N-Demethylation accompanies
-hydroxylation in the metabolic activation of tamoxifen in rat liver cells
David H. Phillips2,
Alan Hewer,
Martin N. Horton
,
Kathleen J. Cole,
Paul L. Carmichael1,
Warren Davis and
Martin R. Osborne
Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton SM2 5NG, UK
 |
Abstract
|
---|
Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via
-hydroxylation leading to modification of the N2-position of guanine in DNA and to a lesser extent the N6-position of adenine. Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifenN2-guanine adduct and the other the equivalent adduct in which the tamoxifen moiety has lost a methyl group. Treatment of rats or rat hepatocytes with N-desmethyltamoxifen gave rise to the second adduct, whereas treatment with tamoxifen or
-hydroxytamoxifen gave rise to both. Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen,
-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the
position was confirmed in experiments in which [
-D2-ethyl]tamoxifen, but not [ß-D3-ethyl]tamoxifen, produced reduced levels of DNA adducts. Tamoxifen N-oxide and
-hydroxytamoxifen N-oxide also gave rise to DNA adducts in rat liver cells, but the adduct patterns were very similar to those formed by tamoxifen and
-hydroxytamoxifen, indicating that the N-oxygen is lost prior to DNA binding. These and earlier results demonstrate that in rat liver cells in vivo and in vitro, Phase I metabolic activation of tamoxifen involves both
-hydroxylation and N-demethylation, which is followed by Phase II activation at the
-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by 32P-postlabelling is achieved with >90% labelling efficiency.
Abbreviations: [
-D2-ethyl]tamoxifen, [3,3-2H2]-(Z)-1-[4-[2-(dimethylamino)-ethoxy]phenyl]-1,2-diphenyl-1-butene; [ß-D3-ethyl]tamoxifen, [4,4,4-2H3]-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene; [D5-ethyl]tamoxifen, [3,3,4,4,4-2H5]-(Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene; PEI, polyethyleneimine; tamoxifen, (Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene.
 |
Introduction
|
---|
Tamoxifen (Figure 1
) is widely used as adjuvant therapy for breast cancer (1) and currently is receiving widespread evaluation for its ability to prevent breast cancer in high risk but healthy women (24). However, it is associated with increased risk of endometrial cancer in women (5) and it is a potent hepatocarcinogen in rats (69). The discovery that it forms DNA adducts in rat liver (10,11) has provided a means with which to elucidate the pathway of metabolic activation of tamoxifen, by characterizing the adducts and determining the ability of various tamoxifen metabolites to form them.
-Hydroxytamoxifen exhibits potent DNA-binding activity both in primary cultures of rat hepatocytes (12,13) and in rat liver in vivo (14). The DNA adduct patterns were indistinguishable from those formed by the parent compound, providing strong evidence that this metabolite is an intermediate in the pathway of activation of tamoxifen to DNA-binding products. Reaction of synthetic tamoxifen derivatives with DNA indicated that
-hydroxytamoxifen would be activated by conversion to an ester (15,16) and recent experiments strongly suggest that the active species is a sulphate ester, whose formation is catalysed by hydroxysteroid sulphotransferase (1721).
Major metabolites of tamoxifen include N-desmethyltamoxifen, tamoxifen N-oxide and 4-hydroxytamoxifen. In theory, activation of tamoxifen by
-hydroxylation could be accompanied by metabolism at other sites in the molecule. In the present study we have examined the DNA-binding properties of a number of tamoxifen metabolites and of deuterated tamoxifen, using improved conditions to resolve and identify the DNA adducts formed in rat hepatocytes in vitro and in rat liver in vivo. Some aspects of this work were published previously in abstract form (22). The involvement of both
-hydroxylation and N-demethylation in the metabolic activation of tamoxifen to DNA-binding products is demonstrated.
 |
Materials and methods
|
---|
Compounds
Tamoxifen was purchased from Sigma (Poole, UK). [D5-Ethyl]tamoxifen was synthesized as previously described (23). [
-D2-Ethyl]tamoxifen and [ß-D3-ethyl]tamoxifen were synthesized by the same procedure, using the appropriately deuterium-labelled iodoethane as a precursor.
