Eurot~. 3". Cancer Vol. 13, pp. 29-32. Pergamon Press 1977. Printed in Great Britain

The Pars Intermedia and Renal Carcinogenesis in Hamsters* j. M. HAMILTON,, P. G. SALUJA+*, A. J. THODY§ and A. FLAKS¶ ~Department of Experimental Pathology and Cancer Research, School of Medicine, University of Leeds, ++Departmentof Anatomy, School of Medicine, University of Manchester, §Department of Dermatology, School of Medicine, University of Newcastle-upon-Tyne, and ¶Department of Biology, University of York, Great Britain A b s t r a c t - - M a l e hamsters, 3 months of age, were treated for 9 months by thrice weekly injections of diethylstilboestrol (DES). All animals developed kidney tumours and histopathological examination showed that the intermediate lobes of the pituitary were hyperplastic and neoplastic. The content of melanocyte-stimulating hormone (MSH) in the pituitary glands of 17 control and 12 DES-treated animals was measured by bioassay and radioimmunoassay. Whan compared with controlpituitaries, the concentration of immunoreactive a-MSH in treated animals was significantly elevated (P < 0-005) with significant positive correlation between pituitary weight and total a-MSH content. The levels of bioactive M S H were also raised. Serum levels of immunoreactive a-MSH were consistently higher in treated animals. The possibility exists that the induction of kidney tumours by oestrogens in the male hamster is mediated via the pituitary gland and that M S H may be of significance in the process of carcinogenesis.

Melanocyte-stimulating hormone (MSH) is produced in the intermediate lobe of the pituitary [6] and, in the present study, the possibility has been examined that increased amounts of MSH are produced by the enlarged intermediate lobe resulting from prolonged oestrogen treatment.

INTRODUCTION THE METHOD by which oestrogens induce renal tumours in the male hamster is uncertain. Kirkman [1] considered the possibility that such hormones acted directly on the kidney since stilboestrol implanted in the spleen and, therefore, in the path of hepatic drainage failed to induce renal tumours. Algard [2] supported this view since he found that organ cultures of renal tumours were oestrogen dependent. Another suggestion was that oestrogeninduced tumours resulted from changes in the pituitary gland. Long-term oestrogen treatment has been shown to cause a significant increase in the number of prolactin-secreting cells while a prolactin-inhibiting drug (CB154), when given in combination with stilboestrol, reduced the incidence and severity of kidney tumours in the hamster [3]. Oestrogen treatment also has been found to induce hyperplastic and neoplastic changeintheintermediate lobe of the pituitary gland of the hamster [3-5].

MATERIAL AND METHODS Fifty-four male Syrian golden hamsters (Mesocricetus auratus), aged 3 months at the beginning of the experiment, were used. The animals were housed individually in a lightand temperature-controlled room, weighed at weekly intervals and maintained on dog-chow and tap water ad libitum, supplemented twice weekly with cabbage. The hamsters were divided randomly into two groups, each of 27 animals. One group consisted of untreated controls while the animals of the second group were treated with diethylstilboestrol (DES). Each animal received one s.c. injection of 0.6 mg DES (Sigma) suspended in 0.2 ml distilled water three times per week. After a total dose of 65 mg DES over a period of 9 months, the animals were finally weighed, killed by cervical dislocation and autopsied.

Accepted 4 June 1976. *This work was made possible by the financial support of the Yorkshire Cancer Research Campaign. 29

30

J. M . Hamilton, P. G. Saluja, A. or. Thody and A. Flaks

The pituitaries of 17 control and of 12 DEStreated hamsters were removed immediately, weighed and placed in separate phials containing 1 ml of 0.25% glacial acetic acid and stored at - 2 0 ° C for M S H assay. Pituitary glands from the remaining treated and control animals were weighed, fixed in 10% formalsublimate and stained by Herlant's tetrachrome method for cytological examination. At post-mortem, testes and kidneys were weighed and fixed in 10% formal-saline together with representative portions of liver, spleen, lungs and brain. The tissues were embedded in paraffin-wax, sectioned at 5/~m and stained with haematoxylin and eosin for histopathological examination. M S H assays The method of Howe and Thody [7] was used for the bioassay of total M S H activity in extracts of pituitary tissue. A radioimmunoassay [8, 9] was used for the measurement of pituitary and serum a - M S H levels. Statistical analysis was done by the 2-tailed Student's t-test using a Data General Corporation Nova 820 computer programmed to correct for inter-group variance difference when computing P values.

