13-cis-Retinoic acid: inhibition of bladder carcinogenesis induced in rats by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Transitional cell carcinoma was induced in the bladders of male Fischer rats by 12 oral doses of the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine. Feeding of 13-cis-retinoic acid after completion of carcinogen treatment diminished the number and severity of cancers and other proliferative lesions of the bladder.

Cancer chemopreventive activity of resveratrol, a natural product derived from grapes.

Resveratrol, a phytoalexin found in grapes and other food products, was purified and shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis. Resveratrol was found to act as an antioxidant and antimutagen and to induce phase II drug-metabolizing enzymes (anti-initiation activity); it mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity); and it induced human promyelocytic leukemia cell differentiation (antiprogression activity). In addition, it inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin cancer model. These data suggest that resveratrol, a common constituent of the human diet, merits investigation as a potential cancer chemopreventive agent in humans.

Histogenesis and dose dependence of N-methyl-N-nitrosourea-induced carcinoma in a localized area of the hamster trachea.

The effect of the number of weekly intratracheal Instillations of N-methyl-N-nitrosourea (MNU) on induction of tracheal tumors was studied in male noninbred Syrian golden hamsters. The histogenesis of metaplastic and neoplastic lesions was also characterized. Treatment of hamsters once weekly for either 6, 8, 10, 12, or 14 weeks with a 0.5% solution of MNU resulted in the induction of a 0, 6, 11, 26, and 42% incidence of carcinoma, respectively, at 6 months after the first MNU treatment. Of the carcinomas induced, 87% were combined epidermoid and adenocarcinomas, whereas 13% were epidermoid carcinomas. In animals killed 1 week following either 1, 3, 6, 8, 10, 12, or 14 treatments, a continuum of metaplastic and neoplastic changes was observed that correlated well with the cancer incidence exhibited at the termination of the study. Mucous cells were found to be of prime importance in the development of the metaplastic and neoplastic tracheal lesions observed.

Carcinogenicity of N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea when applied to a localized area of the hamster trachea.

The carcinogenicity of N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) was studied with the use of a hamster tracheal tumor model system. Hamsters received 15 or 10 once-weekly treatments of either a 0.5 or 0.25% solution of MNU and were killed 9 months after the first intratracheal instillation. Other hamsters received 15 once-weekly treatments of a 0.5, 0.25, or 0.125% solution of ENU and were killed at 6 months. Treatment with MNU resulted in a dose-dependent induction of tracheal carcinomas; 94% of the tumors induced were combined epidermoid and adenocarcinomas. Treatment of hamsters with a 0.5, 0.25, 0.125% solution of ENU induced an 83, 64, and 71% incidence of benign tracheal tumors, respectively (papillomas and polyps). No tracheal carcinomas were induced by ENU. The carcinogenicity of MNU and the reproducibility of tumor induction with the use of the localized tracheal washing, tumor model system were confirmed. Furthermore, the sensitivity of the localized tracheal washing technique for the detection of the tumorigenicity of compounds toward respiratory epithelium was demonstrated.

Effects of 12-O-tetradecanoylphorbol-13-acetate on carcinogen-induced mouse mammary lesions in organ culture.

Mammary glands of young female BALB/c mice develop hyperplastic nodule-like alveolar lesions (NLAL) in response to a 24-h exposure to 7,12-dimethylbenz(a)anthracene (DMBA; 2 micrograms/ml) on Day 3 during a 24-day organ culture. Experiments were conducted to determine if 12-O-tetradecanoylphorbol-13-acetate (TPA), a known tumor promoter, can influence the development of DMBA-induced NLAL in mammary gland organ culture. Mammary glands were incubated with TPA (25 ng/ml) for various periods of time in the presence of appropriate hormone combinations. Results indicated that the presence of TPA in the medium between Days 9 to 14 of the culture period enhanced both the incidence and multiplicity of DMBA-induced NLAL; however, it was ineffective when included in the medium between Days 4 to 9 or 19 to 24 of the organ culture. Toxicity of TPA was evident when it was present during the entire culture period subsequent to DMBA treatment. Computer-assisted image analysis of NLAL in the glands determined the area covered by these lesions within the gland. It was observed that 7.8% of the area was covered by NLAL in DMBA plus TPA-treated glands as compared to 2.5% by DMBA treatment alone. These results provide a model for initiation-promotion studies of mammary carcinogenesis in vitro, as well as a modified approach for quantitative analysis of structural alterations.

