Proliferative changes in the prostate

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

Proliferative

(1989)

101,499-509

Changes in the Prostate’

PARVIZ POUR, TERENCE A. LAWSON, AND SAMUEL

M. COHEN

Eppley Institute for Research on Cancer and Department ofPathology and Microbiology, University ofNebraska Medical Center, Omaha, Nebraska 68105-1065

Received June 30. 1989: accepted August 28, I989 Proliferative Changes in the Prostate. POUR, P., LAWSON, T. A., AND COHEN, S. M. (1989). Toxicol. Appl. Pharmacol. 101,499-509.The prostate of the rat has several lobes which have variable responsiveness to estrogens and testosterone. Testosterone is a major stimulant of cell proliferation in the prostate. Chemical carcinogenesis models in the rat prostate have taken advantage of administering the carcinogen during the peak proliferative period following testosterone administration with subsequent testosterone administered to continue the proliferative stimulus. Invasive adenocarcinomas of the prostate have been induced u&zing such methods. 0 1989 Academic press, hc.

Benign and malignant diseases are common in the human population, but have not been studied experimentally as extensively as other common disorders. With respect to prostatic carcinogenesis, this lack of progress in our understanding of the disease has occurred predominantly because of two reasons: (1) There are large differences in the structure and physiology of the prostate glands between mammalian species, with none in experimental animals identical to the human prostate (Price, 1963); and (2) until recently, there has not been available an appropriate protocol for tumor induction (Handlesman, 1977). There is no organ that shows as much variation between mammalian species as the prostate (Fig. 1). In man, the prostate is a solid and distinct organ which has been arbitrarily divided into lobes, the exact number remaining somewhat controversial. However, it is well accepted that the central zone

’ Presented at the 28th Annual Meeting of the Society of Toxicology, Atlanta, Georgia, February 26-March 3, 1989. 499

surrounding the urethra is related to benign prostatic hyperplasia and hypertrophy, and the neoplastic changes occur peripherally (Mostofi and Price, 1973). In bulls, the prostate has an external body and a diffuse or disseminated gland surrounding the urethra. Dogs have a prostate but no seminal vesicle. In domestic and cottontail rabbits, not only does the male have a prostate, but there is an analogous organ in females, and its presence can be selectively bred (Price, 1963). There are several lobes to the prostate in rats (Fig. 2), including ventral lobes, lateral lobes, dorsal lobes, and anterior lobes (cranial or coagulating glands). In addition, there are paired seminal vesicles present. Each lobe has a distinct histologic and functional pattern (Lee and Holland, 1987). The dorsal and lateral lobes appear to correspond most closely embryologically and structurally to the human prostate (Price, 1963). The remainder of this presentation will concern the rat prostate, since in that species models for prostatic carcinogenesis have been developed. In the past few years, four major models of prostatic carcinogenesis in rats have been 0041-008x/89

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Copyright 0 1989 by Academic Press, Inc. All rightsofreproducfion in any form reserved

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FIG. 1. Comparative anatomy of the male reproductive glands in four species. BL, bladder; BU, bulbourethral glands; D, ductus deferens; PR, prostate; S, seminal vesicle; T, testis; U, ureter; UR, urethra. (Reproduced from Price, 1963).

developed, although each has shortcomings. The first model is essentially the spontaneous disease in aging rats (Ward et al., 1980; Isaacs, 1984). Certain strains of rats, including the AC1 strain and to a lesser extent the Copenhagen strain, appear to be particularly susceptible (Reznik et al., 1981; Bosland, 1987a,b,c). Although there are proliferative changes in the ventral prostate which occur during the first 2 years of life in these strains, neoplasms do not appear to occur until much later, even up to 3 years. In addition, the incidence is relatively low. In these rats, the tumors are predominantly adenocarcinomas and arise from the ventral gland. In addition to these spontaneously arising tumors, transplantable tumors have been available for study for some time, but these have all the inherent shortcomings of transplantable lines (Lubaroff et al., 1980; Pollard, 1980; Noble, 1982). EXPERIMENTAL