-Hydroxytamoxifen (24), N-desmethyltamoxifen (25), tamoxifen N-oxide (26) and
-hydroxytamoxifen N-oxide (27) were kindly provided by Dr Ian Hardcastle (CRC Centre for Cancer Therapeutics at the Institute of Cancer Research). N,N-Didesmethyltamoxifen was a generous gift from Dr Elizabeth Gillam (University of Queensland).
Preparation of adducts from DNA reacted with
-acetoxytamoxifen
-Acetoxytamoxifen was reacted with DNA as described previously (15). This DNA was hydrolysed to nucleosides with DNase I, snake venom phosphodiesterase and alkaline phosphatase and subjected to HPLC. The fractions containing the four normal nucleosides were collected and the total amount of DNA was calculated from the absorbance at 260 nm. The fractions containing the tamoxifennucleoside adducts were collected and the amount of adducts was determined from the absorbance at 275 nm, assuming
= 18 000 (15).
Treatment of rats
Female Fischer F-344 rats, aged 68 weeks, were purchased from Harlan UK Ltd (Bicester, UK). Three animals per group were treated by gavage with 0.012 or 0.12 mmol/kg compound in tricaprylin (1.0 ml/kg) (Sigma, Poole, UK). Animals were killed 24 h later by cervical dislocation and their livers were removed and stored at 80°C prior to DNA isolation. The thawed livers were homogenized and DNA isolated and purified by a phenol/chloroform extraction procedure (28). The liver from each animal was processed and analysed individually.
Isolation and treatment of rat hepatocytes
Primary cultures of hepatocytes were prepared from the livers of uninduced female Fischer F-344 rats, aged 68 weeks, as previously described (12). Test compounds were added dissolved in DMSO (75 µl) to give final concentrations of 1 or 10 µM and the cultures were incubated for 18 h, after which cells were harvested and DNA was isolated (28).
32P-Postlabelling analysis
DNA isolated from rat hepatocytes or liver was subjected to 32P-post-labelling analysis using the nuclease P1 digestion method of sensitivity enhancement and the solvents for TLC on polyethyleneimine (PEI)cellulose of the labelled digests essentially as described elsewhere (13). Solvents for chromatography were: D1, 2.3 M sodium phosphate, pH 5.8; D2, 2.275 M lithium formate, 5.525 M urea, pH 3.5; D3, 0.52 M LiCl, 0.325 M TrisHCl, 5.525 M urea, pH 8.0. Chromatograms were scanned for radioactivity using an InstantImager (Canberra Packard, Pangbourne, UK). Relative levels of DNA modification were calculated from the levels of radioactivity in the DNA adduct spots detected on the postlabelling chromatograms and from the specific activity of the [
-32P]ATP used in the labelling procedure (29).
HPLC analysis of DNA adducts
Prior to HPLC, labelled digests of adducts were chromatographed on PEIcellulose in solvent D1 only. Material was eluted from the origin with 4 M pyridinium formate, pH 4.5. HPLC analysis of tamoxifenDNA adducts was carried out using the system described by Martin et al. (30) with modifications: the HPLC column used was a Jupiter 5µ C18 (250x4.6 mm) column from Phenomenex (Macclesfield, UK); the solvent system was 82% 2 M ammonium formate, pH 4.0 (solvent A), 18% acetonitrile:methanol (6:1 v/v) (solvent B) for 40 min followed by a linear gradient of 1845% solvent B for 20 min. Flow rate was 1 ml/min.
 |
Results
|
---|
DNA binding by deuterated tamoxifen
Primary cultures of rat hepatocytes were treated with tamoxifen and three different deuterated forms of tamoxifen. As expected, the same adduct pattern was observed on TLC with each substance (Figure 2B
E), because although they are isotopically substituted, they are chemically unaltered. However, deuterium substitution at specific positions of the molecule was found to affect the levels of adducts detected (Figure 3A
). The results shown are, in each case, the means ± SD of duplicate determinations of five samples of cells obtained from three separate preparations of rat hepatocytes. [D5-Ethyl]tamoxifen, in which all five hydrogen atoms in the ethyl group are substituted by deuterium, gave rise to only 37% of the adduct level formed by non-deuterated tamoxifen. Similarly, with [
-D2-ethyl]tamoxifen, in which the
position, but not the ß position, is substituted, the binding level was 30% of that produced by the non-deuterated compound. In both cases, this reduction in adduct formation was highly significant (P = 0.0002, two-tailed MannWhitney U-test). Conversely, adduct levels resulting from treatment of cells with [ß-D3-ethyl]tamoxifen were the same as with non-deuterated tamoxifen and significantly different from levels produced by the other two deuterated compounds (P = 0.0002). There was no significant difference between the adduct levels produced by [D5-ethyl]tamoxifen and [
-D2-ethyl]tamoxifen.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3. DNA adduct formation by tamoxifen and deuterium-substituted tamoxifen in rat liver cells in vitro and in vivo. (A) Adducts in primary cultures of rat hepatocytes 18 h after treatment with 10 µM compound; results are means ± SD of duplicate determinations of 36 incubations. (B) Adducts in the livers of female Fischer F-344 rats 24 h after administration of 0.12 mmol/kg compound by gavage; results are means ± SD of duplicate determinations of three animals.
