Induction of invasive carcinomas in the accessory sex organs other than the ventral prostate of rats given 3,2'-dimethyl-4-aminobiphenyl and testosterone propionate

The promotion effects of testosterone propionate (TP) on prostate carcinogenesis were investigated in F344 rats given the prostatic carcinogen, 3,2'-dimethyl-4-aminobiphenyl (DMAB). One group of animals received s.c. DMAB injections at a dose of 50 mg/kg body weight at 2-week intervals for a total of 10 injections along with s.c. implantations of TP-containing Silastic tubes. A second experimental group of rats was given DMAB at the same dose and intervals but each injection of DMAB was combined with 3 prior consecutive daily 100-mg/kg body weight s.c. injections of TP. After cessation of carcinogen administration, animals in these two groups received TP implants from week 21 to the end of the experiment. All surviving animals were killed at week 56 and accessory sex gland tumor incidences were compared to those in DMAB alone and other appropriate control groups. The groups given TP plus DMAB and subsequent long term administration of TP developed lesions of the dorsolateral prostate, seminal vesicles, and coagulating glands which were all invasive adenocarcinomas. Incidences were 84.2% (16 of 19 rats) and 66.7% (12 of 18 rats), respectively. Macroscopic large tumors were induced in 13 animals among which 8 demonstrated metastasis to the abdominal cavity, liver, or lung. None of the control groups except for the group given TP injections plus DMAB had equivalent tumors. Development of carcinomas of the ventral prostate, which were all of in situ type, were not increased by subsequent treatment with TP. These data thus clearly showed that TP can exert strong enhancing effects on tumor development in the dorsolateral prostate, seminal vesicles, and coagulating glands but not in the ventral prostate.     

Development of Androgen-independent Carcinomas from Androgen-dependent Preneoplastic Lesions in the Male Accessory Sex Organs of Rats Treated with 3,2'-Dimethyl-4-aminobiphenyl and Testosterone Propionate

Two kinds of cancer can be induced in rat male accessory sex organs, one a non-invasive carcinoma arising in the ventral lobe and the other an invasive lesion which develops in the dorsolateral and anterior lobe as well as the seminal vesicles. In the present study, one group of male rats were given biweekly s.c. injections of 3,2'-dimethyl-4-aminobiphenyl (DMAB) for 20 weeks for induction of non-invasive carcinomas and the other group received DMAB with 40-week testosterone propionate for induction of invasive carcinomas. Half of the animals in each group were then subjected to bilateral orchiectomy at week 41 to remove testicular androgen, in order to examine the androgen dependence of both types of carcinomas as well as precancerous lesions. Animals were killed at weeks 41, 46 and 60. All parts of the prostate complex showed involution and significant weight reduction after castration, with a complete disappearance of atypical hyperplasias and carcinomas of the ventral prostate. However, in spite of suppression of development of atypical hyperplasias in the anterior prostate and seminal vesicles, the incidence of invasive carcinomas was not changed. Normal epithelial cells and atypical hyperplasias of all parts of the prostate and seminal vesicles and carcinomas of the ventral prostate were immunohistochemically positive for nuclear androgen receptor, while invasive carcinomas that developed in either castrated or non-castrated animals were negative. These findings suggest that in the ventral prostate, both precancerous and cancerous lesions are androgen-dependent, but in the anterior and seminal vesicles, cancerous lesions (invasive carcinomas) are androgen-independent while precancerous lesions are hormone-dependent.     

Development of androgen-independent carcinomas from androgen-dependent preneoplastic lesions in the male accessory sex organs of rats treated with 3,2'-dimethyl-4-aminobiphenyl and testosterone propionate.

