Mutagenesis vol. 18 no. 4 pp. 395-399,
July 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Meeting Report |
The Genotoxicity of Tamoxifen: Extent and Consequences, Kona, Hawaii, January 23, 2003
Carcinogen–DNA Interactions Section, Center for Cancer Research, Building 37, Room 4032, National Cancer Institute, 37 Convent Drive MSC-4255, National Institutes of Health, Bethesda, MD 20892–4255, USA
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The current recommended adjuvant therapy for oestrogen receptor-positive breast cancer typically includes 20 mg/day tamoxifen (Nolvadex®) for 5 years post-operatively. This regimen has been found to reduce the incidence of contralateral breast cancer in breast cancer survivors by 47%, and, when used prophylactically, to reduce new breast cancers in high risk women by 49%. However, epidemiological evidence links tamoxifen therapy to increases in endometrial cancer and thromboembolic events in breast cancer patients. In addition, in tamoxifen-exposed rats dose-related increases in hepatic tamoxifen–DNA adduct formation and liver tumour incidence occur through a classic genotoxic mechanism. In women, endometrial cancers may be the result of genotoxicity, hormonally induced signal transduction and/or other mechanisms. If genotoxicity is relevant to tamoxifen-induced endometrial cancer it may be possible to identify women at risk through detection of tamoxifen–DNA adducts. The aim of this one day conference was to examine the most recent evidence for the occurrence of tamoxifen-induced genotoxicity in women receiving tamoxifen therapy. There were significant experimental differences, as some participants presented evidence for a genotoxic mechanism, while others reported finding insufficient evidence to support a genotoxic mechanism. The discussion was wide ranging and the outcome underscored the need for further investigations, access to more human tissue samples, shared tamoxifen–DNA standards for methodological comparisons and inter-laboratory exchange of human tissue samples.
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Tamoxifen (TAM, Nolvadex®), an oestrogen analogue, has been widely and successfully used for the prevention of breast cancer. The drug received FDA approval for adjuvant treatment in 1977 and for chemoprevention in healthy ‘at risk’ women in 1998. It has been estimated that 700 000 women are presently receiving TAM therapy in the USA. The current recommended dosage for pre- and post-menopausal women with oestrogen receptor-positive breast cancer is 20 mg/day TAM for 5 years. Using this regimen, the incidence of contralateral breast cancer was reduced by 47% (Early Breast Cancer Trialists’ Collaborative Group, 1998
) and new breast cancers in ‘at risk’ women were reduced by 49% (Fisher et al., 1998). However, epidemiological evidence links TAM therapy to increases in endometrial and, rarely, uterine cancer in breast cancer patients (IARC, 1996). The relative risk (RR) of endometrial cancer in patients receiving TAM therapy is estimated to be 1.3–7.5 (Rutqvist et al., 1995; Curtis et al., 1996; Fisher et al., 1998), while prophylactic TAM use is associated with an endometrial cancer RR of 2.5 (Fisher et al., 1998; Cuzick et al., 2003). Therefore, out of 1000 women receiving TAM therapy, two or three are projected to develop endometrial cancer (Jordan and Morrow, 1994). Furthermore, recent studies have revealed TAM use to be associated with a rare increase in uterine sarcoma (Bergman et al., 2000), reported to occur in 
0.01% of women taking the drug (Twombly, 2002).
It has been well established that TAM is a potent rat hepatocarcinogen (Greaves et al., 1993). In rat liver TAM acts as a classical genotoxic chemical carcinogen by forming DNA adducts, which induce mutations in genes required for growth control (Greaves et al., 1993; Carthew et al., 1995; Phillips, 2001; White, 2001; Brown, 2002). Rats exposed to TAM have parallel dose-related increases in hepatic TAM–DNA adducts (White et al., 1992) and liver tumour incidence (Greaves et al., 1993). In contrast, hepatic TAM–DNA adduct levels are low in TAM-exposed mice (White et al., 1992; Martin et al., 1997) and mouse hepatocarcinogenicity does not occur (Tucker et al., 1984).
