Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Cancer by Miso and Tamoxifen, Alone and in Combination

We examined the effects of a Japanese fermented soybean product, miso, and tamoxifen (TAM), alone and in combination, on N -nitroso- N -methylurea (MNU)-induced rat mammary cancer. Seven-week-old female CD/Crj rats received a single i.v. dose (50 mg/kg body weight) of MNU. After administration of MNU, the rats were divided into 4 groups: regular diet (control), 10% miso diet, regular diet+TAM, and 10% miso diet+TAM. TAM was implanted s.c. in the form of pellets containing 2.5 mg at the same time as MNU was administered. All rats were observed for 18 weeks after MNU administration. Incidence (percentage of rats with tumors) and multiplicity (mean tumors/rat) of mammary tumors were 91% and 4.5 in the control, 77% and 2.4 ( P <0.05) in the 10% miso group, 68% and 1.4 ( P <0.01) in the TAM group, and 10% ( P <0.0001 or less) and 0.2 ( P <0.0001) in the 10% miso+TAM group. In the second experiment, the effect of the combination of miso and TAM on established rat mammary tumors was investigated. When the mammary tumors induced by MNU reached 10 to 25 mm, the rats were divided into 3 treatment groups: regular diet, regular diet+TAM, and 10% miso diet+TAM. At 6 weeks after the start of treatment, the mean tumor size in the control and TAM groups was 160% and 141% of the pretreatment value, but a decrease to 85% of the pretreatment value was produced by the combination of miso and TAM, and this was significantly different from both the control and TAM groups ( P <0.01 and P <0.05, respectively). These results indicate that miso is useful in protecting against mammary cancer and it can be expected to have a potent antitumor effect, especially when used in combination with TAM.     

Inhibition of N-methyl-N-nitrosourea- and 7,12-dimethylbenz[a] anthracene-induced rat mammary tumorigenesis by dietary cholesterol is independent of Ha-Ras mutations

Dietary cholesterol has previously been shown to inhibit rat mammary tumorigenesis but the mechanisms remain unclear. Uptake of serum low density lipoprotein cholesterol by tissues leads to down-regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis that catalyzes the formation of mevalonate. In addition to being a precursor of cholesterol, mevalonate is necessary for DNA synthesis and cell proliferation. Isoprenoids, also derived from mevalonate, are required for the post-translational modification of Ras proteins that are mutated in a number of carcinogen-induced rat mammary tumors. The purpose of this study, therefore, was to determine whether inhibition of tumorigenesis by cholesterol is dependent on the frequency of mutations in the Ha-ras gene. Female Sprague-Dawley rats (30/group) were given a single dose of either N-methyl-N-nitrosourea (MNU, 50 mg/kg i.p.) or 7, 12-dimethylbenz[a]anthracene (DMBA, 100 mg/kg intragastrally), carcinogens that produce tumors with either a high (MNU) or low (DMBA) frequency of Ha-ras mutations in codon 12 or 61, respectively. Rats were fed either a control AIN-93G diet or the control diet supplemented with 0.3% cholesterol for 14 weeks. Dietary cholesterol significantly decreased the final tumor incidence in rats given DMBA (83 versus 100%, P < 0.05) or MNU (53 versus 77%, P < 0.05). HMG-CoA reductase activity was higher in mammary tumors than in normal mammary glands, but the activity of this enzyme was reduced by cholesterol feeding only in mammary glands and not in tumors. Tumors induced by MNU had a high frequency of Ha-ras mutations in both the control (65%) and cholesterol-fed (68%) groups. Tumors induced by DMBA had a low frequency of Ha-ras mutations that also did not differ between the control (21%) and cholesterol-fed (18%) groups. These findings show that dietary cholesterol inhibits mammary tumorigenesis induced by either MNU or DMBA and that the inhibition is independent of the type or extent of mutations in the Ha-ras gene.     

Low zinc intake suppressed N-methyl-N-nitrosourea-induced mammary tumorigenesis in Sprague-Dawley rats

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.     