|
|
Analogous experiments were carried out on liver DNA isolated from rats that had been administered the compounds by gavage. The results, shown in Figure 3B
, reproduce those obtained with rat hepatocytes. The adduct level obtained with [D5-ethyl]tamoxifen was 58% of that obtained with non-deuterated tamoxifen (significantly different, P = 0.038, two-tailed MannWhitney U-test). The adduct level obtained with [
-D2-ethyl]tamoxifen was also significantly lower (44%, P = 0.02) whereas the level produced by [ß-D3-ethyl]tamoxifen was not significantly different (118%, P = 0.71) from that formed by the non-deuterated compound.
DNA binding by demethylated tamoxifen metabolites
In a series of experiments in which DNA adduct formation by tamoxifen metabolites was investigated in primary cultures of rat hepatocytes, tamoxifen produced 117 ± 8 adducts/108 nucleotides (Figure 4A
). Adduct formation by N-desmethyltamoxifen was 53% of the level produced by tamoxifen (Figure 4A
). N,N-Didesmethyltamoxifen showed lower DNA adduct-forming activity, 12% of that obtained with tamoxifen. The DNA adduct patterns obtained with these metabolites (Figure 2F and G
) were similar to that obtained with tamoxifen itself.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4. DNA adduct formation by tamoxifen and metabolites in rat liver cells in vitro and in vivo. (A) Adducts in primary cultures of rat hepatocytes 18 h after treatment with 1 or 10 µM compound, as indicated; results are means ± SD of duplicate determinations of 36 incubations. (B) Adducts in the livers of female Fischer F-344 rats 24 h after administration of 0.012 or 0.12 mmol/kg compound by gavage, as indicated; results are means ± SD of duplicate determinations of three animals. n.d., not done.
|
|
Administration of 0.12 mmol/kg tamoxifen to female rats by gavage produced 20.8 ± 0.9 DNA adducts/108 nucleotides in the liver (Figure 4B
). Similar adduct levels were produced by N-desmethyltamoxifen (109%). The DNA adduct patterns on TLC (not shown) were broadly similar to those obtained with rat hepatocytes in vitro (Figure 2B and F
). Because quantities of N,N-didesmethyltamoxifen were limited, the compound was not tested for its DNA-binding activity in vivo.
DNA binding by N-oxidized tamoxifen metabolites
Tamoxifen N-oxide produced adducts in rat hepatocytes at a level 67% of that produced by tamoxifen (Figure 4A
). The adduct pattern on TLC (Figure 2H
) was similar to that produced by tamoxifen.
-Hydroxytamoxifen N-oxide, on the other hand, showed a much higher DNA-binding activity. At 1 µM (a 10-fold lower concentration than that used for tamoxifen, N-desmethyltamoxifen, N,N-didesmethyltamoxifen and tamoxifen N-oxide) adduct formation amounted to 295 ± 38 adducts/108 nucleotides (Figure 4A
). At 10 µM, adduct formation was 12-fold higher, at 3520 ± 404 adducts/108 nucleotides (not shown). The profile of adduct spots seen on TLC (Figure 2I
) was very similar to the patterns produced by tamoxifen and
-hydroxytamoxifen.
For comparison, the DNA-binding activity of
-hydroxytamoxifen in hepatocytes is also shown in Figure 4A
. At 1 µM, the level of adducts produced was similar to that produced by
-hydroxytamoxifen N-oxide and greater than the level resulting from a 10-fold higher concentration of tamoxifen. As previously published (12,13), the adduct pattern obtained with
-hydroxytamoxifen (Figure 2J
) bore a close resemblance to the profile obtained with tamoxifen itself.
