Induction of dorsolateral prostate adenocarcinomas and other accessory sex gland lesions in male Wistar rats by a single administration of N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl after sequential treatment with cyproterone acetate and testosterone propionate.

Groups of 20-25 male Wistar rats (Cpb:WU), nine groups of 4-week-old rats, and nine groups of 8-week-old rats, were given cyproterone acetate (CA) s.c. or by gavage daily for 18 days at a dose of 50 mg/kg/day. Directly following CA treatment, the rats received 3 daily s.c. injections with testosterone propionate (TP) at a dose of 100 mg/kg/day. On the day after the last TP administration, a single dose of one of the following carcinogens was given to 3 groups: N-methyl-N-nitrosourea (MNU), 50 mg/kg i.v.; 7,12-dimethylbenz(a)anthracene, 30 mg/kg i.v.; 3,2'-dimethyl-4-aminobiphenyl, 250 mg/kg s.c. Three other groups received the same carcinogen treatments after 7 days of recovery from the CA administration. The last 3 groups received carcinogen without TP treatment, but immediately after CA pretreatment was stopped. A 25% incidence of invasively growing, metastasizing adenocarcinomas was found in the dorsolateral prostate region of 8-week-old rats that had received MNU after treatment with CA plus TP. In addition, this group had a 5% incidence of carcinoma in situ and a 5% incidence of atypical hyperplasia in the dorsolateral prostate. Lower incidences of adenocarcinoma of the dorsolateral prostate region and of carcinoma in situ and atypical hyperplasia of the dorsolateral prostate were found in other groups that were treated with MNU or 7,12-dimethylbenz(a)anthracene after pretreatment with CA, followed by TP or recovery, but never in rats that had been treated with CA only. In the groups treated with 3,2'-dimethyl-4-aminobiphenyl, which is slowly metabolized, these lesions were also found in groups that were pretreated with only CA. The carcinomas seemed to originate from the dorsolateral prostate and their average latency time was approximately 61 weeks. The 8-week-old rat given a MNU injection after sequential treatment with CA and TP may provide a relevant animal model for human prostatic cancer.

Characterization of adenocarcinomas of the dorsolateral prostate induced in Wistar rats by N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl, following sequential treatment with cyproterone acetate and testosterone propionate.

Carcinomas of the rat prostate induced by a single injection of N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2'-dimethyl-4-aminobiphenyl, after sequential treatment with cyproterone acetate and testosterone propionate, were evaluated as potential animal models for prostatic cancer. All ten carcinomas examined were located in the dorsolateral prostate region and did not involve the distal parts of the seminal vesicles and coagulating glands. The incidence of urinary obstruction leading to the animals' death was 6 of 10 rats, and metastases in the lung, abdominal lymph nodes, and/or liver also occurred in 6 of 10 rats. The tumors were invasive adenocarcinomas, showing frequent perineural invasion and a variable degree of differentiation. There were ultrastructural similarities with human prostatic carcinomas, such as intracellular lumina. Plasma acid phosphatase was increased. Enzyme histochemical analysis revealed similarities with the Dunning R3327H and -HI prostatic carcinomas but was not helpful in determining the site of origin of the tumors. The gross and microscopic appearance of the tumors and the observation of preneoplastic lesions exclusively located in the dorsolateral prostate suggest this lobe as site of origin of the carcinomas. Preneoplastic lesions (n = 9) included atypical hyperplasias (n = 5) and lesions with all histological characteristics of carcinoma except for local invasion and metastases, which were classified as carcinoma in situ (n = 4). Although androgen sensitivity could not be assessed, the observed characteristics of the tumors [their long latency time (46-80 weeks), the presence of preneoplastic lesions, and the short duration of the treatment, leaving the animals intact] all indicate that the present approach is a valid animal model for the study of prostatic carcinogenesis.

Interactive effects of dietary wheat bran and lard on N-methyl-N'-nitro-N-nitrosoguanidine-induced colon carcinogenesis in rats.