Two kinds of cancer can be induced in rat male accessory sex organs, one a non-invasive carcinoma arising in the ventral lobe and the other an invasive lesion which develops in the dorsolateral and anterior lobe as well as the seminal vesicles. In the present study, one group of male rats were given biweekly s.c. injections of 3,2'-dimethyl-4-aminobiphenyl (DMAB) for 20 weeks for induction of non-invasive carcinomas and the other group received DMAB with 40-week testosterone propionate for induction of invasive carcinomas. Half of the animals in each group were then subjected to bilateral orchiectomy at week 41 to remove testicular androgen, in order to examine the androgen dependence of both types of carcinomas as well as precancerous lesions. Animals were killed at weeks 41, 46 and 60. All parts of the prostate complex showed involution and significant weight reduction after castration, with a complete disappearance of atypical hyperplasias and carcinomas of the ventral prostate. However, in spite of suppression of development of atypical hyperplasias in the anterior prostate and seminal vesicles, the incidence of invasive carcinomas was not changed. Normal epithelial cells and atypical hyperplasias of all parts of the prostate and seminal vesicles and carcinomas of the ventral prostate were immunohistochemically positive for nuclear androgen receptor, while invasive carcinomas that developed in either castrated or non-castrated animals were negative. These findings suggest that in the ventral prostate, both precancerous and cancerous lesions are androgen-dependent, but in the anterior and seminal vesicles, cancerous lesions (invasive carcinomas) are androgen-independent while precancerous lesions are hormone-dependent.     

Effects of a soybean isoflavone mixture on carcinogenesis in prostate and seminal vesicles of F344 rats.

Several epidemiological studies have suggested an inverse association between the risk of prostate cancer and intake of soybeans and their products. In vitro data pointing to possible anti-carcinogenic properties of the soybean isoflavone, genistein, led us to investigate the chemopreventive potential of soybean isoflavones in a rat carcinogenesis model induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB) and testosterone propionate (TP). Animals received DMAB s.c. injections at 2-week intervals for the first 20 weeks and implanted silicon tubes containing 40 mg of TP, replaced at 6-week intervals throughout the experiment. The soybean isoflavone mixture consisting of 74% genistein and 21% daidzein was mixed in basal diet (AIN-76A) at concentrations of 100 and 400 ppm and fed to F344 male rats throughout the experiment. Rats treated with carcinogens and administered isoflavone mixture at 100 and 400 ppm developed adenocarcinomas at incidences of 35% and 29%, respectively, in the prostate and seminal vesicles, whereas the figure was 60% for those maintained on control diet. Feeding of the isoflavone mixture at 100 and 400 ppm significantly inhibited the number of argyrophilic nucleolar organizer regions (AgNORs) in adenocarcinomas of the accessory sex glands as compared to those of rats fed control diet. No influence on the development of neoplastic lesions originating in other organs was noted. The results of this study provide evidence that soybean isoflavones may have potential as chemopreventive agents against carcinogenesis in the prostate.     

Effects of a Soybean Isoflavone Mixture on Carcinogenesis in Prostate and Seminal Vesicles of F344 Rats

Several epidemiological studies have suggested an inverse association between the risk of prostate cancer and intake of soybeans and their products. In vitro data pointing to possible anti-carcinogenic properties of the soybean isoflavone, genistein, led us to investigate the chemopreventive potential of soybean isoflavones in a rat carcinogenesis model induced by 3,2'-dimethyl-4-aminobi-phenyl (DMAB) and testosterone propionate (TP). Animals received DMAB s.c. injections at 2-week intervals for the first 20 weeks and implanted silicon tubes containing 40 mg of TP, replaced at 6-week intervals throughout the experiment. The soybean isoflavone mixture consisting of 74% genistein and 21% daidzein was mixed in basal diet (AIN-76A) at concentrations of 100 and 400 ppm and fed to F344 male rats throughout the experiment. Rats treated with carcinogens and administered isoflavone mixture at 100 and 400 ppm developed adenocarcinomas at incidences of 35% and 29%, respectively, in the prostate and seminal vesicles, whereas the figure was 60% for those maintained on control diet. Feeding of the isoflavone mixture at 100 and 400 ppm significantly inhibited the number of argyrophilic nucleolar organizer regions (AgNORs) in adenocarcinomas of the accessory sex glands as compared to those of rats fed control diet. No influence on the development of neoplastic lesions originating in other organs was noted. The results of this study provide evidence that soybean isoflavones may have potential as chemopreventive agents against carcinogenesis in the prostate.     

