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.     

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.     

Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Carcinogenesis by Soy Foods or Biochanin A

We examined the effects of soybeans, a soy product (miso) and biochanin A, an isoflavone derivative, on N -nitroso- N -methylurea (MNU)-induced rat mammary carcinogenesis. Seven-week-old female CD/Crj rats received a single i.v. dose (40 mg/kg body weight) of MNU. After administration of MNU, rats were fed diet containing 0% (control), 2% or 10% soybeans, or 10% miso as a soy-supplemented diet, or 10 or 50 mg/kg biochanin A. All rats were observed for 18 weeks after MNU administration. At 18 weeks, the multiplicity (mean tumors/rat) of palpable mammary tumors was significantly decreased in the 10% soybean (1.1) and 10% miso (1.2) diet groups compared to the control (2.2) ( P <0.05, respectively). In the biochanin A-supplemented diet groups, the incidence (percentage of rats with tumors) was significantly decreased in the 50 mg/kg (32%) diet group compared to the control (80%) ( P <0.01), and the multiplicity was significantly decreased in both the 10 mg/kg (0.7) and 50 mg/kg (0.5) diet groups compared to the control (2.2) ( P <0.01 and P <0.001, respectively). The proliferative cell nuclear antigen labeling index of mammary tumors was significantly decreased in both biochanin A-supplemented diet groups compared to the control. The present results indicate that soybeans, miso, and biochanin A are useful for the prevention of mammary cancer.     

Black tea and mammary gland carcinogenesis by 7,12- dimethylbenz[a]anthracene in rats fed control or high fat diets

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor- bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.      

Ezetimibe reverses the inhibitory effects of dietary cholesterol on mammary tumorigenesis in rats

There are concerns regarding increased cancer incidence in patients treated with ezetimibe, an inhibitor of the absorption of dietary cholesterol. Here we tested the hypothesis that ezetimibe will accelerate mammary tumorigenesis in rats. The drug was administered at a dose of 1 ppm in an AIN‐93G diet that contained 0.3% cholesterol. This experimental diet and control diets that contained either no additions or cholesterol or ezetimibe only, were fed to groups of 30 Sprague‐Dawley rats 3 days after they were treated with 50 mg/kg methylnitrosourea (MNU). All rats were euthanized 22 weeks after MNU administration. Tumor multiplicity was significantly smaller in rats fed cholesterol than those fed no cholesterol (1.84 ± 0.42 vs. 3.86 ± 0.86 respectively, P < 0.05), but was significantly greater in the cholesterol/ezetimibe group than the group fed only cholesterol (3.48 ± 0.59 vs. 1.84 ± 0.42 respectively, P < 0.04). The average weight of tumors/rat was also significantly larger in the cholesterol/ezetimibe group than those fed cholesterol alone (5.67 ± 1.15 vs. 2.56 ± 0.71 respectively, P < 0.04). As expected, ezetimibe prevented the cholesterol raising effect of the dietary cholesterol. These results show that ezetimibe reverses the inhibitory effect of dietary cholesterol on the development of rat mammary tumors.     

Enhancement of mammary carcinogenesis in two rodent models by silymarin dietary supplements

Silymarin is a mixture of polyphenolic flavonoids isolated from milk thistle (Silybum marianum) with anticancer activities reported for several organ sites. The present study tested the efficacy of dietary silymarin against mammary carcinogenesis in two rodent models. In the Sprague-Dawley rat model, female rats were fed a purified diet supplemented with none, 0.03, 0.1, 0.3 or 1% (w/w) of silymarin from 21 days of age (DOA) and carcinogenesis was initiated by a single i.p. injection of 1-methyl-1-nitrosourea (MNU) at 51 DOA. Mammary tumor (MT) development was followed till 110 days after carcinogen injection. In the MMTV-neu/HER2 transgenic mouse mammary carcinogenesis model, homozygous transgenic females were fed a purified diet supplemented with none or 0.3% silymarin, either from 28 or 120 DOA and MT development was followed to approximately 300 DOA. The results showed that dietary silymarin increased the plasma concentration of free and total silibinin, a major component of silymarin, in a dose-dependent manner in the rat, but did not decrease either MT incidence or number. Instead silymarin modestly increased the number of MNU-induced MTs in rats. Similarly, silymarin increased MT incidence and multiplicity and non-MTs in the neu-transgenic mice. In cell culture, treatment of human MCF-7 breast cancer cells with serum-achievable concentrations of silymarin in the rodent models stimulated their growth, in part through an estrogen-like activity. Because silymarin is being used in the treatment of liver cirrhosis and a variety of other human ailments, and is sold as a dietary supplement, our findings add a cautionary note to its application in breast cancer prevention.     

