nancies was not the same in the mice on the diet supplemented with ether extract of whole egg as in those receiving alcohol extract of egg yolk or powdered egg from which the ether extractable part was removed. In the latter 2 groups the incidence of mammary cancer was considerably higher and that of the other types of malignancies (lung adeno- carcinoma, lymphosarcomas, etc.) lower than in Group 2. This difference in incidence of the above two groups of malignancies is probably due to a difference in composition of the extracts used, since it is known that some lipids are more soluble in alcohol and
others more in ether. In the case of Group 4, it is believed that the carcinogenicity is not due to the egg proteins, but to the un- extracted lipids. These and other results in- dicate that there is more than one carcino- genic lipid in eggs.
Some slides were examined by Dr. Stephen S. Sternberg, Sloan-Kettering Inst., and some by Drs. Paul Kotin and Thelma B. Dunn, National Cancer Inst., to whom I am greatly indebted.
1. Szepsenwol, J., PROC. Soc. EXP. BIOL. AND MED., 1963, v112, 1073.
Received May 15, 1964.
P.S.E.B.M., 1964, v116.
Effects of 5-Fluorouracil and Adrenocortical Hormones on Tumors and Adrenals in Rats Bearing Transplanted Tumors .* (29474)
A. CANTAROW, J. J. RUPP AND J. W. GODDARD Departments of Biochemistry and Medicine, Jefferson Medical College, Philadelphia, Pa.
We have found that 5-fluorouracil (5-FU) inhibits fetal growth, liver regeneration, tes- tosterone-induced growth of the seminal vesi- cles, and somatotropin-induced growth of the epiphyseal cartilage(1). The high toxicity of 5-FU for hypophysectomized rats interfered with similar studies of its effect on ACTH- stimulation of adrenal growth. However, the results suggested that this process might not be inhibited by 5-FU. The present study was undertaken for the purpose of investigating this matter further. Observations were ex- tended to tumor-bearing rats because of the possibility that the hypophyseal-adrenocorti- cal mechanism may respond differently in the presence of a tumor than in the normal animal.
Materials and methods. Male rats (Bark- bridge Farms, N. J.), weighing about 150 g, were fed laboratory chow and had free access to water. Tumors were implanted subcutane- ously in the groin by trocar technic, the Walker 256 tumor and the Murphy-Sturm lymphosarcoma in Wistar and the Morris hepatoma 3924C in A X C rats.
(1) In the studies of effects on adrenal weight, treatment with hormones and 5-FU was begun when tumor growth was well established. Cortisone acetate was injected subcutaneously, 5 mg daily for 10 days, ACTH gel subcutaneously, 5 units twice daily for 5 days, and 5-FU intraperitoneally, 25 mg/kg/day for 5 days. In the combina- tion treatment schedules, ACTH or/and 5-FU was/were given on the last 5 days of cortisone administration.
(2) In the studies of effects on tumors, these were allowed to grow for 10 days. The adrenocortical steroids were administered daily from day of implantation, and 5-FU was given daily for the last 5 days. The several dosage schedules are indicated in Table IV.
All animals were sacrificed by exsanguina- tion under light ether anesthesia 24 hours after the last injection.
Results and discussion. Effect of 5-FU on adrenals of normal rats (Table I). Adminis- tration of 5-FU resulted in significant in- crease (49%) of adrenal weight in normal Wistar rats. Body weight was maintained and there was no other evidence of drug toxicity in these animals. Increased adrenal
* Supported by Contract SA-43-ph-2387 from Cancer Chemotherapy National Service Center, Nat. Cancer Inst., N.I.H., U.S.P.H.S.