Efficacy of Targretin on methylnitrosourea-induced mammary cancers: prevention and therapy dose-response curves and effects on proliferation and apoptosis

Various aspects of the chemopreventive and chemotherapeutic properties of the RXR receptor agonist Targretin (LGD 1069) were examined in the methylnitrosourea (MNU)-induced model of mammary cancer. The administration of Targretin at dose levels of 60, 20 or 6.7 mg/kg body wt/day by gavage decreased the number of mammary tumors by 96, 85 and 78%, respectively. When Targretin was administered in the diet at 92 and 275 mg/kg diet cancer multiplicities were reduced by 78 and 92%, respectively. A wider range of dietary doses of Targretin at 15, 50 and 150 mg/kg diet reduced the number of mammary tumors by 38, 55 and 70%, respectively. Treatment of rats with different regimens of Targretin (250 mg/kg diet) yielded cancer multiplicities of 4.3 for non-treated rats, 0.5 for rats treated continuously with Targretin, 2.1 for rats treated with Targretin for 8 weeks followed by 10 weeks of the control diet and 1.6 for rats treated with Targretin alternating 3 days on and 4 days off. Targretin was also examined as a therapeutic agent by treating rats with at least one palpable mammary tumor for 5 weeks. A high dose of Targretin (272 mg/kg diet) caused partial or complete regression of approximately 65% of the cancers over this time period. In contrast, in animals treated with 15 mg Targretin/kg diet only 1 of 12 cancers showed significant regression. Finally, the effect of a limited exposure to Targretin (7 days) on cell proliferation and apoptosis in small mammary tumors was determined. Targretin at 150 mg/kg diet strongly decreased proliferation (75%) and increased apoptosis (300%), while a lower dose of Targretin (15 mg/kg diet, which still prevented 30% of cancers) had no effect on apoptosis but did decrease cell proliferation. Determination of serum IGF1 levels showed that treatment of rats with highly effective doses of Targretin at 272 mg/kg diet or at 60 or 20 mg/kg body wt/day by gavage caused significantly decreased serum IGF1 levels.     

A rapid dual organ rat carcinogenesis bioassay for evaluating the chemoprevention of breast and colon cancer

In this study we evaluated the effect of dietary administration of a high fat, low fiber diet (HRD) with or without 2% phytic acid (PA) on the development of mammary cancer and/or colon cancer in rats exposed to methylnitrosourea (MNU), azoxymethane (AOM) or MNU + AOM. The rats were fed a HRD alone or a HRD + 2% PA. At the end of week 2, the rats were given either a s.c. injection of MNU (50 mg/kg body wt) or one of normal saline (vehicle). At the end of weeks 3 and 4, the rats were given either a s.c. injection of AOM (15 mg/kg body wt per week) or one of normal saline (vehicle). Nine weeks after the injection of MNU or saline, 10 rats from each group were sacrificed and the mammary tumor incidence and the number of colonic aberrant crypt foci (ACF) were compared between different groups. The administration of different diets was continued for an additional 21 weeks and the mammary tumor and colon tumor incidence between different groups were compared. Results showed that rats injected with MNU alone did not develop ACF or colon tumors while those injected with AOM alone did not develop mammary tumors. Linear regression analysis of the number of ACF at 11 weeks versus colonic tumor incidence at 32 weeks, and the linear regression analysis of mammary tumor incidence at 11 weeks versus mammary tumor incidence at 32 weeks, both showed good linear correlation. These results demonstrate the potential value of the short term dual organ carcinogenesis bioassay for screening chemopreventive agents for their relative ability to inhibit the development of mammary cancer and/or colon cancer while on high risk diet.     

Effect of biochanin-a or testosterone on liver-tumors induced by a combined treatment of den and fission neutron in bcf1 mice

