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Mutagenesis Advance Access originally published online on May 31, 2005
Mutagenesis 2005 20(4):297-303; doi:10.1093/mutage/gei038
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© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org

Organ specificity of DNA adduct formation by tamoxifen and {alpha}-hydroxytamoxifen in the rat: implications for understanding the mechanism(s) of tamoxifen carcinogenicity and for human risk assessment

David H. Phillips*, Alan Hewer, Martin R. Osborne, Kathleen J. Cole, Cyd Churchill and Volker M. Arlt

Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton SM2 5NG, UK


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tamoxifen is an anti-oestrogen widely used in the adjuvant therapy of breast cancer and is also used as a prophylactic to prevent the disease in high-risk women. An increased risk of endometrial cancer has been observed in both settings. In rats, tamoxifen potently induces liver carcinomas and also induces uterine tumours when given neonatally. It forms DNA adducts in rat liver via the formation of {alpha}-hydroxytamoxifen, the ultimately reactive form being generated by sulfotransferase. In order to investigate the formation of tamoxifen-derived DNA adducts in other rat tissues, female Fischer F344 or Sprague–Dawley rats were treated with tamoxifen or {alpha}-hydroxytamoxifen by gavage or by intraperitoneal injection, daily for 1, 4 or 7 days, and DNA adducts were detected by 32P-postlabelling analysis. Tamoxifen formed DNA adducts in the liver but not in other tissues (uterus, stomach, kidney, spleen and colon). {alpha}-Hydroxytamoxifen also formed adducts at high levels in liver, but with the exception of single animals (1/8) in which a low level of adducts was detected in the stomach in one case, and in the kidney in the other; it also did not give rise to adducts in other tissues. The results suggest that tamoxifen is a genotoxic carcinogen in rat liver, but a non-genotoxic carcinogen in rat uterus, making it, uniquely, a carcinogen with more than one mechanism of action. Mutagenicity experiments conducted in Salmonella typhimurium strains expressing bacterial or human N,O-acetyltransferase did not provide evidence that either {alpha}-hydroxytamoxifen or {alpha}-hydroxy-N-desmethyltamoxifen undergoes metabolic activation by acetylation. The confinement of ST2A2, the isozyme of hydroxysteroid sulfotransferase that can activate the compounds, mainly to rat liver is the possible reason for the formation of ducts in the liver but not in other organs of the rat.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Tamoxifen is an anti-oestrogenic non-steroidal compound widely used for adjuvant therapy in breast cancer (1Go). Its proven efficacy as a chemotherapeutic agent has led to its prophylactic use in the prevention of breast cancer in healthy women at high risk of developing breast cancer and it has also shown efficacy in this regard (2Go).

Despite these anticarcinogenic properties, tamoxifen is also a carcinogen. Women who take tamoxifen, whether therapeutically or prophylactically, are at significantly increased risk of endometrial cancer (2GoGo–4Go). Tamoxifen is also a potent liver carcinogen in male and female rats (5Go), and induces uterine tumours when administered to neonatal (6Go) and adult rats (7Go).

In rat liver, it has been shown that tamoxifen forms covalent DNA adducts, implying a genotoxic mechanism for its carcinogenicity in this tissue (8Go,9Go). Tamoxifen is initially activated by cytochrome P450 3A enzymes to {alpha}-hydroxytamoxifen (10Go,11Go). N-demethylation of tamoxifen is also mediated by this enzyme subfamily (12Go). Activation of {alpha}-hydroxytamoxifen, with and without concomitant N-demethylation (13GoGoGo–16Go), is mediated by sulfotransferase, specifically by an isoform of hydroxysteroid sulfotransferase, ST2A2 (17GoGo–19Go). The resulting esters generate carbocations that react with DNA; the sites of modification of DNA have been shown, using {alpha}-acetoxytamoxifen as a model electrophile, to be the exocyclic amino groups of guanine and adenine (20Go,21Go). In addition, activation of {alpha}-hydroxylated tamoxifen metabolites by acetyltransferases has been proposed (11Go,22Go), but not substantiated.