| No. of rats | Treatment | Adrenal wt, mg/100 g* ± S.E. |
|---|---|---|
| 25 | None | 17.0 ± .17 |
| 20 | Cortisone, 5 mg/day × 10 | 9.0 ± 1.28 |
| 19 | ACTH, 5 U b.i.d. X 5 | 24.7 ± 1.41 |
| 22 | 5-FU, 25 mg/kg/day × 5 | 25.3 ± 1.94 |
| 17 | Cortisone + 5-FU | 13.0 ± .98 (vs cortisone P <. 01) (vs 5-FU P <. 01) |
| 25 | Cortisone + ACTH | 20.1 ± 1.72 (vs cortisone P <. 01) (vs ACTH P <. 05) |
| 21 | 5-FU + ACTH | 29.3 ± 2.85 (vs ACTH P <. 05) |
| 16 | Cortisone + ACTH + 5-FU | 20.5 ± 1.43 |
* Carcass weight (minus tumor).
hypertrophy after unilateral adrenalectomy has been observed in rats treated with 5-FU, 5-fluorodeoxyuridine, thioguanine, thioguano- sine, and 6-mercaptopurine riboside(2). Gass et al(3) reported enlargement of the adrenals and thymic involution after administration of Myleran, actinomycin D, L-sarcolysin, and 4-aminopyrazolo (3,4-d)pyrimidine. This was interpreted as probably due to a toxic effect of these agents. Perhaps also relevant is the report(4) that an amount of 5-FU that killed 20% of intact dogs killed 80% of adrenalectomized dogs receiving a mainte- nance dose of cortisol. Administration of larger amounts of hormone afforted complete protection to the adrenalectomized animals.
The size of the adrenals was reduced by cortisone (about 47%) and increased by ACTH (about 45%). The increase with 5-FU was comparable to that induced by ACTH. 5-FU induced significant increase (44%) of adrenal weight in animals with simultaneous adrenal suppression by corti-
| No. of rats | Adrenal wt, mg/100 g* ± S.E. | |
|---|---|---|
| Wistar control (no tumor) | 24 | 17.0 ± .17 |
| Walker 256 | 32 | 25.2 ± 2.64 (P <. 01) |
| Murphy lympho- sarcoma | 30 | 26.7 ± 2.28 (P <. 01) |
| A X C control (no tumor) | 24 | 16.3 ± .26 |
| Morris hepatoma 3924C | 20 | 25.7 ± 2.14 (P <. 01) |
* Carcass weight (minus tumor).
sone, and in those with stimulation by ACTH (19%). However, it was without effect in animals receiving both ACTH and cortisone. These observations suggest that 5-FU pro- duces enlargement of the adrenals through the medium of increased secretion of ACTH rather than by enhancing the effect of ACTH.
Effects of cortisone and 5-FU on adrenals of tumor-bearing rats. The adrenal glands were consistently larger than normal in the presence of growing tumors (Table II). Under the experimental conditions employed, there was no significant difference between the Walker tumor, the Murphy-Sturm lymphosarcoma and the Morris hepatoma 3924C with respect to their effect on adrenal size. Similar observations have been reported previously for mice bearing Sarcoma 180(5, 6,7) and rats bearing the Jensen sarcoma (8), Walker tumor (8,9,10), and Murphy lympho- sarcoma (11,12). Association of this enlarge- ment with thymic involution and decreased adrenal lipid and ascorbic acid content (6,7, 12, 13), and reports that it does not occur in hypophysectomized animals(7,9) suggest that it is due to ACTH stimulation. There are reports of increase in urinary and plasma corticosterone (5,11,14), and of increased cor- ticosterone secretion rate(11) which did not differ significantly from that of normal rats subjected to acute operative stress. It has been reported, however, that the patterns of change in nucleic acids and certain enzymes in the adrenal differ from those incident to states of nonspecific stress (15).
