Chemoprevention of breast cancer by tamoxifen: risks and opportunities

Toxicology Letters ELSEVIER

Toxicology

Chemoprevention

Letters

82/83

(1995)

181-186

of breast cancer by tamoxifen: risks and opportunities Lewis L. Smith*, Ian N.H. White

MRC Toxicology

Unit, University of Leicester, Hodgkin

Building, Lancaster Road. Leuzester, LEI

9HN. UK

Abstract The antioestrogen tamoxifen is of proven efficacy in inhibiting the growth of oestrogen receptor positive breast cancers in women. In rats, long-term dosing leads to the development of hepatocellular tumours. Tamoxifen in this species is a genotoxic carcinogen. Metabolic activation by cytochrome P450-dependent enzymes leads to DNA damage detectable by 32P-postlabelling. Factors important in the development of hepatocellular lesions were the nature and quantity of metabolism and promotion/progression of the DNA lesion by agents such as phenobarbital and cell proliferation. No evidence was found for tamoxifen-induced DNA damage in the livers of 7 women taking this drug therapeutically. Keywords:

Tamoxifen:

DNA damage; Genotoxicity:

Liver tumours; Metabolism

1. Introduction Tamoxifen was developed in the late 1960s as an antioestrogen. This drug inhibits oestrogenstimulated cell division but in some tissues it can also exhibit oestrogen-like activities. Tamoxifen is of proven efficacy in inhibiting the growth of oestrogen receptor positive breast cancers in women and is probably one of the safest chemotherapeutic drugs in common use. As adjuvant therapy for breast cancer it has few undesirable side effects. Tamoxifen also has a number of beneficial effects apart from its primary action on breast cancer cells. In treated women, it has a significant action in reducing serum cholesterol [l] and the incidence of fatal myocardial infarction [2]. In post-menopausal individuals it may * Corresponding

0378-4274/95/$09.50 SSDI 0378-4274(

also help to limit the development of osteoporosis (Fig. 1). Clinical trials are currently under way, primarily in the USA and UK, to test the use of tamoxifen as a chemopreventive agent for breast cancer in healthy women. There are unquestionable benefits in the use of this drug for the treatment of women with breast Potential benefits Effecbve ad,uvant therapy for breast cancer

Ireland

Retlnopathy

Llmds osteoporosis in postmenopawal women

Ltd. All rights

reserved

of endometrial

Gl-tract tumors

Lowered blood cholesterol

Fig. 1. Risk/benefit

Science

Promoton tumors

risk of myocardlal mfarctlon Decreased

author.

@ 1995 Elsevier 95)03476-2

Potential risks I

potential

of tamoxlfen.

182

L.L. Smith,

I.N.H.

White

I Toxicology

cancer. Its use as a chemopreventive agent in healthy women is not so clear-cut. Epidemiological evidence from women with breast cancer who have been treated with tamoxifen suggests longterm administration may result in a small increase in the incidence of endometrial [3] or GI tract tumours [4]. Further concerns about the potential safety of tamoxifen were raised when a number of independent laboratories found that long-term administration of tamoxifen to rats at high dose levels gave rise to hepatocellular carcinomas [5,6]. In these studies, there were no reports of tumours of the reproductive system or GI tract. There is also no epidemiological evidence to suggest that there is an increased risk of liver tumours in women taking this drug. However, experience with other carcinogens suggest that the organ or tissue affected is not necessarily the same across species. More evidence is required as to the mechanism of carcinogenic action of tamoxifen in experimental systems to inform better on risk-benefit analysis for women taking tamoxifen. Following the discovery that tamoxifen itself was not genotoxic but could be activated in the rat liver to give genotoxic intermediates [7], our aim has been to define the nature of the activating enzymes system, factors influencing DNA damage and the development of liver tumours. A principal objective of the study was to establish animal models which would permit identification of those factors which contribute to the development of hepatic tumours in the rat but not in the mouse. It might then be possible to determine whether they also operate in humans and if so, establish the potential risk of tamoxifen in women taking this drug. 2. Hepatic DNA damage caused by tamoxifen Since all of the early in vitro tests for tamoxifen genotoxicity, such as the Ames Samonella assay, gave negative results, it was presumed, until a few years ago, that tamoxifen was working via an epigenetic mechanism, the hepatocarcinogenic effect in rats being in some way related to oestrogenic/antioestrogenic potency of this drug. Analogues such as toremifene (Fig. 2) have a similar antioestrogenic potency but do not