Two potential mechanisms of endometrial/uterine tumour induction in women include an oestrogenic pathway and a classical genotoxic pathway. As an oestrogen agonist in the uterus, TAM is able to induce signal transduction resulting in promotion of cellular proliferation. As a genotoxin, TAM can be converted to reactive intermediates capable of binding to DNA; subsequent replication on a damaged DNA template may lead to mutagenesis in critical genes and a heritable loss of growth control.
Finding TAM–DNA adducts in human endometrium/uterus would elucidate the mechanisms underlying TAM-induced carcinogenesis and possibly allow for more accurate human risk assessment. Currently there is considerable controversy in the literature regarding the putative formation of TAM–DNA adducts in human tissues. Using 32P-post-labelling based methodologies several groups have reported the formation of TAM–DNA adducts in human leucocytes (Hemminki et al., 1997; Umemoto et al., 2002) and endometrium (Hemminki et al., 1996; Shibutani et al., 1999, 2000). However, other groups using similar methods have not detected TAM-specific human DNA adducts (Carmichael et al., 1996, 1999; Phillips et al., 1996; Bartsch et al., 2000; Phillips, 2001). Humans exposed to TAM do not demonstrate an increased incidence of liver cancer and an early study reported no 32P-post-labelled TAM–DNA adduct formation in liver biopsies from several patients receiving TAM therapy (Martin et al., 1995).
The aim of this one day conference was to assemble a group of interested investigators in a collegial atmosphere to examine the most recent evidence for TAM genotoxicity in women and to consider the potential relevance of TAM–DNA adduct formation with respect to the endometrial and uterine tumours that occur in women receiving TAM therapy.
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Details regarding the clinical use of TAM were provided by J. Carl Barrett (National Cancer Institute, NIH, Bethesda, MD). In addition to endometrial adenocarcinoma and uterine sarcoma, other potential risks found in women receiving TAM therapy include stroke, pulmonary embolism, deep vein thrombosis and cataracts. A recent study (Veronesi et al., 2003
) demonstrated that TAM protects against breast cancer in women considered to be at high risk for the disease based on height, age at menarche, parity, age at first childbirth and oophorectomy; no protection was observed in women considered to be at low risk. Furthermore, Bernstein et al. (1999) demonstrated an increased risk for endometrial cancer (OR = 9) in obese women who had received oestrogen replacement therapy and were given TAM therapy for 5 years, compared with women who had not taken oestrogen replacement therapy, had a low body mass index (BMI) and received TAM therapy for 5 years (OR = 1). A second study (Ozet et al., 2001) reported that serum leptin levels in women receiving TAM therapy were almost 2-fold higher than those in BMI-matched women not receiving TAM therapy. Such studies suggest that if TAM–DNA adducts contribute to endometrial cancer, the highest levels of TAM–DNA adducts would be likely found in obese women, who have the highest endometrial cancer risk. Although oestrogenic compounds have induced genotoxicity in experimental models, evidence for this as a major mechanism for human cancers is weak. Raloxifen and toremifene, also used for breast cancer adjuvant therapy, form virtually no DNA adducts and do not cause rat liver tumours, although both presumably have oestrogenic effects in the uterus. The status of these drugs with respect to endometrial tumour induction has yet to be reported in the literature.
Classical studies of TAM-induced hepatocarcinogenesis and DNA adduct formation were summarized by Yvonne P. Dragan (National Center for Toxicological Research, FDA, Jefferson, AR). TAM is genotoxic in rat liver, causing increases in proliferation, aneuploidy, strand breaks and spindle aberrations. Chronic TAM exposure in adult rats induces both tumour and DNA adduct formation in liver. TAM is a promoter and forms a low level of DNA adducts in rat kidney, while neither DNA adduct formation nor tumourigenesis appears to occur in the uterus. In contrast, exposure of neonatal mice or rats to TAM does induce uterine tumours in adulthood (Newbold et al., 1997; Carthew et al., 2000).