Cortisone reduced the adrenal weight in
tumor-bearing animals (Table III), some- what more in those with the Walker tumor (63%) than with the lymphosarcoma (46%) or hepatoma (40%). In the case of the Walker tumor the values were lowered to levels approximating those obtained in cor- tisone-treated rats without tumors. This re- sponse to cortisone supports the view that
| 01) | |||||||
|---|---|---|---|---|---|---|---|
| <. | |||||||
| Hepatoma | P | ||||||
| (vs Walker 60 (P <. 01) | 95 (N.S.) | 56 (P <. 01) | |||||
| No. of | rats | 18 | 16 | 14 | |||
| 01) | |||||||
| <. | |||||||
| control*) | P | ||||||
| of | Walker 01) | 01) | |||||
| (% | <. | (N.S.) | <. | ||||
| wt | Lymphosarcoma | (vs (P | 103 | (P | |||
| 54 | 51 | ||||||
| Adrenal | of | ||||||
| No. | rats | 24 | 16 | 14 | |||
| 01) | + | ||||||
| <. | |||||||
| Walker | 37 (P | 114 (N.S.) | 82 (N.S.) | ||||
| No. of | rats | 19 | 11 | 12 | |||
| 10 | 5 | ||||||
| × | × | ||||||
| Treatment | 5 mg/day | mg/kg/day | + 5-FU | ||||
| Cortisone, | 5-FU, 25 | Cortisone | |||||
TABLE III. Influence of Cortisone and 5-Fluorouracil on Adrenal Weight in Tumor-Bearing Rats.
* A control group of 12 untreated tumor-bearing rats was run simultaneously with each treated group.
t N.S. = Not significant.
adrenal enlargement in these animals is due to increased hypophyseal secretion of ACTH rather than to production of a similar sub- stance by the tumor, as has been demon- strated for certain neoplasms in man(16).
The enlarged adrenals of the tumor-bear- ing rats did not increase further in size after administration of 5-FU. Although it is pos- sible that ACTH secretion was already stim- ulated maximally as a result of the presence of the tumor, the fact that adrenal weights as high as 45 mg/100 g were found in ani- mals with double tumor implants indicates that the ACTH-secreting mechanism was capable of considerably greater response to stimulation, at least under these circum- stances.
5-FU overcame cortisone-induced suppres- sion of the adrenals in rats bearing the Walker tumor, as in those without tumors, but not in animals bearing the lymphosar- coma or hepatoma. All 3 tumors induce essentially the same degree of enlargement of the adrenals (Table II), but the ACTH- secreting mechanism is apparently more sensitive both to suppression by cortisone and to stimulation by 5-FU in rats bearing the Walker tumor than in those with the lymphosarcoma or hepatoma.
Effects of corticosteroids and 5-FU on tumor growth (Table IV ). Although the car- cass weights of treated animals were slightly lower than those of controls, there was no consistent difference in this regard between animals bearing the 3 types of tumors. Growth of all 3 tumors was depressed by Decadron (9a-fluoro-118, 17a, 21-trihydroxy- 16a-methyl-1, 4-pregnadiene-3,20-dione) and prednisone (17a, 21-dihydroxy-1, 4-pregna- diene-3, 11, 20-trione) at dosage levels re- garded as metabolically equivalent to 5 mg cortisone and 10 mg corticosterone. In these amounts, the latter 2 hormones inhibited growth of the Walker tumor but not that of the lymphosarcoma or hepatoma. With double this dose of cortisone (10 mg), growth of the lymphosarcoma was inhibited but that of the hepatoma was not affected. The differ- ence in effectiveness of Decadron and pred- nisone as compared to that of cortisone and corticoserone suggests either (a) that the
| Treatment* | % change tumor wtt - Double tumor Lympho- Lympho- Walker sarcoma Hepatoma Walker sarcoma | ||||
|---|---|---|---|---|---|
| Cortisone, 5 mg/day × 10 | -60 | ± | = | -55 | け |
| Cortisone, 10 mg/day × 10 | -55 | -40 | ± | 45 | -45 |
| Corticosterone, 10 mg/day × 10 | -55 | + | + | - | - |
| Decadron, .16 mg/day × 10 | -50 | -70 | -45 | 45 | -60 |
| Prednisone, 1.2 mg/day × 10 | -45 | -45 | -40 | - | - |
| 5-FU, 12.5 mg/kg/day × 5 | ± | -40 | -65 | ± | -50 |
| 5-FU, 25 mg/kg/day × 5 | 士 | -75 | -45 | + | -70 |
| 5-FU, 25 mg + cortisone, 5 mg | -60 | -60 | -55 | -55 | -65 |
| 5-FU, 25 mg + cortisone, 10 mg | -50 | -65 | -60 | - | - |
* 16-24 animals in each group. A control group of 12 untreated tumor-bearing rats was run simultaneously with each treated group.
