Antiestrogens Toremifene and Tamoxifen

Stand 1. Lab, Anim. Sci. No. 3, 1996. Vol. 23

On the safety studies of the antiestrogens toremifene and tamoxifen

by Pil‘kko Hil‘simdki’, Yljjf)‘ IIirsimﬁ/cz'z, Lauri Niemrfrmnl, Sleﬂm Karlsswzz 8.: Hero Mdmyld‘". ‘Turku University Central Hospital, DepL uf Pathology, FllN-ZOSZO, Turku, Finland and 201‘10117Furmos, b’urmos Research, FIN-20101, Turku, Finland‘ Currespundence: Pirkko Hirsiméki, Turku University Central Hospital, Department 01' Pathology BioCity, Tykistiikatu 6. Rt 6, FIN720520 Turku, Finland

Introduction stein 1992). However, during recent years stong Antiestrogens inhibit the binding of estrogens to evidence has been accumulated that tamoxifen is the estrogen receptor (F urr & Jordan 1984, Kallin a genotoxic hepatocarcinogen in the rat. Hepato- er al. 1986). Tamoxifen and toremifene are none cellular carcinomas have been reported both in fe— steroidal triphenylethylene derivatives which male and male rats receiving tamoxifen (Hir- have both antiestrogenic and estrogenic proper— simiz’ki et all 1993, Greaves et a1. 1993, Williams 91 ties (Kangas 1990, Kendall & Rose 1992) depend— (11. 1993, Carﬂmw et al. 19953) but not with ing on species and tissue type studied. The new torelnifene (Hard et a1. 1993, Hirsimdki er al. antiestrogen toremifene differs from tamoxifen 1993). In humans lnng-term medication with only by the substitution of Chlorine atom for a hy— tamoxifen increases the risk of endometrial carci— drogen atom in an ethyl group (Figure 1). Tamoxi- noma 0f the uterus (Formmder et alt 1989, Friedl fen has been used in the therapy of advanced & Jordan 1994). breast cancer for approximately 20 yearst It has In the present review we will deal with the safety been considered as a safe drug and even preven- studies of the both antiestrogens with rodents, es— tive trials have been initiated in which tamoxifen pecially in rats. The risk to humans is also dis— is given to health y women at an increased risk of cussed. developing breast cancer (Fugh-Berman & Ep-

/CH3 /CH3 0 CH2 CH2N\CH OCH 2 CH 2\CH3 N 3

TOHEMIFENE TAMOXIFEN

Figure 1. The formulas of tamoxifen and toremitene.