The formation of TAM–DNA adducts and resulting mutagenicity in different organs of the rat were addressed by Frederick A.Beland (National Center for Toxicological Research, FDA, Jefferson, AR), who presented a study of TAM–DNA adduct formation in young adult female rats given 20 mg/kg body wt TAM. By 32P-post-labelling/HPLC, the major hepatic TAM–DNA adducts were (E)-
-(deoxyguanosin-N2-yl)-tamoxifen (dG-N2-TAM) and (E)--(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-N2-N-desmethyl-TAM). Very minor adducts were formed in rat liver after administration of the metabolites N,N-didesmethyl-tamoxifen and 4-hydroxytamoxifen (4-OH-TAM) (Gamboa da Costa et al., 2001). No TAM–DNA adducts were detected in any other rat tissue, including uterus. Examination of mutagenesis induced by TAM and -OH-TAM in the same animals indicated no significant increase in HPRT mutant frequency in spleen lymphocytes. Both TAM and -OH-TAM induced lacI and cll mutants in livers, but not uteri, of Big Blue rats (Chen et al., 2002; Gamboa da Costa et al., 2002). The mutants observed in Big Blue rat liver DNA were primarily G
T transversions. It was concluded that hepatic TAM metabolic activation in the rat involves -hydroxylation and sulphation, that 4-hydroxylation yields exceedingly low hepatic TAM–DNA adduct levels and that no TAM–DNA adduct formation occurs in non-hepatic rat tissues.
William J.Bodell (University of California, San Francisco, CA) used 32P-post-labelling to investigate TAM–DNA adduct formation in female rats given 20 mg/kg body wt TAM daily for 7 days and found one major and six minor adducts in liver DNA. Studies incubating rat liver microsomes with various cofactors indicated that TAM may be metabolized to metabolite E and 4-OH-TAM, both of which form quinone methides and minor DNA adducts (2–3% of the total) in rat liver DNA (Pathak et al., 1995, 1996). Also, in uteri of female TAM-exposed rats, these investigators detected low levels of a single adduct formed from 4-OH-TAM. What role this pathway might play in human TAM metabolism is a matter of conjecture. However, a DNA adduct with a 32P-post-labelling chromatographic profile the same as that produced by 4-OH-TAM was found in a DNA sample isolated from endometrium of a woman undergoing adjuvant TAM therapy.
A major metabolic pathway for human TAM biotransformation, the sulphation of 4-OH-TAM by the thermostable sulphotransferase 1A1 (SULT1A1), was described by Susan Nowell (National Center for Toxicological Research, FDA, Jefferson, AR). A common polymorphism in this enzyme, a GA nucleotide change resulting in substitution of histidine for arginine in the protein, has been observed in the human population. The altered allele (SULT1A1*2) produces an enzyme that is less thermostable and has 60% lower activity than the common allele (SULT1A1*1). A study of breast cancer patients receiving TAM therapy indicated that improved survival was strongly associated with patients homozygous or heterozygous for the common allele, indicating that efficient sulphation of 4-OH-TAM improves the efficacy of TAM treatment. The mechanistic basis for this is currently under investigation, however, one could speculate that TAM detoxification, accomplished by SULT1A1, reduces the concentration of drug available for genotoxic activation. The data suggest that potential TAM genotoxicity may not be relevant to TAM drug efficacy.
The question of whether or not TAM–DNA adducts are formed in human tissues may be addressed more appropriately in primates than in rats. Laura J.Schild (National Cancer Institute, NIH, Bethesda, MD) described a monkey model in which mature female cynomolgus monkeys were given either vehicle control (n = 1) or 2 mg/kg body wt TAM daily (n = 3) by naso-gastric intubation for 30 days. The tissues were shared by three laboratories and TAM–DNA adducts were measured by: TAM–DNA chemiluminescence immunoassay (TAM-DNA CIA) utilizing an antiserum elicited against DNA modified with dG-N2-TAM (Schild and Poirier) (Divi et al., 1999); an isotope dilution mass spectrometric method consisting of on-line sample preparation coupled with HPLC and electrospray tandem mass spectrometry (ES-MS/MS) (Beland and Marques) (Gamboa da Costa et al., 2003); a 32P-post-labelling/HPLC assay (Shibutani et al., 1999, 2000). For each organ examined, the TAM–DNA adduct levels determined by all three methods in a single sample were remarkably similar, differing by no more than 4-fold. The highest TAM–DNA adduct levels were found in liver and brain cortex, with values ranging from 1.5 to 9.6 TAM–DNA adducts/108 nucleotides (mean, n = 3) for all three methods. The mean values found in uterus and ovary for all three methods were between 0.2 and 0.9 TAM–DNA adducts/108 nucleotides. Although the monkey daily TAM dose was about 6-fold higher than the human daily dose, the data demonstrate that primates have the capacity to metabolize TAM to reactive intermediates that form DNA adducts in the uterus as well as other organs.