Dietary genistein: perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat

Asian women consuming a traditional diet high in soy have a low incidence of breast cancer, yet when they emigrate to the USA the second but not the first generation lose this protection. Accordingly, we hypothesized that early exposure to genistein, a major component of soy, could have a permanent protective effect against breast cancer. Sprague-Dawley CD rats were exposed to genistein from conception to day 21 post-partum in the diet at concentrations of 0, 25 and 250 mg genistein/kg AIN-76A diet. At day 50 post-partum, all animals were treated with 80 mg dimethylbenz[a]anthracene/kg body wt to induce mammary cancers. Dietary genistein resulted in dose-dependent protection against development of mammary tumors (fewer tumors per rat). Analysis of mammary whole mounts showed that 21- and 50-day-old female rats had fewer terminal end buds, terminal ductal structures that were undifferentiated and were most susceptible to carcinogenesis. Bromodeoxyuridine incorporation studies revealed that dietary perinatal genistein resulted in a smaller proliferative compartment for terminal end buds. In rats fed the high genistein dose (250 mg/kg diet) total genistein concentrations in the serum and milk of dams 7 days postpartum were 418+/-198 and 137 pmol/ml, respectively. Total genistein concentrations in stomach milk, serum and mammary glands of 7- day-old offspring were 4439+/-1109 and 726 pmol/ml and 440+/-129 pmol/g, respectively. Total genistein concentrations in the serum and mammary glands of 21-day-old offspring were 1810+/-135 pmol/ml and 370+/-36 pmol/g, respectively. Dietary perinatal genistein did not cause significant toxicity in F0 and F1 females. We conclude that genistein in the diet at 'physiological levels' enhances cell differentiation, resulting in programming of mammary gland cells for reduced susceptibility to mammary cancer, with no observed toxicity to the reproductive tract of F1 females.      

Effects of folate deficiency and supplementation on methylnitrosourea-induced rat mammary tumors.

BACKGROUND: There are metabolic and epidemiologic data consistent with the hypothesis that folate deficiency increases the likelihood of cancer. Conversely, it is also known that folate is necessary for cancer growth, but few experiments in laboratory animals have evaluated the effects of folate deficiency on the development of chemically induced cancers. PURPOSE: Our purpose was to determine the effects of nutritional folate deficiency in female Fischer 344 rats on initiation and early promotion of methylnitrosourea (MNU)-induced mammary cancer. METHODS: Rats (age, 27 days) were fed a folic acid-deficient diet (AIN-76A) supplemented with glycine and succinylsulfathiazole [FA(0)]; the FA(0) diet supplemented with 2 or 40 mg of folic acid per kilogram [FA(2) or FA(40), respectively]; or the FA(0) diet supplemented with 20 mg of folinic acid per kilogram [FL(20)]. At 57 days of age, each diet-treated group (30 rats in each group) received MNU (50 mg/kg) by intravenous injection. Immediately after MNU treatment, all animals were fed the AIN-76A complete diet containing 2 mg of folic acid per kilogram. Control groups were fed the AIN-76A complete diet throughout the entire experiment. RESULTS: After 4 weeks, folate deficiency, but not anemia or growth suppression, was documented by lower folate levels in plasma and red blood cells in the group receiving the FA(0) diet. Cancer multiplicity (i.e., number of mammary cancers per number of tumor-bearing animals) at 180 days after MNU injection was 1.32, 1.90, 2.14, and 2.73 mammary cancers per tumor-bearing animal in the FA(0), FA(2), FA(40), and FL(20) groups, respectively; the value in the FA(0) group was statistically significant compared with the values in the other groups. The time required for 50% of the rats to develop palpable mammary cancer was 170, 142, 100, and 85 days, respectively. The value of 170 days for the FA(0) group was statistically significant compared with the values of 100 and 85 days. Mammary cancer incidence was 63%, 70%, 72%, and 73%, respectively; these percentages were not significantly different. CONCLUSIONS: Folate deficiency suppresses and folate supplementation enhances initiation or early promotion of MNU-induced mammary cancer in rats, even when the folate-deficient rats do not have anemia or growth suppression. IMPLICATION: Since the rat is relatively resistant to folate deficiency anemia, other animal models should be used to test the effect of folate nutriture on carcinogenesis.     