MODELS

Recently, there have been three chemically induced prostatic cancer models in rats, all

three of which are based on the administration of a carcinogen during a burst of proliferative activity hormonally induced in the prostate (Bosland, 1988). It is well known that chemical or surgical castration or highdose estrogen treatment to males leads to marked atrophy of the prostatic gland. This includes a marked reduction in the cell replication of the epithelium, approaching but not reaching zero. One of these models involves the administration of N-methyl-N-nitrosourea (MNU) in chemically castrated rats which had then been treated with testosterone (Bosland et al., 1983). With this procedure, significant incidences of adenocarcinomas were induced in the dorsolateral lobes within 1 year, with several becoming invasive. The other two models which have been developed have utilized the initial finding of a small but significant incidence of prostatic tumors in animals treated with chemicals alone. The first of these is N-nitrosobis(2-oxopropyl)amine (BOP) which we have been studying (Pour, 1981; Pour and Stepan, 19 87), and the second is 3,2’-dimethyl-4-ami-

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FIG. 2. The gross appearance of the male rat reproductive tract and the histologic appearance of the different lobes. A, ampullary gland; BL, bladder; D, ductus deferens; DPR, dorsal prostate; LPR, lateral prostate; UR, urethra; VPR, ventral prostate. (Reproduced from Price, 1963.) (a) The gross appearance of the male rat reproductive system. (b) Ventral prostate (X500). (c) Prostate gland from a 2%day-old female (X250). (d) Coagulating gland (X500). (e) Dorsal prostate (X500). (f) Lateral prostate (X500).

nobiphenyl (DMAB) which has been most extensively studied by Shirai and Ito and their colleagues in Japan (Shirai et al., 1986; Ito et al., 1988) and by the group at the American Health Foundation in New York (Katayama et al., 1982). To enhance the incidence with BOP, bilateral orchiectomy followed by testosterone administration has been utilized to generate a burst of proliferative activity in the prostate during which BOP is administered. High-dose testosterone is then continued to be administered for life. In the DMAB model, Shirai et al. ( 1986) have utilized multiple cy-

cles of high-dose estrogen followed by return to basal levels which generate a burst of proliferative activity in the prostate during which time DMAB is administered, A major difficulty with the DMAB model to this time has been the lack of induction of invasive lesions. Most, if not all, of the tumors induced in rat ventral prostate by DMAB have been in situ. N-NITROSOBIS(2-0XOPROPYL)AMINE The development of the prostatic cancer model in our laboratory utilizing BOP was

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FIG. 3. A preneoplastic, noninvasive, proliferative glandular lesion of the male rat ventral prostate induced by sc BOP weekly for 20 weeks followed by 8 weeks of observation (X 100).

FIG. 4. Squamous metaplasia arising within a proliferative gland of the rat ventral prostate induced by sc BOP weekly for 20 weeks followed by 16 weeks of observation (X 100).

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LABELING INDEX IN DIFFERENT PARTS OF THE PROSTATE FOLLOWING ORCHIEC~OMY AND/OR TESTOSTERONE OR CYA TREATMENT Labeling index Group

subgroup

Treatment’

Coagulating gland

Dorsal prostate

Ventral prostate

Lateral prostate

A

I 2 3 4 1 2 3 4 I 2 3

CYA, T 2X CYA, T 3X CYA, T 4x CYA, T 5X Orch, T 2X Orch, T 3X Orch, T 4X Orch, T 5X T2x T3x T4X Drch Control

44.2 f 3.9 34.6 _c5.2 22.3 f 1.6 22.6 + 2.3 45.3 + 5.8 24.4 + 5.2 24.8 f 4.7 20.3 f 3.4 19.9 + 0.2 11.8+ 1.1 9.0 It 1.4 0.0 7.2 +- 0.3

33.2 ?I 2.8 9.7 c 0.6 10.0 -c 1.4 9.3 + 3.2 31.9 +- 4.3 11.2 I? 1.8 9.7 k 0.6 10.0 + 4.7 13.0 k 1.8 5.2 + 0.8 6.0 + 2.7 0.0 1.8 +- 1.3

7.2 + 2.3 9.7 t 3.0 11.7218 9.5 IL 3.2 20.6 + 4.4 13.0 + 2.6 13.0 + 2.3 11.5 + 3.5 8.4 t 1.8 5.1 20.6 5.0 + 2.3 0.2*0.1 1.2 t 0.0

6.0 + 3.3 9.0 + 4.0 10.2 f 0.8 7.2 + 2.2 22.8 t 4.9 11.7k2.2 11.8k 1.5 11.0 f 3.9 7.7 + 1.4 5.4 + 0.9 6.5 + 2.6 0.1 + 0.0 1.5 to.6

