International Journal ofPancreatology, vol, l 1, no. 2, April 1992 Copyright 1992 by The Humana Press Inc, All rights of any nature whatsoever re~sexved. 0169-4197/92/11:137-146/$2,00 Effects of Orchiectomy, Alone or in Combination with Testosterone, and Cyproterone Acetate on Exocrine Pancreatic Carcinogenesis in Rats and Hamsters M. Meijers, 1 C. J. T. Visser,' J. G. M. Klijn, 2 S. W. J. Lamberts, j A. van Garderen-Hoetmer, x E 1t. de Jong," J. A. Foekens, z and R. A. Woutersen *'1 JDepartment of Biological Toxicology, TNO Toxicology and Nutrition Institute, Zeist, The Netherlands; ~Division of Endocrine Oncology (Department of Medical Oncology), Dr. Daniel den Hoed Cancer Center, 3008 AE Rotterdam, The Netherlands; JDepartment of Internal Medicine, Erasmus University, Rotterdam, The Netherlands; and 4Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, The Netherlands Summary The results of a previous 4-rot study in azaserine-treated rats and BOP-treated hamsters indicated that orchiectomy inhibited pancreatic growth and development of putative preneoplastic lesions in the exo- crine pancreas of rats but not hamsters. This 12-rot study was carried out to investigate the effects of orchiectomy, alone and in combination with testosterone, and of treatment with cyproterone acetate on pancreatic carcinogenesis in azaserine-treated rats and BOP-treated hamsters. Treatment started 4 mo after injection of the carcinogen~ In orchiectomized rats, pancreatic wt was lower than in controls, whereas pancreatic wt of orchiectomized rats treated with testosterone was similar to that of controls. Both orchiectomy and cyproterone acetate caused a decrease in body wt gain and had an inhibitory effect on pancreatic carcinogenesis. Testosterone treatment did not influence the inhibitory effects of orchiectomy on body wt gain and on pancreatic carcinogenesis. In hamsters, neither orchiectomy, alone or in combina- tion with testosterone, nor cyproterone acetate (CA) affected pancreatic growth or pancreatic carcinogenesis. This study indicates that testosterone plays a minor role in the development of pancreatic tumors induced in rats by azaserine but not in that of pancreatic tumors induced in hamsters by BOP. Key Words: Exocrine pancreas; cancer; rat; hamster; orchiectomy; testosterone; cyproterone acetate~ Introduction (1,2). Apart from life-style factors, hormonal factors have been suggested to be responsible for this sex In most countries, the age-adjusted incidence of difference. A higher number of atypical acinar cell pancreatic cancer is higher in men than in women nodules (AACN) and a higher incidence of pancre- Received (September 30, 1991); Revised (October 18, 1991); atic neoplasms have been found in male than in Accepted (November 12, 1991) female rats treated with azaserine (3). Using the AT- *Author to whom all correspondence and reprint requests Pase staining, Bax et al. found that acidophilic loci in should be addressed at: Department of Biological Toxicology, male rats were consistently larger (1.75 times) than TNO Toxicology and Nutrition Instituter PO Box 360, 3700 AJ Zeist, The Netherlands in females and concluded that the sex-related differ- 137 138 Meijers et at. ence in the numbers of acidophilic AACN induced in pancreatic carcinogenesis in hamsters and rats during rat pancreas by azaserine may be ascribed to the dif- a long-term treatment period of 8 mo started 4 mo ficulty in detecting small acidophilic foci in hema- after tumor induction. toxylin and eosin (H&E)-stained sections (4). In rats, the inhibitory effect of castration on growth of Materials and Methods azaserine-induced AACN has been ascribed to decreased serum testosterone levels (5-7). The inhibitory effect Animals and Tumor Induction of castration, however, was not influenced by treat- Two hundred male weanling SPF albino Wistar Bor ment with testosterone. In a previous 4-too study with rats (WISW, Cpb) were obtained from F. Winkelmann azaserine-treated rats, we also found an inhibitory (FRG) and injected ip four times with 30 mg effect of orchiectomy on the development of AACN, azaserine/kg body wt at 19, 26, 33, and 103 d of which was accompanied by a significant decrease in age. One hundred and forty male Syrian golden absolute but not relative pancreatic wt. From the hamsters (Charles River Wiga, Sulzfeld, FRG) were results of that study, we concluded that, apart from each injected sc, once weekly, with 20 mg BOP/kg testosterone, nutritional status and pancreatic growth body wt at 6, 7, and 8 wk of age according to an may play a highly significant role in the development injection protocol described previously (19). BOP of putative pancreatic preneoptastic lesions (8). (Ash Stevens, Inc., 5861 John C. Lodge Freeway, No consistent variations have been found in inci- Detroit, MI 48202) and azaserine (Calbiochem- dence of pancreatic (pre)neoplastic lesions in male Behring Corp., LaJolla, CA) were dissolved freshly and female hamsters induced by sc injections with in 0.9% NaC1 solution. The