Dietary lariciresinol attenuates mammary tumor growth and reduces blood vessel density in human MCF-7 breast cancer xenografts and carcinogen-induced mammary tumors in rats

Lariciresinol is a dietary lignan that accounts for a significant portion of the total phytoestrogen intake from Western foods. Recent epidemiological studies suggest that high dietary intake of lignans and lariciresinol is associated with reduced breast cancer risk. However, no causal relationship between lariciresinol intake and breast cancer development has been established. In this study, we investigated for the first time the effects and possible mechanisms of action of lariciresinol on hormone responsive mammary cancer in vivo in dimethylbenz[a]anthracene induced mammary cancer in rats, and in human MCF-7 breast cancer xenografts in athymic mice. For tumor bearing rats, lariciresinol (3 or 15 mg/kg of body weight) or vehicle was administered p.o. daily for 9 weeks. For E2-maintained ovariectomized athymic mice bearing orthotopic MCF-7 tumors, control diet (AIN-93G) or lariciresinol containing diet (AIN-93G supplemented with 20 or 100 mg of lariciresinol/kg of diet) was administered for 5 weeks. In both models, lariciresinol administration inhibited the tumor growth and tumor angiogenesis. In MCF-7 cells, enterolactone significantly inhibited the E2-stimulated VEGF secretion. Moreover, in MCF-7 xenografts, lariciresinol administration enhanced tumor cell apoptosis and increased estrogen receptor beta expression. Lariciresinol and its further metabolites secoisolariciresinol, enterodiol and enterolactone were found in serum of both rats and athymic mice confirming a similar lignan metabolism pattern as in humans. These findings indicate conceivable importance of dietary lignan lariciresinol in inhibition of breast cancer development.     

Specific inhibitory effect of dietary eicosapentaenoic acid on N-nitroso-N-methylurea-induced mammary carcinogenesis in female Sprague-Dawley rats

We have investigated the effects of two qualitatively different types of unsaturated fatty acids on N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis in female Sprague-Dawley rats. Semipurified diets containing 4.7% eicosapentaenoic acid (EPA) plus 0.3% linoleic acid or 5% linoleic acid were prepared. Animals maintained on these diets were given an i.v. injection of NMU (50 mg/kg body wt) at 50 days of age and killed 20 weeks later. Both tumor incidence and tumor number per rat were significantly lower in the EPA diet group (60.0% and 2.3 +/- 2.5 versus 93.3% and 5.1 +/- 4.5 respectively) for the 5% linoleic acid diet. Furthermore, the average weight of tumor material (total) per rat was significantly lower in the EPA as compared to linoleic acid diet group (2.9 +/- 4.2 g and 11.4 +/- 12.2 g respectively). Analysis of phospholipid fatty acids in the mammary tumors in the EPA diet group showed a higher proportion of C16:0, C18:2, omega-3 fatty acids C20:5 and C22:6 and a lower proportion of C20:4. Furthermore, mammary tumors in rats fed the EPA diet demonstrated significant reduction in prostaglandins. The results thus suggest that inhibition by EPA of NMU-induced mammary carcinogenesis may be mediated via the modulation of lipid metabolism and associated reduction in prostaglandin synthesis.     