Inhibition of DNA synthesis by retinyl acetate during chemically induced mammary carcinogenesis.

The effect of retinoids on DNA synthesis was studied in the female Sprague-Dawley rat. Animals were treated with either solvent, 7,12-dimethylbenz(a)anthracene, or 1-methyl-1-nitro-sourea at 50 days of age and were placed on either placebo or retinyl acetate diet at 57 days of age. [3H]Thymidine incorporation into purified DNA isolated from mammary parenchymal cells was determined. Retinyl acetate effectively inhibited mammary cell DNA synthesis in both 1-methyl-1-nitrosourea- and 7,12-dimethylbenz(a)anthracene-treated animals; however, DNA synthesis in solvent-treated animals was unaffected.

Influence of delayed administration of retinyl acetate on mammary carcinogenesis.

Administration of a dietary retinoid supplement beginning 1 week after carcinogen administration is highly effective in the inhibition of rat mammary carcinogenesis. A study was designed at two carcinogen dose levels to determine to what extent retinoid feeding can be delayed and retain its chemoprotective effect. In the high-dose experiment, groups of 30 virgin female Sprague-Dawley rats received a single i.v. dose of 50 mg N-methyl-N-nitrosourea (MNU) per kg body weight and were fed a dietary supplement of 328 mg retinyl acetate per kg diet beginning at 1, 4, or 8 weeks after MNU administration. In the low-dose experiment, groups of 50 rats received 25 mg MNU per kg, and the retinoid was begun at 1, 4, 8, 12, 16, or 20 weeks post-MNU. Controls at both dose levels received a placebo diet beginning 1 week after carcinogen treatment. At the high MNU dose, retinyl acetate was most effective in inhibition of carcinogenesis when treatment was begun 1 week after MNU administration. Delaying retinyl acetate feeding until 4 weeks post-MNU resulted in slightly reduced chemoprotective efficacy, while an 8-week delay caused a complement loss of anticancer activity. At the low MNU dose, delaying retinyl acetate administration for up to 12 weeks after MNU administration caused no loss of chemopreventive efficacy. A 16-week delay resulted in decreased anticancer activity, while retinoid treatment begun 20 weeks post-MNU had no effect on cancer induction. Retinoid administration can be delayed beyond 1 week and retain its activity against rat mammary carcinogenesis; the length of delay allowable without loss of activity is a function of tumor latency.

Inhibition of carcinogenesis by retinoids.

Retinoids are effective inhibitors of chemical carcinogenesis in the mammary gland and urinary bladder of experimental animals. Modification of the basic retinoid structure has produced retinoids with increased target organ specificity, resulting in increased anticancer activity with reduced systemic toxicity. Combining retinoid treatment with hormonal manipulation results in a synergistic inhibition of mammary carcinogenesis; this combination approach also inhibits development of additional mammary cancers following surgical removal of the first mammary cancer. Retinoids are most effective when administered shortly after the carcinogenic insult. However, even when retinoid treatment is delayed, the compounds are still effective cancer chemopreventive agents for the mammary gland and urinary bladder. The length of time that retinoid exposure can be delayed and retain an anticancer effect is directly related to tumor latency, with a longer delay permissible against tumors with long latent periods.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis by concomitant or postcarcinogen antioxidant exposure.

When administered prior to or at the time of carcinogen exposure, the phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are effective inhibitors of carcinogenesis in several target organs. However, chronic, postcarcinogen administration of BHT apparently enhances tumorigenesis in certain animal models for liver and lung cancer. The present study was performed to determine the effects of BHA and BHT on mammary carcinogenesis when antioxidant exposure is limited to defined periods encompassing or following carcinogen availability. At 50 days of age (Time O), virgin female Sprague-Dawley rats (25/group) were given a single intragastric dose of 8 mg of 7,12-dimethylbenz(a) athracene . Basal diet (Wayne Lab Meal) was supplemented with 5000 or 2500 mg of BHA or BHT/kg by the following protocol: 2 weeks before until 1 week after carcinogen administration; 1 week after carcinogen administration until the end of the study; or none. The experiment was terminated 210 days after 7,12-dimethylbenz(a)anthracene administration, and all mammary tumors were confirmed histologically. When administered by the 2 weeks before to 1 week after schedule, both BHA and BHT were effective inhibitors of mammary carcinogenesis. However, the compounds also were active in chemoprevention when administered by the 1 week after to end protocol. These data indicate that the anticarcinogenic activity of antioxidants is not limited to influences on carcinogen metabolism, since both BHA and BHT inhibited mammary tumor induction when their administration was begun following clearance of the carcinogen from the mammary gland. The anticarcinogenic activity of postcarcinogen administration of BHA and BHT in the mammary gland is in contrast to the apparent tumor-enhancing activity of BHT in the liver and lung.