Letters

8.2183 (1995) 181-186

give liver tumours in rats [6]. There is now good evidence that in the rat, tamoxifen is a genotoxic carcinogen. We have investigated the ability of tamoxifen and toremifene to induce DNA damage using the technique of “‘P-postlabelling. It was established that tamoxifen could cause adduct formation, even after a single dose, that was selective for the liver. In liver DNA, the degree of damage was dependent both on the dose and the length of exposure. Toremifene resulted only in trace levels of such damage [7]. Tamoxifeninduced 32P-postlabelling was detected in mouse liver DNA but the level of adduct formation was about one-third that seen in rats. Two features of the DNA lesions seen in rat liver following tamoxifen treatment may contribute to its hepatocarcinogenic effects. Firstly, following cessation of dosing, the adducts are repaired or eliminated very slowly with a half-life in the order of 3 months [8]. Secondly, with continuous exposure of rats to tamoxifen the extent of DNA damage continues to increase for many months. In adult rats, following 6-12 months exposure to tamoxifen. when there is rapid hepatocellular proliferation in hyperplastic nodules and tumours, the total extent of DNA damage decreases [9], probably as a result of dilution of the adducted DNA by preneoplastic cells. There is strong evidence that cell proliferation plays an important role in the promotion and progression of DNA damage. In a study involving 3 strains of female rat given dietary tamoxifen corresponding approximately to 40 mg/kg/ day, there were only small differences in its concentration and that of its major metabolites in the liver. Similarly, the extent of hepatic DNA damage, determined by 32P-postlabelling at 6 months in all 3 strains were all similar [9]. At 3

Toremlfene

Tamoxlfen

Fig. 2. Chemical

structures

of tamoxifen

and toremifene.

L.L. Smith. I.N.H. White I Toxicology Letters 82183 (1995) 181-186

months after dosing, using either conventional histochemical staining or the markers, yglutamyltranspeptidase or glutathione S-transferase P, the number of positive foci was about lo-fold higher in both Wistar and Lewis rats compared to the Fischer animals. There were also marked strain differences in the time to development of liver tumours with Wistar and Lewis animals being more susceptible. After 11 months, all of the Wistar and Lewis rats had liver carcinomas, while none was seen in the Fischer animals. Such carcinomas were seen in these animals only when they were killed at 20 months. A comparison of the extent of hepatic parenchymal cell division, relative to controls, showed that after 6 months tamoxifen exposure this was depressed in Fischer rats, in contrast to an increase in Wistar and in Lewis rats. It is concluded that the increase in cell proliferation is consistent with the promotion of foci to tumours and the subsequent progression of tumours in the latter 2 strains. Promotion of liver DNA damage initiated by tamoxifen can be achieved by the use of phenobarbital. Wistar rats were dosed with tamoxifen for only 3 months and then returned to a basal diet. Tamoxifen is rapidly cleared from the body with a half-life of about 12 h. This drug could not be detected in the livers of rats 3 months after dosing even though liver DNA lesions detected by “P-postlabelling persisted. In a group a rats promoted with phenobarbital in the drinking water, the majority (12/14) consequently developed liver tumours. Even about one-third of those animals which received no additional promotion after the initial 3 months tamoxifen treatment went on to develop liver tumours in a lifetime study [8]. It was proposed that the persistence of the DNA adducts may account for the ability of phenobarbital to promote a high incidence of liver carcinomas after discontinuation of the tamoxifen dosing. 3. 32P-Postlabelling of DNA from women taking tamoxifen therapeutically Liver DNA samples obtained from 7 women receiving tamoxifen therapeutically or a ‘control group’ not receiving this drug were analyzed