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.     

Effects of dietary folate on the development and progression of mammary tumors in rats

Epidemiologic studies have suggested that dietary intake and blood levels of folate may be inversely related to the risk of breast cancer. However, epidemiologic evidence has not been consistent nor has it provided unequivocal support for this purported inverse relationship. Recent evidence has also raised a concern that folate supplementation may promote carcinogenesis if provided after neoplastic foci are established in the target organ. This study investigated the effect of dietary folate deficiency and supplementation on the development and progression of mammary tumors in the N-methyl-N-nitrosourea (MNU) rat model. Weanling, female Sprague-Dawley rats were fed diets containing 0, 2 (control) or 8 mg folic acid/kg diet during the initiation or the promotion phase of MNU-induced mammary tumorigenesis. At necropsy, all macroscopic mammary tumors were identified and histologically confirmed. Dietary folate deficiency and supplementation provided during the initiation phase did not significantly modulate the development of mammary tumors. In contrast, dietary folate deficiency provided during the promotion phase significantly inhibited the rate of appearance, incidence, mean volume and weight of adenocarcinomas compared with the control and supplemental diets. Folate supplementation provided during the promotion phase did not significantly modulate mammary tumorigenesis compared with the control group. These data indicate that moderate folate deficiency inhibits, whereas dietary folate supplementation at four times the basal dietary requirement does not promote, the progression of MNU-induced mammary neoplastic foci in this rat model. However, the limitations associated with the route and dose of MNU administration preclude a definitive conclusion concerning the effect of folate status on the initiation of MNU-induced mammary tumorigenesis.     

Effect of dietary alfalfa, pectin, and wheat bran on azoxymethane-or methylnitrosourea-induced colon carcinogenesis in F344 rats.

The effect of dietary alfalfa, pectin, and wheat bran on colon carcinogenesis was studied in female inbred F344 rats. Weanling rats were fed semipurified diets containing 0 or 15% alfalfa, pectin, or wheat bran. At 7 weeks of age, all animals except controls were given azoxymethane (AOM) sc at a dose rate of 8 mg/kg body weight/week for 10 weeks or methylnitrosourea (MNU) intrarectally at a dose rate of 2 mg/rat twice a week for 3 weeks. The AOM-treated group was autopsied 40 weeks and the MNU-treated group 30 weeks after the first injection of the carcinogen. No tumors were observed in the colon or other organs of untreated rats fed the various diets. The animals fed the alfalfa diet and treated with MNU had a higher incidence of colon tumors than did those fed the control diet or the diets containing pectin or wheat bran. The incidence of MNU-induced colon tumors did not differ between the animals fed the control diet or the diets containing pectin or wheat bran. However, the incidence of AOM-induced colon tumors in rats fed diets containing pectin or wheat bran was lower than that in rats fed the control diet or the alfalfa diet. These results thus indicate that the effect of fiber in colon carcinogenesis depends on the type of fiber and, possibly, the fiber's mode of action.     

Paternal selenium deficiency but not supplementation during preconception alters mammary gland development and 7,12‐dimethylbenz[a]anthracene‐induced mammary carcinogenesis in female rat offspring