I47 Scum]. J. Lab. Anim. Sci. N0. 3. 1996. Vol. 23

Effects on the liver majority of these tumors were hepatocellular car~ Hepatocarcinogenesis: cinemas with a well differentiated trabecular pat— Tamoxifen has shown to be a strong hepatocarr tern. Hepatocellular carcinomas were observed at einogen in rats in independent toxicity studies all dose levels (Greaves et a1. 1993). (Hard er a1. 1993, Hirsimdki et a1. 1993, Williams Recently it was reported that tamoxifen (420 ppm et all 1993, Greaves et al. 1993, Ahotupa at all in the diet) induced hepatocellular carcinomas in 1994, Carthew er al. 1995a, 1995b). Mice treated all Wistar and Lewis rats in one year, while in with the same dosing regime did not develop ab- Fischer rats the carcinomas developed later, at 20 normal liver pathology (Tucker et al. 1984). months (Carthew er al. 1995b). In Wistar rats We did two separate comparative studies with fe— even 3 months exposure to tamoxifen is sufﬁcient male Sprague—Dawley rats in which tamoxifen to induce liver carcinomas in high frequency induced hyperplastie nodules and hepatocellular (Carthew er al. 1995a). carcinomas at the dose level of 45 mg/kg after one years daily dosing by oral gavage. After a three Mechanism of tamoxifen-mduced hepatocarcirza— month recovery period almost all normal liver genesis was replaced with tumor masses. The equimolar Promotion, initiation or both: dose level of toremifene (48 mg/kg) was not Early arguments proposed that the hepatocarcino- hepatoproliferative (Hirsimc‘iki et al. 1993. Ahoa genie action of tamoxifen would be a result 01‘ its tupa el al. 1994). estrogen agonist action, i.e. tamoxifen would act Williamr et al. (1993) exposed female Sprague- as a tumor promoter (F enliman & Pawles 1987). Dawley rats to tamoxifen for one year. Tamoxifen This was supported by the observation that was given daily by gavage at the doses of 2.8, 11.3 tamoxifen was able to act as a tumor promoter in or 42.5 mg/kg. Tamoxifen induced hepatocellular initiation-promotion studies where diethylnitro- carcinomas between 3 and 6 months of adminis- samine was used as an initiator (Yager et al. 1986, tration. In the high dose group, hepatocellular ad— Dragan er a1. 1991, 1994). lt was suggested that enomas and carcinomas were observed in 71 and tamoxifen lacks of initiating activity when given 29 % of rats respectively. At 12 months the adeno— as a single p.o. dose of 40 mg/kg and that the mas were observed in 50 % and carcinomas in 75 hepatoeareinogenic effect is due to the promotion % of high dose rats. At the mid dose adenomas of spontaneously initiated hepatocytes (Dragan et were observed in 50 % and carcinomas in 10 % of al. 1991). In the work of Ghia andMereto (1989) rats at 12 months. After a 3 month recovery period tamoxifen (400 ppm in the diet for 6 weeks) was at this dose level, 45 % 01 rats exhibited adenomas suggested to act as a complete liver carcinogen and 45 % carcinomas. (induction of foci). In a later study tamoxifen was In another comparative study tamoxifen produced given as an initiator for a longer period (3 months) 100 % incidence of hepatocellular carcinoma at before promotion with phenobarbital. The results the dose level of 22.6 mg/kg at the 12 and 15 suggested that tamoxifen can act as an initiator of month (12 month dosing + 3 month recovery) sac— liver cancer (Carthew et al. 1995a). The initiating rifice intervals and 67 % and 71 % incidence at activity was associated with high-level accumula- the 11.3 mg/kg dose. Toremifene did not produce tion of DNA—adducts with very slow adduct elimi- any hepatoproliferative effects at 12 or 24 mg/kg nation rate (Carthew et ul. 1995a, 1995b). Fur~ dose levels, not in a pilot study at 48 mg/kg (Hard ther, the high incidence of tamoxifen induced can— er a1. 1993). cers, up to 100% at one year, argues against the In a conventional 2-year carcinogenicity bioassay promotion hypothesis (Williams 1995), as does in rats of Zeneca Pharmaceuticals tamoxifen was also the fact that toremifene and tamoxifen have given to female and male rats by gastric intuba» equal estrogen agonism in the liver (Kendall 6’: tion 5, 20 and 35 mg/kg/day. The major finding Rose 1992), but toremifene is non—carcinogen. was a dose-related increase in the incidence of However, promotion is also critical for cancer in- hepatocellular tumors which were first observed duction: in three rat strains (Fischer. Wistar and after 31 weeks of treatment at the high dose. The Lewis) the level of tamoxifen—induced adducts

