroxyethyl]imino tris[hydroxymethyl] methane (Bis-Tris), tricaprylin, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, Folin and Ciocalteu's phenol reagent, micrococcal nuclease, spleen phosphodiesterase, nuclease P1, potato apyrase and horseradish peroxidase were purchased from Sigma (St Louis, MO). T4 polynucleotide kinase, bovine serum albumin and the sodium salt of NADPH were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Carrier-free [{gamma}-32P]ATP was purchased from ICN Pharmaceutical (Irvine, CA). p-Nitrophenol, p-nitrocatechol, 4-dimethylaminoantipyrine and 4-aminoantipyrine were acquired from Aldrich (Milwaukee, WI).

4-Hydroxytamoxifen was obtained as a mixture (~1:1) of the cis and trans isomers by condensation of 4-hydroxy-4'-[2-(dimethylamino)ethoxy]benzophenone with the {alpha}-carbon anion from propylbenzene, followed by dehydration of the resulting diastereomeric carbinols (24). 4-Hydroxytamoxifen quinone methide was prepared by oxidation of cis/trans-4-hydroxytamoxifen with an excess of freshly prepared active manganese dioxide (23). {alpha}-Hydroxytamoxifen was synthesized by the method of Foster et al. (25) and converted to {alpha}-acetoxytamoxifen as described by Osborne et al. (26).

DNA standards
An acetone solution containing 4-hydroxytamoxifen quinone methide was added to salmon testes DNA that had been dissolved in 5 mM Bis-Tris, 0.1 mM EDTA, pH 7.1 (23). The mixture was protected from light and incubated overnight at 37°C. Following evaporation of the organic solvent, decomposition products were extracted sequentially with diethyl ether and n-butanol, both of which had been presaturated with 5 mM Bis-Tris, 0.1 mM EDTA, pH 7.1, and the 4-hydroxytamoxifen-modified DNA was precipitated with NaCl and ethanol. The modified DNA was dissolved in 5 mM Bis-Tris, 0.1 mM EDTA, pH 7.1, and an aliquot was hydrolyzed with DNase I, alkaline phosphatase and snake venom phosphodiesterase (23). Analysis by HPLC indicated two major adducts that have been identified as (E)- and (Z)-{alpha}-(deoxyguanosin-N2-yl)-4-hydroxytamoxifen (23).

{alpha}-Acetoxytamoxifen was reacted with DNA by the procedure of Osborne et al. (26) and the modified DNA was purified as described above. Enzymatic hydrolysis followed by HPLC analysis indicated a profile consisting of one major and three minor adducts that had UV spectra consistent with those reported by Osborne et al. (26,27). The major adduct has been identified as (E)-{alpha}-(deoxyguanosin-N2-yl)-tamoxifen (26), while the minor adducts have been characterized as diasteroisomers of (Z)-{alpha}-(deoxyguanosin-N2-yl)-tamoxifen and {alpha}-(deoxyadenosin-N6-yl)-tamoxifen (27).

Reaction of DNA with 4-hydroxytamoxifen in the presence of horseradish peroxidase
4-Hydroxytamoxifen (5 mg) was incubated with 8 mg of salmon testes DNA, 116 U of horseradish peroxidase and 20 µl of 30% hydrogen peroxide in 8 ml of 5 mM Bis-Tris, 0.1 mM EDTA, pH 7.1. The mixture was protected from light and incubated overnight at 37°C. The decomposition products were extracted and the modified DNA was precipitated as described above.

Treatment of animals
Rats were treated according to the protocol of White et al. (28). Specifically, eight female Sprague–Dawley rats [Crl:COBS CD (SD) BR outbred, 8 weeks old, 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 tricaprylin). Eight additional rats were treated daily for 7 days with 4-hydroxytamoxifen (21 mg/kg, 54 µmol/kg, dissolved in 200 µl tricaprylin) and eight control animals were treated with 200 µl tricaprylin alone. Twenty-four hours following the last treatment, the rats were killed by exposure to carbon dioxide. Half of the animals were used to assess the induction of hepatic cytochrome P-450 and uterine peroxidase activities. The remaining rats were used to determine the DNA adducts formed in vivo.

Hepatic microsomes were prepared by differential centrifugation (29). Uteri were removed as described by Lyttle and DeSombre (30) and uterine extracts were obtained in 250 mM sucrose, 500 mM CaCl2, according to the procedure of Pathak et al. (22). Protein content was determined by the Lowry procedure (31), using bovine serum albumin as the standard. To assess in vivo DNA adduct formation, hepatic nuclei were isolated by the method of Basler et al. (32) and DNA was prepared from the nuclei and whole uteri by slight modifications of the method described in Beland et al. (33).