While the formation and identity of tamoxifen–DNA adducts in rat liver is well established, other organs have received less attention. In the few studies in which DNA binding in extrahepatic tissues has been investigated by 32P-postlabelling, results have been mostly negative (23GoGo–25Go), although in two studies a low level of adduct formation in the kidney was reported (23Go,26Go). Several studies in which rat uterus was investigated reported negative findings (6Go,23GoGoGoGo–27Go), although one study claimed to have evidence for adduct formation in this tissue (28Go). In contrast to these studies is a report using accelerator mass spectrometry, in which [14C]tamoxifen apparently became bound to DNA in liver, intestine, reproductive tract, spleen, lung and kidney of rats dosed orally with the compound (29Go).

The presence of tamoxifen–DNA adducts in human tissues remains controversial and debatable (30Go), with evidence for (31GoGoGo–34Go) and against (35GoGoGo–38Go) the formation of adducts in the endometrium in vivo having been presented. In vitro experiments have also provided conflicting evidence concerning the ability of human endometrial cells to metabolically activate tamoxifen to DNA binding products (36Go,39GoGoGo–42Go).

The published studies on tamoxifen–DNA adduct formation in rat tissues have used several strains of rat, and several routes of administration. In the present study, we have therefore sought to resolve the discrepancies in the results. We have investigated tamoxifen–DNA adduct formation in vivo in six rat organs, following single or multiple treatments by gavage or intraperitoneal injection, using two strains of rat. We also investigated whether {alpha}-hydroxytamoxifen and {alpha}-hydroxy-N-desmethyltamoxifen undergo metabolic activation by N,O-acetyltransferases (NATs), by testing the compounds for mutagenic activity in strains of Salmonella typhimurium expressing human or bacterial forms of NAT. We provide a rational explanation for the organ specificity of tamoxifen–DNA adduct formation in rats, and discuss the implications for human risk assessment.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
Tamoxifen (purity >99%) was purchased from Sigma-Aldrich (Poole, UK). {alpha}-Hydroxytamoxifen (43Go) was synthesized and provided by I.R.Hardcastle. {alpha}-Hydroxy-N-desmethyltamoxifen was prepared by the Kitagawa et al. (22Go) method.

Treatment of animals
Experiment 1: Female Fischer F344 rats (8 weeks old) received a single oral dose by gavage of 0.012 mmol/kg body wt (4.5 mg/kg body wt) tamoxifen (n = 2), 0.12 mmol/kg body wt (45 mg/kg body wt) tamoxifen (n = 3), 0.012 mmol/kg body wt (4.7 mg/kg body wt) {alpha}-hydroxytamoxifen (n = 2), 0.12 mmol/kg body wt (47 mg/kg body wt) {alpha}-hydroxytamoxifen (n = 3) or solvent (tricaprylin) (n = 2). The animals were killed 24 h later and their organs (liver, uterus, stomach, kidney and colon) removed and stored frozen at –80°C prior to DNA isolation.

Experiment 2: Female Fischer F344 rats (8 weeks old) received oral doses by gavage of 0.12 mmol/kg body wt tamoxifen (n = 3), 0.12 mmol/kg body wt {alpha}-hydroxytamoxifen (n = 3) or solvent (tricaprylin) (n = 3) daily for 4 days. The animals were killed 24 h after the final dose and their organs (liver, uterus, stomach, kidney and colon) removed and stored frozen prior to DNA isolation.

Experiment 3: Female Sprague–Dawley rats (8 weeks old,) received either 0.053 mmol/kg b.w. (20 mg/kg body wt) tamoxifen (n = 5), 0.133 mmol/kg body wt (50 mg/kg body wt) tamoxifen (n = 3) or vehicle control (tricaprylin) (n = 4) intraperitoneally daily for 7 days. This protocol was previously used by Pathak et al. (28Go). The animals were killed 24 h after the final dose and their organs (liver, lung, spleen, kidney, colon and uterus) removed and stored frozen prior to DNA isolation.

DNA isolation and 32P-postlabelling analysis
DNA was isolated from thawed homogenized tissues by a phenol–chloroform extraction method using conditions described previously (44Go). 32P-Postlabelling analysis was carried out as described in earlier publications (14Go,24Go,44Go). Briefly, aliquots of DNA (4 µg) were digested for 20 h with micrococcal nuclease (0.14 U) and spleen phosphodiesterase (0.6 mU) at 37°C, followed by nuclease P1 (0.24 U) for 1 h. The digests were then subjected to 32P-postlabelling by incubation with 50 µCi carrier-free [{gamma}-32P]ATP and polynucleotide kinase (6 U) for 30 min. Resolution of the labelled adducts was then carried out on PEI-cellulose thin-layer chromatography (TLC) plates using the following solvents: D1, 2.3 M sodium phosphate (pH 5.8); D2, 2.27 M lithium formate, 5.52 M urea (pH 3.5); D3, 0.52 M LiCl, 0.325 Tris–HCl and 5.52 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 chromatograms and from the specific activity of the [{gamma}-32P]ATP used in the labelling procedure (45Go). Under these conditions, the limit of detection is estimated to be 0.4 adducts/108 nt (46Go).

