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Lipidosis induced in rat uteri by high doses of tamoxifen
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Lipidosis induced in rat uteri by high doses of tamoxifen Niki Ioannidis Anatomisches Institut der Universitat Kiel, Olsha usenstraBe 40, D-24098 Kiel, Germany
Summary. The anti-estrogen ic dru g tamoxifen is an am-
phiphilic cationic compound and might therefore be expected to inte rfe re with int ralysosomal catabolism of polar lipids as has been previously rep orted with several other amphiphilic cationic drugs. The purpose of this study was to investigate whether there is lipido sis induction in the uterus. High oral doses of tam oxifen (100 mgl kg) were administered to 9 adult rats for 6-14 wee ks. Their uteri were examined by light and electron microscopy. Lipidosis-like alte ra tions were see n in the glandular epithelia and in the myometrium. The lumin al epithelium was most seve rely affected. The highest degree of intraepithelial change was already observed afte r a shor t-ter m treatment (6 weeks). The result s support the previously proposed concept of a relat ionship bet ween the amphiphilic cationic characte r of a comp ound and its ability to cause intralysosom al storage of pol ar lipids afte r a high dosage treatm ent of the se drugs in anim als.
Key words: Tamoxifen - Cat ionic amphiphilic dru g Drug-induced lipidosis - Ut erus - Rat
Introduction Ge ne ra lized lipidosis can be indu ced in expe rime nta l animals by treatment with certai n amphiphilic cationic dru gs whith varying ther apeuti c actio ns. These dru gs accumulate in Iysosomes, form indigestible complexes with polar lipids, and disturb the degradat ion of pol ar lipids thu s causing intr alysosom al lipid storage . Drug-induced lipidosis resembles the cytological picture of inherited lipid storage diseases in man. At the ultrastructural level, dru ginduce d lipidosis is characterized by the occurrenc e of lamellat ed and crysta lline- like inclusion bodi es delimit ed Corres pondence to: N. Ioannidi s
Ann Anat (1998) 180: 315-319 © Gustav Fischer Verlag
by a membrane. The inclusions represent Iysosom es filled with undigested polar lipids. The biochemical and cytological feature s of dru g-induc ed lipidosis and the prop osed pathogen etic mechanisms have already been reviewed (Lullrnann et al. 1978; UHlmann -Rauch 1979). The nonste ro idal anti -estrogen tam oxifen is a compound having a cationic amphiphilic character (see Fig. 1). Since the 1970's tamoxifen has been widely used for the endoc rine treatment of advance d br east cancer. Clinic al studies have shown th at this dru g redu ces the incidenc e of contralater al bre ast canc er (Fisher et Redmond 1991) and it has been tested as a preventi ve age nt in healthy pre- and postmenopausal women who were at high risk for this disease (Powles et al. 1990; Jord an 1992). Tamoxifen acts mainl y as an estroge n antagonist in the mammar y gland , but partial oestrogen ago nistic effects in the reproductive tr act have also been obser ved (Nishino 1979; Boc card o et al. 1981; Gottardis et al. 1988). After treatment with tamoxifen, especially with higher doses, retinopathy (KaiserKupfer and Lippm an 1978; Bentley et al. 1992) was observed. Tamoxifen cau ses lipidosis in several tissues of the rat (Lullmann and Lilllmann -Rauch 1981). In matu re rodents treated with ade qua te doses of tamoxifen, a decrease in uterine weight was observed (Greaves et al. 1993; Rumpel et al. 1993). The present experiment was undertaken to discover whether tamoxifen induc es lipidosis in the uterus of the rat afte r long-term high-dosage treatment.
Fig. 1. Chemical stru ctur e of tamoxifen .