32P-post-labeling analyses
32P-post-labeling analyses were conducted by the nuclease P1 enrichment procedure of Reddy and Randerath (34). Briefly, 10 µg DNA was hydrolyzed with micrococcal endonuclease and spleen phosphodiesterase for 3 h at 37°C and then treated for an additional 1 h with nuclease P1. The samples were evaporated in a Speed-Vac concentrator and resuspended in water for labeling with 20 µCi carrier-free [{gamma}-32P]ATP in the presence of T4 polynucleotide kinase. The incubation volume was 20 µl. In preliminary experiments, apyrase was added at the end of the incubation with T4 polynucleotide kinase to destroy excess [{gamma}-32P]ATP. This did not improve the analyses. Likewise, incubations were also conducted with 2 µCi carrier-free [{gamma}-32P]ATP and an incubation volume of 5 µl. Again, the analyses were not improved. Each analysis included DNA standards prepared from reactions with {alpha}-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide.

Aliquots of the labeling mixture were analyzed by HPLC, which was conducted by a modification of the method of Möller and Zeisig (35). Specifically, adducts were separated on a 5 µ DeltaPak C18-100 column (3.9x150 mm; Waters Associates) using a gradient of 100% solvent A for 5 min, followed by a 35 min linear gradient to 100% solvent B and then an isocratic elution with solvent B for 20 min. Solvent A was 1.2 M ammonium formate, 10 mM ammonium phosphate, pH 4.5; solvent B was 24% acetonitrile in 1.2 M ammonium formate, 10 mM ammonium phosphate, pH 4.5. The flow rate was 1 ml/min.

In vitro enzymatic incubations
Uterine peroxidase activity was determined at 510 nm by oxidation of 4-aminoantipyrine in the presence of hydrogen peroxide (36). The hydrogen peroxide was dissolved in 50 mM Bis-Tris, 1 mM EDTA, pH 7.1, instead of the recommended phosphate buffer to prevent interference in the absorbance measurements due to calcium phosphate precipitation.

Hepatic microsomal ethoxyresorufin metabolism (a measure of cytochrome P-450 1A1/1A2) was determined by the procedure of Burke and co-workers (37,38). Analysis of p-nitrophenol metabolism (a measure of cytochrome P-450 2E1) was by the method of Reinke and Moyer (39). Determination of 4-dimethylaminoantipyrine metabolism (a measure of cytochrome P-450 3A4) was based on the procedure of Nash (40), as modified by Cochin and Axelrod (41).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
Organ weight changes in rats administered tamoxifen or 4-hydroxytamoxifen
Female Sprague–Dawley rats were treated by gavage daily for 7 days with tamoxifen, 4-hydroxytamoxifen or the solvent alone. When assessed 24 h after the last treatment, there were no significant differences in liver weights between any of the treatment groups (Table IGo); however, in rats treated with tamoxifen and 4-hydroxytamoxifen, the uterine weights were decreased by 25% (P < 0.05) compared with the solvent control group.


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Table I. Liver and uterus weights in female Sprague–Dawley rats treated with tamoxifen or 4-hydroxytamoxifena
 
4-Hydroxytamoxifen is ~100-fold more potent than tamoxifen as an antiestrogen (42,43), with most of this activity being attributed to the trans-isomer [i.e. (Z)-4-hydroxytamoxifen (42,43)]. The observed decrease in uterine weight with tamoxifen and 4-hydroxytamoxifen is consistent with their antiestrogenic activities (44,45). 4-Hydroxytamoxifen is a relatively minor metabolite of tamoxifen, with the major metabolic pathway being N-demethylation to N-desmethyltamoxifen (46,47). Since tamoxifen and 4-hydroxytamoxifen had nearly identical effects on uterine weight, this indicates that only a small proportion of the administered 4-hydroxytamoxifen reached the uterus.

DNA adduct analyses in rats administered tamoxifen or 4-hydroxytamoxifen
DNA adducts formed in vivo were assessed by 32P-post-labeling analyses in combination with HPLC. Three DNA adducts, with retention times of 44, 46 and 48 min (peaks 1–3) and an approximate ratio of 1:5:4, were detected in liver DNA from rats administered tamoxifen (Figure 2aGo). Liver DNA from rats administered 4-hydroxytamoxifen had two adducts (Figure 2bGo) that corresponded in retention time to the two major adducts detected in liver DNA from rats given tamoxifen. The adduct levels in the liver of 4-hydroxytamoxifen-treated rats were 20-fold lower than those in the tamoxifen-treated animals (Table IIGo) and did not differ significantly from those observed in the control rats, which also had two adducts in the same region of the chromatogram (Figure 2cGo and Table IIGo). Adduct levels in uterus DNA from rats treated with tamoxifen and 4-hydroxytamoxifen did not differ significantly from those detected in control rats (Table IIGo).