Samples in which adduct spots with chromatographic mobilities characteristic of tamoxifen–DNA adducts were detected were also 32P-postlabelled and run on TLC with solvent D1 only, and the origins cut out and extracted with 4 M pyridinium formate. This material was then subjected to HPLC analysis on a Phenomenex Kromosil C18, 250 x 2 mm, 5 µm column, eluted with a linear gradient of 0.2 M ammonium formate (pH 4.2) and methanol, flow rate 0.25 ml/min (37Go), as originally described by Hemminki et al. (31Go).

Bacterial mutagenicity assays
The plate incorporation assay without an exogenous metabolizing system for detecting mutagenic activity was performed, as described (47Go). The following strains of Salmonella typhimurium were used: TA1538, which expresses the endogenous S. typhimurium NAT enzyme; the acetyltransferase-deficient strain TA1538/1,8-DNP; and its derivatives DJ400 (TA1538/1,8-DNP pNAT1) and DJ460 (TA1538/1,8-DNP pNAT2), expressing human NAT1 and human NAT2, respectively (48Go). TA1538/1,8-DNP, DJ400 and DJ460 were generous gifts from P.D.Josephy (University of Guelph, Ontario, Canada). Bacterial cultures were grown in nutrient broth (with addition of 25 µg/ml ampicillin for DJ400 and DJ460) by overnight incubation for 10 h at 37°C with shaking. Test compounds were applied dissolved in DMSO (100 µl). Assays were performed at 40, 80, 160 and 240 nmol/plate of {alpha}-hydroxytamoxifen or {alpha}-hydroxy-N-desmethyltamoxifen. These concentrations were similar to those used in previous mutagenicity studies with {alpha}-hydroxytamoxifen (19Go). Control incubations were treated with DMSO only. In addition, we have used a positive control, 3-nitrobenzanthrone, at 2.5 ng/plate (0.009 nmol), whose mutagenic potency is strongly increased after metabolic activation by human NAT1 and NAT2 (49Go). Revertant colonies were counted after 2 days (TA1538) or 3 days (TA1538/1,8-DNP, DJ400, DJ460) of incubation at 37°C. The results were classified positive if the number of revertants (mean value at dose level) was increased at least 2-fold above the number of spontaneous revertant colonies. Each assay was performed twice, using four plates for controls on each occasion and three plates for treatments. Because of limited amounts of {alpha}-hydroxy-N-desmethyltamoxifen, testing at the highest concentration (240 nmol) was carried out once only.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
DNA adducts in female rats
Female rats were treated with tamoxifen and {alpha}-hydroxytamoxifen in three separate experiments. In the first, Fischer F344 rats received a single dose of the compounds by gavage; in the second, Fischer F344 rats received multiple doses of the compounds by gavage; in the third, an experiment using the protocol previously employed by Pathak et al. (28Go), Sprague–Dawley rats received multiple doses of tamoxifen intraperitoneally. We report here the results of 32P-postlabelling analyses using the nuclease P1 digestion method of sensitivity enhancement; comparable results were obtained using the butanol extraction procedure (data not shown).

Experiment 1: single dose by gavage to female Fischer rats
When rats were administered a single dose of tamoxifen by gavage, tamoxifen-derived DNA adducts were detected in liver DNA by 32P-postlabelling with TLC (Figure 1). At the lower dose (4.5 mg/kg), the mean adduct level was 3.2 adducts/108 nt, while at the 10-fold higher dose (45 mg/kg) it was 58 adducts/108 nucleotides. For {alpha}-hydroxytamoxifen the lower dose (4.7 mg/kg) gave a mean adduct level of 40 adducts/108 nucleotides, while the higher dose (47 mg/kg) gave 647 adducts/108 nucleotides. Adducts were not detected in uterus, stomach, kidney or colon after treatment with tamoxifen, at either dose. With {alpha}-hydroxytamoxifen, no adducts were detected in any of these tissues at the lower dose, but at the higher dose a low level of radioactivity was detected in the chromatographs of stomach DNA from one of the rats (~3 adducts/108 nucleotides) (Figure 1P) and of the kidney DNA of a different rat (~5 adducts/108 nucleotides) (Figure 1Q).