183

using “‘P-postlabelling. In both groups DNA damage was detected but the pattern of postlabelled spots was not the same as those detected in a tamoxifen-treated rat liver DNA. There was no difference in the level of DNA damage (1% 80 adducts/lOx nucleotides) between the 2 groups [8]. The marked difference between the level of hepatic DNA damage in rats which develop liver tumours (3000 adducts/lO’ nucleotides) and women suggests the hazard to humans is considerably less. Several factors may affect this finding. Firstly, in the study only 7 treated human livers were analyzed, compared with the very large numbers of women treated with this drug. Secondly, the individual susceptibility to tamoxifen treatment with respect to carcinogenicity is likely to be influenced by many factors. These may include genetic polymorphisms in Phase 1 or Phase II enzymes responsible for the activation and detoxication of tamoxifen and the balance between these pathways; the efficiency of DNA repair and the extent of cell proliferation. It cannot be excluded that a small number of women given tamoxifen, due to a combination of these factors, produce sufficient DNA damage to result in liver cancer nor can it be certain that tamoxifen does not damage DNA in other cell types of other organs. 4. Mechanisms genotoxic

of activation intermediates

of tamoxifen

to

4.1. Effects of tamoxifen on hepatic drug metabolising systems

Tamoxifen administration to rats causes a 3060-fold increase in the rate of metabolism of benzyloxyresorufin or pentoxyresorufin by liver microsomal preparations [lo]. Smaller increases were seen in the 6p and 16~~ hydroxylation of testosterone as well as the oxidation of testosterone to androstenedione. Western blotting experiments showed a 2-3-fold increase in CYP2B1, CYP2B2 and CYP3Al proteins in liver microsomal fractions. Tamoxifen acts as a weak inducer of these isoenzymic forms but the extent of induction is not nearly so marked as with ‘classical inducers’ such as phenobarbital or dexamethasone. In rat liver microsomal systems in vitro,

184

L.L. Smith. I.N.H. White I Toxicology Letters 82183 (1995) 181-186

Detoxication Activation

N-Desmethyltamoxifen Tamoxifen-3,4-epoxide

0

/Q9cVW,l, c:c

\

Q\

/QoCnfH2N,c”&

/

/ 4-Hydroxytamoxifen

c=c

Tamoxifen

4’-Hydroxytamoxifen Tamoxifen-3:4’-epoxide

Tamoxifen N-Oxide Fig. 3. Metabolic pathways for liver microsomal metabolism of tamoxifen.

tamoxifen is metabolised primarily by N-demethylation, N-oxidation and 4-hydroxylation (Fig. 3). These are Phase I detoxication reactions. Pretreatment of rats with tamoxifen itself or classical inducers such as phenobarbital or dexamethasone stimulated the rate of N-demethylation whereas 4-hydroxylation was depressed. We have concluded that by stimulating its own metabolism tamoxifen may accelerate the rate of its own disposal and could also increase the production of a reactive genotoxic metabolite(s). Human and rat liver microsomal preparations are able to activate [‘4C]tamoxifen in the presence of NADPH to bind irreversibly to microsomal proteins. Protein binding has to be used as an index of metabolic activation and as a surrogate for DNA binding. The extent of binding to DNA appears to be of the order of 50-fold lower than to protein and at the limit of detection using conventional liquid scintillation methods for radioactive detection. Using a panel of 12 human microsomal preparations that had been

characterised for the cytochrome P450 content with respect to 9 CYP isoenzymic forms by Western blotting it was shown that CYP3A4 and CYP2B6 were involved with the metabolic activation of tamoxifen to metabolites which covalently bound to protein [ll]. This study suggested that the same isoenzymic forms involved in the N-demethylation of tamoxifen were also involved in covalent binding whereas 4-hydroxylation reaction was catalysed by CYP2C9. Consistent with the involvement of CYP3A4 and CYP2B6, pretreatment of rats with dexamethasone, phenobarbital or tamoxifen itself caused a significant increase in the rates of protein binding. Although at this stage we cannot formally distinguish if the active metabolites involved in protein and DNA binding are the same, preliminary studies suggest that this is the case. Comparison between species of the binding of tamoxifen to protein in vitro shows rats to be 3.8-fold and mice 17-fold higher than human liver microsomes [ll]. In this respect, the greater