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Fig. 2. HPLC of liver DNA from female Sprague–Dawley rats administered (a) tamoxifen, (b) 4-hydroxytamoxifen or (c) the solvent and DNA modified in vitro with (d) 4-hydroxytamoxifen quinone methide or (e) {alpha}-acetoxytamoxifen. The 32P-post-labeling and elution conditions are outlined in Materials and methods; 10 µg DNA was 32P-post-labeled in (a–c), 2.6 µg DNA was used in (d) and 4.9 µg DNA was assayed in (e).

 

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Table II. DNA adduct levels in liver and uterus of female Sprague–Dawley rats treated with tamoxifen or 4-hydroxytamoxifena
 
The 48 min adduct peak (peak 3) detected in liver DNA (Figure 2aGo) had the same retention time as the major adduct detected from the in vitro reaction of {alpha}-acetoxytamoxifen with DNA (Figure 2eGo). This adduct has been identified as {alpha}-(deoxyguanosin-N2-yl)-tamoxifen (26,27,48; Figure 1Go, pathway A). The retention times of the two peaks eluting at 44 and 46 min (peaks 1 and 2) in liver DNA (Figure 2aGo) corresponded to those of the minor adducts observed with the {alpha}-acetoxytamoxifen DNA standard (Figure 2eGo). Since {alpha}-acetoxytamoxifen gave only one major adduct in vitro, it is presently unclear if the second major adduct (i.e. the 46 min peak, peak 2) observed in liver DNA resulted from a reactive ester of {alpha}-hydroxytamoxifen or from a reactive derivative of another tamoxifen metabolite.

In humans (4953) and rats (46,47,54), the major metabolic pathway for tamoxifen is through N-demethylation to give N-desmethyltamoxifen (Figure 1Go, pathway C). For example, when assessed in breast cancer patients after daily dosing for up to 2.5 years, N-desmethyltamoxifen was found in plasma at approximately twice the concentration of tamoxifen (51). Similar results have been found in human tissues (52) and in rats chronically administered tamoxifen (46). In addition to being found at higher concentrations, N-desmethyltamoxifen has a longer half-life than tamoxifen (50). N-desmethyltamoxifen is further metabolized to a number of products, one of which is {alpha}-hydroxy-N-desmethyltamoxifen [Figure 1Go, pathway C (53,54)]. Given the similarity in structure between {alpha}-hydroxytamoxifen and {alpha}-hydroxy-N-desmethyltamoxifen and the high tissue concentrations of N-desmethyltamoxifen, it is plausible that a reactive metabolite of {alpha}-hydroxy-N-desmethyltamoxifen (e.g. {alpha}-acetoxy- or {alpha}-sulfoxy-N-desmethyltamoxifen) may be responsible for the second major adduct (peak 2) detected in rats treated with tamoxifen. Since submission of this paper, Rajaniemi et al. (55) have presented data in support of this hypothesis.

The contribution of 4-hydroxytamoxifen quinone methide to the hepatic DNA adducts is uncertain. The two peaks observed in the in vitro standard [i.e. (E)- and (Z)-{alpha}-(deoxyguanosin-N2-yl)-4-hydroxytamoxifen; Figure 2dGo] did co-elute with the 44 and 48 min peaks (peaks 1 and 3) observed in liver DNA (Figure 2aGo); however, the low level of hepatic DNA adducts with 4-hydroxytamoxifen (Figure 2bGo and Table IIGo) indicates that 4-hydroxytamoxifen quinone methide may not be an important reactive intermediate in this tissue. Alternatively, it is possible that the low extent of binding was due to the poor absorption of 4-hydroxytamoxifen when given by gavage or to efficient detoxification of 4-hydroxytamoxifen through phase II conjugation reactions. Recently, Hardcastle et al. (56) demonstrated that {alpha},4-dihydroxytamoxifen, an oxidation product of 4-hydroxytamoxifen that may be formed in vivo in equilibrium with 4-hydroxytamoxifen quinone methide (57), reacts 12-fold better than {alpha}-hydroxytamoxifen with DNA at acidic pH. Although the structures of these adducts were not elucidated (56), they are likely to be identical to those generated from 4-hydroxytamoxifen quinone methide. Since these putative metabolites of 4-hydroxytamoxifen have the ability to react with DNA to a significant degree, the low extent of hepatic DNA binding that we detected with 4-hydroxytamoxifen indicates that this metabolic activation pathway is unlikely to make a major contribution to the adducts formed in vivo.