Distribution of retinoic acid-binding proteins in normal and neoplastic mammary tissues.

Cellular retinoic acid-binding proteins were detected in chemically induced mammary tumors using sucrose density gradient analysis. Unlabeled retinoic acid did not displace nonspecific binding in the 5S region but was, however, a competitive inhibitor for the specifically binding 2S component. Mammary gland cytosol fractions from both 1-methyl-1-nitrosourea-treated and untreated as well as from lactating rats contained low levels of retinoic acid-binding proteins. 1-Methyl-1-nitrosourea treatment did not result in the increased number of binding sites. Thus, the increase in the levels of binding proteins in tumors most probably occurred during tumor development and probably was not a result of the carcinogen per se. Retinoids which have been shown to be effective in the chemoprevention of mammary carcinogenesis only partially competed for the binding sites, indicating that they may be metabolized prior to their action as an active chemopreventive agent.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis by retinyl acetate.

The administration of 2.5 mg retinyl acetate daily in the diet to female Sprague-Dawley rats beginning 7 days after the intragastric instillation of either 2.5, 5, or 15 mg 7,12-dimethylbenz(a)anthracene (CMBA) resulted in a reduction in the incidence of benign mammary tumors of 37, 30, and 31%, respectively. An equally significant reduction in the number of tumors was also evident. Although no difference was noted in the percentage incidence of mammary adenocarcinomas between the placebo and 2.5 mg retinyl acetate-treated groups at the 2.5-mg DMBA level, the percentage incidence was reduced by 52 and 39% in these groups at the 5- and 15-mg DMBA dose. Furthermore, the number of adenocarcinomas was also significantly reduced. Although both the percentage incidence and number of tumors were reduced by treatment with 1 mg retinyl acetate, these differences were not statistically significant. Liver histology and liver function tests of rats of the retinyl acetate groups did not differ from that of the control group. Similarly, the estrus cycle of treated animals did not differ from that of control rats. These data indicate that relatively large doses of retinyl acetate significantly inhibit the development of DMBA-induced mammary adenocarcinomas and benign tumors. Furthermore, the suppression of mammary tumorigenesis is apparently not the result of an alteration in either the metabolism of DMBA or estrogen nor to an inhibition of tumor growth resulting from retinyl acetate toxicity. The inhibitory effect of retinyl acetate may be related to the effect of retinoids on epithelial cell differentiation and/or reversal of carcinogen-induced anaplasia.

Modulation of rat mammary carcinogenesis by indomethacin.

Indomethacin, a nonsteroidal antiinflammatory agent which inhibits prostaglandin biosynthesis, has significant activity in inhibiting the growth and/or inducing the regression of transplantable tumors. The present study was designed to determine if, in addition to its chemotherapeutic effects, indomethacin also acts as a cancer chemopreventive agent. Fifty-day-old virgin female Sprague-Dawley rats were given a single intragastric dose of either 8 or 16 mg of 7,12-dimethylbenz(a)anthracene (time 0). Basal diet was supplemented with 25 or 50 mg of indomethacin per kg of diet by the following protocol: (a) -2 to +1 week; (b) +1 week to end; or (c) none. Administration of indomethacin by both protocols resulted in an inhibition of mammary tumorigenesis; however, the effect of -2 to +1 week indomethacin exposure was primarily on the induction of benign mammary tumors, while +1 week to end indomethacin administration inhibited the induction of both benign mammary tumors and mammary cancers. These data indicate that indomethacin has significant protective activity when administered either during the "early" stage (comprising the carcinogen-target cell interaction) or the "late" stage (postcarcinogen tumor development) of mammary carcinogenesis in rats. Possible mechanisms of indomethacin action include both local and systemic effects.

Influence of retinoids on growth and metastasis of hamster melanoma in athymic mice.