Material and methods Mature virgin Wistar rats with initial body weights of 190g-220 g were kept under standard laboratory conditions. They had free access to water and ground chow (Altromin, Altrogge) which contained tamoxifen citrate (Nolvadex, Imperial Chemical Industries, Ltd.) at a concentration of 0.15%. The test diet was freshly mixed every fourth day. Control rats were kept under identical conditions but received drug-free food. Fixed amounts of food were offered in special containers which minimized spilling. The body weights and food consumption were recorded and the resulting daily drug dosages calculated. The tamoxifen-treated rats did not eat as much as the controls (6 g per 100g of body weight per day versus 10 g in the control group). The daily dosages of the drug were approximately 100 mglkg. For the present ultrastructural investigation, 9 rats were treated with tamoxifen between 6 and 14 weeks. 3 animals were treated 6 respectively 12 weeks. Two animals were investigated after 8 and 14 weeks. At the end of the experiments the animals were deeply anesthetized with pentobarbital (100 mg/kg, i. p. injection), killed by opening the left cardiac ventricle. They were then fixed by vascular perfusion (90 minutes) with 3% glutaraldehyd (in 0.1 M phosphate buffer, pH 7.2). The uterine horns were removed and cut into pieces. After postfixation in 2% OS04 for two hours, tissues were embedded in araldite. For light microscopy, semithin sections were stained with toluidine blue. Tissue samples for electron microscopy were stained with uranyl acetate and lead citrate and examined with a Zeiss 900 electron microscope.
Results Tamoxifen caused a gradual loss of body weight. The treated rats lost 10-20% of their initial body weight, while the weight of the controls increased by about 1015%. After 6 weeks of treatment the rats developed a rough coat. In addition, several animals showed focal areas of alopecia. The uterine horns of the treated rats became increasingly atrophic after prolonged treatment. The diameter of the horns, the glands and the myometrium were strongly reduced (Fig. 2). In the control animals occurred as typical cyclical changes vacuoles and drop-shaped inclusions in the luminal epithelial cells. At the ultrastructural level, the luminal epithelium was characterized by the presence of vacuoles containing remnants of nuclei, cytoplasmic organelles and fat droplets also (Fig. 3). Such vacuoles can be interpreted as resulting from processes occurring during late estrus and metestrus, autophagy and heterophagy associated with apoptotic cell death and atrophy of the persistent cells respectively (Ljungkvist 1971; Sandow et al. 1979). In the rats treated with tamoxifen, the endometrium showed no more cyclical changes, that results directly from the antiestrogenic
Fig. 2. a) Uterine hom of a control animal. The small lumina belong to endometrial glands and blood vessels. b) Uterine hom of an animal treated for 6 weeks with tamoxifen (100 mglkg). The diameter of the uterus is reduced. Semithin sections, toluidine blue. a, b) x 550
316
b
property of tamoxifen. The most conspicious cytological glands, the number of abnormal cytoplasmic inclusions feature seen in all cases, was that the luminal epithelial was lower than in the luminal epithelium. At the ultracells were filled with abnormal, intensely stained inclu- structural level, the abnormal cytoplasmic inclusions corsions (Fig.4). In the epithelium of the endometrial responded to lamellated and crystalline-like bodies deliminated by a membrane (Fig. 5). Autophagic vacuoles, apoptotic bodies or normal lysosomes were not observed. The morphological alterations of the luminal epithelium which were seen at the first killed animals (after six weeks of treatment) could not be increased by prolongation of drug administration up to 14 weeks.