A 3 x 3 factorial experiment was conducted to examine how dietary fiber (wheat bran) and fat (lard) interactively affect the genesis of N-methyl-N'-nitro-N-nitrosoguanidine-induced colon cancer in rats. Groups of 30 male 4-week-old Wistar rats were fed ad libitum one of nine experimental diets containing either 15 (low), 27.5 (medium), or 40% (high) energy as fat in combination with 0.7 (low), 2.2 (medium), or 3.8 g (high) fiber/100 kcal for a period of 37 weeks. After 4 weeks, each rat received a total of five weekly intrarectal instillations of 6 mg N-methyl-N'-nitro-N-nitrosoguanidine/kg. The highest colon carcinoma incidence and the highest total number of carcinomas of the colon were observed in the animals fed the medium-fat/medium-fiber diet. The highest number of polyps and a relatively high polyp incidence occurred in the animals on the high-fat/low-fiber diet. An enhancing effect of fat on both the tumor incidence and tumor multiplicity was clearly present for the low-fiber diets, whereas fat had no effect when the fiber content of the diet was high. In general, the results showed a nonlinear dose-response relationship for fiber and fat. These results indicate that both dietary fiber and fat affect colon carcinogenesis in a complex, interactive manner.

Influence of N-methyl-N-nitrosourea, testosterone, and N-(4-hydroxyphenyl)-all-trans-retinamide on prostate cancer induction in Wistar-Unilever rats.

The influence of chemical carcinogen, hormonal stimulation, and chronic dietary administration of the synthetic retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), on the induction of prostate cancer in male Wistar-Unilever rats was determined. Three different tumor induction regimens were used: (a) a single i.v. dose of 50 mg of N-methyl-N-nitrosourea (MNU) per kg body weight, followed by chronic androgen stimulation via s.c. implantation of two silastic capsules containing 40 mg testosterone each; (b) a single i.v. dose of 50 mg of MNU per kg body weight (no testosterone treatment); and (c) chronic androgen stimulation with implanted testosterone capsules (no MNU treatment). In a fourth series of animals, the incidence of spontaneous prostate tumors was determined in groups of rats receiving neither carcinogen nor hormone stimulation. Within each series, parallel groups of animals were fed a control (vehicle-supplemented) diet or control diet supplemented with 4-HPR beginning 1 day after carcinogen administration; retinoid administration was continuous until termination of the study at 450 days. The incidence of accessory sex gland cancer in rats treated sequentially with MNU + testosterone was >60%, in comparison with cancer incidences of <20% in rats receiving MNU only and <5% in rats treated with testosterone only. No spontaneous accessory sex gland tumors were observed in rats receiving no carcinogen and no testosterone. Tumor induction in the accessory sex glands by MNU + testosterone was relatively specific for the prostate: the incidence of carcinoma of the dorsolateral/anterior prostate was more than 5-fold greater than the incidence of cancer present only in the seminal vesicle. 4-HPR conferred no protection against cancer induction in the prostate by any regimen of MNU and/or testosterone. These results demonstrate the importance of both carcinogen exposure and hormone stimulation on the induction of neoplasia in the prostate of Wistar-Unilever rats.

Chemoprevention of rat prostate carcinogenesis by 9-cis-retinoic acid.