The effects of 2-hydroxyethyl retinamide, N-(4-hydroxy-phenyl) all-trans-retinamide, and 13-cis-retinoic acid on the growth and metastasis of a malignant hamster melanoma cell line HM1-F5 was determined in a double blind study using 4- to 5-week-old male NIH Swiss and BALB/c derived athymic nu/nu mice. Mice were fed retinoids (0.75 and 1.0 or 1.5 mmol/kg diet) or a placebo diet ad libitum beginning on the day of s.c. inoculation of 5 x 10(5) HM1-5 cells. Tumor incidence, latency, and growth rate were similar in both strains of mice. All placebo-treated mice had lung metastasis on the day of autopsy, although the total number of metastases was lower in NIH Swiss derived athymic mice. While mean tumor incidence and latency were not significantly altered by any retinoid treatment, tumor growth rate (volume) and final tumor weight were inhibited (P less than 0.05) by 0.75 mmol/kg 13-cis retinoic acid and 1.5 mmol/kg N-(4-hydroxyphenyl) all-trans-retinamide. In contrast, at 1.0 or 1.5 mmol/kg diet, 2-hydroxyethyl retinamide had no significant effect on tumor growth rate. 13-cis retinoic acid, 0.75 mmol/kg, 2-hydroxyethyl, 1.0 mmol/kg, and N-(4-hydroxyphenyl), 1.0 mmol/kg significantly reduced the mean number of metastatic lesions in NIH Swiss derived mice, but N-(4-hydroxyphenyl) all-trans-retinamide also reduced metastatic incidence while 2-hydroxyethyl retinamide and 13-cis retinoic acid had no effect. A concentration of 1.5 mmol/kg diet of 2-hydroxyethyl and N-(4-hydroxyphenyl) all-trans-retinamide significantly reduced the overall number of gross lung metastases in BALB/c and Swiss mice, and mean number of metastases in Swiss mice. Analysis of correlation indicated that the inhibitory effect of high-dose N-(4-hydroxyphenyl) and 2-hydroxyethyl retinamide on metastasis was not associated with (independent of) any inhibitory effect on primary tumor invasiveness or growth rate. Our observations suggest that agents such as retinoids have an antimetastatic potential.

Effect of the duration of retinyl acetate feeding on inhibition of 1-methyl-1-nitrosourea-induced mammary carcinogenesis in the rat.

The effect of the duration of retinoid treatment on the inhibition of 1-methyl-1-nitrosourea-induced mammary carcinogenesis was studied. Female Sprague-Dawley rats were given i.v. injections of 50 mg 1-methyl-1-nitrosourea per kg body weight at both 50 and 57 days of age. Feeding of a placebo diet or diet supplemented with 323 mg retinyl acetate per kg diet (retinoid treatment) was initiated at 10 days after the first carcinogen injection. Retinoid treatment was either continued or discontinued after 60 days postcarcinogen, and the study was terminated at 182 days postcarcinogen. Retinoid treatment between 10 and 60 days postcarcinogen prolonged the cancer latency and reduced the average number of cancers per rat in comparison to that in placebo-treated rats. Continuation or cessation of retinoid treatment in 60-day tumor-bearing rats had no effect on the time of appearance of additional cancers. In 60-day tumor-free rats, continuation of retinoid treatment prolonged cancer latency in comparison to either 60-day tumor-free rats changed to placebo or rats continuously treated with placebo. The cessation of retinoid treatment in 60-day tumor-free rats resulted in a rapid increase in the appearance of cancers; at the termination of the study, the average number of cancers per rat was similar to that of animals fed only the placebo. The data indicated that some rats are more responsive to the retinoid than are others. Retinoid treatment apparently prevented the progression of early neoplastic lesions, and a continuous daily intake of the retinoid appears necessary to sustain the chemopreventive effect under the experimental conditions imposed.

Lifetime dose-response relationships for mammary tumor induction by a single administration of N-methyl-N-nitrosourea.

Dose-response relationships for the induction of mammary tumors by a single i.v. injection of N-methyl-N-nitrosourea (MNU) were studied. At 50 days of age, groups of 20 virgin female Sprague-Dawley rats received single doses of 50, 45, 40, 35, 30, 25, 20, 15, or 10 mg MNU per kg body weight; a group of 10 control rats received 0.85% NaCl solution only. Animals were observed for the appearance of mammary tumors over their life span or until 600 days after carcinogen administration. Both malignant and benign mammary tumors appeared in all groups; however, malignant tumors appeared earlier and at a faster rate than did benign tumors. Incidence of cancer and number of cancers per animal increased with increasing MNU dose; the latent period for cancer increased with decreasing dose. The number of benign tumors induced as a percentage of total tumors increased with decreasing dose, ranging from approximately 10% in groups receiving more than 30 mg MNU per kg to 58% in the group receiving 10 mg/kg. Foci of metastatic mammary carcinoma were found in lungs of animals in several MNU dose groups. Data from the present study indicate that a single i.v. administration of MNU induces mammary cancer in a dose-related fashion, with little toxicity and a short latent period; induced cancers metastasize to distant sites. The single-dose MNU model thus appears to be superior to both the 7,12-dimethylbenz(a)anthracene and multiple-dose MNU models, particularly for use in studies of modification of mammary carcinogenesis.