Fig. 3. Uterine epithelium of control rats (late estrus). a) Light microscopy of the epithelium. Some cells are filled with vacuoles, apoptotic bodies and dense inclusion bodies (arrows). b) The cells contain apoptotic bodies (Ap) , fat droplets (arrowheads) and lysosomes (*). c) Apoptotic body with cell membranes (em), enclosed in a vital epithelial cell. a) x 550, b) x408o, c) x15600
Fig. 4. Endometrium of a rat treated with tamoxifen (100 mg! kg), for 6 weeks. a) Light microscopy of luminal epithelial (Le) and glandular (g) cells filled with inclusion bodies in the apical area. b) Luminal epithelial cells containing many lamellated inclusion bodies. c) Lamellated inclusions at higher magnification. a) x175, b) x5160, c) x26400
317
Disc:ussion Tamoxifen is a non-steroidal derivative of triphenylene which in mammals usually acts as an anti-estrogen, depending on the dose and the species in question (Heel et al. 1978). It inhibits estrogen-stimulated rises in uterine wet weight and vaginal cornification. Jordan et al. (1976) observed an increase in the uterine weight of intact rats after treatment with higher doses of tamoxifen. In our investigation the uteri became more and more atrophic after the treatment with tamoxifen . Observations, such as atrophy of the uteri and the cessation of typical cyclical alterations, agree well with the results of Rumpel et a1. (1993) and can be explained by the anti-estrogenic action of tamoxifen. The remarkable feature in the results of the present study is the high degree of lipidosis in the uterine epithelium and its development within a few weeks. The lamellated and crystalline-like inclusion bodies represent lysosomes filled with aggregated polar lipids in the lamellar and hexagonal phase respectively (Buchheim et al, 1979). The lipidosis results from the impairment of intralysosomal degradation of polar lipids which may come from intra- and extracellular sources. The results concerning the lipidosis observed under light microscopy and at an ultrastructural level are similar to those previously brought about by several other cationic amphiphilic drugs (UHlmann-Rauch 1979; Geist et Lullmann-Rauch 1994). The degree of lipidosis developing in the cells is determined, among other factors, by the load of polar lipids which are degraded physiologically by the lysosomes. We explain the rapid development of marked lipidosis in the rat uterine epithelium as follows: the cellular turnover of the luminal epithelium in cycling rodents is fast; the life span of rat uterine epithelial cells being 5.9 days, with an estrous cycle of 4.5 days (Bertalanffy and Lau 1963). The rapid cell turnover and the physiological reduction of the epithelium during cyclus is associated with apoptotic cell death (Sandow et al. 1979; Pollard et al. 1987), an increase of Iysosomes and the occurrence of numerous autophagic vacuoles (Sandow et al. 1979) filled with cell fragments of the apoptotic cells. In the proestrus of the next cycle the epithelium is free of such vacuoles. It can be assumed that, within those cells that survive one cycle, intralysosomal degradation of the cell fragments has been completed during diestrus. Tamoxifen blocks the degradation of the lipid constituents derived from the sequestered biomembranes and leads to a lysosomal storage. This sequence is repeated a few times only, since the continued drug treatment abolishes cyclic changes in the Fig. 5. Luminal epithelium of a rat treated for 14 weeks with uterine epithelium. Therefore the supply of polar lipids tamoxifen (100 mg/kg). destined for intralysosomal degradation becomes reduced a) The cells are filled with crystalline-like bodies (arrows) and and lipidosis will not continue to increase. small vacuoles (arrowhead). b) Inclusions at higher magnificaIn humans only a retinopathy after tamoxifen treattion, deliminated by a membrane. The vacuoles consist of rough ment with higher doses (240 mgld) (Kaiser-Kupfer and endoplasmatic reticulum (arrowhead). a) x73oo, b) x88000 Lippman 1978) and also with lower doses (40 mgld) (Bentley et al. 1992) has been reported. The ocular alterations have been explained by the cationic amphiphilic character of tamoxifen and had also lipidosis-like charac-
318
ter (Kaiser-Kupfer et al. 1981). Lipidosis-like changes in the human uterus after therapy with tamoxifen have not yet been described, possibly because they were not searched for. Acknowledgements. This work was financially supported by the Deutsche Forschungsgemeinschaft (Lu 172). I wish to thank Sonja Seiter for excellent technical assistance and Heidi Waluk and Heide Siebke for the photographical work.