Preparation and characterization of antibodies against 3,2'-dimethyl-4-aminobiphenyl-modified DNA

Polyclonal antibodies for 3,2'-dimethyl-4-aminobiphenyl (DMAB)–DNAadducts were obtained from the sera of rabbits immunized withN-OH-DMAB-modified DNA. Using the enzyme linked immunosorbentassay (ELISA), these antibodies were shown to recognize DNAmodified by N-OH-DMAB and N-hydroxy-4-aminobiphenyl, but notunmodified DNA, 2-acetylaminofluorene- or 4-nitroquinoline-1-oxide-modifiedDNA, free aminobiphenyl or DMAB derivatives. Using competitiveELISA with DMAB-modified denatured DNA, a 50% inhibition ofantibody antigen binding was caused by 7 fmol of DMAB adductsapplied to each assay well as an inhibitor. Native DNA bearingadducts was associated with 50% inhibition in the range of 22–90fmol/assay well, depending on the levels of modification. Theadducts recognized by the antibody were shown to be stable againsttreatment of heat or alkali denaturation. Optimal conditionsfor the detection of adducts in DNA from the rat organs exposedto DMAB were established by means of competitive ELISA, usingalkaline-denatured DNA as an inhibitor. Thus, as little as 5fmol adducts in 1 µg of DNA could be detected. Indirectimmunofluorescence staining indicated that anti-DMAB-DNA antibodiesbound specifically to nuclear components of rat fibroblast 3Y1cells treated with N-OH-DMAB. The intensity of the fluorescencewas proportional to the dose of carcinogen administered. Theantibodies should be helpful for use in studies on the formationof adducts and their removal in cells and tissues after DMABadministration.     

Rat prostate as one of the target organs for 3,2'-dimethyl-4-aminobiphenyl-induced carcinogenesis: effects of dietary ethinyl estradiol and methyltestosterone

Twenty consecutive weekly sc injections of 50 mg/kg body weight of 3,2'-dimethyl-4-aminobiphenyl (DMAB), a multipotential carcinogen, were given to male F344 rats and subsequently groups of animals were treated with dietary ethinyl estradiol (EE, 2.5 ppm) or methyltestosterone (MT, 300 ppm) for up to 40 weeks. Prostate carcinomas were found in 4 out of 32 rats given DMAB followed by MT and in 2 out of 29 rats given DMAB alone. Atypical hyperplasia of the prostate epithelium in these two groups was found in 22% and 14%, respectively. Neither carcinoma nor atypical hyperplasia was seen in the prostate of animals given DMAB followed by EE. In other organs, tumors were frequently found in the ear duct, skin, and large intestine and less frequently in the lung, preputial glands, small intestine and liver. EE significantly suppressed tumor incidence of the ear duct and sebaceous glands while increasing the incidence of liver tumor and mesothelioma. The present data indicates DMAB to be a useful carcinogen for the induction of prostate carcinomas in rats.     

p-Nonylphenol pretreatment during the late neonatal period has no effect on 3,2'-dimethyl-4-aminobiphenyl-induced prostate carcinogenesis in male F344 rats

The modifying effects of late neonatal administration of p-nonylphenol (NP), a suspected xenoestrogen, on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced prostatic carcinogenesis were investigated in male F344 rats. Three-week-old rats received 25, 250 or 2000 ppm of NP in the diet for 3 weeks prior to DMAB treatment and were sacrificed at 67 weeks of age for histopathological assessment of lesions and Ki-67 immunohistochemical analysis of cell cycle kinetics. Dietary administration of NP during the sexually immature period had no effects on maturation of male sex organs. Incidence, multiplicity and areas of neoplastic lesions in the prostate and seminal vesicles, and Ki-67 labeling indices in normal-looking epithelium were not significantly different among the experimental groups. These results indicate that late neonatal treatment with NP has no modulating effects on DMAB-induced rat prostatic carcinogenesis.     