RESULTS O f the 27 DES-treated animals, 3 died early in the course of the experiment and were discarded. In the remaining animals, statistical analysis failed to show any significant difference in body weight between treated and control animals (P > 0.05) (Table 1) and, consequently, it was possible to make direct comparisons of organ weights. The kidneys of the DES-treated animals were significantly heavier than those of controls (P < 0.0005) (Table 1). This difference in weight was Table 1. Effect of DES treatment on weight* (g) of body, kidneys and pituitary in male hamsters Weight Body Kidneys Pituitary

Control(27)~

DES(24)

133 + 2

138 + 3 N.S.D..+ 0.5199 + 0-0106 1.5176 + 0.2464 < 0.0005§ 0.0032 + 0.0002 0.0369 + 0.0116 < 0.005

*Expressed as the mean weight for the group + S.E.M. tThe number of animals in the group. ;~No significant difference between groups. §The value of P, denoting the significance of difference between groups.

clearly associated with the presence of tumour tissue in the kidneys of all treated animals. In general, kidneys were enlarged, irregular in outline with protruding tumour nodules and showed multiple neoplastic foci of varying size--3 mm to 2 cm in diameter--that were mainly bilateral and cortical in distribution. Lesions were solid, cream coloured or, often, consisted of haemorrhagic cysts intermixed with solid tumour. Central necrosis was prominent in the larger lesions. Four of the animals had haemorrhagic ascites with metastatic deposits on the serosal surfaces of abdominal organs. Histopathologically, a gradation of change from increased basophilia and hyperplasia of tubular epithelial cells to frank neoplasia could be observed within any one kidney. Because of origin from tubular epithelium and their invasive tendencies, the tumours were classified as carcinomas. The pituitary glands from DES-treated animals were larger and significantly heavier than those from control animals (P < 0.005) (Table 1). The anterior lobe was compressed to a thin shell of tissue covering a bulging intermediate lobe. The main alteration lay in the cellular composition of the adenohypophysis with a significant increase in the number of prolactin-secreting cells and a decrease in the number of somatotrophinsecreting cells and in the basophils. Histopathological examination showed that the considerable enlargement of the pituitary was mainly restricted to the intermediate lobe. The cells were hypertrophic and hyperplastic with frequent evidence of mitosis. Neoplastic change had supervened and infiltration of the posterior lobe and infundibular stalk by cells of the intermedia was common with, in some cases, almost total replacement of the posterior lobe. Occasionally, invasion of the anterior lobe was in evidence. Those findings confirm our previous results [3]. The results of the M S H assays are shown in Table 2 and Fig. 1 and 2. The mean level of immunoreactive a - M S H in the pituitary glands of the 17 control animals was 471 + 50 ng/ pituitary. By bioassay, the mean M S H activity in 5 of those pituitaries was equivalent to 3980 + 370 ng a-MSH/pituitary. Thus a - M S H accounted for approximately 10 % of the total bioassayable M S H in the hamster pituitary. There was a significant increase (P < 0.005) in the mean level of immunoreactive a - M S H (16,100 + 430 ng/pituitary; 386 + 69 ng/mg pituitary) in the pituitaries of DES-treated animals when compared with controls (Fig. 1). A significant positive correlation was found

The Pars Intermedia and Renal Carcinogenesis in Hamsters Table 2.

Effect of D E S treatment on a - M S H level * in male hamsters Controls

DES

Pituitary M S H

No. of assays 17 12 Total content 471 + 50 14,810 + 4167 (ng/gland) P < 0"005t Concentration 146 + 14 360 _+ 69 (ng/mg wet wt.) P < 0.005 Serum M S H

No. of assays Concentration (pg/ml)

4 453 + 152

10 3190 + 521

*Expressed as m e a n + S.E.M. ~ T h e v a l u e of P, d e n o t i n g t h e significance of difference b e t w e e n groups.

kt-

18m

o> . -.,.. ~

12-

p < 0.005

~_

P < 0,005

-r _L_

" " :Z;

d~ < =

300

s~ m ~"

450

~

CONTROLS (17)

DES (12)

Fig. 1. Effect of DES treatment on mean pituitary levels of a - M S H in male hamsters. The vertical bars representS.E.M. and the numerals in brackets indicate the number of animals examined. The values of P denote the significance of difference between groups, calculated by two-tailed Student's t-test.

7500

I,

-

o

z o I-

5000

¢:

i-. "-"

z E 0

. 2500

g

CONTROL

Fig. 2.

DES

Effect of DES treatment on serum levelsof a-MSH in male hamsters.