David H.Phillips (Institute of Cancer Research, Sutton, UK) described studies measuring rat DNA adducts by 32P-post-labelling (detection limit 0.1 adduct/108 nucleotides) and attempts to find DNA adducts in human tissues using the same methodological approach. In livers of rats exposed to TAM or -OH-TAM, the major DNA adducts were dG-N2-TAM and dG-N2-N-desmethyl-TAM. In addition, the R isomers of both -OH-TAM and -OH-N-desmethyl-TAM were significantly (8-fold) more active in forming DNA adducts in cultures of rat hepatocytes than their respective S isomers. The lack of TAM–DNA adducts in rat uterus and other organs is consistent with the observation that liver is the only organ expressing SULT2A1 in the rat. In human endometrium and peripheral blood lymphocytes no evidence of human TAM–DNA adduct formation was encountered (Carmichael et al., 1996, 1999; Phillips et al., 1996; Bartsch et al., 2000) and no expression of SULT2A1 has been found in human endometrium (Rubin et al., 1999). Evidence was also presented that -OH-TAM is not metabolically activated by human N-acetyltransferase 1 or 2. The studies performed by Dr Phillips and colleagues do not support the hypothesis that TAM is a genotoxic carcinogen in either adult rat uterus or human endometrium. Further evidence that this drug may not act through a genotoxic mechanism to cause human endometrial cancer was presented in the form of preliminary results from a clinical trial comparing TAM and toremifene. It appears that toremifene, which does not have genotoxic effects, may induce an incidence of endometrial cancer similar to that seen with TAM (K.Holli, personal communication).
The mass spectrometric method (see presentation by L. Schild, above) developed for measurement of TAM–DNA adducts (Gamboa da Costa et al., 2003) was described by M. Matilde Marques (Centro de Química Estructural, Instituto Superior Técnico, Lisbon, Portugal). The assay uses on-line sample preparation coupled with HPLC and ES-MS/MS and has a detection limit of 0.2 dG-N2-TAM adducts/108 nucleotides. The dG-N2-N-desmethyl-TAM adduct is also measurable with a detection limit of 2.0 adducts/108 nucleotides. The assay was validated with a series of DNA samples modified with TAM to different levels in vitro and in DNA from rats exposed in vivo. TAM–DNA adducts were found in liver DNA, but not in uterine DNA, from TAM-exposed rats. A series of human DNA endometrium and breast samples, nine from TAM-exposed women and nine from unexposed women, showed no evidence of measurable TAM–DNA adduct formation.
Contrasting data, presented by Shinya Shibutani (State University of New York, Stony Brook, NY), support the hypothesis that TAM induces human endometrial cancer through a genotoxic mechanism. A combination of HPLC and 32P-post-labelling, with a detection limit that varies between 0.1 and 0.4 adducts/108 nucleotides, was used to examine rat and human samples. The method can separate the trans- and cis-diastereomers of dG-N2-TAM, dG-N2-N-desmethyl-TAM and dG-N2-TAM-N-oxide. Site-specifically modified oligodeoxynucleotides containing a single dG-N2-TAM adduct were prepared by phosphoramidite chemical synthesis and used as absolute standards for the 32P-post-labelling analysis. The method was used to measure dG-N2-TAM adducts in uterus, ovary and liver from cynomolgus monkeys given oral administration of TAM for 30 days (see presentation by L.Schild, above). Microsomal incubations demonstrated that TAM is -hydroxylated by CYP3A2 in rat liver and CYP3A4 in human liver, and subsequently sulphated to form DNA adducts. These investigators (Shibutani et al., 1999, 2000) have reported measurable levels (0.2–18.0 adducts/108 nucleotides) of combined dG-N2-TAM trans- and cis-diastereomers in endometrium from 8 of 16 women receiving TAM therapy, with no adducts in 15 unexposed women. Differences between the TAM–DNA adduct methods employed by the Shibutani and Phillips groups were discussed but not resolved, however, Shibutani proposed to provide his TAM-modified oligomers to be used as internal standards for quantification of TAM–DNA adducts in animal and human tissues.