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  		Fig. 1.. Thin-layer chromatograms of 32P-postlabelled digests of DNA isolated from the organs of female Fischer F344 rats 24 h after treatment by gavage with: (A–E), solvent vehicle (tricaprylin); (F–J), tamoxifen (0.12 mmol/kg body wt); (K–Q), {alpha}-hydroxytamoxifen (0.12 mmol/kg body wt). Putative tamoxifen-derived DNA adducts are circled. Results shown are from Experiment 1 but those shown in A–O are similar to those obtained in Experiment 2, and those in A–J are similar to those obtained in Experiment 3.

 
HPLC analysis of 32P-postlabelled material from the ‘positive’ stomach and kidney DNA samples gave rise to a small peak eluting at 60.5 min, with the same retention time of a much larger peak obtained with liver samples from tamoxifen-treated or {alpha}-hydroxytamoxifen-treated animals (data not shown). DNA from control animals, or treated animals in which TLC spots characteristic of tamoxifen–DNA adducts were not observed, did not give rise to a radioactive peak with this retention time.

Experiment 2: multiple doses by gavage to female Fischer rats
Adducts were detected in all the livers of the rats treated with either tamoxifen (4 x 45 mg/kg/day) or {alpha}-hydroxytamoxifen (4 x 47 mg/kg/day), at levels of 121 ± 91 and 661 ± 249 adducts/108 nt, respectively. Tamoxifen-derived DNA adducts were not detected in the kidney, stomach, colon or uterus of any of the rats treated with either compound. The chromatograms (not shown) were similar to those in Figure 1A–O.

Experiment 3: multiple doses intraperitoneally to female Sprague–Dawley rats
Adducts were detected in the liver of all tamoxifen-treated rats. At the lower dose (7 x 20 mg/kg/day) adduct levels were 345 ± 199 adducts/108 nt (mean ± SD), while at the higher dose (7 x 50 mg/kg/day) they were marginally higher, at 383 ± 235 adducts/108 nt. Tamoxifen–DNA adducts were not detected in any of the other tissues examined, namely colon, lung, kidney, spleen or uterus (as with Experiment 2, the chromatograms were similar to those shown in Figure 1A–J).

Bacterial mutagenicity of tamoxifen metabolites
With the positive control, 3-nitrobenzanthrone, a strong mutagenic response was shown in strains TA1538, which expresses bacterial NAT, and DJ460, which expresses human NAT2 (Table I). A lesser, but still clearly positive, response was shown in strain DJ400, which expresses human NAT1, but no mutagenicity was observed with strain TA1538/1,8-DNP, in which bacterial NAT is deficient (Table I). The background mutation frequencies (revertant colonies/plate) in the S.typhimurium strains TA1538, TA1538/1,8-DNP, DJ400 and DJ460 were 22–30, 6–15, 5–8 and 16–23, respectively. When tested at concentrations of 40, 80, 160 and 240 nmol/plate, {alpha}-hydroxytamoxifen did not induce mutation frequencies that differed from those in plates treated with solvent only (control). Similarly, there was no observable mutagenic activity when these bacterial strains were treated with up to 240 nmol {alpha}-hydroxy-N-desmethyltamoxifen.


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  		Table I.. Mutagenic activity of {alpha}-hydroxytamoxifen and {alpha}-hydroxy-N-desmethyltamoxifen in strains of S.typhimurium

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, from a total of 18 rats treated with tamoxifen, all showed clear evidence of tamoxifen–DNA adduct formation in liver, but none showed any evidence of adduct formation in any of the other tissues examined (kidney, stomach, colon, spleen, lung and uterus). This finding is in general agreement with other studies, where multiple tissues have been investigated and reports on tamoxifen–DNA adduct formation in rat liver have generally been accompanied by evidence for lack of adduct formation in other tissues (23GoGo–25Go).