B

C D E

Group comparison Groups

Coagulating gland

Dorsal prostate

Ventral prostate

Lateral prostate

A vsB AvsC AvsD A vsE B VSC B vsD B vsE C vsD C vsE D vs E

NS” p < 0.000 1 p
NS p
p
p < 0.0002 NS NS NS p
Note. Average of three to four rats/point. For statistical evaluation, the values in groups A I, B 1, C 1, D, and E were compared. Values are given as means + SD. From Pour and Stepan (1987). a CYA, cyproterone, 50 mg/kg body wt injected subcutaneously daily for 2 1 days; T, testosterone, 100 mgkg body wt injected subcutaneously daily for the number of days indicated (2X, 3X, 4X, 5X represents 2,3,4, and 5 injections, respectively); arch, orchiectomy. * NS, not significant.

serendipitous. BOP and its metabolite, N-nitrosobis(2-hydroxypropyl)amine (BHP), are potent pancreatic carcinogens in hamsters (Pour et al., 1975) but do not induce pancreatic tumors in rats (Pour, 1983). However, in the rat, tumors develop in other tissues, including the urinary bladder, urethra, and, most importantly, the prostate. BOP produced an incidence of approximately 33% of

carcinomas of the prostate in rats when administered without any hormonal manipulations (Pour, 198 1, 1983; Pour and Stepan, 1987). The initial change appears to be hyperplasia of the seminalis colliculus, i.e., the opening of the ejaculatory ducts into the prostatic vutricle. In the prostate, most of the proliferative changes occurred later and predominantly in the ventral lobe. The initial

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FIG. 5. Prostatic proliferation in the lateral lobe induced by short-term administration the male rat for 5 days beginning 3 weeks after orchiectomy (X250).

changes in the prostate (Fig. 3) frequently showed nuclear pleomorphism and disorganized cell patterns (Pour, 198 1; Pour and TABLE 2 EFFEC~OFVARIOUSTREATMENTSONDNA SINGLE-STRANDBREAKSINTHERATPROSTATE" Treatment” Orchiectomy Orchiectomy Testosterone Testosterone Orchiectomy BOP Control

+ testosterone + BOP + testosterone + MNU + BOP + MNU + testosterone

Single-strand breaks/ 1O8Da 0.046 0.034 0.032 0.043 0.024 0.024 0.012

+ 0.019’ ? 0.0 12’ Z!I0.008’ + 0.019’ f 0.009’ + 0.016’ +o.007c

” DNA damage was measured as single-strand breaks by alkaline elution analysis of nuclei isolated from the prostate. Prostate tissue was homogenized in a loose-fitting Dounce homogenizer in the buffer of White et al. ( 198 1) and collected by low-speed centrifugation. ’ BOP, N-nitrosobis(2-oxopropyl)amine, 10 mg/kg body wt, sc; MNU, N-methyl-IV-nitrosourea, 50 mg/kg body wt, ip; testosterone, 100 mg/kg body wt, sc. c Significantly greater than the control group, p 4 0.05.

of testosterone to

Stepan, 1987). These lesions occurred as early as 28-30 weeks after injection of the carcinogen. The hyperplasia then progressed, extending to a larger area of the glands and frequently formed a cribriform pattern. At around 32-40 weeks, squamous cell metaplasia began in these areas (Fig. 4) and many of the alveoli became filled with squamous epithelium intermingled with glandular structures. After 40 weeks, invasive squamous cell carcinomas with perineural invasion and regional lymph node metastases appeared. Although the initial development of hyperplasia and squamous cell metaplasia began in the colliculus seminalis, which is an estrogensensitive area (Emmens and Parkes, 1974; Burrows, 1975; Arai et al., 1977), other observations indicated that testosterone played a major role in the proliferative effects in the prostate (Pour, 198 1, 1983; Pour and Stepan, 1987). The ventral prostate is particularly sensitive to effects of testosterone, and previous studies in our laboratory on the effects of

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TABLE 3 TREATMENT GROUPS FORBIOASSAY OFCASTRATION, N-NlTROSOBlS(2-GXOPROPYL)AMINE (BOP), AND TESTOSTERONE IN VARIOUS COMBINATIONS Treatment

