(CANCER RESEARCH 48, 3801-3807, July 1, 1988] Adrenal Regulation of Mammary Tumorigenesis in Female Sprague-Dawley Rats: Incidence, Latency, and Yield of Mammary Tumors1 Julia H. Carter,2 Harry W. Carter, and James Meade Wood Hudson Cancer Research Laboratory, Covington, Kentucky 41011 [J. H. C., H. W. C.J, and Department of Laboratory Medicine, St. Elitabeth Medical Center, Covington and Edgewood, Kentucky 41015 [H. W. C.J ABSTRACT interest to know if these regenerated adrenals were also resistant to a second dose of DMBA. Secondly, Jensen et al. (5) dem Huggins and Morii (J. Exp. Mod., 114: 741, 1961) reported that onstrated that 4 h after exposure to DMBA, incorporation of massive adrenal necrosis occurs in 79 and 100% of female Sprague- [3H]thymidine by adrenals was reduced 56%, suggesting that Dawley rats receiving 20 and 30 mg, respectively, of the mammary carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Here, adrenal ne inhibition of DNA synthesis was a very early manifestation of crosis and regeneration were studied in 158 rats for up to 21 days post- DMBA-induced damage in the adrenal. Pretreatment of ani DMBA by radioautography of the adrenals of animals given 50 ¿iCi mals with drugs that protect adrenals from DMBA-induced [3H]thymidine 30 min before sacrifice. Adrenal cell proliferation was damage prevented inhibition of [3H]thymidine incorporation markedly inhibited 21 days post-DMBA. Regenerated adrenals were into the acid-insoluble fraction of adrenals from DMBA-treated more susceptible to this adrenocorticolytic effect. To investigate if alter rats (6). Although DNA synthesis in adrenals was inhibited 4- ations in adrenal function modify tumorigenesis, animals underwent ad- 20 h after DMBA, it was not known in what cell type or in renalectomies (ADX), hypophysectomies, ovariectomies, and pituitary what zone of the adrenal DNA synthesis was inhibited, nor transplants alone or in combination 6 days after receiving DMBA (20 were later stages of necrosis and regeneration examined. mg/100 g intragastrically) at 50 days of age. To prevent adrenal necrosis, The purpose of these studies initially was to determine if 24 animals were pretreated with metyrapone. Methylprednisolone ace there are persistent long-term abnormalities in adrenal physi tate, 1 mg ¡.m.,was given to 40 animals every 5 days beginning 6 days post-DMBA. There were 50 non-DMBA-treated intact and surgical ology in female Sprague-Dawley rats given carcinogenic doses controls. DMBA was necessary but not sufficient to induce mammary of DMBA. To accomplish this, adrenal parenchymal and inter tumors. No tumors developed in controls or in 46 animals hypophysec- stitial cell proliferation was studied 2-21 days after DMBA in tomized 6 days after DMBA. Metyrapone reduced tumor incidence and the 3 zones of the adrenal cortex by quantitation of cell labeling yield. ADX after DMBA treatment increased the tumorigenic response by radioautography of adrenals from animals given [3H]thymi- and eliminated resistance to tumorigenesis in older rats. Only three dine. Rather than sacrifice all animals, we removed adrenals tumors developed in DMBA-treated rats receiving methylprednisolone surgically for radioautographic studies. To our surprise, breast acetate. Mammary tumorigenesis was increased by pituitary transplant tumors appeared early and grew rapidly in the adrenalectomized 6 days after DMBA to intact and ADX animals. Ovariectomy 6 days survivors, prompting us to investigate further the role of the after DMBA was as effective as methylprednisolone acetate in preventing adrenal and DMBA-induced adrenal necrosis in mammary tumorigenesis; ADX did not overcome either inhibition. We conclude that adrenal hormones inhibit proliferation of initiated mammary cells. carcinogenesis initiated by this drug. The purpose of the ensuing carcinogenesis studies was to evaluate the role of alterations in adrenal function and corti- INTRODUCTION costeroid level after exposure to an initiating dose of DMBA The following experiments began as an inquiry into the extent upon tumor incidence, latency, and yield. While the effect of of adrenal regeneration following DMBAMnduced necrosis in hormones, such as prolactin, on the growth characteristics of Sprague-Dawley rats. Huggins and Morii (1) demonstrated that established DMBA-induced mammary tumors has been well adrenal apoplexy occurred 3 days following p.o. administration characterized (7), the role of hormones on the expansion of the of DMBA to 50-day-old female Sprague-Dawley rats. The population of initiated cells to nodules has been neglected. dosage causing death to one-half of the rats was 27 mg/ml. The optimum dose for induction of adrenal necrosis was 30 mg (18 MATERIALS AND METHODS mg/lût)g body weight). At this dosage all animals survived and developed adrenal hemorrhage and necrosis, while at the lower Animals and