Studies involving exposure of cultured human endometrium to TAM were described by Paul L.Carmichael (Imperial College of Science, Technology and Medicine, London, UK). Using DNA isolated from endometrium and endometrial tissue grown as explant cultures and exposed to TAM, he found no evidence of TAM–DNA adduct formation (Carmichael et al., 1996, 1999). However, in patients receiving TAM therapy, evidence of atypical hyperplasia, proliferative polyposis, atrophy and other events suggest that TAM-induced effects may transcend the known oestrogenic activity. Gene expression, explored in glandular and stromal cells grown out from human endometrium and exposed to TAM, was examined using Affymetrix Human Gene Chip arrays. A large number of changes were observed in exposed compared with unexposed cells. For example, whereas expression of TGFß1 was increased after TAM exposure, the TGFß1 receptor was down-regulated, presumably decreasing the efficacy and cancer protective effects of TGFß1. Dr Carmichael demonstrated that the effects of TAM on human endometrium are highly complex and suggested that the mechanism by which tumours are induced is likely to involve more than simple genotoxicity.
Atsushi Umemoto (University of Tokushima, School of Medicine, Tokushima, Japan) detailed the collection of blood samples from 47 breast cancer patients taking TAM and 20 untreated patients. Leukocyte DNA was analyzed using a 32P-post-labelling/HPLC method able to measure the trans- and cis-diastereomers of dG-N2-TAM, dG-N2-N-desmethyl-TAM and dG-N2-TAM-N-oxide (Umemoto et al., 2001, 2002). The limit of detection for this assay was 0.07 adducts/108 nucleotides. In livers of rats dosed orally with 45 mg/kg body wt TAM daily for 7 days, fractions of the total adducts comprising dG-N2-TAM, dG-N2-N-desmethyl-TAM and dG-N2-TAM-N-oxide were 54, 39 and 0.4%, respectively. For the assay of human samples, 18 or 45 µg of DNA was used per assay and 6 of the 47 patients had measurable TAM–DNA adducts (trans-dG-N2-TAM and trans-dG-N2-N-desmethyl-TAM), with a mean value of 0.26 ± 0.3 adducts/108 nucleotides.
In collaboration with a group at Lawrence Livermore National Laboratory, Elizabeth A.Martin (AstraZeneca, Macclesfield, UK) used accelerator mass spectrometry (AMS) to assess TAM-induced DNA and protein adduct formation in endometrium and myometrium obtained from women undergoing hysterectomy (Martin et al., 2001). A highly sensitive method first developed for carbon dating, AMS quantifies isotopes with a detection limit of a few adducts in 1012 nucleotides. In this study a single dose of 20 mg TAM containing 1.85 MBq was given to women 18 h before hysterectomy, blood was taken before and after surgery and uterine tissue was dissected into endometrium and myometrium. DNA adducts were detected at low levels and were present in both myometrium (0.0492 ± 0.0112 adducts/108 nucleotides) and endometrium (0.0237 ± 0.0077 adducts/108 nucleotides) (mean ± SE, n = 10). The mean value for myometrial TAM–DNA adducts was 2-fold higher than the mean value for endometrial TAM–DNA adducts, but the difference was not significant (P = 0.088). The very low TAM–DNA values may reflect the single TAM dose given. The authors support the hypothesis that this level of DNA damage is insufficient to impact on the carcinogenic process.