To determine the biological effect of biochanin A, miso or NaCl and sexual influence of testosterone on liver tumor induction, male and female BCF1 mice were i.p. injected once with DEN at a dose of 5 mug/g body weight at 15 days of age. In order to shorten the latency of liver tumor occurrence, the whole body of mice were exposed to 2 Gy of Cf-252 fission neutrons at four weeks of age. Three days later, female mice were surgically ovariectomized and given the various doses of testosterone melted into a cholesterol pellet. Male mice were fed on 10 ppm, 20 ppm biochanin A, 10% miso or 2% NaCl supplemented diet for 8 weeks (from 21 to 28 weeks of age) and 4 weeks (from 32 to 36 weeks of age). All mice were sacrificed at 40 weeks of age. Multiplicity of liver tumors was expressed in four different size ranges by <2, 3-5, 6-10 and >10 mm2. Incidence of liver tumors in all experimental groups except in group 1 at 20 weeks were observed at 100%. Average tumor size and multiplicity were smaller at 20 weeks compared to those of 40-week groups. Male groups fed 20 ppm biochanin A and 2% NaCl had an increase in body weight with significant difference from control by p<0.01. Liver weights were more-or-less the same in all groups except an increase was seen in the group of 20 ppm biochanin A (p<0.01). In female groups, both 0.2 mg and 1 mg of testosterone administration resulted in an increase of tumor multiplicities and a decrease of liver weight compared to that of control group with significant differences. In both male and female groups, majority of liver tumor sizes were in the range of 3-5 mm2. Tumor multiplicities and size in less than 2 mm2 in biochanin A groups, 10% miso and 2% NaCl decreased significantly from control group. These findings suggest that 15-20 weeks is the time in which 1-2 mm2 size of liver tumors start to appear. Among others, biochanin A is a component of miso. The potent anti-tumorigenic effect of dietary miso for mouse liver tumorigenesis may be strenghtened by a combination of factors such as the presence of Biochanin A, protease inhibitors and various fermented enzymes.     

Dietary folate deficiency suppresses N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats

Epidemiologic studies have suggested that dietary folate intake is inversely related to breast cancer risk. However, epidemiologic evidence has not been consistent nor has it provided unequivocal support for this purported inverse relationship. This study investigated the effect of dietary folate on N-methyl-N-nitrosourea (MNU)-induced mammary tumorigenesis in rats. Weanling, female Sprague-Dawley rats were fed diets containing either 0 (deficient; n = 22), 2 (basal dietary requirement, control; n = 20) or 8 mg (supplemented; n = 20) folate/kg diet for 30 weeks. At 50 days of age, rats received an i.p. injection of MNU (50 mg/kg body wt). At necropsy, all macroscopic mammary tumors were identified and examined microscopically. The effect of dietary folate on genomic DNA methylation in mammary tumorigenesis was determined by the in vitro methyl acceptance assay. The incidence of mammary adenoma and adenocarcinoma in the folate-deficient group was lower than that of the control and folate-supplemented groups (55 versus 90 and 75%, respectively, P = 0.043). Kaplan-Meier analyses also demonstrated a similar trend in the rates of appearance of either adenoma or adenocarcinoma (P = 0.06). In contrast, folate supplementation did not significantly modulate mammary tumorigenesis compared with the control group. Although mammary tumors were significantly hypomethylated compared with non-neoplastic mammary tissues in each dietary group (P < 0.03), folate status did not significantly affect the extent of DNA methylation. The data suggest that dietary folate deficiency of a moderate degree suppresses, whereas folate supplementation at four times the basal dietary requirement does not significantly modulate, mammary tumorigenesis in this model. The role of folate in mammary tumorigenesis needs to be clarified for safe and effective prevention of breast cancer.     

Decrease in size of azoxymethane induced colon carcinoma in F344 rats by 180-day fermented miso

The present study was designed to investigate the effects of fermented miso (fermented soybean paste) on the induction of colon tumors by azoxymethane (AOM) in male F344 rats. A total of 91 rats, 6 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. The animals were placed on diets one week before the first AOM dose: commercial normal control MF diet or a diet containing 10% 2-year, 180-day fermented, or 3-4-day fermented miso. There were no differences in body and organ weights, and no aberrant crypt foci (ACF) among carcinogen-treated groups at week 25. The rates of tumor incidence were 45%, 85%, 75% and 60% with the 2-year, 180-day, and 3-4-day fermented miso and MF, respectively, and those for colon tumors were 34%, 55%, 60% and 55%, respectively. The size of well-differentiated adenocarcinomas and total (well differentiated and signet ring cell) adenocarcinomas in the 180-day fermented miso group was significantly smaller than that in the 2-year fermented miso and MF+AOM groups. Nuclear staining of beta-catenin in colon tumors was increased for the 3-4-day fermented miso compared to the 180-day fermented miso. Cdx2 staining tendency was decreased in colon tumors and adenocarcinomas compared to normal mucosa and ACF, which stained in 100% of cases. In addition, the PCNA index was significantly reduced in the 180-day group compared with those groups receiving the 3-4-day fermented miso and MF diet. The germinal region was also decreased. The present results indicate that dietary supplementation with 180-day fermented dietary miso could act as a chemopreventive agent for colon carcinogenesis.     