A chemoprevention study was conducted to evaluate the activity of 9-cis-retinoic acid (9-cis-RA) as an inhibitor of prostate carcinogenesis in male Wistar-Unilever (HsdCpb:Wu) rats. After pretreatment with a sequential regimen of cyproterone acetate (50 mg/kg/day for 21 days) and testosterone propionate (100 mg/kg/day for 3 days), groups of 40 rats received a single i.v. injection of N-methyl-N-nitrosourea (MNU; 30 mg/kg body weight). Beginning 2 weeks after carcinogen administration, rats received chronic exposure to testosterone administered in s.c. implanted silastic capsules. The study was terminated at 13 months after MNU administration, and prostate cancer incidence was determined by histopathological evaluation of step sections of accessory sex glands. Continuous dietary administration of 9-cis-RA at 100 mg/kg diet or 50 mg/kg diet beginning 1 week before MNU administration reduced cancer incidence in the dorsolateral + anterior prostate from 65% in dietary controls to 18 and 20%, respectively (P < 0.001 for both comparisons). Similarly, these dose levels of 9-cis-RA reduced the incidence of cancer in all accessory sex glands from 79% in dietary controls to 48 and 33% (P < 0.01 for both comparisons), respectively. Chronic dietary administration of 9-cis-RA induced no gross or organ-specific toxicity in any animal and did not suppress group mean body weight gain. The potent anticarcinogenic activity of 9-cis-RA in the rat prostate, when considered with its apparent lack of toxicity in rodents, suggests that this and other ligands for the retinoid X receptor merit consideration for evaluation in clinical prostate cancer chemoprevention trials.

Chemoprevention of rat prostate carcinogenesis by early and delayed administration of dehydroepiandrosterone.

Two in vivo bioassays were conducted to evaluate the efficacy of dehydroepiandrosterone (DHEA) as an inhibitor of prostate carcinogenesis in rats. Prostate adenocarcinomas were induced in male Wistar-Unilever rats by a sequential regimen of cyproterone acetate and testosterone propionate, followed by a single i.v. injection of N-methyl-N-nitrosourea (MNU) and chronic androgen stimulation. In the first experiment, DHEA (1000 or 2000 mg/kg diet) was administered continuously to rats beginning 1 week before MNU exposure. In the second experiment, continuous administration of DHEA (2000 mg/kg diet) was begun either 1 week before, 20 weeks after, or 40 weeks after MNU exposure. Controls received basal diet without added DHEA. Studies were terminated at 13 months after MNU administration, and prostate cancer incidence was determined by histopathological evaluation of step sections of accessory sex glands. In the first study, continuous dietary administration of DHEA beginning 1 week before MNU resulted in a dose-related inhibition of prostate cancer induction. In the second experiment, comparable reductions in prostate cancer incidence were observed in groups exposed to DHEA beginning 1 week before, 20 weeks after, and 40 weeks after carcinogen exposure. These data demonstrate that nontoxic doses of DHEA confer significant protection against prostate carcinogenesis in rats. The efficacy of delayed administration of DHEA suggests that the compound confers protection against later stages of prostate cancer induction and can suppress the progression of existing preneoplastic lesions to invasive disease.

The role of steroid hormones in prostate carcinogenesis.

Carcinoma of the prostate is the most frequently diagnosed malignancy and the second leading cause of death as a result of cancer in men in the United States and in many other Western countries. Notwithstanding the importance of this malignancy, little is understood about its causes. The epidemiology of prostate cancer strongly suggests that environmental factors, particularly diet and nutrition, are major determinants of risk for this disease, and evidence is mounting that there are important genetic risk factors for prostate cancer. Human prostate carcinomas are often androgen sensitive and react to hormonal therapy by temporary remission, followed by relapse to an androgen-insensitive state. These well-established features of prostate cancer strongly suggest that steroid hormones, particularly androgens, play a major role in human prostatic carcinogenesis, but the precise mechanisms by which androgens affect this process are unknown. In addition, the possible involvement of estrogenic hormones is not entirely clear. The purpose of this overview is to summarize the literature about steroid hormonal factors, androgens and estrogens, and prostate carcinogenesis. From these literature observations, a multifactorial general hypothesis of prostate carcinogenesis emerges with androgens as strong tumor promoters acting via androgen receptor-mediated mechanisms to enhance the carcinogenic activity of strong endogenous genotoxic carcinogens, such as reactive estrogen metabolites and estrogen- and prostatitis-generated reactive oxygen species and possible weak environmental carcinogens of unknown nature. In this hypothesis, all of these processes are modulated by a variety of environmental factors such as diet and by genetic determinants such as hereditary susceptibility and polymorphic genes that encode for steroid hormone receptors and enzymes involved in the metabolism and action of steroid hormones.