A series of 23 samples of human endometrial DNA, 10 from unexposed women and 13 from breast cancer patients receiving TAM therapy, were assayed by TAM-DNA CIA. The results were reported by Miriam C.Poirier (National Cancer Institute, NIH, Bethesda, MD). The same samples had previously been subjected to 32P-post-labelling by David Phillips, who found no evidence for TAM–DNA adducts. Replicate batches of these samples were assayed by TAM-DNA CIA, and initially a few samples from TAM-exposed patients appeared positive. However, DNAs shipped at different times gave inconsistent results when assayed on multiple occasions, and finally it was impossible to identify more than one sample as clearly positive. This sample was assayed twice with a mean value of 1.35 dG-N2-TAM/108 nucleotides. The difficulties encountered with these experiments underscore the need for an additional source of human endometrial samples. However, if TAM–DNA adducts are confirmed in human endometrium, it appears unlikely that a large fraction of DNA samples will contain measurable adducts.
Inter-laboratory trial of TAM–DNA adducts in rat liver
A final presentation, by Laura J.Schild (National Cancer Institute, NIH, Bethesda, MD), detailed the results of an inter-laboratory comparison, in which rats were fed 500 p.p.m. TAM in the diet for 6 weeks and liver TAM–DNA adducts were measured by four different laboratories. Participating investigators included David H.Phillips, Frederick A.Beland, Karen Brown and Laura J.Schild, who used 32P-post-labelling, HPLC with ES-MS/MS, 32P-post-labelling/HPLC and TAM-DNA CIA, respectively. Values for measurement of the dG-N2-TAM adduct in five replicate rat livers varied by less than 3-fold. The mean values were 3.6, 6.9, 3.1 and 2.5 dG-N2-TAM adducts/106 nucleotides for 32P-post-labelling, HPLC with ES-MS/MS, 32P-post-labelling/HPLC and TAM-DNA CIA, respectively. Considering that four laboratories employed their respective assays without the use of a shared standard for comparison, the TAM–DNA adduct values were remarkably close. Greater variation was observed for the determination of the dG-N2-N-desmethyl-TAM adduct. Although the DNA samples used for this study were quite highly modified, the data indicate that the different methods give very similar results. The study will be repeated using lower doses and the results of the complete investigation published elsewhere.
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The purposes of this conference were: (i) to explore the evidence that tamoxifen (Nolvadex®) therapy leads to the formation of TAM–DNA adducts in any human tissue; and (ii) to consider whether or not such adduct formation might play a role in the induction of endometrial and uterine tumours that occur in women receiving TAM therapy. The conference participants expressed a wide diversity of views with regard to both major points of discussion, and it was clear that our knowledge of TAM–DNA adduct formation in any human tissue is incomplete. Studies reporting the presence of TAM–DNA adducts in human endometrium were criticized for lack of specificity, discrepancies in diastereomeric TAM–DNA adduct conformation between animals and humans, adduct levels near the limit of assay detection, inability to reproduce the same measurement with different sample batches and the fact that some investigators knew the treatment status of the patient before the sample was assayed. There was, however, some general agreement on a logical approach to the resolution of these issues. Progress has been significantly hampered by the shortage of endometrial tissue from women receiving TAM therapy. There is a need to obtain additional human endometrial and myometrial DNA samples and to share human samples among different laboratories. There was also a consensus that it would be useful to have a TAM–DNA standard, modified to a known level in the range expected for human samples, which could be shared by all investigators and used as a positive control when assaying human samples. A consortium of laboratories (all are invited) will pursue the rat liver DNA investigations, which, along with a known standard, will allow for further comparison and validation of the different relevant methodologies.
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The meeting was supported by generous grants from the Office of Women’s Health, NIH, and the Center for Cancer Research, NCI, NIH, Bethesda, MD. We are most grateful to Ms Linda Amspaugh for handling the administrative organization of the conference and to Ms Bettie Sugar for editorial assistance.
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1To whom correspondence should be addressed. Tel: +1 301 402 1835; Fax: +1 301 402 8230; Email: poirierm{at}exchange.nih.gov
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Received on March 17, 2003; accepted on March 28, 2003.
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