CORTICOSTEROID ACTIVITY OF A TRANSPLANTABLE ADRENOCORTICAL CARCINOMA IN OSBORNE-MENDEL RATS
A. S. MULAY Laboratory of Pathology, Nat. Cancer Inst., N. I. H., Bethesda, Maryland
ABSTRACT
An adrenocortical carcinoma, arising in an Osborne-Mendel rat, was tested by a standardized stress procedure, for its ability to secrete mineralocorticoids. The tumor was grown in adrenalectomized male rats and then stress was in- duced by intraperitoneal injection of 5 ml. of KCI solution per 100 gm. body weight. The dosage of KCI was regulated by changing the molality of the solu- tion. This tumor offered complete protection to rats against 0.16 m. KCI, a dose lethal to comparable rats without tumor. This gives qualitative proof of cor- ticoid secretion by the tumor.
R EPORTS on functional activity of adrenocortical tumors of rats have been made by Cohen et al. (1), Iglesias and Mardones (2), Houssay et al. (3), and Snell and Stewart (4). The transplantable adreno- cortical adenocarcinoma under study was one of the 4 observed in a group of female Osborne-Mendel rats fed low protein, low riboflavin, semisyn- thetic diet, containing 0.06% p-dimethylaminoazobenzene (5). This paper reports on the functional activity of this tumor as determined by a stand- ardized stress procedure of potassium intoxication.
MATERIALS AND METHODS
Bilateral adrenalectomy was performed on male, Osborne-Mendel rats through a paravertebral incision. Anesthesia was produced by pentobarbital given intraperi- toneally, supplemented with ether inhalation. The rats were about two months old and weighed 200 to 250 gm. at the time of operation. One month later tissue from the adrenocortical carcinoma was transplatned into half of the rats; the others served as controls. These operated rats received a 0.9% saline in place of drinking water. When the transplants grew and were visible as subcutaneous nodules, the saline was replaced with tap water. The carcinoma was also transplanted into intact male rats about 3 months old. All rats were fed a diet of Purina chow pellets.
Stress was produced by the intraperitoneal injection of potassium chloride (KCI) solution at 5 ml./100 gm. body weight, to rats fasted over night (18 hours). The dose of KCI was regulated by changing the molar concentration of the KCI solution used for injections. The dose is expressed as moles (m) of KCI per liter of solution. When the transplanted tumors were 2 cm. or more in diameter, stress was induced with various doses of KCI in adrenalectomized and intact tumor-bearing rats, and at the same time in comparable controls. Some rats were subjected to the stress treatment after removal of the tumors. The time interval between adrenalectomy and induction of stress was
Received December 4, 1959.
the same for tumor-bearing rats and their comparable controls. All animals were ob- served for toxic effects for two hours after the KCI stress, and survivors were saved for at least 24 hours before they were killed. After each stress experiment, the rat was rested for at least one week before it was used for another stress experiment. After removal of a tumor at least a week was allowed for recovery before that rat was sub- jected to KCI stress. All rats were autopsied at the end of the experiments. The peri- renal fat was examined for accessory adrenal bodies, and all organs examined for tumor metastases and other lesions. Tumors, adrenal glands, and accessory adrenal bodies were weighed and specimens of these tissues were taken for histologic examination.
RESULTS
The response of the intact and the adrenalectomized rats to various doses of KCI is listed in Table 1. As these stress experiments were per- formed 3 to 4 months after adrenalectomy; congenital, accessory adrenal bodies in some of the adrenalectomized rats had developed into macro-
| No. of rats | Treatment | KCI molality | Millimoles of K administered per 100 gm. body wt. | % mortality |
|---|---|---|---|---|
| 15 | None | 0.20 | 1 | 100 |
| 10 | None | 0.18 | 0.9 | 40 |
| 20 | None | 0.16 | 0.8 | 0 |
| 8 | Adrenalectomized | 0.16 | 0.8 | 100 |
| 6 | Adrenalectomized | 0.14 | 0.7 | 100 |
| 11 | Adrenalectomized | 0.10 | 0.5 | 91 |
| 12 | Adrenalectomized, accessory adrenal bodies | 0.16 | 0.8 | 100 |
| 14 | Adrenalectomized, accessory adrenal bodies | 0.10 | 0.5 | 86 |
scopic glands a millimeter or more in diameter, at the time the stress was applied. In 3 cases they weighed about 30 mg. each. The response of the rats with accessory adrenal bodies to KCI stress was similar to that of comparable rats without these bodies.