It should also be noted that a low level of adducts occurred in control liver (Figure 2cGo) with the same retention times as those detected in the livers of rats treated with tamoxifen (Figure 2aGo) and 4-hydroxytamoxifen (Figure 2bGo). Therefore, the hepatic DNA adducts detected following administration of tamoxifen and 4-hydroxytamoxifen may not arise from these compounds but rather may be due to these antiestrogens altering the formation of endogenous DNA adducts. Such a possibility has been suggested for the tamoxifen DNA adducts reported to be formed in human endometrial samples (58).

White et al. (28) have used 32P-post-labeling analyses in combination with TLC to examine the DNA adducts in female F344/n rats treated by gavage daily for 7 days with tamoxifen at doses of 5–45 mg/kg. Multiple adducts were detected in liver DNA, with one adduct accounting for >80% of the total. At the same dose used in the current experiment (i.e. 20 mg/kg), the total adduct level was ~75 adducts/108 nucleotides. Considering the difference in strains of rats and also the uncertainties and interlaboratory variations that occur with 32P-post-labeling analyses (59), this value is reasonably similar to the 170 adducts/108 nucleotides that we found (Table IIGo). In contrast to the work of White et al. (28), who reported one major and multiple minor adducts, we detected two adducts of nearly equal intensity, accompanied by a third minor adduct, in the livers of rats administered tamoxifen (Figure 2aGo). This discrepancy may be due to differences in the chromatographic systems (i.e. TLC versus HPLC) because, in other work from the same laboratory (60), two major adducts were found in liver DNA from tamoxifen-treated rats when the 32P-post-labeling analyses were conducted using HPLC.

White et al. (28) did not detect binding to uterus DNA, even at a tamoxifen dose of 45 mg/kg. In contrast, uterine DNA adducts have been detected by Pathak et al. (22) in Sprague–Dawley rats treated with seven 20 mg/kg i.p. injections of tamoxifen. In their work, the binding to uterus DNA was ~37-fold lower than that found in the liver, which is nearly identical to the 35-fold difference that we detected (Table IIGo). Although Pathak et al. (22) found multiple adducts in the liver, only one adduct was detected in the uterus; this adduct also appeared to be in the liver, but accounted for only a very minor amount (~3%) of the total binding. Through comparison with in vitro experiments, they suggested that this adduct arose from peroxidase activation of 4-hydroxytamoxifen. A similar metabolic activation pathway has been proposed to occur in the livers of mice treated with tamoxifen (21). While our observations are consistent with a relatively minor role of 4-hydroxytamoxifen activation in rat liver (vide supra), our experiments to date do not allow us to draw definite conclusions concerning activation pathways in the uterus. When we incubated 4-hydroxytamoxifen with DNA in vitro in the presence of horseradish peroxidase, we found an adduct profile by HPLC (data not shown) that was virtually identical to the one obtained from 4-hydroxytamoxifen quinone methide. This observation indicates that peroxidases have the ability to mediate the oxidation of 4-hydroxytamoxifen to a DNA-binding electrophile. This activation pathway may not be important in rat uterus because the adduct levels in the uteri of the tamoxifen- and 4-hydroxytamoxifen-treated rats did not differ from controls.

Induction of hepatic and uterine enzyme activities
To determine if repeated oral dosing with tamoxifen or 4-hydroxytamoxifen affected cytochrome P-450 activities, hepatic microsomal preparations were assayed for the induction of cytochrome P-450 1A1/1A2 (as measured by ethoxyresorufin deethylase activity), 2E1 (as measured by p-nitrophenol hydroxylase activity) and 3A4 (as measured by 4-dimethylaminoantipyrine demethylase activity). These assays were selected in view of studies indicating that the N-demethylation of tamoxifen to N-desmethyltamoxifen is catalyzed by cytochrome P-450 1A1 (61) and members of the 3A family (62,63), while ring oxidation to give 4-hydroxytamoxifen appears to be catalyzed primarily by cytochrome P-450 2D6 (64) and to a lesser extent by cytochrome P-450 2E1 (65). In addition, incubation of tamoxifen with cell lines containing cytochrome P-450 2E1, 3A4 or 2D6 leads to genotoxicity, although the specific genotoxic metabolites were not identified (65). None of the activities was altered to a significant extent by treatment with either tamoxifen or 4-hydroxytamoxifen (data not shown). White et al. (66) did observe an ~2-fold induction in hepatic microsomal ethoxyresorufin deethylase activity in F344 rats treated daily by gavage with tamoxifen. Our failure to find induction of ethoxyresorufin deethylase activity could be due to the difference in rat strains or to the fact that White et al. (66) administered a 2.5-fold greater dose.