148 Scaml. J. Lab. Anim. Sci. "In. 3. 1996. Vol 23 was equal at the 6 months time point, however the arations was studied the covalent binding to mi— proliferation index in Fischer rats was depressed crosonial protein was mostly correlated With when compared to other two strains. This resulted CYP3A4 and CYPZBG isoenzyme content (White to early carcinomas in Wistar and Lewis rats (6-11 et al. 1995). Pretreatment with CYP3A inducers, months) and late ones (20 months) in Fischer rats including tamoxifen, increases the in vitro cova— (Carthew gt al. 1995b). To conclude: tamoxifen is lent binding to the rat microsomal preparations likely to be a complete liver carcinogen. (Mani et al. 1994, White et al. 1995). The level of covalent bindin g into mouse microsomal prepare: A genotoxie mechanism seems likely: tions was markedly higher when compared to hu— During recent few years a vast amount of evi- man and rat microsomes (Mani et al. 1994, White dence has been cutnulated from several independ- et al. 1995). This suggests that the covalent bind ent laboratories to suggest that tamoxifen expo— ing to mierosotnes does not directly predict the sure of the animals leads to marked genotoxieity. DNA-damage level as e.g. higher DNA-adduct DNA adducts are formed in vivo and in vitro, also levels are achieved in rats than in mice (White et human microsomes are active (see genotoxicity al. 1992). chapter). The high level DNA accumulation of The elastogenicity of tamoxifen correlates with adducts formed, their slow disappem‘ence and cor— e.g. CYPZE and 3A expression (White et a]. 1992, relation with the adduct level and tumor induction Styles et al. 1994). Further. liver microsomes rate suggest that the DNA adducts are involved in from phenobarbital pretreated rats yield 3-6-fold the cancer initiation process (White at al. 1992, higher DNA—adduet levels in vitro than control Montadon et a1. 1994, Carthew el al. 1995a). The mierosomes and the DNA-adduct level is de— noncareinogenic toremifene produces no signifi- creased by CYP inhibitors (Pathak & Badell cant amounts of adducts (White et a]. 1992, Hard 1994). However, in mice pretreatment with phe- at (1]. 1993, Montadon Let (Il. 1994). Sargent et al. nobarbital or B-naphthoflavone before tamoxifen (1994) showed that a single in viva injection of exposure DNA-adduct formation in viva did not tamoxifen even at the very low dose level (0.3 increase (Randerath at al. 1994a). On the other mg/kg) can produce a variety of clastogenie eff hand, there is evidence that phase II metabolic feats. High frequency of p53 mutations have been enzymes could be involved in tamoxifen activa- found in hepatocarcinomas induced by tamoxifen tion as sulphotransferase inhibition with pen, (Vancmsem et al. 1994). These ﬁndings support tachlophenol increases DNA adduct formation in the genotoxic mechanism as the cause of hepato- mouse liver (Randerath et al. 1994a). Additional carcinogenicity of tamoxifen. _ experimentation, e.g. in vitro works with speciﬁc Evidence is accumulating that the species respon- CYP inhibitors, is needed to elucidate the CYP sible for the genotoxic effect are reactive metabo- isoenzyme(s) responsible for tamoxifen activa- lites of tamoxifen, although the ultimate molecu- tion to DNA-binding species. le(s) bound to DNA have not yet been verified (Lim 61 ul. 1994, Pathak & Bodell 1994, PhilIips Extrahepatic tumor induction el al. 1994, Randerarh et al. 1994b). Experimental endometrial data: Endometrial histopathologic data which was col- CYP enzyme involvement in tamoxifen activation: lected from several long-term comparative toxe Tamoxifen is activated in vz'tro by rat, mouse and icity studies with tamoxifen and toremifene in human microsomal cytochrome P450 isoenzymes Sprague-Dawley rats (Mdmyla' el al. 1996) show (CYPs) into reactive metabolites that bind cova— that a daily dose of tamoxifen (45 mg/kg p.o.) lently to microsomal protein. This activation is induced endometrial preneopIastic lesions in 10 mainly mediated by the CYP3A isoenzyme (Mani % and squamous cell carcinomas in 2 % of the & Kupfer 1991, Mani er a1. 1994). A higher rats. The neoplasias occurred during recovery CYPBA activity in some human individuals can periods. Equimolar dose levels of toremifene did lead to increased activation (Mani et al. 1994). not produce any such lesions although the here When a panel of 12 human liver microsomal prep- monal effects of the drugs were equal suggesting

