The Prostate 36:168–171 (1998) Dihydrotestosterone Prevents Spontaneous Adenocarcinomas in the Prostate-Seminal Vesicle in Aging L-W Rats Morris Pollard* Lobund Laboratory, University of Notre Dame, Notre Dame, Indiana BACKGROUND. Gene-regulated mechanisms govern tumor development, but the actual development of tumors can be suppressed or promoted by epigenetic factors. Lobund-Wistar (L-W) rats are genetically predisposed to development of spontaneous and induced metastasizing moderately differentiated adenocarcinomas in the prostate-seminal vesicle (P-SV) complex. In L-W rats with one slow-release subcutaneous implant of dihydrotestosterone (DHT) (5a-Androstan-17b-ol-3-one), the development of induced P-SV tumors 14 months later was significantly suppressed, with involution of testes, aspermia, and absence of detectable serum testosterone. The tumor-suppressive effect of DHT was confirmed. Spontaneous P-SV tumors developed in 57 of 220 control L-W rats (26%) at an average age 20 months. METHODS. At age 12 months, 70 L-W rats were administered an implant of 40 mg of DHT, and 75 untreated rats served as controls. All rats that developed palpable P-SV tumors were autopsied, and surviving rats were autopsied at age 24 months. RESULTS. At age 24 months, 9 of 70 DHT-treated rats (12.8%) and 20 of 75 DHT-free control rats (26.6%) had developed P-SV tumors spontaneously at average age 20.5 and 20 months, respectively. CONCLUSIONS. Slow-release implants of DHT administered to L-W rats at age 12 months reduced by 50% the development of spontaneous P-SV tumors by age 24 months. Prostate 36:168–171, 1998. © 1998 Wiley-Liss, Inc. KEY WORDS: dihydrotestosterone; prostate-seminal vesicle cancer; spontaneous tumors; prevention of P-SV tumors INTRODUCTION In 1987, we reported that testosterone propionate (TP) promoted, but that dihydrotestosterone (DHT) did not promote, the development of cancers in the accessory sex glands [prostate and seminal vesicles (P-SV)] of Lobund-Wistar (L-W) rats . Compared with TP-treated rats, the testes in DHT-treated rats were significantly reduced in size, levels of testosterone in their serums were significantly reduced; and within the time-frame of 14 months, there was no microscopic evidence of spermatogenesis. The ducts and acini in the prostatic lobes were lined by single layers of columnar and cuboidal cells. Among control rats, 24% of rats that were treated with multiple implants of TP developed large metastasizing tumors. The DHTtreated rats showed neither gross nor microscopic evidence of a tumorigenic effect . The inhibitory effect © 1998 Wiley-Liss, Inc. of DHT on induced P-SV tumors was confirmed in Fischer  and in L-W rats . L-W rats, at risk of developing P-SV tumors that were induced by a combination of methylnitrosourea (MNU) plus TP  were administered DHT or estradiol or were castrated, at a time midway in the estimated latency period . Within a time-frame of 14 months, the tumorigenic process was significantly inhibited by DHT, by estradiol and by castration; however, the same treatments were of no benefit in rats that were treated after palpable P-SV tumors had developed . Contract grant sponsor: The Coleman Foundation; Contract grant sponsor: The Scully Trust; Contract grant sponsor: University of Notre Dame. *Correspondence to: Morris Pollard, Lobund Laboratory, University of Notre Dame, Notre Dame, IN 46556. Received 10 November 1997; Accepted 23 March 1998 DHT Prevents Spontaneous P-SV Tumors DHT, metabolized from TP by the action of 5areductase, was described as the trophic androgen in initiating proliferative changes and tumors in the prostate gland [6–8]. This finding was confirmed in men whose tumor-free status was associated with deficiency in 5a-reductase , and in men who had been castrated when young . The inhibitory effects of DHT reported by us [1,5], contrasting with the reported trophic effect of DHT (6–8), was clarified: DHT (5a-Androstan-17b-ol-3-one) inhibited the development of induced P-SV tumors, but DHT propionate promoted tumor development . The trophic effect was also demonstrated with DHT-benzoate (Sigma Chemical Company, St. Louis, MO) . Metastasizing P-SV adenocarcinomas developed spontaneously in 26% of L-W rats, in an average latency period of 26 months . Further examinations of 220 aged L-W rats revealed that 57 rats (26%) developed large spontaneous P-SV tumors in average latency of 26 months; and that smaller tumors could be detected earlier by palpation at average age 20 months . This long latency period and low frequency rate simulated the natural history of prostate cancer in man. It was of interest to determine whether DHT would inhibit the development of spontaneous P-SV tumors from estimated midlife span. L-W rats at 12 months of age were treated with a single subcutaneous implant of DHT, and they were observed until they reached 24 months of age. The results of tumor development in DHT-treated and in untreated control rats is described below. MATERIALS AND METHODS Animals Pathogen-free L-W rats were propagated at random in isolated air-conditioned rooms with 12/12 hour light-dark cycles. They were maintained in plastic cages on a bedding of wood shavings and fed a whole grain diet (L-485, TekLad, Madison, WI). Although not intentionally inbred through 56 generations, L-W rats did not reject reciprocal skin transplants. The care of our experimental animals was and is in accord with the United States Public Health Service bulletin Guide for The Care and Use of Laboratory Animals. This facility is AAALAC-accredited. Experimental Design At age 12 months, 70 male L-W rats were implanted subcutaneous with 40 mg of DHT (Sigma Chemical Company), which was enclosed in a slow-release silicone membrane . This implant of DHT exerted an antigonadotropic effect for >14 months [1,5]. Seventy- 169 five control rats and 70 DHT-treated rats were fed ad libitum whole grain diet L-485 (TekLad). They were examined at frequent intervals for body weights, for changes in sizes of palpable testes, and for P-SV tumors. Rats with palpable P-SV tumors were killed by inhaled halothane and exsanguination from the heart; then