In addition to measuring hepatic microsomal activities, assays were conducted on uterine peroxidase activity. Treatment with tamoxifen or 4-hydroxytamoxifen decreased uterine peroxidase activity by ~50% (Table IIIGo), a variation that was marginally significant (P = 0.06 by one-way ANOVA). This result differs from that reported by Pathak et al. (22) who found a nearly 10-fold induction of peroxidase activity in Sprague–Dawley rats treated i.p. with tamoxifen. This difference in response could be due to the difference in the route of administration.


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Table III. Uterine peroxidase activity in female Sprague–Dawley rats treated with tamoxifen or 4-hydroxytamoxifena
 

    Conclusions
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 
In this study, we confirm extensive hepatic DNA adduct formation by tamoxifen. As reported by others (26,67), an adduct resulting from {alpha}-hydroxytamoxifen is clearly present. This was accompanied by a second major adduct, the identity of which is currently unknown. DNA adducts could be detected in uterine DNA from rats treated with tamoxifen but the levels did not differ from those observed in control animals. Although sequential oxidation of tamoxifen to 4-hydroxytamoxifen and 4-hydroxytamoxifen quinone methide (and/or {alpha},4-dihydroxytamoxifen) has been proposed as a potential metabolic activation pathway for tamoxifen, the low extent of DNA binding we detected with 4-hydroxytamoxifen suggests that this is not a major pathway. This observation is important in view of the fact that 4-hydroxytamoxifen is a potent antiestrogen currently undergoing clinical trials along with its 4-phosphate derivative (68).


    Acknowledgments
 
We thank Cindy Hartwick for helping prepare this manuscript. This work was supported in part by a grant from the Office of Women's Health of the US Food and Drug Administration, by a Postgraduate Research Program administered by Oak Ridge Institute for Science and Education and by a grant from Program Praxis XXI, Fundacião para a Ciência e Tecnologia, Portugal.


    Notes
 
2 To whom correspondence should be addressed Email: fbeland{at}nctr.fda.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 References
 