Adenocarcinomas of the prostate induced by N-nitroso-N-methylurea in rats pretreated with cyproterone acetate and testosterone.

Prostatic adenocarcinomas were induced in 5 out of 20 Wistar rats upon a single administration of 50 mg/kg N-nitroso-N-methylurea (NMU). The rats were pretreated with a daily dose of 50 mg/kg cyproterone acetate for 3 weeks followed by 3 daily injections of 100 mg/kg testosterone. All tumours developed in the dorsolateral prostate and were invasively growing. In 2 cases distant metastases were found. Three proliferative lesions classified as carcinomas in situ were also found in the dorsolateral prostate. A total of 7/20 animals (35%) carried an adenocarcinoma and/or a carcinoma in situ. In addition, 6 epithelial hyperplasias were observed in the dorsolateral and 1 in the ventral prostate of non-tumour-bearing rats. The method described may provide a good animal model for cancer of the prostate and lead to a better understanding of prostatic carcinogenesis.

The role of stromal cells in prostate cancer development and progression.

Prostate cancer is one of the most common malignancies in males, and tumor progression critically determines its clinical significance. Prostatic stromal cells may be critically involved in growth and progression of prostate cancer. There is substantial evidence that the stromal component of the embryological tissue of origin, the urogenital sinus, is essential in directing outgrowth and prostatic differentiation of the epithelial anlage of the prostate. The presence of a stromal androgen receptor is required for this effect, and humoral factors, such as keratinocyte growth factor, have been shown to be able to mediate it in a paracrine fashion. The adult prostate is also under control of multiple steroid hormone and paracrine peptide factors, and there is evidence that the prostatic stroma plays a major role in mediation of androgen effects on prostatic epithelium. Normal seminal vesicle mesenchyme can cause differentiation of the Dunning R3327H prostate carcinoma. Normal rat prostatic fibroblasts influence the in vivo and soft agar growth of epithelial cells derived from chemically/hormonally induced rat prostate carcinomas, as do fibroblasts that are isolated from these tumors. Both growth-enhancing and growth-inhibiting effects were observed, apparently depending on the stage of progression of both cell types as well as on whether fibroblasts were derived from the same or a different tumor than the epithelial cells. These findings indicate that stromal cells critically influence epithelial prostate cancer growth, and they suggest that these effects can significantly vary in different tumors as well as in different stages of tumor progression.

Animal models for the study of prostate carcinogenesis.

Human prostate carcinogenesis has been viewed as a multi-step process involving progression from low histologic grade, small, latent carcinoma to large, higher grade, metastasizing carcinoma. However, recent data suggest that a variety of pathogenetic pathways may exist. The precise etiology and pathogenesis of human prostate cancer remain largely undefined. It is difficult to investigate stages in the development of human prostate cancer, but some animal models provide opportunities in this regard. Short-term treatment of rats with chemical carcinogens produces a low incidence (5-15%) of prostate cancer, provided that prostatic cell proliferation is enhanced during carcinogen exposure. Chronic treatment with testosterone also produces a low prostate carcinoma incidence. A high carcinoma incidence can only be produced by chronic treatment with testosterone following administration of carcinogens such as N-methyl-N-nitrosourea (MNU) and 3,2'-dimethyl-4-aminobiphenyl (DMAB). Testosterone markedly enhances prostate carcinogenesis even at doses that do not measurably increase circulating testosterone. Thus, testosterone is a strong tumor promoter for the rat prostate. All such MNU- or DMAB-initiated and/or testosterone-promoted tumors are adenocarcinomas; most originate from the dorsolateral and anterior, but not ventral, prostate lobes. These tumors share a number of important characteristics with human prostate cancer. A high frequency (70%) of activation of the K-ras gene by a G35 to A mutation occurs in these carcinomas. Another high incidence prostate carcinogenesis model, representing a different pathogenetic pathway, involves chronic administration of estradiol-17 beta to rats in combination with low-dose testosterone. The resulting carcinomas are low-grade and originate exclusively from periurethral ducts of the dorsolateral and anterior prostate. While it is unknown whether testosterone is a tumor promoter in this system, preliminary studies indicate the formation of a DNA adduct in the target tissue, which suggests that estradiol-17 beta acts as a tumor initiating agent in this system. The high incidence models mentioned earlier are adequate for the study of chemoprevention of prostatic carcinogenesis. Analysis of shifts in the relative incidence of metastasizing carcinoma, grossly apparent but not-metastasizing carcinoma, microscopic-size carcinoma, and carcinoma in situ or atypical hyperplasia may allow study of the modifying effects of potential chemopreventive agents on tumor progression in these animal models of prostatic carcinogenesis.