Dietary iodine deficiency as a tumor promoter and carcinogen in male F344/NCr rats

Groups of 6-wk-old male F344/NCr rats received a single i.v. injection of either vehicle or N-nitrosomethylurea (Cas: 684-93-5) (MNU) at a dose of 41.2 mg/kg body weight. Two wk later, groups of rats were placed on iodine-deficient, iodine-adequate, or commercial (Wayne Lab Blox) diets, or one of these diets and without previous MNU injection. Animals were sacrificed at either 52 or 77 wk, or when they became moribund. Carcinogen-treated rats on the iodine-deficient diet for up to 52 wk had significantly increased thyroid gland weights and increased incidences of both thyroid follicular cell carcinoma (90%) and diffuse pituitary thyrotroph hyperplasia (90%) at 52 wk. The majority of the follicular carcinomas were transplantable and invasive into the mammary fat pad of weanling F344/NCr rats. No other tumors induced by MNU were affected by the iodine-deficient diets. Rats fed the iodine-deficient diet without MNU injection had a 40% incidence of thyroid follicular adenomas at 52 wk and 60% at 77 wk, and a 10% incidence of follicular carcinomas at 77 wk. Thus this experiment provided evidence that the iodine-deficient diet is a potent promoter of thyroid tumors initiated by MNU and carcinogenic by itself. In addition, pituitary tumors were found in 29 of the 58 rats treated with the carcinogen alone, compared to only 3 of the 20 rats in the control groups. The vast majority of these pituitary tumors contained prolactin that was demonstrable by the avidin:biotin:peroxidase complex immunocytochemical technique.     

Fatty acid synthase is a potential molecular target for the chemoprevention of breast cancer

The purpose of this investigation was to determine whether fatty acid synthase (FAS) is a potential molecular target for the chemoprevention of breast cancer by evaluating the effect of the FAS inhibitor triclosan on rat mammary carcinogenesis. At 50 days of age, 60 female Sprague-Dawley rats received 50 mg/kg methylnitrosourea (MNU) i.p. to initiate mammary carcinogenesis. One week later, half of the rats were fed triclosan at a level of 1000 p.p.m. in an AIN-93G diet for the remainder of the experiment. The other 30 control rats were fed an AIN-93G diet without triclosan. Twelve weeks after MNU treatment, 70% of control rats had mammary adenocarcinomas compared with only 43.3% of the triclosan group (P < 0.05). The control rats had an average of 2.7 +/- 0.3 tumors/rat compared with 1.8 +/- 0.3 in the triclosan group (P < 0.05). Western analysis showed that the tumors in the control rats expressed high levels of FAS. Immunohistochemistry showed that sections of tumors that stained strongly for FAS also showed strong staining for proliferating cell nuclear antigen. Furthermore, at biologically relevant dose levels, triclosan inhibited the activity of FAS in mammary tumor homogenates. These results indicate that triclosan suppresses rat mammary carcinogenesis by inhibiting FAS and suggest that FAS is a promising molecular target for breast cancer chemoprevention.     