For intact Osborne-Mendel rats the minimum lethal dose of KCI was 0.2m and the maximum tolerated dose was 0.16m. For adrenalectomized rats these values were 0.14m and less than 0.1m respectively. Two rats with unilateral adrenalectomy tolerated a KCI dose of 0.16m.
The response of the tumor-bearing rats, adrenalectomized and intact, to KCI stress is listed in Table 2. Adrenalectomized rats with tumor growth survived the maximum dose of KCI (0.16m) tolerated by intact control rats. The same dose of KCI, a week after the removal of the tumor, was lethal to these animals. A dose of 0.2 or 0.18m KCI was not tolerated by any of the tumor-bearing rats, irrespective of the size of the tumor. The smallest tumor weighed 3.5 gm. and the largest one was 40 gm. The re- sponse of the intact rats with transplant growth to KCI stress was similar to those of the comparable adrenalectomized rats. These rats also did not
| No. of rats | Treatment | KCI molality | Millimoles of K administered per 100 gm. body wt. | % mortality |
|---|---|---|---|---|
| 15 | Adrenalectomized | 0.16 | 0.8 | 0 |
| 12 | Adrenalectomized | 0.20 | 1.0 | 100 |
| 8 | Adrenalectomized | 0.18 | 0.9 | 1.00 |
| 3 | Adrenalectomized, tumor removed | 0.16 | 0.8 | 100 |
| 10 | Adrenalectomized, accessory adrenals | 0.16 | 0.8 | 0 |
| 2 | Adrenalectomized, accessory adrenals, tumor removed | 0.16 | 0.8 | 100 |
| 10 | Intact | 0.16 | 0.8 | 0 |
| 5 | Intact | 0.18 | 0.9 | 100 |
| 2 | Intact, tumor removed | 0.16 | 0.8 | 100 |
tolerate the maximum tolerated dose of KCI (0.16m) after removal of the tumor. Adrenal glands of the tumor-bearing rats were atrophied, reduced in size, and weighed about half as much as the adrenal glands of the con- trol rats.
DISCUSSION
The adrenocortical carcinoma under study induces atrophy of the ad- renal glands of the tumor-bearing rat (5). This suggested secretion of physiologically active steroid hormones by the tumor. Polyuria and nitremia observed in these tumor-bearing rats pointed to a possible secre- tion of mineralocorticoids by this carcinoma.
The importance of the adrenal cortex in potassium metabolism was shown by chemical studies of the blood and tissues by Zwemer and Trus- kowski (6) and a quantitative relationship between a dose of cortical hor- mone and the percentage of survival of adrenalectomized mice following injection of KCI was established by Truskowski and Duszynska (7). On the basis of these studies, Kolmer (8) suggested a potassium tolerance test for clinical use to determine the degree of adrenocortical deficiency in doubtful cases. The rat was used as a test animal by Feil and Dorfman (9) to evaluate the potency of cortin like material excreted in urine.
Potassium tolerance test evaluates the effective concentration of corti- costeroid (mineralocorticoids) in the test animal. In this study the carci- noma suspected of secreting corticosteroid was grown in the test animal. Thus effective concentration of corticosteroid in these animals was a re- sultant of the rate of secretion and the rate of its destruction or elimina- tion. The response of Osborne-Mendel rats, intact and adrenalectomized, to KCI stress was similar to that obtained by Collins (10) in albino rats. Adrenalectomized rats with tumor tolerated 0.16m KCI, a dose always lethal to comparable rats without tumor, giving a qualitative proof of corticosteroid secretion by the tumor. Inability of the test rats to tolerate
the same dose of KCI after removal of the tumor added strength to this proof. Tumor-bearing rats did not tolerate any dose of KCI higher than 0.16m, irrespective of the size of the tumor. This means that corticoid concentration in tumor-bearing rat is maintained at the same level as in intact control rats, and suggests that other factors besides the secretory activity of the tumor play a part in regulating this level. The largest ac- cessory adrenal bodies (30 mg.) did not protect the rat against 0.16m dose of KCI.
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