References Bentley CR, Davies G, Aclimandos W (1992) Tamoxifen retinopathy: a rare but serious complication. Br Med J 304: 495-496 Bertalanffy FD, Lau C (1963) Mitotic rates, renewal times and cytodynamics of the female genital tract epithelium in the rat. Acta Anat 54: 39-81 Boccardo F, Bruzzi P, Rubagoni A, Nicolo G, Rosso R (1981) Estrogen-like action of tamoxifen on vaginal epithelium in breast cancer patients. Oncology 38: 281-285 Buchheim W, Drenckhahn D, Lillimann-Rauch R (1979) Freezefracture studies of cytoplasmic inclusions occuring in experimental lipidosis as induced by amphiphilic cationic drugs. Biochim Biophys Acta 575: 71-80 Fisher B, Redmond C (1991) New perspective on cancer of the contralateral breast: a marker of assessing tamoxifen as a preventive agent. J Nat Cancer Inst 83: 1278-1280 Geist SH, Lullmann-Rauch R (1994) Experimentally induced lipidosis in uterine and vaginal epithelium of rats. Ann Anat 176:3-9 Gottardis MM, Robinson SP, Satyatyswaroop PG, Jordan VG (1988) Contrasting actions of tamoxifen on endometrial and breast tumor growth in the athymic mouse. Cancer Res 48: 812-815 Graeves P, Goonetilleke R, Nunn G, Topham J, Orton T (1993) Two-year carcinogenity study of tamoxifen in Alderley Park Wistar-derived rats. Cancer Res 53: 3919-3924 Heel RC, Brogden RN, Speight TM, Avery GS (1978) Tamoxifen: A review of its pharmacological properties and therapeutic use in the treatment of breast cancer. Drugs 16: 1- 24
Jordan VC (1976) Antiestrogenic and antitumor properties of tamoxifen in laboratory animals. Cancer Treatm Rep 60: 1409-1419 Jordan VC (1992) The role of tamoxifen in the treatment and prevention of breast cancer. Curr Probl Cancer 16: 134-176 Kaiser-Kupfer MI, Lippman ME (1978) Tamoxifen retinopathy. Cancer Treat Rep 62: 315-320 Kaiser-Kupfer MI, Kupfer C, Rodrigues MM (1981) Tamoxifen retinopathy. A clinicopathological report. Ophthalmology 88: 89-93 Ljungkvist I (1971) The attachment of rat uterine luminal epithelium. III. The effect of estradiol, estron and estriol on the morphology of the luminal epithelium of the spayed, virgin rat. Acta Soc Med Ups 76: 139-157 Lillimann H, Lullmann-Rauch R, Wassermann 0 (1978) Lipidosis induced by amphiphilic cationic drugs. Biochem Pharmacol 27: 1103-1108 Lullmann-Rauch R (1979) Drug induced lysosomal storage disorders. In: Dingle IT, Jacques PJ, Shaw IH (eds) Lysosomes in Applied Biology and Therapeutics. Elsevier North-Holland, Amsterdam, Vol 6, pp 49-130 Lullmann H, Lullmann-Rauch R (1981) Tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses. Toxicol Appl Pharmacol61: 138-146 Nishino Y (1979) Pharmakologie der Antiostrogene, Gynakologie 12: 199-211 Pollard JW, Pacey J, Cheng SVY, Jordan EG (1987) Estrogens and cell death in murine uterine luminal epithelium. Cell Tissue Res 249: 533-540 Powles TJ, Hardy JR, Ashley SE, Farrington GH, Cosgrove D, Davey JR, Dowsett M, McKinna JA, Wash AG, Sennett HD, Tillyer CR, Treleaven JG (1989) A pilot trial to evaluate the acute toxicity and feasibility of tamoxifen for prevention of breast cancer. Br J Cancer 60: 126-131 Rumpel E, Kuhnel W, Michna H (1993) Ostrogene und antiostrogene Effekte am Uterus intakter Ratten nach Langzeitbehandlung mit Tamoxifen. Ann Anat Suppl 175: 45 Sandow BA, West NB, Normann RL, Brenner RM (1979) Hormonal control of apoptosis in hamster uterine luminal epithelium. Am J Anat 156: 15-36
Accepted February 24,1998
319
Lipidosis induced in rat uteri by high doses of tamoxifen Niki Ioannidis Anatomisches Institut der Universitat Kiel, Olsha usenstraBe 40, D-24098 Kiel, Germany
Summary. The anti-estrogen ic dru g tamoxifen is an am-
phiphilic cationic compound and might therefore be expected to inte rfe re with int ralysosomal catabolism of polar lipids as has been previously rep orted with several other amphiphilic cationic drugs. The purpose of this study was to investigate whether there is lipido sis induction in the uterus. High oral doses of tam oxifen (100 mgl kg) were administered to 9 adult rats for 6-14 wee ks. Their uteri were examined by light and electron microscopy. Lipidosis-like alte ra tions were see n in the glandular epithelia and in the myometrium. The lumin al epithelium was most seve rely affected. The highest degree of intraepithelial change was already observed afte r a shor t-ter m treatment (6 weeks). The result s support the previously proposed concept of a relat ionship bet ween the amphiphilic cationic characte r of a comp ound and its ability to cause intralysosom al storage of pol ar lipids afte r a high dosage treatm ent of the se drugs in anim als.