  1. Early Breast Cancer Trialists' Collaborative Group (1998) Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet, 351, 1451–1467.[ISI][Medline]
  2. Ault,A. and Bradbury,J. (1998) Experts argue about tamoxifen prevention trial. Lancet, 351, 1107.[Medline]
  3. Greaves,P., Goonetilleke,R., Nunn,G., Topham,J. and Orton,T. (1993) Two-year carcinogenicity study of tamoxifen in Alderley Park Wistar-derived rats. Cancer Res., 53, 3919–3924.[Abstract]
  4. Hard,G.C., Iatropoulos,M.J., Jordan,K., Radi,L., Kaltenberg,O.P., Imondi,A.R. and Williams,G.M. (1993) Major difference in the hepatocarcinogenicity and DNA adduct forming ability between toremifene and tamoxifen in female CrI:CD (BR) rats. Cancer Res., 53, 4534–4541.[Abstract]
  5. Williams,G.M., Iatropoulos,M.J., Djordjevic,M.V. and Kaltenberg,O.P. (1993) The triphenylethylene drug tamoxifen is a strong liver carcinogen in the rat. Carcinogenesis, 14, 315–317.[Abstract]
  6. Magriples,U., Naftolin,F., Schwartz,P.E. and Carcangiu,M.L. (1993) High-grade endometrial carcinoma in tamoxifen-treated breast cancer patients. J. Clin. Oncol., 11, 485–490.[Abstract]
  7. Seoud,M.A.-F., Johnson,J. and Weed,J.C.Jr (1993) Gynecologic tumors in tamoxifen-treated women with breast cancer. Obstet. Gynecol., 82, 165–169.[Abstract]
  8. Fisher,B., Costantino,J.P., Redmond,C.K., Fisher,E.R., Wickerham,D.L., Cronin,W.M. and other NSABP contributors (1994) Endometrial cancer in tamoxifen-treated breast cancer patients: findings from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14. J. Natl Cancer Inst., 86, 527–537.[Abstract]
  9. van Leeuwen,F.E., Benraadt,J., Coebergh,J.W.W. et al. (1994) Risk of endometrial cancer after tamoxifen treatment of breast cancer. Lancet, 343, 448–452.[ISI][Medline]
  10. Cook,L.S., Weiss,N.S., Schwartz,S.M., White,E., McKnight,B., Moore,D.E. and Daling,J.R. (1995) Population-based study of tamoxifen therapy and subsequent ovarian, endometrial, and breast cancers. J. Natl Cancer Inst., 87, 1359–1364.[Abstract]
  11. Cuenca,R.E., Giachino,J., Arrendondo,M.A., Hempling,R. and Edge,S.B. (1996) Endometrial carcinoma associated with breast carcinoma: low incidence with tamoxifen use. Cancer, 77, 2058–2063.[ISI][Medline]
  12. Curtis,R.E., Boice,J.D.Jr, Shriner,D.A., Hankey,B.F. and Fraumeni,J.F.Jr (1996) Second cancers after adjuvant tamoxifen therapy for breast cancer. J. Natl Cancer Inst., 88, 832–834.[Free Full Text]
  13. IARC (1996) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Some Pharmaceutical Drugs. Tamoxifen. IARC Scientific Publications no. 66, IARC, Lyon, pp. 253–365.
  14. MacMahon,B. (1997) Overview of studies on endometrial cancer and other types of cancer in humans: perspectives of an epidemiologist. Semin. Oncol., 24 (suppl. 1), S1-122–S1-139.
  15. Hemminki,K., Rajaniemi,H., Lindahl,B. and Moberger,B. (1996) Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res., 56, 4374–4377.[Abstract]
  16. Hemminki,K., Rajaniemi,H., Koskinen,M. and Hansson,J. (1997) Tamoxifen-induced DNA adducts in leucocytes of breast cancer patients. Carcinogenesis, 18, 9–13.[Abstract]
  17. Shibutani,S., Dasaradhi,L., Sugarman,S., Grollman,A.P. and Pearl,M. (1998) Tamoxifen-derived DNA adducts in endometrial samples obtained from patients treated with tamoxifen. Proc. Am. Assoc. Cancer Res., 39, 636.
  18. Randerath,K., Bi,J., Mabon,N., Sriram,P. and Moorthy,B. (1994) Strong intensification of mouse hepatic tamoxifen DNA adduct formation by pretreatment with the sulfotransferase inhibitor and ubiquitous environmental pollutant pentachlorophenol. Carcinogenesis, 15, 797–800.[Abstract]
  19. Randerath,K., Moorthy,B., Mabon,N. and Sriram,P. (1994) Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite. Carcinogenesis, 15, 2087–2094.[Abstract]
  20. Pongracz,K., Pathak,D.N., Nakamura,T., Burlingame,A.L. and Bodell,W.J. (1995) Activation of the tamoxifen derivative metabolite E to form DNA adducts: comparison with the adducts formed by microsomal activation of tamoxifen. Cancer Res., 55, 3012–3015.[Abstract]
  21. Moorthy,B., Sriram,P., Pathak,D.N., Bodell,W.J. and Randerath,K. (1996) Tamoxifen metabolic activation: comparison of DNA adducts formed by microsomal and chemical activation of tamoxifen and 4-hydroxytamoxifen with DNA adducts formed in vivo. Cancer Res., 56, 53–57.[Abstract]
  22. Pathak,D.N., Pongracz,K. and Bodell,W.J. (1996) Activation of 4-hydroxytamoxifen and the tamoxifen derivative metabolite E by uterine peroxidase to form DNA adducts: comparison with DNA adducts formed in the uterus of Sprague–Dawley rats treated with tamoxifen. Carcinogenesis, 17, 1785–1790.[Abstract]
