The specific objectives of this project include: 1) to determine the influence of type of diet and dietary ingredients in delaying the development of mammary tumors, 2) to determine the analogue and nontoxic dose of retinoid that can prevent/delay mammary cancer development, and 3) to determine the combination effects of different dietary supplements and therapeutic agents on delaying mammary tumor development. For this purpose transgenic mouse line TG.NK with c-neu the human breast cancer oncogene homologue of erbb2 that develops palpable mammary tumors after 20 weeks of age were a) fed different diets to delay the development of tumors, b) treated with selected doses of retinoids to prevent/delay tumor development and c) the most effective dietary supplements and therapeutic agents will be evaluated in combination to prevent mammary cancer for 12 months or longer. Nonpurified diets decreased the incidence and multiplicity, and delayed the development of mammary tumors as compared to a purified diet. Increasing the fiber content of nonpurified diet decreased the tumor incidence further. There is approximately 18- week interval between weaning and development of palpable mammary masses to evaluate intervention strategies to delay or prevent the development of mammary cancer in TG.NK mouse model. Fiber from nonpurified cereal ingredients appears to be highly beneficial in delaying the development of mammary cancer in TG.NK mouse and this observation is in agreement with human epidemiological findings. Therefore, the TG.NK transgenic mouse with oncogene c-neu (erbb2), appears to be a useful animal model for evaluation of dietary intervention strategies. NTP-2000 diet containing the retinoid analogue 4-Hydroxyphenyl retinamide (4-HPR) at 5 mmol/kg or an arotinoid Ro 40-8757 at 2 and 3 mmol/kg for 26 weeks. The 4-HPR at 5 mmol/kg diet delayed the development of palpable tumors up to 24 weeks, but by 26 weeks, the incidence was not significantly different from the NTP- 2000 diet control group. However, the 4-HPR diet markedly decreased the average weight of the tumors at 26 weeks. The 4-HPR diet also caused a significant increase in body weight without an increase in food consumption. The mice may tolerate higher doses of 4-HPR. Arotinoid Ro 40-8757 at both doses, inhibited the development of mammary tumors for the duration of the study. However, the Ro 40-8757 at 3 mmol/kg appeared to be toxic as indicated by a significant depression of the average body weight with alopecia and skin scaling in some mice. The 4-HPR at 7 mmol/kg diet delayed the development of palpable tumors up to 24 weeks, but by 26 weeks, the incidence markedly increased and was closer to the NTP-2000 diet control group. However, the 4-HPR diet markedly decreased the average weight of the tumors at 26 weeks with no decrease in multiplicity but caused a significant increase in liver weight without an effect on body weight. Arotinoid Ro 40-8757 at 1.5 and 2.5 mmol/kg diet caused a dose-related inhibition of mammary tumor development with significant decrease in the incidences, multiplicity and tumor weights as compared to the NTP-2000 diet control. The arotinoid at 2.5 mmol/kg diet caused marked decrease in number and branching of mammary ducts. Arotinoid also caused a dose-related and significant increase in liver weights without significant effect on body weights. The arotinoid but not 4-HPR at the doses tested decreased the circulating levels IGF-1. However, the IGF-1 levels did not show an association with the size, incidence or absence of tumors when evaluated for other treatment groups or for all mice in the study irrespective of treatment. The oncogene erbb2 (c-neu) expression was more prominent in the small and slow growing tumors of the mice treated with the arotinoid than in the tumors of the control group, indicating that erbb2 expression alone may not be adequate for growth of mammary tumors. The delay in onset of mammary tumors by the arotinoid may be related to the delay in development of mammary glands. Melatonin when given either by gavage or in diet at approximately 50 to 200 mg/kg body weight decreased the incidence and delayed the development of mammary tumors. Linolenic acid when administered by gavage as flaxseed oil did not decrease the incidence or delay the development of mammary tumors in the TG.NK mouse model. Silymarin in diet at approximately 80 to 320 mg/kg body weight did not delay or prevent, but may have promoted the development of mammary cancer in the TG.NK mouse model.Curcumin and indole-3-carbinol when given in diet did not delay the development of mammary cancer in the TG.NK mouse model. Recent study with the Tg.NK mouse model indicated that low dose of soy isoflavones may have the potential to delay the development of mammary tumors. However, higher doses (5 to 10 times) may have the potential to promote mammary cancer. In this study we exposed mice starting at 4 weeks of age, which is close to adult exposure. However, the normal human exposure to soy isoflavones will be during all stages of life including in utero, neonatal, prepuberty and adult. Therefore, it is necessary to expose the Tg.NK mice to different concentrations of soy isoflavones (by diet) during all stages of life to determine the beneficial effects and adverse effects of soy isoflavones on mammary cancer.The study to determine the effects of soy isoflavone exposure during all stages of life on mammary cancer development is scheduled to start soon.Studies with other phytoestrogens such as resveratrol are in progress with the Tg.NK transgenic mouse model.Current and future plans include studies with dietary supplements, therapeutic agents, and least toxic combinations of the above products to prevent the development of mammary cancer. Collaborative studies using this animal model in an European Union Project """"""""The Role of Phytoestrogens in the Prevention of Breast Cancer""""""""(EU Phytoprevent Project) with the National Institute of Public Health and Environment (RIVM) of the Netherlands and Danish Vterinary and Food Administration (VFA) of Denmark are in progress.
