Dihydrotestosterone Prevents SpontaneousAdenocarcinomas 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 actualdevelopment of tumors can be suppressed or promoted by epigenetic factors. Lobund-Wistar(L-W) rats are genetically predisposed to development of spontaneous and induced metas-tasizing 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 monthslater was significantly suppressed, with involution of testes, aspermia, and absence of detect-able serum testosterone. The tumor-suppressive effect of DHT was confirmed. SpontaneousP-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 wereautopsied, 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 controlrats (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 monthsreduced by 50% the development of spontaneous P-SV tumors by age 24 months. Prostate36:168–171, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: dihydrotestosterone; prostate-seminal vesicle cancer; spontaneous tu-mors; 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 theaccessory sex glands [prostate and seminal vesicles(P-SV)] of Lobund-Wistar (L-W) rats [1]. Comparedwith TP-treated rats, the testes in DHT-treated ratswere significantly reduced in size, levels of testoster-one in their serums were significantly reduced; andwithin the time-frame of 14 months, there was no mi-croscopic evidence of spermatogenesis. The ducts andacini in the prostatic lobes were lined by single layersof columnar and cuboidal cells. Among control rats,24% of rats that were treated with multiple implants ofTP developed large metastasizing tumors. The DHT-treated rats showed neither gross nor microscopic evi-dence of a tumorigenic effect [1]. The inhibitory effect

of DHT on induced P-SV tumors was confirmed inFischer [2] and in L-W rats [3].

L-W rats, at risk of developing P-SV tumors thatwere induced by a combination of methylnitrosourea(MNU) plus TP [4] were administered DHT or estra-diol or were castrated, at a time midway in the esti-mated latency period [5]. Within a time-frame of 14months, the tumorigenic process was significantly in-hibited by DHT, by estradiol and by castration; how-ever, the same treatments were of no benefit in ratsthat were treated after palpable P-SV tumors had de-veloped [5].

Contract grant sponsor: The Coleman Foundation; Contract grantsponsor: The Scully Trust; Contract grant sponsor: University ofNotre Dame.*Correspondence to: Morris Pollard, Lobund Laboratory, Universityof Notre Dame, Notre Dame, IN 46556.Received 10 November 1997; Accepted 23 March 1998

The Prostate 36:168–171 (1998)

© 1998 Wiley-Liss, Inc.

DHT, metabolized from TP by the action of 5a-reductase, was described as the trophic androgen ininitiating proliferative changes and tumors in theprostate gland [6–8]. This finding was confirmed inmen whose tumor-free status was associated with de-ficiency in 5a-reductase [9], and in men who had beencastrated when young [10]. The inhibitory effects ofDHT reported by us [1,5], contrasting with the re-ported trophic effect of DHT (6–8), was clarified: DHT(5a-Androstan-17b-ol-3-one) inhibited the develop-ment of induced P-SV tumors, but DHT propionatepromoted tumor development [3]. The trophic effectwas also demonstrated with DHT-benzoate (SigmaChemical Company, St. Louis, MO) [11].

Metastasizing P-SV adenocarcinomas developedspontaneously in 26% of L-W rats, in an average la-tency period of 26 months [12]. Further examinationsof 220 aged L-W rats revealed that 57 rats (26%) de-veloped large spontaneous P-SV tumors in averagelatency of 26 months; and that smaller tumors couldbe detected earlier by palpation at average age 20months [11]. This long latency period and low fre-quency rate simulated the natural history of prostatecancer in man. It was of interest to determine whetherDHT would inhibit the development of spontaneousP-SV tumors from estimated midlife span. L-W rats at12 months of age were treated with a single subcuta-neous implant of DHT, and they were observed untilthey reached 24 months of age. The results of tumordevelopment in DHT-treated and in untreated controlrats is described below.

MATERIALS AND METHODS

Animals

Pathogen-free L-W rats were propagated at randomin isolated air-conditioned rooms with 12/12 hourlight-dark cycles. They were maintained in plasticcages on a bedding of wood shavings and fed a wholegrain diet (L-485, TekLad, Madison, WI). Although notintentionally inbred through 56 generations, L-W ratsdid not reject reciprocal skin transplants. The care ofour experimental animals was and is in accord withthe United States Public Health Service bulletin Guidefor The Care and Use of Laboratory Animals. This facilityis AAALAC-accredited.

Experimental Design

At age 12 months, 70 male L-W rats were implantedsubcutaneous with 40 mg of DHT (Sigma ChemicalCompany), which was enclosed in a slow-release sili-cone membrane [1]. This implant of DHT exerted anantigonadotropic effect for >14 months [1,5]. Seventy-

five control rats and 70 DHT-treated rats were fed adlibitum whole grain diet L-485 (TekLad). They wereexamined at frequent intervals for body weights, forchanges in sizes of palpable testes, and for P-SV tu-mors. Rats with palpable P-SV tumors were killed byinhaled halothane and exsanguination from the heart;then they were examined for gross and microscopiclesions. All surviving rats were killed for similar ex-aminations when they reached age 24 months. Tissuespecimens from DHT-treated and from control ratswere examined for histologic changes. The resultswere assessed for significance by Student’s t- test andby x2 test.

RESULTS

The DHT-treated and the control rats were in ex-cellent physical condition, except that those with largeP-SV tumors lost weight. When rats were autopsied at24 months of age, the implants of DHT were one-quarter full.

