Virchows Arch [Cell Pathol] (1983) 42:227-233

VirchowsAMdvB 9 Springer-Verlag 1983

Nafoxidine Administered to Newborn Female GR Mice Arrests the Development of Their Mammary Glands Judy M. Strum Department of Anatomy, University of Maryland, School of Medicine. 655 West Baltimore Street, Baltimore, Maryland 21201, USA

Summary. Mice of the GR strain develop many hormone-dependent mammary tumors in response to estrogen and progesterone stimulation. Since this strain is so sensitive to steroid hormones, we administered a single dose of the antiestrogen Nafoxidine to female GR mice within 24 hours after their birth. This treatment arrested the development of their mammary glands and when the mice were adults, 10 weeks old, they did not cycle normally but were in a state of persistent estrus. Whole mounts of mammary glands from Nafoxidine-treated mice revealed cystic areas within some ducts and bulbous swellings at the ends of others. No hyperplastic alveolar nodules (HAN) were identified in the glands. In contrast, a single dose of 17 fl estradiol administered within 24 h after birth, resulted in a highly branched gland displaying typical end buds, a few alveoli and more HAN than were observed in glands of control adult mice of the same strain. Thus Nafoxidine treatment not only arrested the development of the mammary glands in female GR mice (causing them to appear "masculinized") but it also produced abnormalities within the glands. Key words: Nafoxidine- Mammary gland - Estrogen - Mice

Introduction The compound Nafoxidine is commonly considered to act as an antiestrogen or estrogen antagonist (Jacobi and Lloyd 1978), but in some tissues it functions as an atypical estrogen (Clark et al. 1973, 1974, 1977). For example, in the immature rat uterus Nafoxidine greatly stimulated the growth of the epithelium and produced uterine tumors (Clark and McCormack 1980). This estrogenic effect of Nafoxidine produced changes similar to those seen after chronic exposure to estrogen, where preneoplastic and neoplastic changes were induced in certain organs of the reproductive tract (Hertz 1974). However, Nafoxidine acted as an antiestrogen in tissues such as ovary where

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it caused the production of cysts (Clark and McCormack 1977). A variety of other reproductive tract abnormalities have been reported following Nafoxidine treatment (Clark and McCormack 1977), but to our knowledge the mammary glands of such animals have not been examined. Since the mammary gland in newborn animals is sensitive to estrogens and androgens, we wished to administer Nafoxidine just after birth and examine its effects on the subsequent development, growth and function of the mammary gland. The GR mouse strain was selected as the animal model because it is highly sensitive to steroid hormones and readily develops hormoneresponsive mammary tumors during pregnancy (Mulbock 1965; van Nie and Thung 1965). Other newborn GR mice were injected with 17 fl estradiol for comparison with Nafoxidine-treated animals. Our results indicate that Nafoxidine treatment of newborn female mice arrests the development of their mammary glands and also produces abnormalities within various parts of the epithelium.

Materials and Methods A total of 16 newborn GRS/AJs mice (GR mice) less than 24 h old, were injected in the nape of the neck with 25 ~tg Nafoxidine (1-[2-[p-(3,4-dihydro-6-methoxy-2-phenly-l-naphthyl) phenoxy]ethyl]hydrochloride (U-11100A) (manufactured by the Upjohn company and obtained through the National Cancer Institute, NIH, Bethesda, Maryland, USA). The Nafoxidine was administered as a suspension in warm water. Mice used as controls were females the same age as the Nafoxidine-treated group. At 4 weeks of age the mice were weaned and separated according to sex. When the female mice were 10 weeks of age, vaginal smears were taken to establish the various phases of the estrous cycle, and it was found that all Nafoxidine-treated mice were in persistent estrus. When the mice were 12 weeks old the treated and control groups were killed and their abdominal mammary glands were removed and fixed for whole mount preparations and for light and electron microscopy. Since the Nafoxidine-treated mice were found to be in constant estrus, their mammary glands were compared with control glands removed at the estrus stage of the cycle. Another group of newborn GR mice (24 h old) were injected in the nape of the neck with 20 ~tg 17 fl estradiol dissolved in sesame oil. The mice were weaned and separated according to sex when they were 1 month old. Vaginal smears of these female mice, taken at 10 weeks of age, indicated that most of them did not cycle normally and about half of them were in persistent estrus. Three mice in persistent estrus were killed at 12 weeks of age and whole mounts were made of their abdominal mammary glands. These were compared with glands from Nafoxidine-treated and control mice. Mammary gland tissue was also removed and fixed for light and electron microscopy. All tissue samples were placed overnight in 2% paraformaldehyde-2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 (Karnovsky 1965). They were then transferred to 0.2 M sodium cacodylate buffer, pH 7.3. For whole mount preparations, the glands were stretched out on glass microscope slides prior to fixing them in paraformaldehyde-glutaraldehyde. After 2 to 3 days in 0.2 M buffer, the whole mount preparations were placed into acetone (two changes, 24 h each) and transferred to 0.1% methylene blue in saline (for overnight staining). The glands were then dehydrated through increasing concentrations of ethanol and photographed when they were in 100% ethanol. These whole mount preparations were used to evaluate the extent of epithelial development within the gland. For light and electron microscopy, pieces of tissue were transferred from the 0.2 M buffer to 1% OsO4 in 0.1 M sodium cacodylate buffer, pH 7.4, where they were post-fixed for one hour at 4~ C. They were then dehydrated through a graded series of ethanol, stained en block with 2% uranyl acetate and embedded in Epon 812. Semi-thin sections were cut and stained with Toluidine blue-O for