Lack of modification by naturally occurring antioxidants of 3,2'-dimethyl-4-aminobiphenyl-initiated rat prostate carcinogenesis

The modifying effects of 6 naturally occurring antioxidants on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-initiated rat prostate carcinogenesis were investigated in male F344 rats. Animals were pretreated with DMAB in a 20-week initiation protocol and then administered basal diet containing 0.8% catechol, 0.8% resorcinol, 0.8% hydroquinone, 2 ppm selenium, 2% gamma-orysanol or 1% alpha-tocopherol for 40 weeks. The experiment was terminated at week 60 for histopathological assessment of lesion development. Atypical hyperplasias and carcinomas of the prostate were observed in the ventral lobe in all groups treated with DMAB. However, the incidences of these lesions were not significantly different between carcinogen control and antioxidant-treated groups. There were also no significant increases or decreases in the incidences of tumors in any other organs.     

Dose Dependence of N-Hydroxy-3,2'-dimethyM-aminobiphenyl-induced Rat Prostate Carcinogenesis

Groups of F344 rats were administered biweekly intraperitoneal injections of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMAB) at a dose of 5, 10 or 20 mg/kg body weight or DMAB, the parent compound, at a dose of 25 mg/kg body weight, for a total of 10 times. Prostate carcinomas in the ventral lobe developed in a N-OH-DMAB dose-dependent manner (0, 17.6 and 66.7%, respectively) with limited tumor yields in other organs. Although intraperitoneal administration of DMAB was similarly found to induce prostate tumors, it also caused severe chemical peritonitis, which resulted in a high mortality. The present data confirmed that intraperitoneal administration of N-OH-DMAB provides a relatively specific induction method for models of prostate carcinogenesis.     

Dose dependence of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl-induced rat prostate carcinogenesis.

Groups of F344 rats were administered biweekly intraperitoneal injections of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMAB) at a dose of 5, 10 or 20 mg/kg body weight or DMAB, the parent compound, at a dose of 25 mg/kg body weight, for a total of 10 times. Prostate carcinomas in the ventral lobe developed in a N-OH-DMAB dose-dependent manner (0, 17.6 and 66.7%, respectively) with limited tumor yields in other organs. Although intraperitoneal administration of DMAB was similarly found to induce prostate tumors, it also caused severe chemical peritonitis, which resulted in a high mortality. The present data confirmed that intraperitoneal administration of N-OH-DMAB provides a relatively specific induction method for models of prostate carcinogenesis.     

Selective induction of rat urinary bladder tumors by simultaneous administration of 3,2'-dimethyl-4-aminobiphenyl (DMAB) and butylated hydroxyanisole or butylated hydroxytoluene is associated with increased DMAB-DNA adduct formation

Modification of 3,2'-dimethyl-4-aminobiphenyl (DMAB) multi-organ carcinogenesis by simultaneous treatment with butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) was studied using young and old male F344 rats. Animals, 4 or 54 weeks old, were given DMAB (s.c. injection of 50 mg/kg body wt once a week for 10 weeks) along with BHA (2.0% in diet for 11 weeks) or BHT (1.0% in diet for 11 weeks). The experiments were terminated 55 weeks after the commencement. Combined administration of BHA or BHT with the carcinogen resulted in development of urinary bladder tumors in greater than 90% of both young and old rats thus treated, whereas no tumors were induced in animals given DMAB alone. In contrast, the appearance of preneoplastic lesions in the liver and pancreas was reduced by BHA or BHT treatment. Tumor development (less than 30% incidence) was also evident in the small and large intestines, prostate, preputial glands, skin/subcutis and ear duct, with no modification by BHA or BHT. No ageing effects were evident. The formations of DMAB-DNA adducts, evaluated by the enzyme-linked immunosorbent assay and immunohistochemical staining, correlated well with tumorigenesis in the urinary bladder, liver and pancreas. The selective enhancement of urinary bladder tumor induction by BHA and BHT appeared to be due to both increased DMAB-DNA adduct formation caused by metabolic alteration of DMAB in the liver and increased DNA synthesis in the urothelial cells.     