Prevention of methylnitrosourea-induced mammary cancers by 9-cis-retinoic acid and/or vitamin D3

Two cancer chemopreventive agents, vitamin D3 and 9-cis-retinoic acid (9-cis-RA), were evaluated alone and in combination in the methylnitrosourea (MNU)-induced mammary cancer model. In this study, female Sprague-Dawley rats received MNU (50 mg/kg BW) at 50 days of age. Vitamin D3 and 9-cis-RA were administered in the diet beginning three days later. The groups were: Group 1, vehicle only; Group 2, 9-cis-RA (60 mg/kg diet); Group 3, vitamin D3 (10 microg/kg diet); Group 4, vitamin D3 (3.3 microg/kg diet); Group 5, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (10 microg/kg diet); and Group 6, 9-cis-RA (60 mg/kg diet) plus vitamin D3 (3.3 microg/kg diet). Animals were observed daily for signs of toxicity and were palpated 2x/week for mammary tumors. The study was terminated 150 days after treatment with MNU. The average number of mammary cancers was 6.7 in the animals receiving only the carcinogen. 9-cis-RA alone caused a 23% decrease in mammary cancer multiplicity, while vitamin D3 alone actually caused slight increases of 17 and 16% at 10 and 3.3 microg/kg diet dose levels, respectively. When the agents were given in combination, however, the 9-cis-RA plus the high dose of vitamin D caused a statistically significant decrease (44%) in mammary cancer number, while the 9-cis-RA plus the low dose resulted in a 37% decrease. Thus, low doses of these agents that were not effective in preventing mammary cancer when given alone appeared to be active when given in combinations. Possible interactions between the retinoic acid receptors and vitamin D receptor may be responsible for the observed inhibition of mammary carcinogenesis.     

Chemoprevention of MNU-induced mammary tumors in the mature rat by 4-HPR and tamoxifen.

The chemopreventive efficacies of the retinoid all-trans-N-(4-hydroxyphenyl)-retinamide (4-HPR) and the anti-estrogen tamoxifen citrate were evaluated against N-methyl-N'-nitrosourea (MNU) induced mammary cancer in 120-day old female Sprague-Dawley rats. The agents were tested alone and in combination. They were administered in a modified AIN-76A diet, beginning 60 days prior to a single i.v. dose of 50 mg MNU/kg-bw and continuing until the end of the study, 180 days post-carcinogen treatment. At 782 mg/kg diet, 4-HPR alone significantly inhibited the induction of mammary adenocarcinomas compared with carcinogen controls. At 0.250 mg/kg diet, tamoxifen alone reduced tumor incidence compared with carcinogen controls. At 0.125 mg/kg diet, tamoxifen was ineffective. Combinations of 782 mg 4-HPR/kg diet with either 0.250 or 0.125 mg tamoxifen/kg diet were effective in inhibiting MNU-induced adenocarcinomas. The reductions in tumor incidence were greater for these combinations than for either agent alone. 4-HPR and 0.250 mg tamoxifen/kg diet decreased tumor incidence 81% (p less than 0.005), whereas 4-HPR and 0.125 mg tamoxifen/kg diet decreased tumor incidence 72% (p less than 0.005) compared with carcinogen controls. The combination of 391 mg 4-HPR/kg diet and 0.500 mg tamoxifen/kg diet was also tested and was effective in reducing tumor incidence.     

Chemopreventive effects of the aromatase inhibitors vorozole (R-83842) and 4-hydroxyandrostenedione in the methylnitrosourea (MNU)-induced mammary tumor model in Sprague-Dawley rats

The chemopreventive activity of the aromatase inhibitors vorozoleand 4-hydroxyandrostenedione were determined in the methylnitrosourea(MNU)-induced model of rat mammary tumorigenesis. Vorozole (5and 2.5 mg/kg body wt) and 4-hydroxyandrostenedione (15 and6 mg/rat) were administered daily (by gavage) to virgin femaleSprague-Dawley rats starting at an age of 43 days. Seven dayslater animals were given a single dose of MNU. Following treatmentwith MNU, animals continued to be treated with vorozole and4-hydroxyandrostenedione daily until the end of the experiment(100 days post MNU treatment). Vorozole at either dose provedto be a profound inhibitor of MNU-Induced mammary tumors. Vorozoledecreased tumor incidence from 100% to 10%, while simultaneouslydecreasing tumor multiplicity from 5 tumors per annual to 0.1tumors per animal. This chemopreventive effect was accompaniedby significant increases In body weight gain in the animalstreated with vorozole when compared with control rats. In contrast,neither dose of 4-hydroxyandrostenedione had any effect on tumorincidence and only the higher dose slightly decreased tumormultiplicity.     