It was of particular interest to determine whether tamoxifen formed DNA adducts in rat uterus, because administration of tamoxifen to neonatal and adult rats induces tumours in this organ (7Go,50Go). In one of our experiments (experiment 3) we followed the exact protocol used in a previous study in which an adduct was reported to be formed in this tissue (28Go). We found no adducts in this tissue with either single or multiple doses of tamoxifen, administered intraperitoneally or by gavage. Thus our results are in contrast to the one positive report of adduct formation in rat uterus (28Go), but in agreement with other studies showing a lack of adduct formation either in neonatal (6Go,25Go) or adult rats (23Go,25Go). Studies of in vivo mutagenic activity support this: in BigBlue rats, tamoxifen and {alpha}-hydroxytamoxifen increased the mutation frequency in lacI in the liver, but not in the uterus (51Go).

The much higher (5–10-fold) DNA binding activity in rat liver, described here, of {alpha}-hydroxytamoxifen compared with tamoxifen is a similar effect to that observed in previous in vivo (14Go,15Go,52Go) and in vitro (14Go,44Go,53Go) studies, and reaffirms the evidence that {alpha}-hydroxytamoxifen is a key intermediate in the activation pathway of tamoxifen to DNA binding products (9Go). Although we occasionally detected putative adducts at low level in extrahepatic tissues (one stomach, one kidney in Experiment 1) these results were not reproduced in other animals or in subsequent experiments. Importantly, we obtained no positive results with uterine DNA from any rats treated with {alpha}-hydroxytamoxifen. da Costa et al. (25Go) also did not detect adducts in the uterus, spleen, thymus or bone marrow of rats treated with {alpha}-hydroxytamoxifen.

It should be noted that {alpha}-hydroxytamoxifen has a weak propensity to bind to DNA without further metabolic activation, and this activity increases at acidic pH (20Go). Thus, although we cannot be certain that the low levels of adducts detected in the stomach of one rat, and the kidney of another, following a high dose of {alpha}-hydroxytamoxifen, are in fact due to binding of this compound, conditions in these isolated cases may have favoured formation of a low level of adducts, perhaps because of acidic activation of the compound and/or a lack of adequate detoxification, for example by glucuronidation (54Go). Nevertheless, the binding levels in these instances were <1% of that occurring in the liver and animals treated with the lower dose of {alpha}-hydroxytamoxifen consistently failed to show any evidence of DNA adduct formation in tissues other than the liver.

While studies using 32P-postlabelling analysis to detect tamoxifen–DNA adducts indicate a lack of adduct formation in extrahepatic tissues, a study that used accelerator mass spectrometry reported tamoxifen-related adducts in multiple tissues of rats (29Go). The evidence came from the association of radioactivity with DNA isolated from the tissues of rats administered 14C-labelled tamoxifen, but chromatographic analysis was not presented to support these assertions, except in the analysis of liver DNA, where some of the radioactivity was associated with normal nucleotides (29Go). Where radioactively labelled test compounds are used, it has been recommended that evidence for DNA binding should include chromatographic separation of putative labelled adducts from normal nucleotides to verify that the incorporation of radiolabel is indeed due to the chemical bonding of the test compound (55Go). There is evidence that it is possible for a drug to co-precipitate with DNA, and to be non-extractable from it, even when it is not covalently bound (56Go). Thus it cannot be assumed that association of radiolabelled compound with DNA is the result of covalent binding (56Go). It has yet to be determined by chromatographic analysis whether or not the association of radioactivity with endometrial DNA, detected by accelerator mass spectrometry, from women administered a dose of 14C-labelled tamoxifen is the result of covalent binding or of some non-covalent interaction (34Go).

Although it has been suggested that activation of tamoxifen metabolites by acetylation may occur (11Go,22Go), no evidence to support this hypothesis has been advanced as yet. The bacterial mutagenicity experiments conducted in the present study do not provide evidence for activation by acetyltransferases. Previously, it was demonstrated that {alpha}-hydroxytamoxifen was mutagenic in a strain of S.typhimurium expressing rat hydroxysteroid sulfotransferase (ST2A2), at doses of 20 nmol and above (19Go), but in the present study both {alpha}-hydroxytamoxifen and {alpha}-hydroxy-N-desmethyltamoxifen, at doses up to 240 nmol, were inactive in the assay using the same parental bacterial strain engineered to express human NATs. Since NATs are expressed in many extrahepatic tissues, activation of tamoxifen by this route would be expected to result in DNA adducts in multiple tissues in rats; the fact that hydroxylated metabolites of tamoxifen are activated only by an isoform of sulfotransferase that is expressed predominantly in the liver is a plausible explanation for the detection of tamoxifen-derived DNA adducts in rat liver, but not in other tissues. Indeed, it has been shown that although the female rat liver expresses higher levels of this enzyme than male rat liver, exposure to tamoxifen results in enzyme induction in the latter (18Go), with the result that males and females are equally susceptible to DNA adduct formation (18Go) and liver tumour formation when administered tamoxifen in the long term (5Go).