  23. Marques,M.M. and Beland,F.A. (1997) Identification of tamoxifen–DNA adducts formed by 4-hydroxytamoxifen quinone methide. Carcinogenesis, 18, 1949–1954.[Abstract]
  24. Olier-Reuchet,C., Aitken,D.J., Bucourt,R. and Husson,H.-P. (1995) Synthesis of tamoxifen and 4-hydroxytamoxifen using super-base-metalated propylbenzene. Tetrahedron Lett., 36, 8221–8224.[ISI]
  25. 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.[ISI][Medline]
  26. Osborne,M.R., Hewer,A., Hardcastle,I.R., Carmichael,P.L. and Phillips,D.H. (1996) Identification of the major tamoxifen–deoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res., 56, 66–71.[Abstract]
  27. Osborne,M.R., Hardcastle,I.R. and Phillips,D.H. (1997) Minor products of reaction of DNA with {alpha}-acetoxytamoxifen. Carcinogenesis, 18, 539–543.[Abstract]
  28. White,I.N.H., de Matteis,F., Davies,A., Smith,L.L., Crofton-Sleigh,C., Venitt,S., Hewer,A. and Phillips,D.H. (1992) Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells. Carcinogenesis, 13, 2197–2203.[Abstract]
  29. Howard,P.C., McManus,M.E. and Koop,D.R. (1994) The role of cytochrome P450 2C3 in rabbit liver microsomal metabolism of 1-nitropyrene and 3-nitrofluoranthene. J. Biochem. Toxicol., 9, 71–78.[ISI][Medline]
  30. Lyttle,C.R. and DeSombre,E.R. (1977) Uterine peroxidase as a marker for estrogen action. Proc. Natl Acad. Sci. USA, 74, 3162–3166.[Abstract]
  31. Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265–275.[Free Full Text]
  32. Basler,J., Hastie,N.D., Pietras,D., Matsui,S.-I., Sandberg,A.A. and Berezney,R. (1981) Hybridization of nuclear matrix attached deoxyribonucleic acid fragments. Biochemistry, 20, 6921–6929.[ISI][Medline]
  33. Beland,F.A., Fullerton,N.F. and Heflich,R.H. (1984) Rapid isolation, hydrolysis and chromatography of formaldehyde-modified DNA. J. Chromatogr., 308, 121–131.[Medline]
  34. Reddy,M.V. and Randerath,K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 1543–1551.[Abstract]
  35. Möller,L. and Zeisig,M. (1993) DNA adduct formation after oral administration of 2-nitrofluorene and N-acetyl-2-aminofluorene, analyzed by 32P-TLC and 32P-HPLC. Carcinogenesis, 14, 53–59.[Abstract]
  36. Worthington,C.C. (1993) Worthington Enzyme Manual. Worthington Biochemical Corp., Freehold, NJ, pp. 293–299.
  37. Burke,M.D. and Mayer,R.T. (1974) Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Dispos., 2, 583–588.[ISI][Medline]
  38. Burke,M.D., Thompson,S., Elcombe,C.R., Halpert,J., Haaparanta,T. and Mayer,R.T. (1985) Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem. Pharmacol., 34, 3337–3345.[ISI][Medline]
  39. Reinke,L.A. and Moyer,M.J. (1985) p-Nitrophenol hydroxylation. A microsomal oxidation which is highly inducible by ethanol. Drug Metab. Dispos., 13, 548–552.[Abstract]
  40. Nash,T. (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J., 55, 416–421.[ISI]
  41. Cochin,J. and Axelrod,J. (1959) Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmacol. Exp. Ther., 125, 105–110.[ISI]
  42. Osborne,C.K., Wiebe,V.J., McGuire,W.L., Ciocca,D.R. and DeGregorio, M.W. (1992) Tamoxifen and the isomers of 4-hydroxytamoxifen in tamoxifen-resistant tumors from breast cancer patients. J. Clin. Oncol., 10, 304–310.[Abstract]
  43. Jordan,V.C., Koch,R., Langan,S. and McCague,R. (1988) Ligand interaction at the estrogen receptor to program antiestrogen action: a study with nonsteroidal compounds in vitro. Endrocrinology, 122, 1449–1454.[Abstract]
  44. Löser,R., Seibel,K., Roos,W. and Eppenberger,U. (1985) In vivo and in vitro antiestrogenic action of 3-hydroxytamoxifen, tamoxifen and 4-hydroxytamoxifen. Eur. J. Cancer Clin. Oncol., 21, 985–990.[ISI][Medline]
  45. Carthew,P., Edwards,R.E., Nolan,B.M., Martin,E.A. and Smith,L.L. (1996) Tamoxifen associated uterine pathology in rodents: relevance to women. Carcinogenesis, 17, 1577–1582.[Abstract]
  46. Dragan,Y.P., Fahey,S., Street,K., Vaughan,J., Jordan,V.C. and Pitot,H.C. (1994) Studies of tamoxifen as a promoter of hepatocarcinogenesis in female Fischer F344 rats. Breast Cancer Res. Treat., 31, 11–25.[ISI][Medline]
  47. Lien,E.A., Solheim,E. and Ueland,P.M. (1991) Distribution of tamoxifen and its metabolites in rat and human tissues during steady-state treatment. Cancer Res., 51, 4837–4844.[Abstract]
  48. Dasaradhi,L. and Shibutani,S. (1997) Identification of tamoxifen–DNA adducts formed by {alpha}-sulfate tamoxifen and {alpha}-acetoxytamoxifen. Chem. Res. Toxicol., 10, 189–196.[ISI][Medline]