+ All stated numerical values (calculated to nearest 5%) are statistically significant (P <0.01).
lymphosarcoma and hepatoma may differ from the Walker tumor in their ability to metabolize the natural but not the syn- thetic types of hormone or (b) that the tumoristatic action of these substances is not related quantitatively to other recognized metabolic activities of adrenocortical steroids.
Growth of the hepatoma and lymphosar- coma was inhibited by 5-FU, but not that of the Walker tumor. Simultaneous adminis- tration of cortisone did not alter the tumori- static effect of 5-FU on the lymphosarcoma and hepatoma, nor did 5-FU alter that of cortisone on the Walker tumor. In the case of these 3 tumors, therefore, sensitivity to 5-FU is associated with relative resistance to cortisone, and vice versa.
To explore this apparent relationship fur- ther, the effects of 5-FU and steroids were investigated on growth of the Walker tumor and lymphosarcoma implanted simultaneously in the same animal, one in each groin. In untreated animals both tumors grew essen- tially as in single implantations. Each ex- hibited the same type of response to ad- ministration of 5-FU, cortisone and Decad- ron as when present alone (Table IV). A similar reciprocal relationship between tumor sensitivity to adrenocortical steroids and to 5-FU has been reported for corticoid-sensi- tive and corticoid-resistant strains of the P-1798 mouse lymphoma(17). These dif- fered from the tumors in the present study in that the cortisone-resistant subline was resistant also to Decadron and prednisone
(18). However, the experimental design also differed in that the steroids were adminis- tered only after the tumors had become well established.
The Walker tumor, Murphy lymphosar- coma and hepatoma 3924C exhibit equally high levels of incorporation of uracil and fluorouracil into RNA(19). The difference in their sensitivity to 5-FU is therefore attributable to difference at some point in the metabolic pathway from uridine to DNA. Acquired resistance to 5-FU has been re- ported to be accompanied in one line of Ehrlich ascites tumor by decreased uridine kinase activity(20), and in another by in- ability of 5-fluoro-2’-deoxyuridylic acid to in- hibit thymidylate synthetase activity (21), presumably owing to alteration of the en- zyme. There is no information as to the comparative sensitivity to adrenocortical steroids of these FU-sensitive and -resistant tumor lines.
No explanation is apparent for the peculiar association of sensitivity to one of these agents with resistance to the other. How- ever, its existence in several types of tumors suggests that this relationship is not merely fortuitous. It is interesting, too, that the ACTH-secreting mechanism is apparently more responsive to FU-stimulation and to cortisone-depression in animals bearing the tumor most sensitive to cortisone (Walker) than in those bearing the tumors (lympho- sarcoma and hepatoma) relatively resistant to cortisone.