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Fig. 1-3. Whole mount preparations of mammary glands from adult female GR mice Fig. 1. Control mouse : note extensive branching and peripheral development of ducts, x 4 Fig. 2. Mouse treated neonatally with Nafoxidine: only major ducts are evident and gland appears "masculinized". • 4 Fig. 3. Mouse treated neonatally with 17 fl estradiol: branching and development are similar to controls and a small HAN is present (arrow). x 6.5

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evaluation by light microscopy. Thin sections were then cut from selected blocks, stained with uranyl acetate (Watson 1958) and lead citrate (Reynolds 1963) and examined in a Philips EM201 electron microscope.

Observations

Whole mount preparations of mammary glands from control vs. Nafoxidine-treated female GR mice demonstrated the marked effect this compound had on the differentiation of the gland (compare Figs. 1, 2). Since the Nafoxidine-treated mice were all found to be in persistent estrus, their glands were compared with glands of control mice in the estrus phase of the cycle. Mammary glands from Nafoxidine-treated female mice usually had only a major duct system with a few slender branches (Fig. 2). In contrast, the mammary glands from control mice had an extensive duct system (Fig. 1) and occasionally end buds and alveoli were observed. Two hyperplastic alveolar nodules (HAN) were also identified in glands from control mice, but no HAN were identified in the mammary glands of Nafoxidine-treated mice. GR mice treated neonatally with 17 fl estradiol did not cycle normally and vaginal smears often revealed persistent estrus. However, a few mice displayed partial cycling (from estrus to diestrus) and whole mounts of their glands showed many major ducts with a large number of end buds. Whole mounts of glands from mice in persistent estrus revealed an appearance very similar to glands from control mice (Fig. 3). However, estrogentreated glands displayed more HAN and more alveoli than those from control mice. Light microscope observations of whole mounts from Nafoxidinetreated mice often revealed bulbous abnormal swellings at the ends of some ducts (Fig. 4). Occasionally ducts with cystic swellings were also encountered. However, HAN were not observed in the whole mounts of mammary glands from Nafoxidine-treated GR mice. In contrast, HAN were frequently observed in glands from 17 fl estradiol-stimulated mice (Fig. 5) and they were also occasionally seen in control mice. In sections observed by both light and electron microscopy, the large ducts within Nafoxidine- and 17 fl estradiol-treated glands were similar to those within control glands. An incomplete layer of myoepithelial cells was present underlying the epithelial cells which were adjacent to the lumens. In Nafoxidine-treated mice the duct epithelial cells showed little secretory activity, but they produced mammary tumor virus (MTV) (Fig. 6). Type A virus particles were clustered in the cytoplasm of many cells (Fig. 6) and mature MTV was observed within the lumens of the ducts (Fig. 7). Glands from control and estradiol-stimulated mice also contained MTV within the lumens of end buds and ducts. In control and 17 fl estradiolstimulated mice, casein-like particles were observed in the cells of the end buds (Fig. 8), but such particles were not seen in mammary glands from Nafoxidine-treated mice.

Fig. 4. Bulbous swellings (arrows) are present at ends of ducts in whole mount of gland from Nafoxidine-treated mouse, x 26 Fig. 5. HAN (arrow) are common in glands from mice treated neonatally with 17 fl estradiol. x 26 Fig. 6. Electron micrograph of epithelial cells lining duct in mammary gland from Nafoxidinetreated mouse. Mammary tumor virus particles (V) are synthesized by these cells and released into the lumen (L). x 16,600 Fig. 7. Mature virus particles (arrow) are present in ducts of gland from Nafoxidine-treated mouse, x 44,000 Fig. 8. Casein-like particles (arrow) are present in terminal end buds of control and 17 fl estradiol-treated mice. x 48,100