  49. 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 patients on long-term therapy for breast cancer. Drug Metab. Dispos., 23, 377–382.[Abstract]
  50. Buckley,M.M.-T. and Goa,K.L. (1989) Tamoxifen. A reappraisal of its pharmacodynamic and pharmacokinetic properties, and therapeutic use. Drugs, 37, 451–490.[ISI][Medline]
  51. Daniel,P., Gaskell,S.J., Bishop,H., Campbell,C. and Nicholson,R.I. (1981) Determination of tamoxifen and biologically active metabolites in human breast tumours and plasma. Eur. J. Cancer Clin. Oncol., 17, 1183–1189.[ISI][Medline]
  52. Murphy,C., Fotsis,T., Pantzar,P., Adlercreutz,H. and Martin,F. (1987) Analysis of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen levels in cytosol and KCl-nuclear extracts of breast tumours from tamoxifen treated patients by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM). J. Steroid Biochem., 28, 609–618.[ISI][Medline]
  53. Poon,G.K., Chui,Y.C., McCague,R., Lønning,P.E., Feng,R., Rowlands, M.G. and Jarman,M. (1993) Analysis of phase I and phase II metabolites of tamoxifen in breast cancer patients. Drug Metab. Dispos., 21, 1119–1124.[Abstract]
  54. Jarman,M., Poon,G.K., Rowlands,M.G., Grimshaw,R.M., Horton,M.N., Potter,G.A. and McCague,R. (1995) The deuterium isotope effect for the {alpha}-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D5-ethyl]tamoxifen. Carcinogenesis, 16, 683–688.[Abstract]
  55. Rajaniemi,H., Mäntylä,E. and Hemminki,K. (1998) DNA adduct formation by tamoxifen and structurally-related compounds in rat liver. Chem. Biol. Interact., 113, 145–159.[ISI][Medline]
  56. Hardcastle,I.R., Horton,M.N., Osborne,M.R., Hewer,A., Jarman,M. and Phillips,D.H. (1998) Synthesis and DNA reactivity of {alpha}-hydroxylated metabolites of nonsteroidal antiestrogens. Chem. Res. Toxicol., 11, 369–374.[ISI][Medline]
  57. Potter,G.A., McCague,R. and Jarman,M. (1994) A mechanistic hypothesis for DNA adduct formation by tamoxifen following hepatic oxidative metabolism. Carcinogenesis, 15, 439–442.[Abstract]
  58. Orton,T.C. and Topham,J.C. (1997) Correspondence re: K. Hemminki et al., Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients. Cancer Res., 56, 4374–4377, 1996. Cancer Res., 57, 4148.[Abstract]
  59. Phillips,D.H. and Castegnaro,M. (1993) Results of an interlaboratory trial of 32P-postlabelling. In Phillips,D.H., Castegnaro,M. and Bartsch,H. (eds) Postlabelling Methods for Detection of DNA Adducts. IARC Scientific Publication no. 124, IARC, Lyon, pp. 35–49.
  60. Phillips,D.H., Hewer,A., White,I.N.H. and Farmer,P.B. (1994) Co-chromatography of a tamoxifen epoxide–deoxyguanylic acid adduct with a major DNA adduct formed in the livers of tamoxifen-treated rats. Carcinogenesis, 15, 793–795.[Abstract]
  61. Furr,B.J.A. and Jordan,V.C. (1984) The pharmacology and clinical uses of tamoxifen. Pharmacol. Ther., 25, 127–205.[ISI][Medline]
  62. Mani,C., Gelboin,H.V., Park,S.S., Pearce,R., Parkinson,A. and Kupfer,D. (1993) Metabolism of the antimammary cancer antiestrogenic agent tamoxifen. I. Cytochrome P-450-catalyzed N-demethylation and 4-hydroxylation. Drug Metab. Dispos., 21, 645–656.[Abstract]
  63. Jacolot,F., Simon,I., Dreano,Y., Beaune,P., Riche,C. and Berthou,F. (1991) Identification of the cytochrome P-450 IIIA family as the enzymes involved in the N-demethylation of tamoxifen in human liver microsomes. Biochem. Pharmacol., 41, 1911–1919.[ISI][Medline]
  64. Dehal,S.S. and Kupfer,D. (1997) CYP2D6 catalyzes tamoxifen 4-hydroxylation in human liver. Cancer Res., 57, 3402–3406.[Abstract]
  65. Styles,J.A., Davies,A., Lim,C.K., De Matteis,F., Stanley,L.A., White,I.N.H., Yuan,Z.-X. and Smith,L.L. (1994) Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s. Carcinogenesis, 15, 5–9.[Abstract]
  66. White,I.N.H., Davies,A., Smith,L.L., Dawson,S. and de Matteis,F. (1993) Induction of CYP2B1 and 3A1 and associated monooxygenase activities by tamoxifen and certain analogues in the livers of female rats and mice. Biochem. Pharmacol., 45, 21–30.[ISI][Medline]
  67. Brown,K., Brown,J.E., Martin,E.A., Smith,L.L. and White,I.N.H. (1998) Determination of DNA damage in F344 rats induced by geometric isomers of tamoxifen and analogues. Chem. Res. Toxicol., 11, 527–534.[ISI][Medline]
  68. Jordan,V.C. (1995) `Studies on the estrogen receptor in breast cancer'—20 years as a target for the treatment and prevention of cancer. Breast Cancer Res. Treat., 36, 267–285.[ISI][Medline]
Received July 8, 1998; revised October 26, 1998; accepted November 4, 1998.