In rat liver in vivo, tamoxifen N-oxide gave rise to an adduct level 66% of that produced by tamoxifen (Figure 4B
). The adduct pattern on TLC was, again, indistinguishable from that of the parent compound (not shown). Because only limited quantities of
-hydroxytamoxifen N-oxide were available, the compound was not tested for its DNA-binding activity in vivo.
We also tested the DNA-binding potential of
-hydroxytamoxifen in vivo. At 0.012 mmol/kg, i.e. one tenth of the dose of tamoxifen, adduct formation was 54.5 adducts/108 nucleotides, more than twice the level produced by tamoxifen at the higher dose (Figure 4B
). At an equimolar dose, the adduct level obtained with
-hydroxytamoxifen was 647 ± 140 adducts/108 nucleotides, 31 times that obtained with tamoxifen (not shown).
HPLC analysis of DNA adducts
Following a preliminary TLC step to remove unincorporated radioactivity and residual unmodified nucleotides, the labelled adducts derived from experiments with rat hepatocytes and with rats treated in vivo were subjected to HPLC analysis. DNA from tamoxifen-treated hepatocytes gave rise to several radioactive peaks, of which peaks 2 and 3 were predominant (Figure 5
). The amount of radioactivity in peak 3 was approximately twice that in peak 2 (ratio 32:68). A minor peak 1 eluted prior to these peaks. A complex pattern of minor peaks was also evident that eluted between 50 and 60 min.
-Hydroxytamoxifen produced a very similar elution profile, with the same major peaks 2 and 3 and minor peak 1. N-Desmethyltamoxifen gave rise to peak 2 but not 3 and also produced a small amount of 1. N,N-Didesmethyltamoxifen gave rise to a major adduct peak that co-eluted with peak 1 produced by tamoxifen, N-desmethyltamoxifen and
-hydroxytamoxifen. Tamoxifen N-oxide and
-hydroxytamoxifen N-oxide both produced elution profiles showing the same peaks as those produced by tamoxifen and
-hydroxytamoxifen.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5. HPLC radioactivity profiles of 32P-labelled digests of rat hepatocyte DNA after treatment with tamoxifen or one of its metabolites. Chromatography conditions were as described in the text.
|
|
HPLC analysis of the 32P-post-labelled products of the reaction of
-acetoxytamoxifen with DNA (15) gave a major peak that co-eluted with peak 3 formed by tamoxifen and a minor peak, <5% of the size of peak 3, eluted with the retention time of peak 2 (data not shown). A cluster of minor peaks also eluted between 50 and 60 min.
HPLC analysis of rat liver DNA adducts produced elution profiles (Figure 6
) that were very similar to those obtained from the experiments with primary cultures of rat hepatocytes. Peak 2 was produced by tamoxifen, N-desmethyltamoxifen, tamoxifen N-oxide and
-hydroxytamoxifen, peak 3 by tamoxifen, tamoxifen N-oxide and
-hydroxytamoxifen, and peak 1 (the major product formed by N,N-didesmethyltamoxifen in vitro) was a minor product of all four compounds. In DNA from tamoxifen-treated rats, the ratio of peak 2 to peak 3 was 29:71, very similar to that in DNA from rat hepatocytes treated in vitro. Varying amounts of radioactive material also eluted in later fractions, between 50 and 60 min.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6. HPLC radioactivity profiles of 32P-labelled digests of liver DNA from rats treated by gavage with tamoxifen or one of its metabolites. Chromatography conditions were as described in the text.
|
|
Efficiency of 32P-postlabelling of tamoxifenDNA adducts
The recovery of 32P-labelled products on TLC following 32P-postlabelling analysis, using the nuclease P1 digestion method of sensitivity enhancement, was determined using DNA that had been reacted with
-acetoxytamoxifen and the level of modification determined as described in Materials and methods. Values for the levels of DNA adducts formed were >90% of the expected values, indicating that tamoxifenDNA adducts are labelled efficiently by the procedure used.