Key words: Tamoxifen - Cat ionic amphiphilic dru g Drug-induced lipidosis - Ut erus - Rat
Introduction Ge ne ra lized lipidosis can be indu ced in expe rime nta l animals by treatment with certai n amphiphilic cationic dru gs whith varying ther apeuti c actio ns. These dru gs accumulate in Iysosomes, form indigestible complexes with polar lipids, and disturb the degradat ion of pol ar lipids thu s causing intr alysosom al lipid storage . Drug-induced lipidosis resembles the cytological picture of inherited lipid storage diseases in man. At the ultrastructural level, dru ginduce d lipidosis is characterized by the occurrenc e of lamellat ed and crysta lline- like inclusion bodi es delimit ed Corres pondence to: N. Ioannidi s
Ann Anat (1998) 180: 315-319 © Gustav Fischer Verlag
by a membrane. The inclusions represent Iysosom es filled with undigested polar lipids. The biochemical and cytological feature s of dru g-induc ed lipidosis and the prop osed pathogen etic mechanisms have already been reviewed (Lullrnann et al. 1978; UHlmann -Rauch 1979). The nonste ro idal anti -estrogen tam oxifen is a compound having a cationic amphiphilic character (see Fig. 1). Since the 1970's tamoxifen has been widely used for the endoc rine treatment of advance d br east cancer. Clinic al studies have shown th at this dru g redu ces the incidenc e of contralater al bre ast canc er (Fisher et Redmond 1991) and it has been tested as a preventi ve age nt in healthy pre- and postmenopausal women who were at high risk for this disease (Powles et al. 1990; Jord an 1992). Tamoxifen acts mainl y as an estroge n antagonist in the mammar y gland , but partial oestrogen ago nistic effects in the reproductive tr act have also been obser ved (Nishino 1979; Boc card o et al. 1981; Gottardis et al. 1988). After treatment with tamoxifen, especially with higher doses, retinopathy (KaiserKupfer and Lippm an 1978; Bentley et al. 1992) was observed. Tamoxifen cau ses lipidosis in several tissues of the rat (Lullmann and Lilllmann -Rauch 1981). In matu re rodents treated with ade qua te doses of tamoxifen, a decrease in uterine weight was observed (Greaves et al. 1993; Rumpel et al. 1993). The present experiment was undertaken to discover whether tamoxifen induc es lipidosis in the uterus of the rat afte r long-term high-dosage treatment.
Fig. 1. Chemical stru ctur e of tamoxifen .