Breast cancer is a global public health problem and accumulating evidence indicates early‐life exposures as relevant factors in the disease risk determination. Recent studies have shown that paternal nutrition can influence offspring health including breast cancer risk. Selenium is a micronutrient with essential role in central aspects of embryogenesis, male fertility and cancer and that has been extensively studied as a chemopreventive agent in several breast cancer experimental models. Thus, we designed an animal study to evaluate whether paternal selenium deficiency or supplementation during preconception could affect the female offspring mammary gland development and breast cancer susceptibility. Male Sprague‐Dawley rats were fed AIN93‐G diet containing 0.15 ppm (control diet), 0.05 ppm (deficient diet) or 1 ppm (supplemented diet) of selenium for 9 weeks and mated with control female rats. Mammary carcinogenesis was induced with 7,12‐dimethylbenz[a]anthracene (DMBA) in their female offspring. Paternal selenium deficiency increased the number of terminal end buds, epithelial elongation and cell proliferation in the mammary gland of the female rat offspring and these effects were associated with higher susceptibility to DMBA‐induced mammary tumors (increased incidence and higher grade tumors). On the other hand, paternal selenium supplementation did not influence any of these parameters. These results highlight the importance of father's nutrition including selenium status as a relevant factor affecting daughter's breast cancer risk and paternal preconception as a potential developmental stage to start disease preventive strategies.     

Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis, with a partial contribution of its component isoflavones

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.     

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.     

Anticarcinogenic activity of quinacrine in the rat mammary gland

Mammary carcinogenesis studies were conducted to determine thechemopreventive activity of quinacrine, an antimalarial drugwhich suppresses the production of arachidonic acid from phospholipidthrough inhibition of phospholipase A₂. Beginning 1 week aftera single i.v. dose of N-methyl-N-nitrosourea (MNU), female Sprague—Dawleyrats were fed a semi-purified diet supplemented with 0 or 75mg quinacrine/kg diet. Quinacrine reduced cancer incidence andcarcinoma multiplicity in rats administered 20 mg MNU/kg bodywt, but had no inhibitory activity in rats treated with 50 mgMNU/kg. These data suggest that pathways of arachldonic acidmeta-bolism in addition to cyclooxygenase present useful targetsfor mammary cancer chemoprevention. However, the chemo-preventiveactivity of quinacrine may be limited by toxicity.     

4-(hydroxyphenyl)retinamide selectively inhibits the development and progression of ductal hyperplastic lesions and carcinoma in situ in mammary gland.

In most previous chemoprevention studies on inhibition of mammary carcinogenesis, the formation of palpable tumors has been used as an end-point. Little is known about whether chemopreventive agents may similarly or selectively suppress hyperplastic and premalignant stages of the neoplastic process. In this study, we evaluated the effect of 4-(hydroxyphenyl)retinamide (4-HPR) on the development and progression of hyperplastic lesions and carcinoma in situ (CIS) in the N-methyl-N-nitrosourea (MNU) mammary carcinogenesis model in rats. 4-HPR was used as the chemopreventive agent because of its proven inhibitory effect on both the early and late phases of mammary carcinogenesis. Treatment with 4-HPR (2.0 mM/kg diet), beginning 2 days after MNU administration and administered continuously for 10 weeks, suppressed all mammary gland lesions (hyperplasia, CIS and invasive carcinoma) in 35% of animals. In the remaining 65%, 4-HPR allowed the development of hyperplastic lesions, alone or combined with CIS, and/or invasive carcinomas (CA). 4-HPR also increased by 2-fold the ratio between CIS and CA (0.75 per animal in control versus 1.5 in 4-HPR-treated animals), suggesting that it may also suppress the transition of CIS into CA. 4-HPR, when administered beginning 4 weeks after MNU administration [when hyperplastic and premalignant (CIS) lesions are present in the mammary gland], inhibited the frequency of terminal end bud hyperplasia (TEBH) and CA but did not significantly suppress ductal hyperplasia, ductal alveolar hyperplasia, alveolar hyperplasia and CIS. In these animals, 4-HPR induced partial disintegration of mostly peripheral areas of lesions, including carcinomas. Taken together, our data indicate that 4-HPR selectively suppresses the development and progression of hyperplastic lesions and CIS in TEBs. Furthermore, it appears that, in addition to mammary carcinomas, TEBH and CIS could also be used as end-point biomarkers in breast cancer chemoprevention studies.     