149 Seand. J. Lab. Anni). Set. No. 3. 1996, Val. 23

that a nonhormonal mechanism in the tamoxi— tamoxifen in vitro, resulted in induction of un- fen—induced endometrial carcinogenesis is possi— scheduled DNA synthesis, when cells from ble in the rat. Grea ves et al. (1993) did not report tamoxifen-pretreated rats were used (White et a1. any endometrial changes in their study. The use 1992) but toremifene did not have this effect of different strain of rats (Wistar derived Alder- (White et al.‘ 1993). ley Park rats), lower dose levels and omission of recovery groups may explain the discrepancy. Clastogenicity: Anyhow, further large-seale and mechanistic Toreinifene has not shown any elastogenie activi- studies are warranted. ty in in vivo mouse mieronucleus test 01' in vitro in cultured human lymphocytes (Orion—Farmos, un- Human endometrial data: published results). Tamoxifen induced a signifi- Numerous clinical studies show that long-term cant increase in mieronueleus formation in a dose tamoxifen therapy increases the incidence of sec— dependent manner in human lymphoblastoid cell ondary endometrial cancer (Il'rz'edl & Jordan cultures that express several human cytochrome 1994). In a recent report two Swedish and (me P450 isoenzymes (White et a1. 1992). In this test Danish trial (5000 patients) were evaluated to- system toremit‘ene also gave positive results but gether showing that tamoxifen—thcrapy increased was weaker in inducing genotoxieity than tamoxi- the risk for uterine endometrial cancer in average fen (Styles el al. 1994). Sargent et a1. (1994) indi— 4-fold (Fomartder et a1. 1989, 1993, Rutqvist er cared that a single dose of tamoxifen (0.3—35 mg/ al. 1995). This tumorigenieity is generally be- kg p.o.) induces aneuploidy, premature condensa- lieved to result from the estrogen agonist action of tion, chromosomal breakage and mitotic spindle the drug on the endometrium but a genotoxie disruption in female Sprague—Dawley rats. component in the mechanism is also possible. Thus far there is no evidence that toremifene DNA adduct formation: would cause endometrial neoplasias after long— Mani & Kupfer (1991) were the first to show that term therapy however more follow-up data will be tamoxifen is metabolized in vitro to a reactive needed. metabolite which is covalently bound to micro- semal protein. Han & Liehr (1992) reported that Gastrointestinal cancers: tamoxifen induced DNA-adduet formation in vivo The recent clinical cumulative data from three in rat and hamster liver and rat kidney. Even a sin— Scandinavian tamoxifen trials show that tamoxi— gle i.p. dose of 5 mg/kg of tamoxifen citrate was fen increases the risk of colorectal and stomach able to induce hepatic adduct formation in hain— cancers, relative risk 2— and 3-fold, respectively ster liver. As this kind of DNA-reaetivity is com— (quvist er al. 1995). 1t is possible that metabo- mon to several genotoxie carcinogens (Williams lism of tamoxifen in the gut forms some reactive & Weisbm‘ger 1991), this ﬁnding opened a new species which might cause local damage. No ani- era in tamoxifen studies the target being human mal data show any increase in gastrointestinal risk assessment: what is the relevance of the cancers. DNA-reaetivity in human therapy? In rat liver tamoxifen induced high levels of Genozoximy DNA adducts in a dose—response manner (White Mutagenieity: et ul. 1992, Hard at al. 1993, Montadon & Wil- Although being a potent hepatocarcinogen tamoxi- liams 1994). The adduets are slowly eliminated fen has not shown Inutagenic activity in the con- so that daily dosing of the animals caused adduet ventional Ames test or in several other in vizro tests accumulation (White at al. 1992, Carthew er al. (Greaves er a1. 1993). Neither has toremifene not 1995a, 1995b). Toremifene did not cause adduets shown any niutagenic activity in bacterial tests. (Hard at at, 1.993, Mamadon & Williams 1994). In formal safety studies tamoxifen has not been In another study it produced one faint adduct shown to induce unscheduled DNA synthesis. spot and insignificant total amount of adducts However, exposure of rat hepatocytes to (l4()-fold less adduets than with equimolar Scand. J. Lab. Anim. Sci. No. 3. 1996. Vol. 23