 |
Discussion
|
---|
Adoption of a HPLC procedure modified from that developed by Martin et al. (30) has significantly improved the resolution of 32P-labelled tamoxifen-derived DNA adducts, compared with our previous procedure (15). We have used this system to make qualitative comparisons of the adducts formed by tamoxifen and its metabolites in rat liver in vivo and in rat hepatocytes in vitro. Two main peaks are obtained from DNA from tamoxifen-treated rat hepatocytes and liver (Figures 5 and 6
). The later eluting of these (peak 3, Figures 5 and 6
) co-elutes with the bisphosphate of the major product of the reaction of
-acetoxytamoxifen with DNA, product TG1 (15), which is the trans adduct of the N2-position of deoxyguanosine bonded to the
position of tamoxifen. We have found that 32P-postlabelling of the synthetic adducts of the reaction of
-acetoxytamoxifen with DNA results in >90% incorporation of radiolabel, meaning that the values calculated for the DNA-binding activities of tamoxifen and its metabolites, in this study and in previous ones (1113,15,17,20,3133), are probably close to the true values and not significant underestimations.
The other main DNA adduct (peak 2, Figures 5 and 6
) derives from N-desmethyltamoxifen. Although not demonstrated directly here, there is every expectation that this metabolite is metabolically activated by
-hydroxylation and esterification to give rise to adducts identical to those previously characterized (15,34) except for the loss of a methyl group. The key intermediate,
-hydroxy-N-desmethyltamoxifen, has been detected as a metabolite of tamoxifen incubated with rat liver microsomes (35). Indeed, the recent elegant characterization by mass spectrometry of the two main tamoxifenDNA adducts formed in rat liver (36) shows directly that the end results of the activation process are exactly what would be expected from this pathway. Although a minor product of the reaction of
-acetoxytamoxifen with DNA co-eluted with peak 2, a pathway involving
-hydroxytamoxifen only cannot account for the much larger size of peak 2, relative to peak 3, seen with DNA from tamoxifen-treated liver cells or for the specific production of peak 2 by N-desmethyltamoxifen. Further improvement of the HPLC procedure is probably desirable.
As peak 2 is produced both by N-desmethyltamoxifen and by
-hydroxytamoxifen, it appears that both N-demethylation followed by
-hydroxylation and
-hydroxylation followed by N-demethylation lead to DNA adduct formation. The current experiments also reveal the presence of a minor adduct peak on HPLC that is more polar than the two major adducts and that co-elutes with the major product formed by N,N-didesmethyltamoxifen. Thus demethylation of tamoxifen can result in the loss of both methyl groups and the results again indicate that
-hydroxylation can both precede and follow this biotransformation.
Deuterium substitution at positions involved in metabolic activation of a carcinogen is expected to result in reduction of DNA adduct levels, because of the greater bond energy of the CD bond, compared with the CH bond (37) (the deuterium isotope effect). The present experiments with deuterium-substituted tamoxifen extend and confirm our earlier studies (31). In those experiments deuteration of the molecule at both the
and ß positions reduced the genotoxicity of tamoxifen in two systems: DNA adduct formation in rat hepatocytes and micronucleus formation in MCL-5 cells. The magnitude of the reduction (2- to 3-fold) was similar to what would be expected from the observed reduced rate of metabolism at the
position (35). In the current study, specific deuterium labelling at either the
or the ß position has demonstrated that, as predicted from the proposed activation mechanism for tamoxifen (38), the reduced genotoxicity is specific to substitution at the
position; the magnitude of the effect is similar whether deuterium substitution is at the
position only or at both carbon atoms of the ethyl group, but no reduction in DNA-binding potential occurs with the ß-deuterated compound. This is equally the case in in vitro experiments in primary cultures of rat hepatocytes and in vivo experiments in which DNA adducts are formed in rat liver following oral administration of the drug.
-Hydroxytamoxifen demonstrates very high DNA-binding activity in rat liver in vivo, with adduct levels 31 times higher than those obtained with tamoxifen itself. This enhanced level is comparable with the 16-fold enhancement reported by Brown et al. (14), who administered the compounds i.p. It also lies within the range of increased binding activity of the metabolite, 25- to 50-fold relative to tamoxifen itself, observed previously in rat hepatocytes in vitro (12,13).
Tamoxifen N-oxide can be further metabolised to
-hydroxytamoxifen N-oxide, but the N-oxide can also undergo reduction to regenerate tamoxifen (39). It would seem from the current experiments that
-hydroxytamoxifen N-oxide can be reduced to
-hydroxytamoxifen and then form DNA adducts by this pathway. This conclusion is based on consideration of the possible structures of the DNA adducts that result from these metabolites. A more direct demonstration of these effects could be obtained from analysis of the metabolites formed in the incubation medium, but these confirmatory experiments have yet to be performed.