2-Deoxyglucose as an energy restriction mimetic agent: effects on mammary carcinogenesis and on mammary tumor cell growth in vitro

Dietary energy restriction (DER) is a potent inhibitor of carcinogenesis, but chronic DER in human populations is difficult to sustain. Consequently, interest exists in identifying energy restriction mimetic agents (ERMAs), agents that provide the health benefits of DER without reducing caloric intake. The selection of a candidate ERMAs for this study was based on evidence that DER inhibits carcinogenesis by limiting glucose availability. The study objective was to determine if 2-deoxyglucose (2-DG), a glucose analogue that blocks its metabolism, would inhibit mammary carcinogenesis. Pilot studies were done to establish a dietary concentration of 2-DG that would not affect growth. For the carcinogenesis study, ninety 21-day-old female Sprague-Dawley rats were injected i.p. with 50 mg of 1-methyl-1-nitrosourea per kilogram of body weight. Following injection, animals were ad libitum fed AIN-93G diet containing 0.00%, 0.02%, or 0.03% (w/w) 2-DG for 5 weeks. 2-DG decreased the incidence and multiplicity of mammary carcinomas and prolonged cancer latency (P < 0.05). The 0.02% dose of 2-DG had no effect on circulating levels of glucose, insulin, insulin-like growth factor-I, IGF binding protein-3, leptin, or body weight gain. Using MCF-7 human breast cancer cells to investigate the signaling pathways perturbed by disruption of glucose metabolism, 2-DG reduced cell growth and intracellular ATP in a dose- and time-dependent manner (P < 0.01). Treatment with 2-DG increased levels of phosphorylated AMP-activated protein kinase and Sirt-1 and reduced phosphorylated Akt (P < 0.05). These studies support the hypothesis that DER inhibits carcinogenesis, in part, by limiting glucose availability and that energy metabolism is a target for the development of ERMA for chemoprevention.     

Effect of dietary iron deficiency or excess on the induction of mammary carcinogenesis by 1-methyl-1-nitrosourea

The hypothesis that low body iron stores are protective against cancer whereas high body stores promote tumor occurrence was examined in the 1-methyl-1-nitrosourea (MNU)-induced experimental model for breast cancer. Twenty-one-day-old female Sprague-Dawley rats were randomized into one of three experimental groups and fed a formulation of AIN-76A diet modified to be low in iron (2 p.p.m.), or the same diet supplemented with an adequate (120 p.p.m.) or excess (1200 p.p.m.) amount of iron provided as FeSO4.7H2O. Rats were maintained on their respective diets throughout the experiment which was terminated 32 weeks post carcinogen administration. Rats were injected i.p. with either 25 mg MNU/kg body wt or the saline-solvent in which MNU was dissolved at 50 days of age. In the first 14 weeks, dietary iron deficiency resulted in a low hematocrit and a decrease in weight gain. The appearance of mammary tumors was markedly suppressed in this group compared to those given an adequate or excess level of iron. It has been reported in the literature that reduction in weight gain due to food restriction at a period immediately after carcinogen administration severely inhibits the subsequent development of tumors. Thus the low tumor incidence in the iron-deficient rats during this time frame could be attributed to the combined effects of low hematocrit and depressed weight gain. For the period between week 14 and week 32, the hematocrit in the iron-deficient animals was maintained at a normal level, and the body wt of these rats was comparable to that of the controls given an adequate level of iron. The rate of tumor appearance in the iron-deficient group during the second half of the experiment was similar to that of the iron-adequate group in the first half of the experiment. In other words, it appeared that once hematocrit and body wt gain were restored to normal in the iron-deficient animals, tumor incidence was only minimally affected by low dietary iron. In the second half of the experiment, the tumor incidence in the adequate iron group seemed to have plateaued, whereas it continued to rise in the excess iron group. Thus excess iron appears to be more prominent than iron deficiency in modification of mammary carcinogenesis, especially when the confounding effects of low hematocrit and reduced weight gain are taken into consideration in the latter case.