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Estrogen‐induced thyroid follicular cell adenomas in C57BL/6 mice a



David L. Greenman , Benjamin Highman , James Chen , b

Winslow Sheldon & George Gass

b c


National Center for Toxicological Research, and Pathology Services Project , Food and Drug Administration , Jefferson, AR, 72079 b

National Center for Toxicological Research, and Pathology Services Project , Food and Drug Administration , Jefferson, Arkansas c

Department of Pharmacology , Oral Roberts School of Medicine , Tulsa, Oklahoma Published online: 20 Oct 2009.

To cite this article: David L. Greenman , Benjamin Highman , James Chen , Winslow Sheldon & George Gass (1990) Estrogen‐induced thyroid follicular cell adenomas in C57BL/6 mice, Journal of Toxicology and Environmental Health: Current Issues, 29:3, 269-278, DOI: 10.1080/15287399009531390 To link to this article:

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ESTROGEN-INDUCED THYROID FOLLICULAR CELL ADENOMAS IN C57BL/6 MICE David L. Greenman, Benjamin Highman, James Chen, Winslow Sheldon, George Gass

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Food and Drug Administration, National Center for Toxicological Research, and Pathology Services Project, Jefferson, Arkansas

Diethylstilbestrol (DES) was fed chronically to C57BL/6 mice at concentrations of 0, 5, 10, 20, 40, 160, 320, or 640 ppb in order to define the dose-response curve for neoplastic responses. The incidence of thyroid follicular cell adenomas was higher in control females than in males and was increased at mid-level doses of DES, especially in males. None were found in mice fed 640 ppb DES, probably because these mice died from other causes before follicular cell adenomas had developed. In both sexes, DES fed at 160 or 320 ppb significantly shortened time-to-onset of these tumors, and 40 ppb increased their probability late in life. It is concluded that DES has a causal relationship to thyroid neoplasia in C57BL/6 mice, and similarities between this and the human disease suggest that C57BL/6 mice may be an appropriate model for human thyroid neoplasia.

INTRODUCTION The age-adjusted annual incidence rates of thyroid neoplasms have been reported to be up to seven times higher in human females than in males (Cutler and Young, 1975; Ron et al., 1987), and an association between estrogens and thyroid cancer has been reported (McTiernan et al., 1984). To our knowledge, thyroid neoplasia never has been reported to be an estrogen-associated lesion in laboratory animals. The results reported here are from a study designed to quantitatively examine the neoplastic dose-response curves to chronic diethylstilbestrol (DES) exposure in a series of mouse genotypes having several different neoplastic endpoints. C57BL/6 mice were used to examine the dose-response curve, primarily for pituitary neoplasms. Although this strain responded to DES with pituitary tumors, as expected, the data reported here are related to The authors thank Mrs. Ruth York for preparation of the manuscript. Current address for C. Cass is Department of Pharmacology, Oral Roberts School of Medicine, Tulsa, Oklahoma. Requests for reprints should be sent to David L. Greenman, National Center for Toxicological Research, Jefferson, AR 72079.

269 Journal of Toxicology and Environmental Health, 29:269-278, 1990 Copyright © 1990 by Hemisphere Publishing Corporation



the unexpected occurrence of thyroid adenomas, apparently in response to chronic DES administration.

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MATERIALS AND METHODS DES [4,4'-(1,2-diethyl-1,2-ethenediyl)-bis-phenol] (CAS 56-53-1), obtained from Sigma Chemical Company (St. Louis, Mo.), was found without impurities when analyzed by GC and HPLC. Test diets, formulated with an autoclavable cereal-based diet (Purina 5010), were mixed with DES in a V-blender after autoclaving and regrinding. DES was dissolved in 95% ethanol and the solution was mixed with feed at a ratio of 5/95 (v/w). Control diets were mixed with the same proportion of ethanol. Excess ethanol was removed from feed by continuing to operate the blender under vacuum at 82°C. Generally diets were used within 10 wk of mixing. All batches of feed were analyzed for DES by either electron-capture GC or HPLC (King et al., 1977). The average concentrations of DES found were within 2% of the target concentrations. Bioassays of the control diet never detected estrogenic activity (-




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Time (Days) on Experiment FIGURE 3. Time-to-onset distributions of thyroid follicular cell adenomas in C57BL/6 mice continuously fed throughout life, beginning 1-2 wk after weaning.


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vations suggested a possible association of thyroid neoplasia with estrogens: (1) thyroid follicular cell adenomas were more frequent among female than male controls or low-dose animals despite the somewhat longer lifespan of males, and (2) the dramatic reduction in lifespan associated with 320 and 640 ppb DES exposures could have prevented appearance of the late-occurring thyroid adenomas in these high dose groups. Thus, incidence data would not reflect a consistent positive dose-reponse trend. Indeed, subsequent analyses showed a clearly dosedependent shortening of time-to-onset of thyroid adenomas. It thus seems reasonable to conclude that thyroid adenomas in this strain were estrogen-induced. The life-shortening effect of high levels of estrogen exposure may account for previous failure to detect estrogen-induced thyroid neoplasia since earlier studies in this strain were carried out only at high levels of exposure. Although growth inhibition can dramatically reduced tumor development, this factor probably was secondary to life-shortening in causing a reduced incidence of thyroid neoplasia in our study at 320 and 640 ppb. In females, 640 ppb DES did both depress growth and prevent thryroid adenomas. However, females fed 320 ppb DES grew as well as, if not better than, controls, yet had far fewer thyroid adenomas. Furthermore, growth in males was about equally depressed by 160, 320, and 640 ppb DES, yet the highest frequency of thyroid adenomas was found in those fed 160 ppb and none were present in those fed 640 ppb. The current study gives limited information concerning the mechanism of estrogen-induced thyroid neoplasia. Thyroid neoplasms, induced by goitrogens or radioactive iodine in the rat (Lindsay et al., 1966; Nadler et al., 1970), require the presence of the pituitary gland and are thought to be induced by excessive thyroid-stimulating hormone (TSH) secretion (McTiernan et al., 1984). Therefore, one might speculate that induction of thyroid adenomas in the current study was mediated through an effect on the pituitary gland since pituitary adenomas also were induced by DES in these mice. Indeed, comparisons of the frequencies of pituitary and thyroid adenomas in the current study (Greenman, 1988) are consistent with the suggestion that pituitary adenomas were causally related to thyroid adenomas. (1) Female control and low-dose groups had higher frequencies of both pituitary and thyroid adenomas than males. (2) Pituitary adenomas were more frequent than thyroid adenomas in every treatment group of both sexes. (3) Shortening of time-tothyroid adenomas was found only in those dose groups that also had a shortening of time-to-pituitary adenomas. Although the majority of pituitary adenomas in C57BL/6 mice reportedly are associated with high circulating levels of prolactin (Schechter et al., 1981), the possibility that estrogen-induced pituitary adenomas secrete high levels of TSH should be explored. Indeed, some human pituitary adenomas secrete excessive levels of TSH and are associated with evidence of hyperthyrodism