Material and methods Mature virgin Wistar rats with initial body weights of 190g-220 g were kept under standard laboratory conditions. They had free access to water and ground chow (Altromin, Altrogge) which contained tamoxifen citrate (Nolvadex, Imperial Chemical Industries, Ltd.) at a concentration of 0.15%. The test diet was freshly mixed every fourth day. Control rats were kept under identical conditions but received drug-free food. Fixed amounts of food were offered in special containers which minimized spilling. The body weights and food consumption were recorded and the resulting daily drug dosages calculated. The tamoxifen-treated rats did not eat as much as the controls (6 g per 100g of body weight per day versus 10 g in the control group). The daily dosages of the drug were approximately 100 mglkg. For the present ultrastructural investigation, 9 rats were treated with tamoxifen between 6 and 14 weeks. 3 animals were treated 6 respectively 12 weeks. Two animals were investigated after 8 and 14 weeks. At the end of the experiments the animals were deeply anesthetized with pentobarbital (100 mg/kg, i. p. injection), killed by opening the left cardiac ventricle. They were then fixed by vascular perfusion (90 minutes) with 3% glutaraldehyd (in 0.1 M phosphate buffer, pH 7.2). The uterine horns were removed and cut into pieces. After postfixation in 2% OS04 for two hours, tissues were embedded in araldite. For light microscopy, semithin sections were stained with toluidine blue. Tissue samples for electron microscopy were stained with uranyl acetate and lead citrate and examined with a Zeiss 900 electron microscope.
Results Tamoxifen caused a gradual loss of body weight. The treated rats lost 10-20% of their initial body weight, while the weight of the controls increased by about 1015%. After 6 weeks of treatment the rats developed a rough coat. In addition, several animals showed focal areas of alopecia. The uterine horns of the treated rats became increasingly atrophic after prolonged treatment. The diameter of the horns, the glands and the myometrium were strongly reduced (Fig. 2). In the control animals occurred as typical cyclical changes vacuoles and drop-shaped inclusions in the luminal epithelial cells. At the ultrastructural level, the luminal epithelium was characterized by the presence of vacuoles containing remnants of nuclei, cytoplasmic organelles and fat droplets also (Fig. 3). Such vacuoles can be interpreted as resulting from processes occurring during late estrus and metestrus, autophagy and heterophagy associated with apoptotic cell death and atrophy of the persistent cells respectively (Ljungkvist 1971; Sandow et al. 1979). In the rats treated with tamoxifen, the endometrium showed no more cyclical changes, that results directly from the antiestrogenic
Fig. 2. a) Uterine hom of a control animal. The small lumina belong to endometrial glands and blood vessels. b) Uterine hom of an animal treated for 6 weeks with tamoxifen (100 mglkg). The diameter of the uterus is reduced. Semithin sections, toluidine blue. a, b) x 550
316
b
property of tamoxifen. The most conspicious cytological glands, the number of abnormal cytoplasmic inclusions feature seen in all cases, was that the luminal epithelial was lower than in the luminal epithelium. At the ultracells were filled with abnormal, intensely stained inclu- structural level, the abnormal cytoplasmic inclusions corsions (Fig.4). In the epithelium of the endometrial responded to lamellated and crystalline-like bodies deliminated by a membrane (Fig. 5). Autophagic vacuoles, apoptotic bodies or normal lysosomes were not observed. The morphological alterations of the luminal epithelium which were seen at the first killed animals (after six weeks of treatment) could not be increased by prolongation of drug administration up to 14 weeks.