L.L. Smith. I.N.H. White I Toxtcology Letters 82183 (1995) 181-186

activity in the mouse microsomal preparations reflects the higher levels of overall metabolism. Although the liver was the main site of activation, binding in microsomal preparations from normal human breast tissue could also be detected, although rates were some 7-fold lower than in human liver. 4.2. Clastogenicity in Crespi cell lines lymphoblastoma-derived Using a human MCL-5 cell line which the human isoenzymes CYPlAl, lA2, 2A6, 3A4 and 2El are functionally expressed, tamoxifen gave a positive result in a micronucleus assay [7]. Using similar cell lines which express individual cytochrome P45Os suggested that the isoenzymes CYP2El and CYP3A4 were capable of metabolising tamoxifen to genotoxic intermediates as judged by a positive micronucleus test. The Crespi cells CYPlAl, CYPlA2, expressing CYP2D6, CYP2A6 or CYP2B6 did not give positive results over the range of concentrations used with the former 2 isoenzymes [12]. It should be noted that because it has not been established the extent to which the various isoenzymes are expressed within the cells, it is not possible to be categoric that negative results reflect the response in either rodent or human tissues. These results do show that the human P45Os have the ability to activate tamoxifen and at concentrations normally found in the serum of women taking tamoxifen therapeutically (~300 ng/ml). 4.3. Identity of tamoxifen active metabolite(s); role of epoxides Studies using liquid chromatography with on line electrospray mass spectrometry have detected the presence of a number of metabolites formed from tamoxifen in microsomal incubation mixtures which correspond formally to the addition of oxygen to the drug. A number of metabolites such as tamoxifen N-oxide with known retention times on HPLC can be assigned to the peaks observed. Two additional peaks, believed to represent aromatic 3,4-epoxide and 3’,4’-epoxides of tamoxifen have been described (Fig. 3). Although not sufficiently stable to be isolated in quantities sufficient for chemical analysis, in the

185

presence of acid they are converted to the corresponding dihydrodiols, consistent with an epoxide structure. Formation of these epoxides are detected using rat, mouse and human liver microsomal preparations [13]. Other putative active metabolites have been proposed such as a yet unidentified product formed as a result of activation of 4-hydroxytamoxifen [14] or cu-hydroxyethyltamoxifen [15]. Support for cY-hydroxylation of the ethyl group as a major pathway of tamoxifen activation comes from the observation of a reduced genotoxicity of [D5When ethyl]-tamoxifen. a-hydroxyethyltamoxifen was prepared chemically and added to rat hepatocytes in culture, the extent of DNA damage, as assessed by “‘P-postlabelling, was in the order of 58fold higher than with tamoxifen itself [16]. The identity of the active metabolite would help to locate which tissues such as endometrium or GI tract, have the potential to activate tamoxifen. 5. Opportunities The molecular mechanism of action of tamoxifen in breast cancer appears to be a complex mixture of antagonism of the mitogenic action of oestradiol at the level of the oestrogen receptor and cellular effects which may include: (1) inhibition of protein kinase C and calmodulin-dependent CAMP phosphodiesterase and (2) modulation of growth factors including insulin-like growth factor 1 and transforming growth factor beta [17]. Several new antioestrogenic drugs are being developed. Some are based on the triphenylethylene structure of tamoxifen and, like toremifene and droloxifene, do not give rise to liver tumours in rats in life-time bioassays. Raloxifene has a benzothiaphene structure which has antioestrogenic potential [18], while the substituted steroid, ICI 182 780 is a potent specific pure antioestrogen which may offer advantages in breast cancer treatment compared with partial agonists like tamoxifen. These latter analogues, structurally unrelated to tamoxifen, will not have the potential for metabolic activation to genotoxic intermediates. The potential of these newly developed antioestrogens to bring

186

L.L.

Smith,

I.N.H.