Hormonal factors in carcinogenesis of the prostate and testis in humans and in animal models.

The etiology of human testicular tumors is poorly defined. With the possible exception of prenatal estrogen exposure, no specific chemical exposures have been associated with testicular cancer risk in men. Prenatal as well as postnatal estrogen treatments induce testicular tumors in some mouse strains, but not in other mouse strains or in rats. Prenatal estrogen exposure also causes cryptorchid testes in mice and possibly rats. Cryptorchidism is a consistent risk factor for testicular cancer in men, and estrogen- or surgically-induced cryptorchidism is associated with Leydig cell tumorigenesis in mice. In rats, however, surgically induced cryptorchidism inhibits Leydig cell tumor formation. Overall, it appears that the mouse is the most appropriate species as animal model for testicular tumorigenesis in humans. Any of the following hormonal exposures can cause testicular tumor formation in rodents: 1) chronic exposure to estrogenic compounds of adult mice and hamsters; 2) prenatal exposure to estrogenic compounds of mice and possibly humans; and 3) any treatment or condition that induces cryptorchidism in mice and humans. The mechanisms whereby these treatments or conditions may cause testicular tumorigenesis are poorly understood. Undefined local testicular factors appear to be dominant in tumorigenesis in cryptorchid human and rodent testes. Pituitary factors, most likely LH and perhaps prolactin, play a critical but poorly defined role in estrogen-induced and spontaneous testicular tumorigenesis in rodents. In the mouse, estrogen receptor-mediated mechanisms seem to be involved in induction of testicular tumors by prenatal estrogen exposure, and a direct, perhaps estrogen receptor-mediated, inhibiting effect of estrogens on the action of müllerian inhibiting substance is probably central in the induction of cryptorchidism in this species.

Use of animal models in defining efficacy of chemoprevention agents against prostate cancer.

Animal models are crucial in preclinical efficacy testing of chemoprevention agents. The most feasible, realistic, and potentially effective target for prostate cancer chemoprevention is progression from prostatic intraepithelial neoplasia (PIN) to histologic cancer and from histologic to clinically manifest cancer. There are transgenic mouse models for prostate cancer and models for PIN, but these have not yet been fully developed and evaluated for chemoprevention studies. Human prostate cancer xenografts in mice and transplantable Dunning rat prostate carcinomas can be used to assess tumor growth inhibition. Several Dunning tumors metastasize, enabling detection of inhibition of metastases. Detection of inhibitory effects on de novo prostate cancer development requires induction of a high cancer incidence and similarity of induced tumors to human prostate carcinomas. Transgenic mice with oncogenes expressed in a prostate-specific fashion, combined chronic treatment of NBL rats with estradiol-17beta and testosterone, and sequential treatment of rats with carcinogens such as N-methyl-N-nitrosourea (MNU) and chronic testosterone treatment all lead to a high incidence of prostatic adenocarcinomas. PIN occurs mostly in the former two models, and metastases are frequent in some transgenic models and the MNU-testosterone rat model. The latter model has been applied to chemoprevention agent efficacy testing. In 8 control groups, the carcinoma incidence was 77% in all accessory sex glands combined, 51% for small tumors confined to dorsolateral/anterior prostate, and 25% for large tumors of uncertain origin in the prostate area. This model was predictive of the lack of antiprostate cancer efficacy of N-(4-hydroxyphenyl)all-trans-retinamide in humans. Thus, rats given MNU and chronic testosterone represent a relevant and reliable model for efficacy testing of chemoprevention agents. In conclusion, there are now adequate animal models for prostate cancer proven to be suitable for preclinical chemoprevention studies.