they were examined for gross and microscopic lesions. All surviving rats were killed for similar examinations when they reached age 24 months. Tissue specimens from DHT-treated and from control rats were examined for histologic changes. The results were assessed for significance by Student’s t- test and by x2 test. RESULTS The DHT-treated and the control rats were in excellent physical condition, except that those with large P-SV tumors lost weight. When rats were autopsied at 24 months of age, the implants of DHT were onequarter full. Compared with data in control rats (Table I), changes were noted in the DHT-treated rats recorded here and are described in Ref. 1: (a) their palpable testes were significantly reduced in size within 1 month, and, in rats that developed P-SV tumors, the testes remained small for the duration of the experiment. (b) Serum testosterone levels were below the level of detection (0.1 ng/ml ml serum) by the RIA test used . (c) At autopsy, the average weight of the prostate complex in tumor-free rats was marginally less than the weight in control rats. (d) The weight of testes in DHT-treated rats was reduced by ∼70%, and the seminiferous tubules were free of spermatozoa. (e) The ducts and acini in the prostate and in the seminal vesicles were clean and lined with single layers of columnar and cuboidal cells. The connective tissue stromata were relatively sparse. These changes were also recorded and are described in Ref. 1. (f) There was little structural evidence of the prostatic intraepithelial neoplasia (PIN) described by Bostwick and Shrigley . (g) Palpable P-SV adenocarcinomas developed spontaneously in 9 of 70 DHT-treated rats (12.8%) in an average latency time of 20.5 months, and in 20 of 75 control rats (26.6%) in an average latency time of 20 months. Also, pituitary glands and Leydig cells in the testes were not altered in appearance and size in the DHTtreated rats, and there were no cytolytic changes in the involuted testes. In a separate experiment, 1 month after removal of the DHT implant from six rats, their small testes were restored to original size, and their breeding potency, previously negative, was restored . 170 Pollard TABLE I. Comparison of DHT-treated and Untreated Control L-W Rats† DHT treated 547 0.0* 3.06 ± 0.59* 0.31* Negative* Sparse 9 of 70 (12.8%)* 20.5 months Results Controls Average body weight/g Serum testosterone in ng/ml Average prostate complex/g Average testes weight/g Spermatogenesis PIN in prostate complex P-SV adenocarcinomas Average latent period/months 521 1.4 3.89 ± 0.82 1.38 Positive Positive 20 of 75 (26.6%) 20 months † Male L-W rats, age 12 months, were administered one subcutaneous implant of 40 mg of DHT enclosed in a slow-release silicone membrane. DHT-treated and control untreated rats were autopsied on appearance of tumors, and survivors were autopsied at age 24 months. The information listed was derived from tumor-free DHT-treated and age-related control untreated rats. *Statistically significant. DISCUSSION There is growing consensus that gene-regulated mechanisms govern predisposition to prostate cancer ; but the actual development of tumors can be suppressed or promoted by factors in the environment. Suppression of tumorigenesis in susceptible animals would be manifested by absence of tumors, by reduced incidence of tumors, and/or by prolongation of the latency time. A recent review on prostate cancer recommended that three transplantable cell lines derived from metastatic tumors in men and one cell line (Dunning) from a rat serve as models for studies on pathogenesis of prostate cancer . Pathogenesis is defined as ‘‘the organization and development of a disease.’’ We can agree that transplantable malignant and metastatic tumor cells had basically completed the sequence of pathogenic events leading to malignancy; thus, malignant tumors are not models for studies on pathogenesis. Decades of research on transplanted tumors have contributed little to our knowledge of the tumorigenic process; and the research rarely produced information that was confirmed through trials in animals with autochthonous tumors. This finding was exemplified in the following trials in the L-W rat: (a) cyclophosphamide (CPA) suppressed transplanted PA-III cells, but CPA was inactive against the development of induced P-SV tumors ; (b) 4-hydroxyphenyl retinamide very significantly suppressed metastatic spread of transplanted PA-III cells, but the effects on induced autochthonous P-SV tumors was marginal [17,18]. Transplantation models are being replaced by more authentic models of autochthonous tumors, which are needed to assess data that would be prerequisite to clinical trials. L-W rats are genetically susceptible to the development of spontaneous and induced metastasizing adenocarcinomas in the prostate-seminal vesicle (P-SV) complex. The development of induced P-SV tumors was suppressed by early treatments with antiandrogenic agents and procedures , and with antigonadotropic DHT. The incidence of induced P-SV tumors was reduced in L-W rats by antiangiogenic linomide  and by tamoxifen . Metastatic spread of autochthonous P-SV tumor cells was suppressed by viable BCG organisms, only if BCG had been inoculated intravenously . The latency periods, but not the incidence of induced tumors, was prolonged by feeding experiments with a soybean-derived high isoflavone diet . The incidence of spontaneous tumors was suppressed by life-long moderate dietary restriction ; and in the experiment reported here on DHT the incidence of spontaneous tumors was reduced by 50% when treatment was initiated at 12 months of age. The evidence is accumulating that the natural history of the disease in L-W rats resembles facets of the epidemiologic pattern of prostate cancer in man  and that the predicted altered ‘‘hormonal environment’’ may provide for early intervention in prostate cancer . The practicality of controlling prostate cancer in man by an implant of DHT may not attract volunteers, but DHT did prevent the hormone-influenced disease in rats without overt persistent damage to the host. 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