Inhibition of mammary cancer by retinyl methyl ether.

Daily feeding of the synthetic retinoid, retinyl methyl ether, beginning one week after the oral administration of 7,12-dimethylbenz(a)anthracene to female Sprague-Dawley rats, inhibited the incidence of mammary cancer and diminished the number of mammary tumors, both malignant and benign, caused by 7,12-dimethylbenz(a)anthracene. Retinyl methyl ether also markedly increased the latent period for appearance of mammary cancers. Retinyl methyl ether caused no evident toxicity and did not affect weight gain in these experiments. This synthetic retinoid was superior to the natural retinoid, retinyl acetate, for inhibition of mammary carcinogenesis.

Synergistic effect of chronic prolactin suppression and retinoid treatment in the prophylaxis of N-methyl-N-nitrosourea-induced mammary tumorigenesis in female Sprague-Dawley rats.

Two hundred forty Sprague-Dawley rats were treated i.v. with 2.5 or 1.25 mg of N-methyl-N-nitrosourea (MNU) per 100 g body weight at 50 and 57 days of age. At 60 days of age, rats given either dose were divided into 4 groups (30 rats/group) and treated as follows: Group 1, controls; Group 2, 0.4 mg 2-bromo-alpha-ergocryptine (CB-154) per 100 g body weight injected s.c. once daily; Group 3, retinyl acetate (328 mg/kg diet) fed daily; and Group 4, CB-154 and retinyl acetate treatments combined. Rats that received the 2.5-mg dose of MNU were treated for 129 days; those that received the 1.25-mg dose of MNU were treated for 175 days. The rats that were treated with the high dose of MNU were maintained without any treatment for an additional 13 weeks, after which they were sacrificed. The rats that were treated with the low dose of the carcinogen were sacrificed immediately after treatment. All rats were palpated once weekly for palpable mammary tumors. The number of rats with mammary tumors and the total number of mammary tumors at cessation of treatments were, respectively, as follows. MNU (2.5 mg): Group 1, 22 of 30 (73%), 82: Group 2, 11 of 30 (37%), 17; Group 3, 11 of 30 (37%), 19: Group 4, 2 of 30 (7%), 2. MNU (1.25 mg): Group 1, 8 of 30 (27%), 14; Group 2, 4 of 30 (13%), 5; Group 3, 3 of 30 (10%), 4; Group 4, 0 of 30, (0%), 0. Thus, chronic CB-154 treatment or retinyl acetate feeding markedly reduced the percentage of rates bearing mammary tumors and the total number of mammary tumors. The combined treatments were superior to either treatment alone, inasmuch as mammary tumorigenesis was nearly completely blocked in the rats of Group 4 that received the 2.5-mg dose of MNU and was totally blocked in the rats of Group 4 that received the 1.25-mg dose of MNU. Retinyl acetate feeding or CB-154-induced prolactin suppression appear to be equally effective treatments in the prophylaxis of MNU-induced mammary tumorigenesis in rats; the combined modality, however, appears to be far superior than either treatment alone.

Effect of delay in administration of 13-cis-retinoic acid on the inhibition of urinary bladder carcinogenesis in the rat.

The effect of a delay in starting 13-cis-retinoic acid treatment on the inhibition of urinary bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine was studied in male Fischer 344 rats. Animals received a total p.o. dose of either 1200, 1800 or 2400 mg N-butyl-N-(4-hydroxybutyl)nitrosamine over a period of six weeks. At either one, five, and nine weeks after the last N-butyl-N-(4-hydroxybutyl)nitrosamine intubation, animals were started on a diet supplemented with 13-cis-retinoic acid (240 mg/kg of laboratory chow) or continued on laboratory chow. Animals were killed at one year after the first carcinogen intubation for histological evaluation of the bladder. Feeding of 13-cis-retinoic acid reduced the incidence, average number, and severity of transitional cell carcinomas as well as hyperplasia and cellular atypia. Furthermore, even a nine-week delay in starting the retinoid feeding did not diminish the ability of 13-cis-retinoic acid to inhibit bladder carcinogenesis.