Some functional characteristics of adrenal medullary tumors in aged male Wistar rats.

Neither analysis of the urinary catecholamine metabolites vanillymandelic acid and 4-hydroxy-3-methoxyphenylglycol nor blood pressure measurements allowed the detection of adrenal medullary tumors or hyperplasia in 115 aged male Wistar rats (70 rats, 24 months of age and 45 rats, 30 months of age). Histochemical examination of the adrenal glands demonstrated that the 55 hyperplastic and 25 neoplastic lesions of the medulla usually had little or no chromaffinity. Chromaffinity was found comparable to normal medulla in only three tumors examined histochemically. Of these tumors, two were partially and one completely chromaffin-positive, but all three were small and did not result in elevated blood pressure or catecholamine metabolite excretion values. These observations indicate that excessive catecholamine synthesis and release is not a feature of adrenomedullary tumors and hyperplasia occurring spontaneously in aged male Wistar rats.

Differential effects of diethylstilbestrol and estradiol-17 beta in combination with testosterone on rat prostate lobes.

Treatment of Noble rats with separate silastic implants containing testosterone (T) and estradiol-17 beta (E2) for 16 weeks has previously been shown to induce multifocal epithelial dysplasia, a putative preneoplastic lesion, consistently in the dorsolateral prostate (DLP) but not in the ventral prostate (VP). We now studied effects of diethylstilbestrol (DES) substituted for E2 on these prostate lobes under the same conditions of exogenous androgen support. Three-week treatments with one 1-cm-long silastic implant of E2 or DES were approximately equipotent in changing target-organ weights and plasma prolactin. Accordingly, rats received for 16 weeks one 1-cm-long E2 or DES implant and two 2-cm-long T implants. In contrast to T + E2, T + DES induced widespread multifocal VP dysplasia and less or no DLP dysplasia. A serum-free explant-culture assay was used to determine uptake and metabolic disposition of 3H-labeled 5 alpha-dihydrotestosterone (DHT), T, and E2. Dysplastic VP explants incubated with 1.7 microM 1 beta-3H-labeled DHT and T accumulated more 3H-labeled steroid, metabolized 69 and 50% less substrate to terminal hydroxylated metabolites, and thereby formed and retained up to eight times as much estrogenic metabolite 5 alpha-androstane-3 beta,17 beta-diol (3 beta-androstanediol) and its lipoidal derivative than control VP. Experimental DLP explants did not form or retain more 3 beta-[3H]androstanediol than control DLP irrespective of treatments. Control VP metabolized [2-3H]E2 more actively to estrone than DLP. Dysplastic VP, however, metabolized one-half and accumulated five times as much E2 as VP and did not release more 3H as a marker of the 2,3-catechol estrogen pathway. These data suggest that differential target-tissue bioavailability of the estrogen component of the protracted dual-hormone stimulus determines in which prostate lobe dysplasia develops.

Selective depletion of spinal noradrenaline inhibits post-decapitation convulsions in rats.

Local administration of 6-hydroxydopamine in the subarachnoidal space of the spinal cord in rats resulted in a selective long-lasting depletion of spinal noradrenaline, but not of dopamine, and prevented the occurrence of post-decapitation convulsions.