tamoxifen dose) (White e! a]. 1992). Preliminary Summary results suggest that in women on tamoxifen ther- The safety studies of the two triphenylethylene apy no increase in hepatic adduct level can be antiestrogen dings, tamoxifen and toremifene, are observed (Martin et al. 1994). However, more discussed. Tamoxifen has been shown to be a studies are needed and also other possible target strong hepatocarcinogen in rats in several inde- tissues (especially endometrium and gut) must pendent toxicity studies but the new antiestrogen be studied with respect of adduct accumulation. toremifene is not a liepatocareinogen. A genotoxie Interestingly, in vim; human liver mierosomes mechanism is involved in tamoxifen—induced he- are also capable activating tamoxifen to DNA- patocareinogenesis and the species responsible reaetive species (Pathak & Bodell, 1994). Stud- for this genotoxic effect are apparently reactive ies in mice have indicated that there are at least metabolites of the drug. The activation of tamoxi- two different metabolic pathways leading to fen to genotoxie metabolite(s) may be mediated tamoxifen activation to DNAa‘eutive species by cytochrome P450 isoenzyines (mainly (Runderath et al. 1994b). CYP3A). Tamoxifen induces high levels of DNA adducts in experimental animals in vivo and in Conclusions and human risk assessment virro. Toremifene produces no signiﬁcant amount Tamoxifen is a potent liver carcinogen in the rat. of adducts. The evidence suggests that tamoxifen is active in In long—term studies tamoxifen induces also en— both in initiation and promotion phases of car- dometrial preneoplastic lesions and some squa- cinogenesis, i.e. tamoxifen is a complete liver care mous cell carcinomas in the rat. Equimolar dose cinogen. The structurally closely related anties- levels of toremifene do not produce these lesions. trogen, toremifene, has not carcinogenic property. In humans tamoxifen increases the risk for en- The mechanism of cancer initiation seems to be dometrial cancers and possibly also the risk for mediated by the DNAvreactivity of the products gastrointestinal cancers. of tamoxifen metabolism via CYP enzymes. The small difference in the chemical structure makes References toremifene safe in this respect. Some evidence Ahotupa M, P Hi'rsimiiki, R Pdrssim’n & E suggests that endometriuni would be another tar— Mii/ztylﬁ: Alterations of drug metabolizing and get tissue of tamoxifen's carcinogenic action in antioxidant enzyme activities during tamoxi- the rat. fen—indueed hepatoeareinogenesis in the rat. As experimental data suggest tamoxifen to be a Carcinogenesis 1994, 15, 8637868. genotoxic hepatoearcinogen it is potentially also a Carlhew P, EA Martin, INH White, F De Mallets, human cancer hazard. However. up to now there RE Edwards, BM Dorman, RT Heydon & LL is no published clinical evidence that tamoxifen Smith: Tamoxifen induces short-term cumula— would cause liver cancer in man in spite of its glo- tive DNA damage and liver tumors in rats: bal use. Instead, tamoxifenincreases the risk for promotion by phenobarbital. Cancer Res. endotnetrial and possibly also gastrointestinal 1995a, 55, 544-547. cancers. More studies in patients on tamoxifen Carthew P, K] Rich, EA Martin, F De Manet's, C- therapy will be needed in order to get an answer to K Lim, MM Manson, MF W F csting, INH White the major guestions: Is there an increase in cova— & LL Smith; DNA damage as assessed by 32P- lent binding or DNA-damage in human liver in postlabelling in three rat strains exposed to certain susceptible individuals (e.g. high hepatic dietary tamoxifen: the relationship between CYP 3A activity)? Is there any DNA damage def cell proliferation and liver turnout formation. tectable in the suggested extrahepatie target tis- Carcinogenesis 1995b, 16, 1299-1304. sues? If a clear DNA—reaetivity is verified in hu- Dragan YP, YXu & HC Pitot: Tumour promotion man tissues the risk versus benefit of tamoxifen- as a target for estrogen/antiestrogen effects in therapy should be carefully reevaluated. In pref rat hepatocarcinogenesis. Prev. Med. 1991, 20, ventive trials the use of safer antiestrogens would 15-26. be justified. Dragan YP, 5' Fahey, K Street et aL: Studies of Scand. J. Lab. Anim. Sci. No. 3. 1996. Vol. 23