Group 1 2 3 4 5 6 7 8 9 10 11 12

BOP ( 10 mg/kg ig) 1X weekly for 20 weeks. BOP (10 mg/kg ig) 1X weekly for 20 weeks, then testosterone pellet for life. BOP (10 m&kg ig) 1X weekly for 20 weeks, testosterone pellet from Week 0 of life. BOP (20 mg/kg sc) 1X daily for 3 days. Testosterone0 1X daily for 5 days, BOP (20 mg/kg sc) 1 X daily for 3 days.’ Testosterone’ 1X daily for 5 days, BOP (20 mg/kg sc) 1X daily for 3 days!, testosterone pellet for life. Castrate, 3 weeks later testosterone” 1X daily for 5 days, BOP (20 mg/kg sc) 1X daily for 3 days. b Castrate, 3 weeks later testosterone’ 1X daily for 5 days, BOP (20 mg/kg sc) 1X daily for 3 daysb, testosterone pellet for life. Testosterone” 1X daily for 5 days, BOP (20 mg/kg ig) 1X daily for 3 day@, testosterone pellet for life. Castrate, 3 weeks later testosterone” 1X daily for 5 days, BOP (20 mg/kg ig) 1X daily for 3 daysb, testosterone pellet for life. Testosterone pellet at 20 weeks for life. Testosterone pellet from Week 0 for life.

Note. From Pour and Stepan (1987). ” Testosterone was given sc at a dose of 100 mg/kg. b BOP treatment started after the second testosterone injection.

castration on BOP carcinogenesis indicated that castration completely inhibited the induction of the prostatic lesions. However, the induction of squamous cell carcinomas, a rare type of tumor in the human prostate, was not of much value to the study of the human disease. EFFECTS

OF TESTOSTERONE

To develop a better model of prostatic carcinogenesis in the rat, we explored the effects of hormonal manipulation on the proliferation of the prostatic epithelium in the rat and the effect of administering carcinogens during the phases of these manipulations (Pour, 198 1; Pour and Stepan, 1987). Castration was performed either by orchiectomy or by daily administration of cyproterone acetate (CYA) at a daily dose of 50 mg/kg. Beginning 3 weeks aBer castration, the rats were administered testosterone at a dose of 100 mg/kg per day for 5 days. The labeling index following a 1-hr pulse of [3H]thymidine was evaluated 2,3,4, and 5 days after the beginning of testosterone administration. In addition, the

effect of testosterone without previous castration and the effect of castration itself were evaluated. The labeling index was determined for each lobe of the prostate, and the results are summarized in Table 1. It is readily apparent that testosterone has a significant effect on stimulating the proliferation of the prostatic epithelium (Fig. 5), and this is increased even further in rats treated with testosterone after castration. Also, there is a significant difference in the rate of cell replication between different lobes of the prostate. Nevertheless, this study indicated that the peak of DNA synthesis occurred 2 days after the beginning of testosterone treatment. To evaluate the correlation between DNA damage in prostatic cells upon the nature of the carcinogen on the one hand and the hormonal manipulations on the other hand, we evaluated DNA strand breaks via administration of testosterone, BOP, or MNU, alone or in different combinations. As can be seen in Table 2, both carcinogens increased the rate of DNA damage. Interestingly, testosterone itself also had an effect.

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TABLE 4 PATTERNS OF INDUCTION OF PR~STATIC PROLIFERATION LESIONS BY N-NITR~~~~~~(~-OX~PR~PYL)AMINE (BOP)” ALONE OR INCOMBINA~ON WITH TESTOSTERONE (T) IN MRC RATS

Group 1 2 3 4 5

6 7 8 9 10

11 12

Treatment” BOP 20x BOP 20x, T T, BOP 20X, T BOP 3x T,BOP3x T, BOP 3x, T Orch, T, BOP 3X Orch, T, BOP 3X, T T, BOP 3x, T Orch, T, BOP 3X, T T at 20 weeks T for life

Effective number ofrats 24 23 28 13 27 24 29 26 28 28 16 27

Hyperplasia no. (%)b

Total carcinoma no. (%)’ 0 4(17) 11 (39) 1 (8) 1 (4) 16 (67) 1 (3) 19 (73) 17 (61)

l(4) 11 (48) 20 (7 I) 0 3(11) 2 1 (86) 0 21 (81) 23 (82) 23 (82)

18 (64)

2(13) 24 (89)

2u3) 4(15)

Adenocarcinoma no. (%) 0 0 3(11) 0

0 5 (21) 0 4(15)

6G’l) l(4) l(6) 0

Note. From Pour and Stepan (1987). a BOP was given sc except in groups 1, 2, 3, 9, and 10 in which rats received BOP ig. Details of the treatment regimen are given in Table 3. ’ Number (%) of rats with that particular lesion. ’ Includes all types of carcinomas. These values were used for statistical comparison of tumor incidence.