Enhancing Effect of Cadmium on Rat Ventral Prostate Carcinogenesis Induced by 3,2'-Dimethyl-4-aminobiphenyl

The effects of cadmium given at different stages during 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced rat prostate carcinogenesis were investigated using male F344 rats. Animals were given 10 subcutaneous injections of 50 mg/kg body weight of DMAB or the corn oil vehicle at two-week intervals. In addition, cadmium was administered at doses of 0, 10, or 30 /μmol/kg body weight as single intramuscular injection on the 1st day of the experiment or one day after the last injection of DMAB at week 20. Two further groups were subjected to administration of cadmium at 10 μmol/kg at week 20 and then 5 μmol/kg at week 40, or 10 μmol/kg at week 20 and then 5 μmol/kg at weeks 30, 40 and 50. At the termination, 60 weeks after the beginning of the experiment, the incidences and multiplicity of ventral prostate carcinomas in the groups given cadmium plus DMAB demonstrated a consistent tendency for increase over control values (groups receiving DMAB or cadmium alone). The numbers of carcinomas per rat and per unit area of prostate section were significantly elevated in the two groups given low doses of cadmium after cessation of DMAB administration. Cadmium alone also induced a few prostate carcinomas. The influence on development of prostate tumors did not appear to be a result of the induced severe testicular atrophy because serum testosterone levels were not affected. The results indicate that cadmium and DMAB can act synergistically to cause rat prostate carcinogenesis.     

Organ dependent enhancement of rat 3,2'-dimethyl-4-aminobiphenyl (DMAB) carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): positive effects on the intestine but not the prostate

In order to evaluate tumor enhancing effects of the heterocyclic carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), doses of 100 and 300 p.p.m. PhIP were given for 40 weeks to male F344 rats, which initially received 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB shows a similar carcinogenic organ spectrum to that of PhIP, including the prostate and colon. PhIP alone at a dose of 300 p.p.m. resulted in the development of prostate and intestine cancers. Furthermore, among the DMAB-treated group, enhancement of intestinal carcinogenesis by 300 p.p.m. PhIP was observed. However, no prostate enhancement was demonstrated in the DMAB + PhIP group. Since PhIP-DNA adduct formation in the prostate epithelial cells in a satellite experiment was not affected by pre-treatment with DMAB, it is speculated that the contradictory findings between the intestine and prostate may be due to the specific biological effects of PhIP. Taking into account previous data, that PhIP clearly enhanced rat 1,2-dimethylhydrazine-initiated colon tumorigenesis, the potential of PhIP to enhance colon carcinogenesis may be initiator dependent.     

Suppressive Effects of Dietary Genistin and Daidzin on Rat Prostate Carcinogenesis

High intake of phytoestrogens through soybeans and their products is thought to be associated with low incidences of prostate and/or breast cancer in Asian countries. Possible chemopreventive effects of genistin or daidzin on rat prostate carcinogenesis were therefore investigated. Male F344 rats were given 10 biweekly subcutaneous injections of 3,2'-dimethyl-4-aminobiphenyl (DMAB) and then either genistin or daidzin in the diet at a concentration of 0.1% for 40 weeks. Other groups of rats given DMAB were treated with genistin or daidzin together with a high dose of testosterone propionate (TP). Both genistin and daidzin reduced the numbers of ventral prostate carcinomas (P<0.05), with a tendency for decrease in incidence. Invasive carcinomas which developed in the anterior prostate and seminal vesicles with TP were, however, not influenced by the two isoflavones. Thus, the present data suggest that genistin and daidzin possess anti-cancer effects at relatively early stages of prostate cancer development, providing experimental support for epide-miological findings.     

Suppressive effects of dietary genistin and daidzin on rat prostate carcinogenesis.