Enhanced inhibition of mammary carcinogenesis by combined treatment with N-(4-hydroxyphenyl)retinamide and ovariectomy.

Bilateral ovariectomy and dietary administration of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) are both effective inhibitors of chemical carcinogenesis in the rat mammary gland. The present study was designed to determine whether an enhanced inhibitory effect is obtained with combined ovariectomy and 4-HPR administration, compared to either treatment alone. In separate experiments, 50-day-old virgin female Sprague-Dawley rats received either a single i.v. injection of 50 mg N-methyl-N-nitrosourea per kg body weight or a single intragastric dose of 20 mg 7,12-dimethylbenz(a)anthracene. The experimental design was the same in both the N-methyl-N-nitrosourea and 7,12-dimethylbenz(a)anthracene experiments: Group 1, 25 intact rats, placebo diet; Group 2, 25 intact rats, supplement of 782 mg 4-HPR per kg diet; Group 3, 50 ovariectomized rats, placebo diet; Group 4, 50 ovariectomized rats, supplement of 782 mg 4-HPR per kg diet. Feeding of the 4-HPR supplement was begun 7 days after carcinogen administration; ovariectomy was performed 7 days post-7,12-dimethylbenz(a)anthracene or 14 days post-N-methyl-N-nitrosourea. In both experiments, combined ovariectomy plus 4-HPR was significantly more active in suppressing mammary cancer induction than was either manipulation alone. 4-HPR was a more effective inhibitor of carcinogenesis in ovariectomized rats than in intact animals. These data indicate that 4-HPR is highly effective in inhibiting ovarian hormone-independent cancers and suggest that retinoid inhibition of mammary carcinogenesis does not involve an influence on ovarian hormone action.

Inhibition of rat mammary gland chemical carcinogenesis by dietary dehydroepiandrosterone or a fluorinated analogue of dehydroepiandrosterone.

The chemopreventive efficacy of p.o. administered dehydroepiandrosterone (DHEA), DHEA plus N-(4-hydroxyphenyl)retinamide (4-HPR), or 16 alpha-fluoro-5-androsten-17-one (DHEA analogue 8354) was examined in rats treated with N-methyl-N-nitrosourea (MNU; 50 mg/kg body weight, i.v.) at 50 days of age. Semipurified diet (AIN-76A) containing each steroid alone, or DHEA plus 4-HPR, was administered during initiation (-1 week to +1 week post-MNU), promotion/progression (+1 week post-MNU to termination), or both phases (-1 week post-MNU to termination) of the carcinogenic process. Neither DHEA nor DHEA analogue 8354 (0.2%, w/w) significantly affected the initiation of mammary cancer when administered alone; however, DHEA (0.2%, w/w) plus 4-HPR (1 mmol/kg diet) significantly reduced cancer multiplicity (26%) when given during initiation. All three treatments were strongly effective when given during promotion/progression, significantly reducing mammary cancer multiplicity by 77% (DHEA), 84% (DHEA/4-HPR), and 66% (DHEA analogue 8354), relative to carcinogen controls. Cancer incidence was significantly inhibited by DHEA (33% inhibition) and DHEA/4-HPR (24% reduction) during promotion/progression. However, the most effective chemopreventive treatment encompassed both phases of carcinogenesis. Thus, under these conditions, DHEA (0.2% or 0.1%, w/w) reduced cancer incidence (52% and 32% reductions, respectively) and multiplicity (91% and 86% reductions, respectively). Further reduction in mammary cancer incidence was observed in animals that received DHEA (both doses) plus 4-HPR (1 and 0.5 mmol/kg diet, respectively). DHEA analogue 8354 (0.2% or 0.1%, w/w) given for the duration of the study reduced only cancer multiplicity (61% and 56% reductions, respectively). Tumor-related mortality was significantly lower in rats that received long-term treatment with DHEA or DHEA/4-HPR, when compared with carcinogen controls. Except for a slight, but significant, postcarcinogen decrease in the mean body weights of rats treated concomitantly with DHEA (plus or minus 4-HPR) and MNU, additional gross manifestations of steroid-induced toxicity were not observed.