Effect of retinyl acetate and 4-hydroxyphenylretinamide on initiation of chemically-induced mammary tumors

The anti-promotional effect of retinoids on chemically-induced mammary carcinogenesis in the rat is well established. The present studies were performed to determine the effect of long-term feeding of retinyl acetate and 4-hydroxyphenylretinamide (4-HPR) on initiation of mammary tumors induced by MNU or DMBA. Retinyl acetate (328 mg/kg of diet) or 4-HPR (782 mg/kg of diet) was added to the diet of female Sprague-Dawley rats for two months prior to the administration of the carcinogens. In the MNU model, a 50% increase in the number of mammary adenocarcinomas was observed in rats pretreated with retinyl acetate, while pretreatment with 4-HPR resulted in a 93% increase in the number of cancers. Continued treatment with 4-HPR throughout the study, however, caused a reduction in cancer number. In the DMBA mode, pretreatment with these retinoids significantly increased the number of benign mammary tumors, but not mammary cancers. These data suggest that newly synthesized retinoids should be evaluated for chemopreventive activity against mammary cancer initiation as well as for their anti promotional activity.     

Prevention by long-term fermented miso of induction of colonic aberrant crypt foci by azoxymethane in F344 rats.

The present study was designed to investigate the effects of fermented miso in the diet on the induction of aberrant crypt foci (ACF) by azoxymethane (AOM) in male F344 rats. A total of 50 rats, 8 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. Rats were fed a normal control MF solid diet, or solid diet containing 10% long-term fermented (aged), medium- or short-term fermented miso, or 2.2% NaCl for 5 weeks, starting one week before the first AOM dosing. It was found that, compared to the control (MF) diet, the long-term fermented diet significantly decreased (by 22.2%) ACF/colon, but increased (by 18.2%) the number of aberrant crypts (Acs)/focus. The latter was also increased by the medium-term fermented diet (by 25.3%). The PCNA labeling index was only affected by the short-term fermented diet (36.9% increase) and by 2.2% NaCl diet (27.2% increased). The present results indicate that aged or completely fermented miso supplemented into the diet, could act as a chemopreventive agent for colon carcinogenesis.     

Dietary effects of conjugated docosahexaenoic acid on N-methyl-N-nitrosourea-induced mammary carcinogenesis in female Sprague-Dawley rats

The dietary effects of conjugated docosahexaenoic acid (CDHA) were examined in an N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis model. Female Sprague-Dawley rats were administered 50 mg/kg MNU intraperitoneally at 49 days of age. A powdered AIN-76A diet containing 0, 0.2 or 1.0% CDHA was fed to the rats from 21 to 49 days of age (before MNU; pre-initiation phase) or from 49 days to 40 weeks of age (after MNU; post-initiation phase). Rats were sacrificed when their largest mammary tumor was > or =1 cm in size or when they reached 40 weeks of age. All histologically detected mammary carcinomas were evaluated. In rats that received CDHA after MNU, development of mammary carcinoma > or =1 cm was inhibited, and there was a significant decrease in the final mammary cancer incidence and multiplicity, compared with rats that did not receive CDHA. Consumption of the 0.2% CDHA diet after MNU significantly prolonged latency. Suppression of mammary cancer yield by consumption of a CDHA diet after MNU administration was not dose-dependent. In rats that received CDHA before MNU, suppression of mammary cancer was not observed. These results indicate that CDHA administration in the post-initiation period suppressed mammary carcinogenesis, whereas CDHA administration in the pre-initiation period was ineffective.     

Effect of carcinogen dose and age at administration on induction of mammary carcinogenesis by 1-methyl-1-nitrosourea.