-Hydroxytamoxifen N-oxide may also form DNA adducts without the loss of the N-oxygen, because it gave rise to significantly more radioactive material in the later fractions of the chromatograms (between 50 and 60 min) than did tamoxifen, N-desmethyltamoxifen and
-hydroxytamoxifen.
Minor products of the reaction of
-acetoxytamoxifen with DNA include adducts with guanine in which the tamoxifen moiety is cis about the central double bond and trans and cis adducts formed with adenine residues in DNA (34). In the HPLC profiles obtained with rat liver DNA and rat hepatocyte DNA, the cluster of minor peaks observed to elute after the major peaks (Figures 5 and 6
) may contain such products and/or N-oxidized adducts (see above). Preliminary experiments (not shown) indicate that some minor 32P-labelled products of the reaction of
-acetoxytamoxifen with DNA elute in this region of the chromatogram. Experiments are in progress to determine the nature of the rat liver DNA products eluting between 50 and 60 min and whether they include the expected minor products of the reaction of tamoxifen activated at the
position and corresponding demethylated or N-oxide products.
In conclusion, these and earlier studies demonstrate that tamoxifen is activated in rat liver cells via
-hydroxylation, with a proportion of the pathway involving either prior or subsequent N-demethylation and, to a lesser extent, N,N-didemethylation (Figure 7
). N-Oxidation of tamoxifen appears to be reversible, so that although tamoxifen N-oxide and
-hydroxytamoxifen N-oxide give rise to DNA adducts, some of these adducts appear to lack oxygen at the N position. 4-Hydroxylation, on the other hand, is not a major pathway leading to DNA adducts in rat liver cells (30,40,41), despite the fact that 4-hydroxytamoxifen is a major metabolite and despite the prediction (38) and demonstration by chemical means (40,42) that reactive intermediates can be generated from it.
 |
Acknowledgments
|
---|
We thank Dr Karen Brown and colleagues for communicating their results to us prior to publication. This work was supported by The Cancer Research Campaign. W.D. gratefully acknowledges the receipt of a research studentship from the Institute of Cancer Research.
 |
Notes
|
---|
1 Present address: Imperial College School of Medicine, Division of Biomedical Sciences, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, UK 
2 To whom correspondence should be addressed Email: davidp{at}icr.ac.uk 
Deceased. Formerly of CRC Centre for Cancer Therapeutics, Institute of Cancer Research,Cotswold Road, Sutton SM2 5NG, UK 
 |
References
|
---|
-
Early Breast Cancer Collaborative Group (1998) Tamoxifen for early breast cancer: an overview of the randomized trials. Lancet, 351, 14511467.[ISI][Medline]
-
Fisher,B., Costantino,J.P., Wickerham,D.L. et al. (1998) Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl Cancer Inst., 90, 13711388.[Abstract/Free Full Text]
-
Powles,T., Eeles,R., Ashley,S., Easton,D., Chang,J., Dowsett,M., Tidy,A., Viggers,J. and Davey,J. (1998) Interim analysis of the incidence of breast cancer in the Royal Marsden Hospital tamoxifen randomised chemoprevention trial. Lancet, 352, 98101.[ISI][Medline]
-
Veronesi,U., Maisonneuve,P., Costa,A., Sacchini,V., Maltoni,C., Robertson,C., Rotmensz,N. and Boyle,P. (1998) Prevention of breast cancer with tamoxifen: preliminary findings from the Italian randomised trial among hysterectomised women. Italian Tamoxifen Prevention Study. Lancet, 352, 9397.[ISI][Medline]
-
IARC (1996) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 66, Some Pharmaceutical Drugs. IARC, Lyon.