Carcinogen. Female Sprague-Dawley CD rats were dose of 20 mg (12 mg/100 g body weight), which had previously obtained from Charles River Breeding Laboratories, Wilmington, MA. been shown to be carcinogenic (2), 79% of the animals devel DMBA (Eastman Organic Chemicals, Rochester, NY) was dissolved in oped adrenal necrosis. Interestingly, the extent of DMBA- sesame oil (20 mg/ml) by gentle warming and 1 ml/100 g body weight induced hemorrhage and necrosis was considerably less in ad was administered i.g. (No. 9 French catheter) to animals that had been deprived of food but not water for 5 h. The average total dose of DMBA renal glands which had regenerated after enucleation (1). Since for 50-day-old rats was 32 mg. Animals were given 0.9% saline con DMBA-induced adrenal necrosis is followed by adrenal regen taining 6% glucose to drink for 6 days after DMBA treatment to eration beginning 6 or 7 days post-DMBA (1,3, 4), it was of compensate for toxic effects of drug (decreased food consumption and Received 11/3/87; revised 3/21/88; accepted 4/6/88. adrenal necrosis). The costs of publication of this article were defrayed in part by the payment Radiological Studies of Adrenal Regeneration. To quantify cellular of page charges. This article must therefore be hereby marked advertisement in proliferation in the adrenal, 50 animals were given DMBA (20 mg/100 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Major portions of this work were performed at the Cancer Research Institute g body weight) at 50 days of age. At intervals of 2, 3, 4, 6, 8, 14, and of the New England Deaconess Hospital, Boston, MA, and were supported by 21 days after DMBA, an average of 6 animals per group were given 50 the Atomic Energy Commission [Contracts AT (30-l)-3777 and AT (30-1)- fiCi [3H]thymidine (Schwarz Laboratories, Inc., Mt. Vernon, NY) s.c. 3779]. and were adrenalectomized 30 min later. Eighteen control animals 2To whom requests for reprints should be addressed, at Wood Hudson Cancer received [3H]thymidine at 50 or 60 days of age but no DMBA. Research Laboratory, 1830 Greenup Street, Covington, KY 41011. Adrenals from animals given [3Hjthymidine were fixed for 24 h in 3The abbreviations used are: DMBA, 7,12-dimethylbenz(a)anthracene; i.g., intragastrically; ADX, adrenalectomy; PitX, hypophysectomy; OVX, ovariec- neutral 10% formalin. After embedding in paraffin, 5 sections were cut tomy; l'i! Ir, pituitary transplant. from each pair of adrenals at 5 ^m and mounted on 5 slides. Slides 3801 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. ADRENAL REGULATION OF MAMMARY TUMORIGENESIS were deparaffinized, rehydrated, and dipped in NTB2 emulsion (East receive DMBA but which either were adrenalectomized or received a man Kodak Co., Rochester, NY). Emulsion-coated slides were exposed pituitary transplant. Animals in other surgical treatment groups re for 4 weeks in the Con-Rad/Joftes fluid emulsion autoradiography ceived DMBA at 50 or 70 days of age and underwent surgery 6 days system in a CO: atmosphere and a relative humidity below 15%. They thereafter. Surgery was performed under ether anesthesia and animals were developed using Kodak 19 developer (3 min), Kodak SBSa stop received 2.5 mg of hydrocortisone acetate s.c. and 200,000 units of bath (15 s), and Kodak acid fixer (twice clearing time). Solutions were penicillin i.p. postoperatively. Surgical procedures included ADX, PitX, maintained at 18°Cthroughout development. Developed slides were OVX, and PitTr, alone or in combinations of two procedures. Hypo- washed for 12 to 18 h, stained with hematoxylin and eosin, dehydrated, physectomy was by the parapharyngeal route. Pituitary transplant and mounted in balsam. animals received a single pituitary from a 56-day-old donor which was The thymidine-labeling index was measured by counting 2500 cells transplanted to the subcapsular space of the left kidney. per zone of each adrenal of each rat (i.e., 500 cells per zone in each of Observation of Animals. All animals were housed at 26.5°Cand given 5 tissue sections) and was expressed as the mean number of labeled 0.9% saline ad libitum as these conditions were required for survival of cells per 100 x 100%. Cells which showed 5 or more silver granules adrenalectomized animals. Animals were palpated twice weekly begin over the nucleus were considered to be labeled and in S phase. Only ning 4 weeks post-DMBA. When an animal developed numerous viable cells away from necrotic areas were counted. The number of nodules, or large, life-threatening nodules, it was killed. Animals in labeled interstitial and parenchyma! cells was recorded. S phase is some groups were killed as early as 65 days (see Table 1). Remaining considered a more accurate assessment of regeneration than mitotic animals in tumor-bearing groups were sacrificed by 150 days. Hypophy- index. sectomized animals were maintained for up to 200 days.