On the basis of these preliminary studies, we performed a long-term carcinogenesis bioassay utilizing surgical orchiectomy as the method for castration since CYA causes adrenal atrophy in addition to the testicular atrophy (Pour and Stepan, 1987). We also utilized BOP as the carcinogen rather than MNU. Moreover, since the peak of DNA synthesis occurred between 2 and 5 days posttestosterone treatment, BOP was administered daily for 3 days during this time. After BOP administration, testosterone treatment was continued for life by means of replaceable subcutaneous pellets. Appropriate control groups were also included in the study. In addition, a comparison of intragastric to subcutaneous injection of the BOP was made. The various treatments are listed in Table 3 and the results of the experiment are listed in Table 4. As in our previous study (Pour, 1983), BOP by itself induced a significant incidence of prostatic carcinomas, but they were again predominantly of squamous cell type and de-

veloped in the ventral lobe. The noninvasive lesions were either glandular or a mixture of glandular and squamous differentiation. In contrast, the treatment of castrated rats with BOP and testosterone produced a high incidence of adenocarcinomas of the prostate (Fig. 6). In contrast to the lesions induced with BOP only, most of these tumors arose in the dorsal lobe and coagulating glands. Interestingly, BOP plus testosterone without prior castration also resulted in a significant incidence of adenocarcinomas. Somewhat surprisingly, testosterone alone induced a low incidence of prostatic malignancies. The latter finding, confirming the studies by Pollard (Pollard et al., 1982; Pollard and Luckert, 1986) and Noble (1982) correlates well with our above-mentioned alkaline elution study. Histologically, the prostate of rats treated with testosterone chronically (Fig. 7) showed hypercellular glands with numerous cytoplasmic vacuoles (Pour, 198 1; Pour and Stepan, 1987). There was also glandular distension in the ventral and dorsal lobes, with

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FIG. 6. Adenocarcinoma of the male rat dorsal prostate induced by BOP administered during the active proliferative phase following testosterone administration. BOP administration was then followed by longterm testosterone to continue the proliferative stimulus. From a rat in Group 6 after 64 weeks of the experiment (X 130).

focal sclerosis in the interstitium of the lateral lobes. In sclerotic areas, there was budding of small groups of dysplastic cells and distortion of the glands, and several in situ changes were found in these areas. Squamous cell carcinomas occurred in the ventral prostate, whereas the glandular adenocarcinomas occurred predominantly in the dorsal prostate and the coagulating glands. These data clearly show that BOP is a prostatic carcinogen in the rat and that adenocarcinemas can be induced if testosterone is used as an agent to continue to alter hormonal milieu and to provide a stimulus for additional proliferation (Pour, 198 1; Pour and Stepan, 1987). Continuous testosterone

treatment resulted in a greater number of adenocarcinomas than the short period of testosterone treatment. Noteworthy is the different location of the lesions when highdose testosterone is given with the BOP compared to BOP alone. Immunohistochemical examination of the tumor tissue showed that there were large amounts of both testosterone and estrogen present in tumor cells (Pour, 198 1; Pour and Stepan, 1987). The specific hormonal relationships to the development of these tumors remain unknown, but clearly the development of adenocarcinomas is dependent on the hormonal milieu. Whether testosterone acts as a proliferation stimulating agent to

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FIG. 7. Long-term administration of testosterone (64 weeks) produces proliferation of the rat ventral prostate with the appearance of vacuolization of the epithelial cells (X 100).

compliment the effects of BOP or whether a change in the ratio of androgen to estrogen, and possibly to prolactin, remains unknown and is an area warranting additional studies. ACKNOWLEDGMENTS The authors gratefully acknowledge the comments and technical assistance of Kathy Stepan, and the assistance of Dr. Tsuneo Masui, Jan Leemkuil, Deboraha Coleman, and Ginni Philbrick with the preparation of this manuscript. This research was supported in part by USPHS Grants CA34473 and CA36727 from the National Cancer Institute.

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