-
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, 315317.[Abstract]
-
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, 39193924.[Abstract]
-
Hirsimäki,P., Hirsimäki,Y., Nieminen,L. and Payne,B.J. (1993) Tamoxifen induces hepatocellular carcinoma in rat liver: a 1-year study with two antiestrogens. Arch. Toxicol., 67, 4954.[ISI][Medline]
-
Carthew,P., Rich,K.J., Martin,E.A., De Matteis,F., Lim,C.-K., Manson,M.M., Festing,M.F.W., White,I.N.H. and Smith,L.L. (1995) DNA damage as assessed by 32P-postlabelling in three rat strains exposed to dietary tamoxifen: the relationship between cell proliferation and liver tumour formation. Carcinogenesis, 16, 12991304.[Abstract]
-
Han,X. and Liehr,J.G. (1992) Induction of covalent DNA adducts in rodents by tamoxifen. Cancer Res., 52, 13601363.[Abstract]
-
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, 21972203.[Abstract]
-
Phillips,D.H., Carmichael,P.L., Hewer,A., Cole,K.J. and Poon,G.K. (1994)
-Hydroxytamoxifen, a metabolite of tamoxifen with exceptionally high DNA-binding activity in rat hepatocytes. Cancer Res., 54, 55185522.[Abstract]
-
Phillips,D.H., Carmichael,P.L., Hewer,A., Cole,K.J., Hardcastle,I.R., Poon,G.K., Keogh,A. and Strain,A.J. (1996) Activation of tamoxifen and its metabolite
-hydroxytamoxifen to DNA-binding products: comparisons between human, rat and mouse hepatocytes. Carcinogenesis, 17, 8894.
-
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, 527534.[ISI][Medline]
-
Osborne,M.R., Hewer,A., Hardcastle,I.R., Carmichael,P.L. and Phillips,D.H. (1996) Identification of the major tamoxifendeoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen. Cancer Res., 56, 6671.[Abstract]
-
Dasaradhi,L. and Shibutani,S. (1997) Identification of tamoxifenDNA adducts formed by
-sulfate tamoxifen and
-acetoxytamoxifen. Chem. Res. Toxicol., 10, 189196.[ISI][Medline]
-
Davis,W., Venitt,S. and Phillips,D.H. (1998) The metabolic activation of tamoxifen and
-hydroxytamoxifen to DNA binding species in rat hepatocytes proceeds via sulphation. Carcinogenesis, 19, 861866.[Abstract]
-
Shibutani,S., Dasaradhi,L., Terashima,I., Banoglu,E. and Duffel,M.W. (1998) Alpha-hydroxytamoxifen is a substrate of hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen DNA adducts. Cancer Res., 58, 647653.[Abstract]
-
Glatt,H., Bartsch,I., Christoph,S. et al. (1998) Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems. Chem. Biol. Interact., 109, 195219.[ISI][Medline]
-
Glatt,H., Davis,W., Meinl,W., Hermersdorfer,H., Venitt,S. and Phillips,D.H. (1998) Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture. Carcinogenesis, 19, 17091713.[Abstract]
-
Shibutani,S., Shaw,P.M., Suzuki,N., Dasaradhi,L., Duffel,M.W. and Terashima,I. (1998) Sulfation of alpha-hydroxytamoxifen catalyzed by human hydroxysteroid sulfotransferase results in tamoxifenDNA adducts. Carcinogenesis, 19, 20072011.[Abstract]
-
Phillips,D.H., Carmichael,P.L., Cole,K.J. and Hewer,A. (1995) Modulation of DNA binding of tamoxifen and its metabolites in rat hepatocytes. Proc. Am. Assoc. Cancer Res., 36, 147.
-
Horton,M.N., Jarman,M. and Potter,G.A. (1994) Synthesis of [D5-ethyl]tamoxifen; a mechanistic probe of tamoxifen induced hepatic DNA adduct formation. J. Labelled Compounds Radiopharm., 34, 767772.[ISI]
-
Foster,A.B., Jarman,M., Leung,O.-T., McCague,R., Leclercq,G. and Devleeschouwer,N. (1985) Hydroxy derivatives of tamoxifen. J. Med. Chem., 28, 14911497.[ISI][Medline]
-
Jarman,M. and McCague,R. (1985) The use of octafluorotoluene and pentafluoropyridine in the synthesis of pure Z- and E-isomers of derivatives of tamoxifen {1,2-diphenyl-1-[4-(2-dimethylaminoethoxy)phenyl]butene}. J. Chem. Res. (S), 116117; (M), 13421388.