High intake of phytoestrogens through soybeans and their products is thought to be associated with low incidences of prostate and / or breast cancer in Asian countries. Possible chemopreventive effects of genistin or daidzin on rat prostate carcinogenesis were therefore investigated. Male F344 rats were given 10 biweekly subcutaneous injections of 3,2'-dimethyl-4-aminobiphenyl (DMAB) and then either genistin or daidzin in the diet at a concentration of 0.1% for 40 weeks. Other groups of rats given DMAB were treated with genistin or daidzin together with a high dose of testosterone propionate (TP). Both genistin and daidzin reduced the numbers of ventral prostate carcinomas (P < 0.05), with a tendency for decrease in incidence. Invasive carcinomas which developed in the anterior prostate and seminal vesicles with TP were, however, not influenced by the two isoflavones. Thus, the present data suggest that genistin and daidzin possess anti-cancer effects at relatively early stages of prostate cancer development, providing experimental support for epidemiological findings.     

Beef Tallow, but Not Perilla or Corn Oil, Promotion of Rat Prostate and Intestinal Carcinogenesis by 3,2-Dimethyl-4-aminobiphenyl

The modifying effects of three kinds of fat (corn oil, beef tallow or perilla oil, each at 20% in the diet) on F344 rat prostate carcinogenesis induced by 3,2-dimethyl-4-aminobiphenyl (DMAB) were investigated. Non-invasive carcinomas of the ventral prostate were induced by DMAB alone and invasive carcinomas of the other prostate lobes and seminal vesicles by DMAB and testosterone propionate (TP). Eight groups of F344 rats were initiated with 50 mg/kg body weight of DMAB at 2-week intervals for the first 20 weeks, four also receiving TP, extended until week 60. The animals received basal chow powder diet or one of three high fat diets throughout the experiment (60 weeks). One further group served as a non-carcinogen-treated control maintained on basal chow powder diet. Beef tallow significantly increased the development of ventral prostate carcinomas with DMAB alone (from 15 to 45%, P<0.05), while perilla oil reduced the incidence of prostatic intraepithelial neoplasia (PIN) in the ventral lobe of rats given DMAB + TP (from 70 to 10%, P<0.01), but not in those given DMAB alone. No other effects of high fats were observed regarding PIN or invasive cancers of the dorsolateral and anterior prostate or seminal vesicles. A satellite experiment demonstrated that all high fat diets for 4 weeks increased the 5-bromo-2-deoxyuridine (BrdU) labeling index of prostate epithelial cells, suggesting that a high fat intake, irrespective of the fatty acid composition, may accelerate cell kinetics in the prostate. Of the three high fat diets, beef tallow was also found to increase intestinal carcinogenesis. Thus, the present data revealed carcinogenesis in the prostate and intestine to be promoted by beef tallow.     

Different carcinogenic responses in a variety of organs, including the prostate, of five different rat strains given 3,2'-dimethyl-4-aminobiphenyl

Tumorigemc response in the prostate of F344, ACI, Lewis, CDand Wistar rat strains to 3,2'-dimethyl-4-amtnobiphenyl (DMAB)was examined in relation to development of other types of tumors.Rats of each strain aged 6 weeks were divided into two groupsreceiving DMAB S.C. at a dose of 50 mg/kg body wt once everyother week for 10 times, with or without 1 week dietary ethynylestradiol (EE) pretreatment. The experiment was terminated atweek 60, carcinomas of the ventral prostate, all of microscopicsize, being respectively found in 50, 17, 21, 15 and 0% of F344,ACI, Lewis, CD and Wistar strain animals treated with EE plusDMAB. The tumor yield correlated well with DMAB–DNA adductfor mation. One invasive adenocarcinoma also developed in theperlurethral part (occupying both of lateral and dorsal areas)of the prostate. The final survival rates were 46, 24, 65, 4and 0% in F344, ACI, Lewis, CD and Wistar rats respec tively.DMAB administration without EE pretreatment resulted in similarincidences of prostate tumors and mor talities. Tumors arosein >14 dIfferent sites with strain dependency, lesions predominatingin the skin/subcutis of ACI and F344, preputial gland of F344,urinary bladder of ACI, and mammary glands of CD rats respectively.Consideration of mortality and the relative incidence of prostatecancer and other types of tumors indicates the F344 rat strainto be the most appropriate for investigation of DMAB prostatecarcinogenesis.