White

I Toxicology

about the enzyme inhibition and growth factor modulation described above may be central in optimizing their effects as therapeutic agents for the effective treatment of breast cancer in women. References [l] Powles, T.J., Jones, A.L.. Ashley, S.E., O‘Brien, M.E.R.. Tidy, VA., Treleavan, J., Cosgrove, D., Nash, A.G., Sacks, N., Baum. M., McKinna, J.A. and Davey, J.B. (1994) The Royal Marsden Hospital pilot tamoxifen chemoprevention trial. Breast Cancer Res. Treat. 31. 73-82. [2] McDonald, C.C. and Stewart, H.J. (1991) Fatal myocardial infarction in the Scottish adjuvant tamoxifen trial. Br. Med. J. 303, 435-437. [3] Fisher, B., Costantino, J.P., Redmond, C.K., Fisher, E.R., Wickerham. D.L., Cronin, W.M. and NSABP Contributors (1994) Endometrial cancer in tamoxifentreated breast cancer patients: findings from the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14. J. Natl. Cancer Inst. 86. 527-537. [4] Rutqvist, L.E., Johansson, H.. Signomklao, T., Johansson, U., Fornander, T. and Wilking, N. (1995) Adjuvant tamoxifen therapy for early stage breast cancer and second primary malignancies. J. Natl. Cancer Inst. 87. 645-651. [5] Greaves, P., Goonetilleke, R., Nunn, G., Topham, J. and Orton, T. (1993) Two-year carcinogenicity study of tamoxifen in Alderly Park Wistar-derived rats. Cancer Res. 53, 3919-3924. [6] Hard, G.C., Iatropoulos, M.J., Jordan, K., Radi, L., Kaltenberg, O.P.. Imondi, A.R. and Williams, G.M. (1993) Major differences in the hepatocarcinogenicity and DNA adduct forming ability between toremifene and tamoxifen in female Crl: CD(BR) rats. Cancer Res. 53, 4534-4541. [7] White, I.N.H., De Matteis, F., Davies, A., Smith. L.L., Crofton Sleigh, C., Venitt, S., Hewer, A. and Phdlips, 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. [8] Carthew, P., Martin, E.A., White, I.N.H., De Matteis, F., Edwards, R.E., Dorman, B.M., Heydon, R.T. and Smith, L.L. (1995) Tamoxifen induces short-term cumulative DNA damage and liver tumors in rats: promotion by phenobarbital. Cancer Res. 55, 544547.

Letters

82183 (1995) 181-186

[91 Carthew, P., Rich, K.J., Martin, E.A., De Matteis, F., Lim, C.K., Manson, M.M., Festing, M., White, I.N.H. and Smith, L.L. (1995) DNA damage as assessed by 32P-postlabelling in three rat stains exposed to dietary tamoxifen: the relationship between cell proliferation and liver tumour formation. Carcinogenesis, 16, 12991304. [lOI Martin, E.A., Rich, K., White, I.N.H., Woods, K.L., Powles, T.J. and Smith, L.L. (1995) “P-Postlabelled DNA adducts in liver obtained from women treated with tamoxifen. Carcinogenesis, 16, 1651-1654. Pll White, I.N.H., Davies, A., Smith, L.L., Dawson, S. and De Matteis, F. (1993) Induction of CYP2Bl and 3A1, and associated monooxygenase activities by tamoxifen and certain analogues in the livers of female rats and mice. Biochem. Pharmacol. 45, 21-30. [121 White, I.N.H., De Matteis, F., Gibbs, A.H., Lim, C.K., Wolf, C.R., Henderson, C. and Smith, L.L. (1995) Species differences in the covalent binding of [‘4C]tamoxifen to liver microsomes and the forms of cytochrome 450 involved. Biochem. Pharmacol. 49. 1035-1042. [I31 Lim, C.K., Yuan, Z., Lamb, J.H., White, I.N.H., De Matteis. F. and Smith, L.L. (1994) A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes. Carcinogenesis 15, 589-593. [I41 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 cY-hydroxylation of the ethyl group as a major pathway of tamoxifen activation to a liver carcinogen. Carcinogenesis 15, 1487-1492. [I51 Pathak, D.N., Pongracz, K. and Bodell, W.J. (1995) Microsomal and peroxidase activation of rl-hydroxytamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen. Carcinogenesis 16, 11-15. WI Colletta, A.A.. Benson. J.R. and Baum, M. (1994) Alternative mechanisms of action of anti-oestrogens. Breast Cancer Res. Treat. 31. 5-9. 1 Draper, M.W., Flowers, D.E., Neild, J.A., Huster, W.J. [171 and Zerbe, R.L. (1995) Antiestrogenic properties ot raloxifene. Pharmacology 50, 209-217. (181 DeFriend, D.J., Howell. A., Nicholson, R.I., Anderson, E., Dowsett, M., Mansel, R.E., Blarney, R.W., Bundred, N.J., Robertson, J.F., Saunders, C., Baum, M., Walton, P., Sutcliffe, F. and Wakeling, A.E. (1994) Investigation of a new pure antiestrogen (ICI 182780) in women with primary breast cancer. Cancer Res. 54, 408-414.