-
Foster,A.B., Griggs,L.J., Jarman,M., van Maanen,J.M. and Schulten,H.R. (1980) Metabolism of tamoxifen by rat liver microsomes: formation of the N-oxide, a new metabolite. Biochem. Pharmacol., 29, 19771979.[ISI][Medline]
-
McCague,R. and Seago,A. (1986) Aspects of metabolism of tamoxifen by rat liver microsomes. Identification of a new metabolite: E-1-[4-(2-dimethylaminoethoxy)-phenyl]-1,2-diphenyl-1-buten-3-ol N-oxide. Biochem. Pharmacol., 35, 827833.[ISI][Medline]
-
Gupta,R.C. (1984) Non-random binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl Acad. Sci. USA, 81, 69436947.[Abstract]
-
Reddy,M.V. and Randerath,K. (1986) Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 15431551.[Abstract]
-
Martin,E.A., Heydon,R.T., Brown,K., Brown,J.E., Lim,C.K., White,I.N.H. and Smith,L.L. (1998) Evaluation of tamoxifen and
-hydroxytamoxifen 32P-post-labelled DNA adducts by the development of a novel automated on-line solid-phase extraction HPLC method. Carcinogenesis, 19, 10611069.[Abstract]
-
Phillips,D.H., Potter,G.A., Horton,M.N., Hewer,A., Crofton-Sleigh,C., Jarman,M. and Venitt,S. (1994) Reduced genotoxicity of [D5-ethyl] tamoxifen implicates
-hydroxylation of the ethyl group as a major pathway of tamoxifen activation to a liver carcinogen. Carcinogenesis, 15, 14871492.[Abstract]
-
Pace,P., Jarman,M., Phillips,D.H., Hewer,A., Bliss,J.M. and Coombes,R.C. (1997) Idoxifene is equipotent to tamoxifen in inhibiting mammary carcinogenesis but forms lower levels of hepatic DNA adducts. Br. J. Cancer, 76, 700704.[ISI][Medline]
-
Osborne,M.R., Hewer,A., Davis,W., Strain,A.J., Keogh,A., Hardcastle,I.R. and Phillips,D.H. (1999) Idoxifene derivatives are less reactive to DNA than tamoxifen derivatives, both chemically and in human and rat liver cells. Carcinogenesis, 20, 293297.[Abstract/Free Full Text]
-
Osborne,M.R., Hardcastle,I.R. and Phillips,D.H. (1997) Minor products of reaction with DNA of
-acetoxytamoxifen. Carcinogenesis, 18, 539543.[Abstract]
-
Jarman,M., Poon,G.K., Rowlands,M.G., Grimshaw,R., Horton,M.N., Potter,G.A. and McCague,R. (1995) The deuterium isotope effect for the
-hydroxylation of tamoxifen by rat liver microsomes accounts for the reduced genotoxicity of [D5-ethyl]tamoxifen. Carcinogenesis, 16, 683688.[Abstract]
-
Rajaniemi,H., Rasanen,I., Koivisto,P., Peltonen,K. and Hemminki,K. (1999) Identification of the major tamoxifenDNA adducts in rat liver by mass spectroscopy. Carcinogenesis, 20, 305309.[Abstract/Free Full Text]
-
Keefer,L.K., Lijinsky,W. and Garcia,H. (1973) Deuterium isotope effect on the carcinogenicity of dimethylnitrosamine in rat liver. J. Natl Cancer Inst., 51, 299302.[ISI][Medline]
-
Potter,G.A., McCague,R. and Jarman,M. (1994) A mechanistic hypothesis for DNA adduct formation by tamoxifen following hepatic oxidative metabolism. Carcinogenesis, 15, 439442.[Abstract]
-
Mani,C., Hodgson,E. and Kupfer,D. (1993) Metabolism of the antimammary cancer antiestrogenic agent tamoxifen. II. Flavin-containing monooxygenase-mediated N-oxidation. Drug Metab. Dispos., 21, 657661.[Abstract]
-
Osborne,M.R., Davis,W., Hewer,A.J., Hardcastle,I.R. and Phillips,D.H. (1999) 4-Hydroxytamoxifen gives DNA adducts by chemical activation, but not in rat liver cells. Chem. Res. Toxicol., 12, 151158.[ISI][Medline]
-
Beland,F.A., McDaniel,L.P. and Marques,M.M. (1999) Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo. Carcinogenesis, 20, 471477.[Abstract/Free Full Text]
-
Marques,M.M. and Beland,F.A. (1997) Identification of tamoxifenDNA adducts formed by 4-hydroxytamoxifen quinone methide. Carcinogenesis, 18, 19491954.[Abstract]
Received March 17, 1999;
revised June 14, 1999;
accepted June 14, 1999.