Compared with data in control rats (Table I),changes were noted in the DHT-treated rats recordedhere and are described in Ref. 1: (a) their palpabletestes were significantly reduced in size within 1month, and, in rats that developed P-SV tumors, thetestes remained small for the duration of the experi-ment. (b) Serum testosterone levels were below thelevel of detection (0.1 ng/ml ml serum) by the RIA testused [5]. (c) At autopsy, the average weight of theprostate complex in tumor-free rats was marginallyless than the weight in control rats. (d) The weight oftestes in DHT-treated rats was reduced by ∼70%, andthe seminiferous tubules were free of spermatozoa. (e)The ducts and acini in the prostate and in the seminalvesicles were clean and lined with single layers of co-lumnar and cuboidal cells. The connective tissue stro-mata were relatively sparse. These changes were alsorecorded and are described in Ref. 1. (f) There waslittle structural evidence of the prostatic intraepithelialneoplasia (PIN) described by Bostwick and Shrigley[13]. (g) Palpable P-SV adenocarcinomas developedspontaneously in 9 of 70 DHT-treated rats (12.8%) inan average latency time of 20.5 months, and in 20 of 75control rats (26.6%) in an average latency time of 20months.

Also, pituitary glands and Leydig cells in the testeswere not altered in appearance and size in the DHT-treated rats, and there were no cytolytic changes in theinvoluted testes. In a separate experiment, 1 monthafter removal of the DHT implant from six rats, theirsmall testes were restored to original size, and theirbreeding potency, previously negative, was restored[11].

DHT Prevents Spontaneous P-SV Tumors 169

DISCUSSION

There is growing consensus that gene-regulatedmechanisms govern predisposition to prostate cancer[14]; but the actual development of tumors can be sup-pressed or promoted by factors in the environment.Suppression of tumorigenesis in susceptible animalswould be manifested by absence of tumors, by re-duced incidence of tumors, and/or by prolongation ofthe latency time. A recent review on prostate cancerrecommended that three transplantable cell lines de-rived from metastatic tumors in men and one cell line(Dunning) from a rat serve as models for studies onpathogenesis of prostate cancer [15]. Pathogenesis isdefined as ‘‘the organization and development of adisease.’’ We can agree that transplantable malignantand metastatic tumor cells had basically completed thesequence of pathogenic events leading to malignancy;thus, malignant tumors are not models for studies onpathogenesis. Decades of research on transplanted tu-mors have contributed little to our knowledge of thetumorigenic process; and the research rarely producedinformation that was confirmed through trials in ani-mals with autochthonous tumors. This finding wasexemplified in the following trials in the L-W rat: (a)cyclophosphamide (CPA) suppressed transplantedPA-III cells, but CPA was inactive against the devel-opment of induced P-SV tumors [16]; (b) 4-hydroxy-phenyl retinamide very significantly suppressedmetastatic spread of transplanted PA-III cells, but theeffects on induced autochthonous P-SV tumors wasmarginal [17,18]. Transplantation models are being re-placed by more authentic models of autochthonoustumors, which are needed to assess data that would beprerequisite to clinical trials.

L-W rats are genetically susceptible to the develop-ment of spontaneous and induced metastasizing ad-enocarcinomas in the prostate-seminal vesicle (P-SV)complex. The development of induced P-SV tumorswas suppressed by early treatments with antiandro-genic agents and procedures [5], and with antigonado-tropic DHT. The incidence of induced P-SV tumorswas reduced in L-W rats by antiangiogenic linomide[3] and by tamoxifen [19]. Metastatic spread of autoch-thonous P-SV tumor cells was suppressed by viableBCG organisms, only if BCG had been inoculated in-travenously [20]. The latency periods, but not the in-cidence of induced tumors, was prolonged by feedingexperiments with a soybean-derived high isoflavonediet [21]. The incidence of spontaneous tumors wassuppressed by life-long moderate dietary restriction[22]; and in the experiment reported here on DHT theincidence of spontaneous tumors was reduced by 50%when treatment was initiated at 12 months of age. Theevidence is accumulating that the natural history ofthe disease in L-W rats resembles facets of the epide-miologic pattern of prostate cancer in man [23] andthat the predicted altered ‘‘hormonal environment’’may provide for early intervention in prostate cancer[24]. The practicality of controlling prostate cancer inman by an implant of DHT may not attract volunteers,but DHT did prevent the hormone-influenced diseasein rats without overt persistent damage to the host.

There are unanswered questions in this report onDHT: (a) what is the optimal dosage of DHT; (b) willthe incidence of tumors be further reduced by earliertreatment of rats with DHT; (c) where in the hypotha-lamic-pituitary-testicular axis is the functional linkageinterrupted by DHT; (d) are the results on induced

TABLE I. Comparison of DHT-treated and Untreated ControlL-W Rats†

DHT treated Results Controls

547 Average body weight/g 5210.0* Serum testosterone in ng/ml 1.43.06 ± 0.59* Average prostate complex/g 3.89 ± 0.820.31* Average testes weight/g 1.38Negative* Spermatogenesis PositiveSparse PIN in prostate complex Positive9 of 70 (12.8%)* P-SV adenocarcinomas 20 of 75 (26.6%)20.5 months Average latent period/months 20 months

†Male L-W rats, age 12 months, were administered one subcutaneousimplant of 40 mg of DHT enclosed in a slow-release silicone membrane.DHT-treated and control untreated rats were autopsied on appearance oftumors, and survivors were autopsied at age 24 months. The informationlisted was derived from tumor-free DHT-treated and age-related controluntreated rats.*Statistically significant.

170 Pollard

P-SV tumors comparable to results on spontaneoustumors; and (e) why was esterified DHT active butnonesterified DHT inactive in the development of P-SV tumors [3]? Acknowledging the limits of confi-dence in model systems, evidence in accumulatingthat environmental (epigenetic) factors can modifygene-directed prostate-related cancers.

ACKNOWLEDGMENTS

I acknowledge, with thanks, the important contri-butions to this work by Phyllis H. Luckert, and byValerie Schroeder of the Freimann Life Science Center.

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