The objective of the work reported in this paper was to determine if the tumorigenic response to 1-methyl-1-nitrosourea (MNU) in the mammary gland varied with age of administration and was dose dependent when the carcinogen was injected prior to 50 days of age. Using a recently developed method for mammary tumor induction, MNU was injected i.p. at doses ranging from 25 to 75 mg/kg at 35 days of age or 50 mg/kg at 28, 35 or 42 days of age. Treatment with MNU resulted in induction of both benign and malignant mammary tumors. The incidence of mammary gland adenocarcinomas was 100% at and above the 50 mg/kg dose of MNU, irrespective of the age at which carcinogen was administered. The number of cancers increased in proportion to carcinogen dose, whereas cancer latency decreased as the MNU dose increased. In rats injected with 50 mg MNU/kg body weight at 28, 35 or 42 days of age, differences among groups in cancer incidence, number or latency were not statistically significant. Metastases of mammary neoplasms to lung, liver and spleen were observed in rats injected with MNU at 35 or 42 days of age. These data indicate (i) the dose responsiveness of MNU-induced mammary carcinogenesis in rats initiated prior to 50 days of age; (ii) the lack of effect of age at initiation if prior to 50 days on final tumor outcome; and (iii) that the age at which MNU is injected may affect the metastatic potential of the mammary carcinomas that are induced.     

Inhibitory Effects of Nabumetone, a Cyclooxygenase-2 Inhibitor, and Esculetin, a Lipoxygenase Inhibitor, on N-Methyl-N-nitrosourea-induced Mammary Carcinogenesis in Rats

We investigated the modifying effects of nabumetone, a relatively selective cyclooxygenase-2 inhibitor, and esculetin, a lipoxygenase inhibitor, on N -methyl- N -nitrosourea(MNU)-induced mammary carcinogenesis in female Sprague-Dawley rats. A total of 124 rats, 6 weeks old, were divided into 6 groups. At 50 days of age, groups 1, 2, and 3 were treated with MNU (50 mg/kg body weight) by subcutaneous injection. From the age of 8 weeks, groups 2 and 4 were given 0.03% nabumetone in the diet and groups 3 and 5 were given 0.03% esculetin in the diet. All rats were necropsied at the termination (25 weeks after the start of experiment). The incidence and multiplicity of neoplasms in group 2 were significantly smaller than those in group 1 ( P <0.005 and P <0.001, respectively). The incidence of neoplasms in group 3 was also significantly smaller than that in group 1 ( P <0.05). These results indicate that the intake of nabumetone or esculetin during the time corresponding to the post initiation phase has a chemopreventive effect on MNU-induced mammary carcinogenesis in rats.     

Mammary tumor induction by N-methyl-N-nitrosourea in genetically resistant Copenhagen rats.

The Copenhagen rat is completely resistant to mammary cancer induction by N-methyl-N-nitrosourea (MNU) when the carcinogen is administered during sexual development, a period when other strains of rats are normally susceptible to mammary gland carcinogenesis. Here we administered 30 mg/kg MNU i.p. to two groups of neonatal (2-3-day-old) Copenhagen rats. One group (group B, 18 animals) received no further treatment, while the other group (group C, 17 animals) received a second dose of 30 mg/kg MNU via the tail vein at 50 days of age. About 30% of the rats in group B and about 70% of those in group C developed mammary carcinomas before they were 1 year of age. About one-half of the tumors in both groups were cribriform adenocarcinomas and one-half were adenosquamous carcinomas. The latter tumor type has not been observed previously in susceptible rat strains. The ability to induce these mammary tumors in the Copenhagen rat suggests that the putative mammary carcinoma suppressor gene is functionally inactive in neonatal animals or is inactivated when these animals are treated with MNU.     

Inhibition by long-term fermented miso of induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine in CD (SD) rats

The present study was designed to investigate the effects of fermented miso in the diet on the induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in male CD (SD) rats. A total of 120 animals, 6 weeks of age, were divided into 6 groups and given MNNG (100 ppm) in the drinking water for 16 weeks. Starting 1 week before the carcinogen treatment the rats were fed a normal control MF solid diet, or the same diet containing 10% long-term fermented, medium- or short-term fermented miso, or 1% NaCl until the end of the MNNG exposure period. They were then maintained on the MF control diet and normal tap water until the autopsy time point at 52 weeks. The long-term fermented miso significantly reduced the size of the gastric tumors as compared with the other groups. The present results thus indicate that dietary supplementation with long-term fermented miso could act as a chemopreventive agent for gastric carcinogenesis.