Fig. 3. Uterine epithelium of control rats (late estrus). a) Light microscopy of the epithelium. Some cells are filled with vacuoles, apoptotic bodies and dense inclusion bodies (arrows). b) The cells contain apoptotic bodies (Ap) , fat droplets (arrowheads) and lysosomes (*). c) Apoptotic body with cell membranes (em), enclosed in a vital epithelial cell. a) x 550, b) x408o, c) x15600
Fig. 4. Endometrium of a rat treated with tamoxifen (100 mg! kg), for 6 weeks. a) Light microscopy of luminal epithelial (Le) and glandular (g) cells filled with inclusion bodies in the apical area. b) Luminal epithelial cells containing many lamellated inclusion bodies. c) Lamellated inclusions at higher magnification. a) x175, b) x5160, c) x26400
317
Disc:ussion Tamoxifen is a non-steroidal derivative of triphenylene which in mammals usually acts as an anti-estrogen, depending on the dose and the species in question (Heel et al. 1978). It inhibits estrogen-stimulated rises in uterine wet weight and vaginal cornification. Jordan et al. (1976) observed an increase in the uterine weight of intact rats after treatment with higher doses of tamoxifen. In our investigation the uteri became more and more atrophic after the treatment with tamoxifen . Observations, such as atrophy of the uteri and the cessation of typical cyclical alterations, agree well with the results of Rumpel et a1. (1993) and can be explained by the anti-estrogenic action of tamoxifen. The remarkable feature in the results of the present study is the high degree of lipidosis in the uterine epithelium and its development within a few weeks. The lamellated and crystalline-like inclusion bodies represent lysosomes filled with aggregated polar lipids in the lamellar and hexagonal phase respectively (Buchheim et al, 1979). The lipidosis results from the impairment of intralysosomal degradation of polar lipids which may come from intra- and extracellular sources. The results concerning the lipidosis observed under light microscopy and at an ultrastructural level are similar to those previously brought about by several other cationic amphiphilic drugs (UHlmann-Rauch 1979; Geist et Lullmann-Rauch 1994). The degree of lipidosis developing in the cells is determined, among other factors, by the load of polar lipids which are degraded physiologically by the lysosomes. We explain the rapid development of marked lipidosis in the rat uterine epithelium as follows: the cellular turnover of the luminal epithelium in cycling rodents is fast; the life span of rat uterine epithelial cells being 5.9 days, with an estrous cycle of 4.5 days (Bertalanffy and Lau 1963). The rapid cell turnover and the physiological reduction of the epithelium during cyclus is associated with apoptotic cell death (Sandow et al. 1979; Pollard et al. 1987), an increase of Iysosomes and the occurrence of numerous autophagic vacuoles (Sandow et al. 1979) filled with cell fragments of the apoptotic cells. In the proestrus of the next cycle the epithelium is free of such vacuoles. It can be assumed that, within those cells that survive one cycle, intralysosomal degradation of the cell fragments has been completed during diestrus. Tamoxifen blocks the degradation of the lipid constituents derived from the sequestered biomembranes and leads to a lysosomal storage. This sequence is repeated a few times only, since the continued drug treatment abolishes cyclic changes in the Fig. 5. Luminal epithelium of a rat treated for 14 weeks with uterine epithelium. Therefore the supply of polar lipids tamoxifen (100 mg/kg). destined for intralysosomal degradation becomes reduced a) The cells are filled with crystalline-like bodies (arrows) and and lipidosis will not continue to increase. small vacuoles (arrowhead). b) Inclusions at higher magnificaIn humans only a retinopathy after tamoxifen treattion, deliminated by a membrane. The vacuoles consist of rough ment with higher doses (240 mgld) (Kaiser-Kupfer and endoplasmatic reticulum (arrowhead). a) x73oo, b) x88000 Lippman 1978) and also with lower doses (40 mgld) (Bentley et al. 1992) has been reported. The ocular alterations have been explained by the cationic amphiphilic character of tamoxifen and had also lipidosis-like charac-
318
ter (Kaiser-Kupfer et al. 1981). Lipidosis-like changes in the human uterus after therapy with tamoxifen have not yet been described, possibly because they were not searched for. Acknowledgements. This work was financially supported by the Deutsche Forschungsgemeinschaft (Lu 172). I wish to thank Sonja Seiter for excellent technical assistance and Heidi Waluk and Heide Siebke for the photographical work.