Tamoxifen is thus somewhat unusual as a DNA damaging agent in this respect, by forming DNA adducts essentially in a single organ (notwithstanding some equivocal, non-reproducible, evidence for low-level adduct formation in other tissues). Most genotoxic carcinogens display DNA-damaging activity in multiple mammalian tissues, even though they may have a more limited number of target organs for carcinogenicity.

The implication of these studies is that tamoxifen is a genotoxic carcinogen in rat liver, but a non-genotoxin in rat uterus. Its potent uterotrophic activity provides a plausible mechanism for its activity in inducing uterine tumours in rats when administered neonatally (57Go). Thus tamoxifen may be a unique example of a carcinogen with more than one mechanism of carcinogenicity in one species, in this case the rat.

Such a conclusion clearly has implications for regulatory testing of chemicals and for extrapolation of experimental data from rodent studies to human risk assessment. With tamoxifen there is again an unusual situation in that large numbers of women have already been exposed to tamoxifen as an adjuvant therapy for breast cancer (1Go) and as a prophylactic in breast cancer prevention trials (2Go). In both these clinical settings, tamoxifen increases the risk of developing endometrial cancer (1Go,2Go,4Go).

While some investigators have claimed to have obtained evidence for the presence of tamoxifen-DNA adducts in human endometrium (32GoGo–34Go), we, along with others, have reported an absence of such damage in women taking the drug (36GoGo–38Go). The positive and negative studies involved different human subjects, but differences in results are not likely to be due to different limits of detection by different investigators (58Go). The issue of whether tamoxifen forms DNA adducts in the tissue and, if it does, whether it is at a sufficient level to imply that tamoxifen is a genotoxic carcinogen in humans is still a matter of controversy (8Go,9Go,30Go,34Go). The presence of low levels of tamoxifen–DNA adducts in cynomolgus monkeys has also been reported (59Go,60Go), but there are no data on the carcinogenicity of tamoxifen in this species to aid interpretation of this result. There is conflicting evidence on the extent to which tamoxifen metabolites are substrates for human sulfotransferases (19Go,61Go,62Go). However, it is noteworthy that SULT2A1, the human isoform equivalent to rat ST2A2, is not expressed in human endometrium at any stage in the oestrus cycle (63Go).

It has recently been reported that some endometrial tumours occurring in women who have taken tamoxifen or toremifene contain mutations in K-ras (64Go). Toremifene does not induce liver tumours in rats (65Go) and forms few, if any, DNA adducts under experimental conditions in which tamoxifen forms adducts at high levels (24Go,66Go). Thus it is not clear by what mechanism these mutations have arisen in human endometrium, but their occurrence with both agents implies that it may not be a consequence of direct covalent modification of DNA. Furthermore, a recent study found similar genetic alterations in tamoxifen and non-tamoxifen-associated endometrial carcinomas arising in women with breast cancer (67Go).

In conclusion, the present study demonstrates the reproducible formation of tamoxifen-DNA adducts in liver, but not in other tissues, of rats. The lack of adducts in rat uterus suggests that carcinogenesis in this organ is by a non-genotoxic mechanism, as opposed to a genotoxic mechanism of carcinogenesis in rat liver. {alpha}-Hydroxytamoxifen and {alpha}-hydroxy-N-desmethyltamoxifen are not substrates for human NATs, and while there is controversy concerning the existence of tamoxifen–DNA adducts in human endometrium, no enzymatic mechanism for the metabolic activation of tamoxifen in this tissue has yet been advanced.


   Acknowledgments
 
This study was supported by Cancer Research UK.


   Notes
 
* To whom correspondence should be addressed. Tel: +44 020 8722 4016; Fax: +44 020 8722 4052; Email: david.phillips{at}icr.ac.uk


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received on March 17, 2005; revised on April 25, 2005; accepted on April 28, 2005.


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