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Discussion

The effect of Nafoxidine on neonatal mammary glands has not heretofore been reported, although this compound produced marked abnormalities in the ovary, vagina and uterus of rats when it was administered neonatally (Clark and McCormack 1977). Since the mammary gland of the GR mouse is highly sensitive to estrogen, we wished to examine what effect this atypical estrogen would have on its early development and subsequent function. The GR mouse strain is unusual in that during pregnancy the mice often develop estrogen-progesterone dependent mammary tumors. Moreover, the gland is so sensitive to hormones it need not be "primed" (as other mammary glands need be) in order to differentiate in culture (Harbell et al. 1977). In addition, hormone independent mammary tumors also arise in this strain from HAN. Since tumors and hyperplasias were characteristically found in the reproductive tract of rats after Nafoxidine treatment, we anticipated that the compound might produce similar changes in the mammary glands of GR mice. However this was not the case. Nafoxidine arrested the development of the ductal tree in GR mammary glands so that end buds and alveoli did not develop. However, bulbous swellings were observed at the ends of some ducts. However, HAN were not identified in glands from Nafoxidine-treated mice, probably because the terminal portions of the ductal tree did not develop in these animals. In contrast, 17 fl estradiol stimulated ductal growth in the glands and more HAN were found in these glands than in the glands of control mice. Moreover, whereas 17 fl estradioltreated mice were able to bear litters, the Nafoxidine-treated mice were sterile. The cystic areas observed in some ducts from Nafoxidine-treated mice suggest an antiestrogen effect of the compound, since estrogen is required for the normal growth of ducts within the mammary gland. However, our results also indicate that Nafoxidine is acting as an atypical estrogen in these female mice by apparently inducing masculinization of the hypothalamic-hypophyseal axis as was suggested in the studies of Clark and McCormack (1977). This in turn is reflected in a "masculinized" mammary gland. Whether or not the abnormalities induced by Nafoxidine result in a greater incidence of mammary tumors in older mice remains to be determined, but in a preliminary study only 1 of 9 treated mice developed a tumor within a year following treatment. Typically, 2 or 3 mice in a control population of 9 would develop tumors by 12 months of age. In a previous study we showed that the hormone-dependent mammary tumors in this strain which arise during pregnancy, probably have their origins within the smallest ducts and/or alveoli (Strum 1981) whereas the hormone independent mammary tumors arise from HAN. Since Nafoxidine inhibits the development of both HAN and the distal ends of the mammary tree this might be a partial explanation as to why tumors were not common in the treated mice. Acknowledgements. The assistance.

author wishes to thank Ms. Marietta McAtee for excellent technical

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This research was supported by PHS Grant Number R01 CA20764 awarded by the National Cancer Institute, DHHS. Nafoxidine (NSC-70735) was a gift from the Drug Liaison and Distribution Section, Investigational Drug Branch, NCI, Bethesda, Maryland, USA.

References Clark JH, McCormack S (1977) Clomid or nafoxidine administered to neonatal rats causes reproductive tract abnormalities. Science 197: | 64-165 Clark JH, McCormack S (1980) The effect of Clomid and other triphenylethylene derivatives during pregnancy and the neonatal period. J Steroid Biochem 12:47-53 Clark JH, Anderson JN, Peck EJ, Jr (1973) Estrogen receptor-antiestrogen complex: atypical binding by uterine nuclei and effects on uterine growth. Steroids 22:707-718 Clark JH, Anderson JN, Peck EJ, Jr (1974) Oestrogen receptors and antagonism of steroid hormone action. Nature 251:446-448 Clark, JH, Paszko Z, Peck EJ, Jr (1977) Nuclear binding and retention of the receptor estrogen complex: relation to the agonistic and antagonistic properties of estriol. Endocrinology 100:91-96 Harbell JW, Bowman PD, Shannon JM, Daniel CW (1977) Long-term organ culture of mouse mammary gland. In vitro 13: 490-496 Hertz R (1974) The estrogen-cancer hypothesis. Cancer 38:534-540 Jacobi JM, Lloyd HM (1978) Anti-oestrogenic effects of Nafoxidine on the synthesis of DNA by the rat pituitary gland. J Endocrinol 76:555-556 Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27:137 A Muhlbock O (1965) Note on a new inbred mouse strain. Eur J Cancer 1:123-124 Reynolds ES (1963) The use of lead citrate at high pH as an electron - opaque stain in electron microscopy. J Cell Biol 17:208-212 Strum JM (1981) Autoradiographic and ultrastructural observations of plaques and hormonedependent mammary tumors in GR mice. Exp Mol Pathol 35:219-230 van Nie R, Thung PJ (1965) Responsiveness of mouse mammary tumors to pregnancy. Eur J Cancer 1:41-50 Watson ML (1958) Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478 Received October I / Accepted November 16, 1982