References Bentley CR, Davies G, Aclimandos W (1992) Tamoxifen retinopathy: a rare but serious complication. Br Med J 304: 495-496 Bertalanffy FD, Lau C (1963) Mitotic rates, renewal times and cytodynamics of the female genital tract epithelium in the rat. Acta Anat 54: 39-81 Boccardo F, Bruzzi P, Rubagoni A, Nicolo G, Rosso R (1981) Estrogen-like action of tamoxifen on vaginal epithelium in breast cancer patients. Oncology 38: 281-285 Buchheim W, Drenckhahn D, Lillimann-Rauch R (1979) Freezefracture studies of cytoplasmic inclusions occuring in experimental lipidosis as induced by amphiphilic cationic drugs. Biochim Biophys Acta 575: 71-80 Fisher B, Redmond C (1991) New perspective on cancer of the contralateral breast: a marker of assessing tamoxifen as a preventive agent. J Nat Cancer Inst 83: 1278-1280 Geist SH, Lullmann-Rauch R (1994) Experimentally induced lipidosis in uterine and vaginal epithelium of rats. Ann Anat 176:3-9 Gottardis MM, Robinson SP, Satyatyswaroop PG, Jordan VG (1988) Contrasting actions of tamoxifen on endometrial and breast tumor growth in the athymic mouse. Cancer Res 48: 812-815 Graeves P, Goonetilleke R, Nunn G, Topham J, Orton T (1993) Two-year carcinogenity study of tamoxifen in Alderley Park Wistar-derived rats. Cancer Res 53: 3919-3924 Heel RC, Brogden RN, Speight TM, Avery GS (1978) Tamoxifen: A review of its pharmacological properties and therapeutic use in the treatment of breast cancer. Drugs 16: 1- 24
Jordan VC (1976) Antiestrogenic and antitumor properties of tamoxifen in laboratory animals. Cancer Treatm Rep 60: 1409-1419 Jordan VC (1992) The role of tamoxifen in the treatment and prevention of breast cancer. Curr Probl Cancer 16: 134-176 Kaiser-Kupfer MI, Lippman ME (1978) Tamoxifen retinopathy. Cancer Treat Rep 62: 315-320 Kaiser-Kupfer MI, Kupfer C, Rodrigues MM (1981) Tamoxifen retinopathy. A clinicopathological report. Ophthalmology 88: 89-93 Ljungkvist I (1971) The attachment of rat uterine luminal epithelium. III. The effect of estradiol, estron and estriol on the morphology of the luminal epithelium of the spayed, virgin rat. Acta Soc Med Ups 76: 139-157 Lillimann H, Lullmann-Rauch R, Wassermann 0 (1978) Lipidosis induced by amphiphilic cationic drugs. Biochem Pharmacol 27: 1103-1108 Lullmann-Rauch R (1979) Drug induced lysosomal storage disorders. In: Dingle IT, Jacques PJ, Shaw IH (eds) Lysosomes in Applied Biology and Therapeutics. Elsevier North-Holland, Amsterdam, Vol 6, pp 49-130 Lullmann H, Lullmann-Rauch R (1981) Tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses. Toxicol Appl Pharmacol61: 138-146 Nishino Y (1979) Pharmakologie der Antiostrogene, Gynakologie 12: 199-211 Pollard JW, Pacey J, Cheng SVY, Jordan EG (1987) Estrogens and cell death in murine uterine luminal epithelium. Cell Tissue Res 249: 533-540 Powles TJ, Hardy JR, Ashley SE, Farrington GH, Cosgrove D, Davey JR, Dowsett M, McKinna JA, Wash AG, Sennett HD, Tillyer CR, Treleaven JG (1989) A pilot trial to evaluate the acute toxicity and feasibility of tamoxifen for prevention of breast cancer. Br J Cancer 60: 126-131 Rumpel E, Kuhnel W, Michna H (1993) Ostrogene und antiostrogene Effekte am Uterus intakter Ratten nach Langzeitbehandlung mit Tamoxifen. Ann Anat Suppl 175: 45 Sandow BA, West NB, Normann RL, Brenner RM (1979) Hormonal control of apoptosis in hamster uterine luminal epithelium. Am J Anat 156: 15-36
Accepted February 24,1998
319