Summary. Administration of 5-fluorouracil (5-FU) caused enlargement of the adrenal glands in Wistar and A X C rats. Results of simultaneous administration of cortisone and/or ACTH suggest that this enlargement is due to stimulation of secretion of ACTH rather than to enhancement of its activity. The enlarged adrenals of animals bearing the Walker tumor, Murphy-Sturm lymphosar- coma or Morris hepatoma 3924C did not enlarge further under treatment with 5-FU. They were reduced in size by administration of cortisone, more so in the case of the Walker tumor. The suppressive effect of cor- tisone was not overcome by simultaneous administration of 5-FU in rats bearing the lymphosarcoma or hepatoma but was in those with the Walker tumor. The ACTH- secreting mechanism in the latter is appar- ently more sensitive both to suppression by cortisone and to stimulation by 5-FU. The Walker tumor was sensitive to the tumori- static action of cortisone and relatively re- sistant to that of 5-FU, whereas the converse was the case for the lymphosarcoma and hepatoma. These 3 tumors therefore exhibit a reciprocal relationship between sensitivity to cortisone and to 5-FU similar to that reported for corticosteroid-sensitive and re- sistant strains of mouse lymphoma P-1798.
1. Paschkis, K. E., Bartuska, D., Zagerman, J., Goddard, J. W., Cantarow, A., Cancer Research, 1959, v19, 1196.
2. Palma, V., Atti Acad. Med. Lombarda, 1961, v16, 409.
3. Gass, G. H., Umberger, E. J., Davis, K. J., Davis, L. J., Cancer Chemother. Rep., 1959, v3, 16.
4. Tipton, J., Regan, W. J., Surgery, 1963, v53, 495.
5. Hilf, R., Burnett, F. F., Borman, A., Cancer Research, 1960, v20, 1389.
6. Savard, K., Science, 1948, v108, 381.
7. Savard, K., Homburger, F., PROC. Soc. ExP. BIOL. AND MED., 1949, v70, 68.
8. Haven, F. L., Bloor, W. R., Randall, C., Cancer Research, 1949, v9, 511.
9. Ball, H. A., Samuels, L. T., PROC. Soc. ExP. BIOL. AND MED., 1938, v38, 441.
10. Begg, R. W., Cancer Research, 1951, v11, 341.
11. Millar, F. K., Toal, J. N., Brooks, R. H., Davis, J. O., White, J., Am. J. Physiol., 1963, v205, 189.
12. Murphy, J. B., Sturm, E., Cancer Research, 1948, v8, 139.
13. Hilf, R., Breuer, C., Borman, A., ibid., 1961, v21, 1439.
14. Peric-Golia, L., Jones, R. S., PROC. Soc. ExP. BIOL. AND MED., 1963, v113, 317.
15. Hilf, R., Cowett, V. W., Johnson, M. L., Bor- man, A., Cancer Research, 1962, v22, 449.
16. Meador, C., Liddle, G. W., Island, D. P., Nicholson, W. E., Lucas, C. P., Nuckton, J. G., Leutscher, J. A., J. Clin. Endocrinol., 1962, v22, 693.
17. Lampkin-Hibbard, J. M., J. Nat. Cancer Inst., 1962, v28, 569.
18. ----- , ibid., 1960, v24, 1353.
19. Cantarow, A., Williams, T. L., Paschkis, K. E., Cancer Research, 1962, v22, 1021.
20. Reichard, P., Sköld, O., Klein, G., Révész, L., Magnusson, P. H., ibid., 1962, v22, 235.
21. Heidelberger, C., Kaldor, G., Mukherjee, K. L., Danneberg, P. B., ibid., 1960, v20, 903.
Received May 15, 1964. P.S.E.B.M., 1964, v116.
Influence of Gonads and Adrenals on Growth of Solid Ehrlich Tumor. (29475)
MITSUO KODAMA* (Introduced by George E. Moore) Department of Medicine, Roswell Park Memorial Institute, Buffalo, N. Y.
The induction and further development of neoplasms are often affected by the hormonal state of the host(1,2). The growth of solid Ehrlich tumor, a mammary cancer by origin, is much greater in male mice than in female
mice, and the sex difference is reversed by castration(3). The present study attempts to elucidate the relationship of the gonads and adrenals to these observations.
Materials and methods. All animals were Ha/ICR Swiss mice, propagated at Roswell Park Memorial Inst. by cousin mating. Hy-
* Present address: Nagoya University School of Med., Showa-ku, Nagoya, Japan.