31

between pituitary weight and total content of a-MSH (r=0-6; P < 0.02). Three pituitaries from DES-treated animals bioassayed for M S H were found to contain markedly increased activity (equivalent to 119,260 + 47,840ng e-MSH/pituitary). The immunoreactiveJbioactive ratio in the pituitaries of 2 of those animals was higher than normal and suggested a preferential increase in the a-MSH content of the glands. To measure serum levels of immunoreactive a-MSH it was necessaryto pool sera from several animals so that ultimately there were insufficient numbers for statistical analysis. However, the values illustrated in Fig. 2 indicate that serum a-MSH levels were consistently higher in DES-treated than in control animals. There have been several reports that prolonged oestrogen treatment of the hamster leads to hyperplastic and neoplastic changes in the intermediate lobe of the pituitary [3-5]. The present results confirm those findings but also show that the enlarged intermediate lobes are capable of producing and releasing increased amounts of MSH. Various forms of M S H exist and, in the normal hamster, Penny and Thody (unpublished data) have estimated that c~-MSH accounts for approximately 10% of the total bioas~ayable MSH of the pituitary. That figurd was confirmed in the present study. However, in 2 of the 3 DES-treated hamsters, the pituitaries of which had also been bioassayed, a greater proportion of a-MSH was found indicating that the hyperplastic and neoplastic intermediate lobes were preferentially producing a-MSH. That may have been a common feature of all the enlarged lobes since most of them contained exceedingly high levels of immunoreactive a-MSH. Hamilton et al. [3] have reported that a prolactin inhibitor (CB154), when given concurrently with stilboestrol, reduced the incidence and severity of kidney turnouts. The effect of CB154 may be related to interference with prolactin secretion although Hamilton et al. (unpublished data) failed to induce renal tumours with prolonged prolactin treatment and was unable to demonstrate that, when given concurrently with DES, prolactin influenced the induction or severity of the kidney tumours. However, Penny and Thody (unpublished data) have found recently that, in the rat, CB154 inhibits the secretion of MSH and that finding suggests the possibility of a relationship between renal carcinogenesis and M S H secretion. There is emerging evidence that indicates that M S H acts on the kidney.

32

& M . Hamilton, P. G. Saluja, A. J. Thody and A. Flaks

Orias and M c C a n n [10] reported that a-MSH and fl-MSH induced natriuresis in the rat while increased levels of immunoreactive fl-MSH have been found in patients with chronic renal failure [11, 12]. Smith and Shuster (personal communication) consider that, in

man, the kidney may metabolise MSH-like peptides. Such metabolites may occur in the hamster and under conditions of increased M S H secretion may lead to neoplastic change in the kidney. T h a t possibility must now be examined.

REFERENCES 1.

2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12.

H. KIRKMAN,Estrogen-induced tumors of the kidney in the Syrian hamster. Nat. Cancer Inst. Monogr. 1, 1 (1959). F . T . ALOARD,Hormone-induced tumours, or. nat. Cancer Inst. 25, 557 (1960). J. M. HAMILTON, A. FLAKS, P. G. SALUJAand S. MAGUIRE, Hormonally induced renal neoplasia in the male Syrian hamster and the inhibitory effect of 2-bromo-a-ergocryptine methanesulfonate, d. nat. Cancer Inst. 54, 1385 (1975). E. VASQUEz-LoPEZ, The reaction of the pituitary gland and related hypothalamic centres in the hamster to prolonged treatment with oestrogens. J. Pathol. 56, 1 (1944). A.A. KONEFF,M. E. SIMPSONand H. M. EVANS,Effects of chronic administration of diethylstilboestrol on the pituitary and other endocrine organs of the hamster. Anat. Rec. 94, 169 (1946). A. HowE, The mammalian pars intermedia: a review of its structure and function, or. Endocr. 59, 385 (1973). A. HOWE and A. J. THODY,The in vitro assay of pituitary MSH. or. Physiol. (Lond.) 209, 5 (1970). A . J . THODY, R. J. PENNY and M. D. CLARK, A radioimmunoassay for amelanocyte-stimulating hormone in the rat. d. Endocr. 64, 62 (1975). A . J . THODY, R. J. PENNY, M. D. CLARKand C. TAYLOR, Development of a radioimmunoassay for a-melanocyte-stimulating hormone in the rat. or. Endocr. 67, 567 (1975). R. ORIASand S. M. MCCANN, Natriuresis induced by alpha and beta melanocyte-stimulating hormone (MSH) in rats. Endocrinology 90, 700 (1972). J . J . H . GILKES,R. A. J. EADY, L. H. REESE, D. D. MUNRO and J. F. MOORHEAD, Plasma immunoreactive melanotrophic hormones in patients on maintenance haemodialysis. Brit. med. J. l, 656 (1975). A . G . SMITH, S. SHUSTER,J. g. COMARSH,N. A. PLUMMER,A. J. THODY, F. ALVAREZ-UDEand D. N. S. KERR, Plasma immunoreactive B-melanocytestimulating hormone and skin pigmentation in chronic renal failure. Brit. med. d. 1, 658 (1975).