animals were allocated nitrosamine derivatives. Pour et al. did not find a by a computerized randomization procedure to four difference between male and female hamsters in the different groups, each of which consisted of either 50 incidence of pancreatic tumors after injection with rats or 35 hamsters. The rats were housed in stain- N-nitrosobis (2-oxopropyl)amine (BOP) (9,10). less-steel cages, fitted with wiremesh floors and fronts, Kokkinakis, on the contrary, found that tumor inci- and the hamsters in macrolon cages on softwood bed- dence, multiplicity, and Size were generally larger in ding. The animals were kept under standard labora- female than in male hamsters after administration of tory conditions, five animals per cage, and fed a high N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine fat/high protein diet during the first 4 mo of the study (HPOP) (11). Pancreatic (H2T) ceils transplanted in in order to enhance the development of ductular lesions hamster cheek pouches developed faster into ductat in the exocrine pancreas. adenocarcinomas in intact females and castrated males than in ovariectomized females and intact males, Treatment respectively, pointing to an inhibitory effect of tes- tosterone or an enhancing effect of estrogen (12). In order to mimic the human situation, treatment SchaUy and co-workers, however, found that the started when early (pre)neoplastic lesions had already development of pancreatic tumors induced in male developedin the pancreas, i.e. 4 mo after the last injec- hamsters by BOP was inhibited by medical or surgi- tion with carcinogen. The animals received one of the cal castration (13-18). Furthermore, in a previously following treatments: group A, saline (controls); group conducted 4-mo study, we did not find any effect of B, surgical castration; group C, surgical castration plus castration or Luteinizing Hormone Releasing Hor- testosterone; and group D, CA. Testosterone was mone (LHRH) treatment on the development of early administered as testosterone proprionate by silastic (pre)neoplastic lesions induced in hamster pancreas implants as described previously (20). Rats received by BOP (8). This experiment was performed to evalu- two, 3-cm implants per animal, and hamsters, in view ate the effects of castration, either alone or in combi- of lower body wt, received one implant per animal nation with testosterone h-eatment, and of treatment once at start of treatment. Cyproterone acetate was with the antiandrogen cyproterone acetate (CA) on used as Androcur tablets (50 rag) dispersed in tap International Journal of Pancreatology Volume 11, 1992 Sex Steroid Hormones 139 water. The animals were given CA by gavage daily at using initial body wt as covariable. The number of a dose of 50 mg/kg, as described previously (21). borderline and other (pre)neoplasfic lesions were sta- During treatment, the animals were fed the TNO tistically evaluated with a generalized linear model Institute's basal diet, which is Iow in fat (5%) and (26). With such a model, the effects of type of treat- compounded from natural feed ingredients. Body wt rnent (control, surgical castration, surgical castration were recorded weekty during the first 3 mo and once plus testosteror~, or CA) were assessed. For rats, the a month during the rest of the experiment. The gen- influence of time of sacrifice (interim kill, terminal eral condition and behavior of the animals were autopsy) and possible interaction between type of checked daily. treatment and time of sacrifice were assessed as well. A Poisson error distribution was used for evaluation of Autopsy and Histological Analysis the lesions. In rats, an interim kill was performed on 20 ani- Evaluation of the prevalence (incidence) of tu- mals of each group after 4 mo of treatment. Terminal mors (terminal autopsy animals only) was done with autopsy on rats and hamsters was performed after 8 a generalized linear model. The difference with the mo of treatment on days 371 (rats), and 363 and 364 model used for lesions was the error distribution. (hamsters) after the last injection with carcinogen. The With respect to prevalence, the binomial distribu- animals were anesthetized by ether, exsanguinated by tion was appropriate. cannulating the abdominal aorta, autopsied, and then Endocrine Investigations examined for gross pathological changes. From each animal, the pancreas, liver, testes (if present), adrenals, Blood sampling for hormone estimations was per- and pituitary were excised and weighed. These formed at the start of the treatment period (n = 19- organs were fixed in 10% buffered formalin. The 20), after 4 mo (n = 10) and after 8 mo (n = 10) of pancreata were completely processed for microscopy treatment at the end of the experiment by exsang- by conventional methods, step-sectioned at 5 ~m uination. In groups of five animals, blood sampling (about 5 per pancreas), stained with H&E, and exam- was also performed after 2 and 6 mo by orbita ined by light microscopy. About 200 mm2 of pancre- punctions.