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(Kourides et al., 1977), even though the majority of them secrete high prolactin levels (Mukai, 1983). While up to 20% of the mice with thyroid tumors apparently did not have pituitary tumors, this does not rule out the possibility that chronic DES may stimulate pituitary TSH secretion. The role of sex hormones in human thyroid neoplasia is clearly suggested by much higher incidence rates in females than in males (Cutler and Young, 1975; Ron et al., 1987; Weiss, 1979; Preston-Martin et al., 1987) during the period between puberty and female menopause, with no difference before puberty and a gradual loss of the difference after menopause. Although oral contraceptive use and postmenopausal estrogen use seem to increase the risk of thyroid neoplasia among women, this increased risk is only marginal (McTiernan et al., 1984). Thus, the role of estrogens, as a causal factor in human thyroid tumorigenesis, has been suggested but is not strongly supported by epidemiological evidence. The current study, for the first time, gives experimental evidence that an estrogen may play a causal role in thyroid tumorigenesis. The C57BL/6 mouse seems to mimic the human condition and may give important clues concerning the causation of the human neoplasm.

REFERENCES Cutler, S. J., and Young, J. S., eds. 1975. Third National Cancer Survey: Incidence Data (NCI Monograph No. 41). Bethesda, Md.: NCI. Frith, C. H., Highman, B., and Konvicka, A. 1976. Advances in automation for experimental pathology. Lab. Anim. Sci 26:171-185. Greenman, D. L. 1988. Dose-response relationships between estrogenicity and carcinogenicity: rangefinding study. NCTR Technical Report for Experiment 035. Jefferson, Ark.: National Center for Toxicological Research. Kaplan, E. L., and Meier, P. 1958. Non-parametric estimation from incomplete observations. J. Am. Stat. Assoc. 53:457-481. King, J., Nony, C. R., and Bowman, M. C. 1977. Trace analysis of diethylstilbestrol (DES) in animal chow by parallel high-speed liquid chromatography, electron-capture gas chromatography, and radioassays. J. Chromatogr. Sci. 15:14-21. Kodell, R. L., Haskin, M. C., Shaw, G. W., and Gaylor, D. W. 1983. CHRONIC: A SAS procedure for statistical analysis of carcinogenesis studies. J. Stat. Comput. Simul. 16:287-310. Kourides, I. A., Ridgway, E. C., Weintraub, B. D., Bigos, S. T., Gershengorn, M. C., and Maloof, F. 1977. Thyrotropin-induced hyperthyroidism: Use of alpha and beta subunit levels to identify patients with pituitary tumors. J. Clin. Endocrinol. Metab. 45:534. Lindsay, S., Nichols, C. W., and Chaikoff, I. L. 1966. Induction of benign and malignant thyroid neoplasms in the rat. Arch. Pathol. 81:308-316. McTiernan, A. M., Weiss, N. S., and Daling, J. R. 1984. Incidence of thyroid cancer in women in relation to reproductive and hormonal factors. Am. J. Epidemiol. 12:423-435. Mukai, K. 1983. Pituitary adenomas: Immunocytochemical study of 150 tumors with clinicopathologic correlation. Cancer 52:648-653. Nadler, N. J., Mandavia, M., and Goldberg, M. 1970. The effect of hypophysectomy on the experimental production of rat thyroid neoplasms. Cancer Res. 30:1909-1911. Preston-Martin, S., Bernstein, L., Pike, M. C., Maldonado, A. A., and Henderson, B. E. 1987. Thyroid cancer among young women related to prior thyroid disease and pregnancy history. Br. J. Cancer 55:191-195.



Ron, E., Kleinerman, R. A., Boice, J. D., Livolsi, V. A., Flannery, J. T., and Fraumeni, J. F. 1987. A population-based case control study of thyroid cancer. JNCI 79:1-12. Schechter, J. E., Felicio, L. S., Nelson, J. F., and Finch, C. E. 1981. Pituitary tumorigenesis in aging female C57BL/6J mice: A light and electron microscopic study. Anat. Rec. 199:423-432. Tarone, R. E. 1975. Tests for trend in life table analysis. Biometrika 62:679-682. Weiss, W. 1979. Changing incidence of thyroid cancer. JNCI 62:1137-1142.

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Received January 1, 1988 Accepted October 8, 1989

6 mice.

Diethylstilbestrol (DES) was fed chronically to C57BL/6 mice at concentrations of 0, 5, 10, 20, 40, 160, 320, or 640 ppb in order to define the dose-r...
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