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Phillips DH, GA Potter, MN Horton, A Hawaii. C Vancutscm PM, P Lazarus & GM Williams: Fre- (,‘mfrmz-tS‘leigh, M Jarman. & S 1’33nitt.‘ Ree quent and specific mutations of the rat p53 duced genotoxieity of (D5—ethyl)-tarnoxifen gene in hepatoeareinomas induced by implicates a-hydroxylation of the ethyl group tamoxifen. Cancer Res. 1994, 54, 3864-3867. as a major pathway of tamoxifen activation to White INH, F De Matteis, A Davies, LL Smith, C liver carcinogen. Carcinogenesis 1994, 15, Crofton Sleigh, S Venitt, A Hewer & D11 Phil- 1487—1492. lips: Genotoxie potential of tamoxifen and an- Randeralh K , J Bi, N Mabon, P Sriram & B Moor» alogues in female Fischer F344/N rats, DBA/Z thy: Strong intensiﬁcation of mouse hepatic and C57B1/6 mice and human MCL—5 cells. tamoxifen DNA adduct formation by pretreat- Carcinogenesis 1992, 13, 2197-2203. ment with the sulfotransferase inhibitor and White INH, A Davies, R flakes, DH Phillips & F ubiquitous environmental pollutant pentachlo- De Matteis: A comparison of the genotoxie rophenol. Carcinogenesis 1994a, 15, 797-800. potential of tamoxifen and toremifene in fe— Randeraz‘h K, B Moorthy, N Mahon & P Sriram: male F344/TOX rats. Hum. Exp. Toxicol. Tamoxifen: evidence by 32P-postlabeling and 1993, 12, 569. use of metabolic inhibitors for two distinct White INH, FDeManeis,A11 Gibbs, CK Lim, CR pathways leading to mouse hepatic DNA ad- Wolf, C Henderson & LL Smith: Species dif- duct formation and identification of 4ehydrox- ferences in the covalent binding of 14C ytamoxifen as a proximate metabolite. Car- tamoxifen to liver microsomes and the forms cinogenesis 1994b, [5, 2087—2094. of cytochrome P450 involved. Biochem. Phar- Rutqvist LE, H Johansson, T Signomklao, U 10- macol, 1995, 49, 1035—1042. hansson, T Fornander & N Wilking: Adjuvant William; GM; Tamoxifen experimental carcino- tamoxifen therapy for early stage breast can- genicity studies. —lmplications for human ef- cer and second primary malignancies. J. Natl. fects. Proc. Soc. Exp. Biol. Med. 1995, 208, Cancer Inst. 1995, 87, 645—651. 1417143. Sargent, LM, YP Dragan, N Bahnub. JE Wiley, C11 Williams GM, MJ latropoulos, MV Djm‘djavic & Sattlcr, P Schroeder, GL Saltler, VC Jordan & 0P Kattenberg: The triphenylethylene drug HC Pilot: Tamoxifen induces hepatic aneu- tamoxifen is a strong liver carcinogen in the ploidy and mitotic spindle disruption after a rat. Carcinogenesis 1993, 14, 315-317. single in. viva administration to female Sprague— Williams GM & JH Weisburger: Chemical car— Dawley rats. Cancer Res. 1994, 54, 3357—3360 cinogenesis. In: Amdur M.O., Doull, J. & Styles JA, A Davies, CK Lim, 1" De Matteis, LA Klaassen. C.D., Eds. Casarett and Doull’s Toxi- Stanley, INH White, Z-X Yuan & L Smith: Ge- cology The Basic Sciences of Poisons 4th Ed. notoxieity of tamoxifen, tamoxifen epoxide New York: Pergamon Press 1991 , pp. 127-200, and toreniifeiie in human 1yphoblastoid cells Yager JD, BD Roebuck, TL Paluszcyk & VA Mam» containing human cytochrome P450s. Car- 011: Effects of ethinyl estradiol and tamoxifen cinogenesis 1994, 15, 5-9. on liver DNA turnover and new synthesis and Tucker MJ, HK Adams & JS Patterson: Tamoxi- appearance of gamma glutamyltranspepti- fen. In: Laurence. D. R. McLean, A. E. M. & dase-positive foci in female rats. Carcinogene— Weatherall, M. (eds), Safety testing of new sis 1986, 7, 200772014. drugs. 1984, Academic Press, New York, pp. 125—161.