{THESIS This is to certify that the thesis entitled EFFECTS OF DIETARY FAT 0N MAMMARY TUMOR DEVELOPMENT AND GROWTH AND RELATION TO THE ENDOCRINE SYSTEM presented by Charles Frederic Aylsworth has been accepted towards fulfillment of the requirements for Ph.D. degree in Physiology Date August 14, 1981 0—7 639 LIBRARY Michigan State University OVERDUE FINES: 25¢ per day per item RETURNIKS LIBRARY MATERIALS: N Piace in book return to remove charge from circulatton records EFFECTS OF DIETARY FAT ON MAMMARY TUMOR DEVELOPMENT AND GROWTH AND RELATION TO THE ENDOCRINE SYSTEM BY Charles Frederic Aylsworth A DISSERTATION Submitted to Michigan State University al fulfillment of the requirements for the degree of in parti DOCTOR OF PHILOSOPHY Department of Physiology 1981 Ros m\\® Pr‘ CO] ABSTRACT EFFECTS OF DIETARY FAT ON MAMMARY TUMOR DEVELOPMENT AND GROWTH AND RELATION TO THE ENDOCRINE SYSTEM By Charles Frederic Aylsworth l. Rats fed the 20.0% high fat (HF) diet consistently consumed less of their diet than rats fed the 4.5% control fat (CF) diet. After 4 days of dietary treatment, no differ— daily caloric consumption or body weight were ences in It was con- observed during the 4 week treatment period. cluded that the effects of HF diets on mammary tumor develop- ment are not mediated by an increased caloric intake in rats fed the HF diet. 2. The effects of HF diet consumption on serum pro— lactin levels during the estrous cycle of intact female rats and during the progesterone-induced surge of prolactin in ovariectomized, estrogen—primed rats were determined. No differences in serum prolactin levels were observed at any time during the estrous cycle or in the induced surge of prolactin in rats fed either the HF or the CF diet. It was concluded that the mechanism by which HF diet consumption Charles Frederic Aylsworth stimulates mammary tumor development does not involve alter— ations in serum prolactin levels. 3. The influence of estrogen and prolactin upon the stimulatory action of high dietary fat during mammary tumor development was examined. Female Sprague-Dawley rats, 55 days of age, were injected with DMBA, placed on either the CF or the HF diet, and subjected to various drug and endo- crine manipulations to maintain serum estrogen and prolactin at controlled levels. Sham operated and ovariectomized rats treated with both haloperidol and estradiol benzoate which were fed the HF diet showed a significant stimulation of mammary tumor development when compared with similarly treated rats fed the CF diet. These results demonstrate that HF diets can increase mammary tumor development in rats which have controlled levels of estrogen and prolactin, and suggest that HF diets may act directly on the incipient mammary tumor tissue to sensitize it to circulating hormone levels. 4. Promotional aspects of stimulated mammary tumori- genesis by HF diet were examined. rats were injected with DMBA and placed on high fat and low Female Sprague-Dawley fat diets for variable lengths of time at different periods during early tumorigenesis. Rats fed the HF diet for equal time intervals, but at different times during tumorigenesis, showed similar development of mammary tumors. Increasing the duration of HF diet treatment resulted in increased mammary tumor development, suggesting a time dose-response I114 Charles Frederic Aylsworth relationship. Removal of the HF diet treatment reversed the stimulatory effects of the HF diet on mammary tumor development. These results suggest that dietary fat may act as classical promotors to enhance mammary tumorigenesis. The effects of a high fat diet on the growth of 5. Female rats with established mammary tumors were studied. established DMBA—induced mammary tumors were placed on a HF and CF diet approximately 12 weeks after DMBA administra- Mammary tumor growth was significantly stimulated tion. in rats fed the HF in rats fed the HF diet. Furthermore, there was an increased number of newly palpable tumors diet, These data at the end of the 5 week treatment period. suggest that a HF diet can stimulate mammary tumor develop* ment and growth even during the later stages of tumorigenesis. 6. The effects of a HF diet on specific prolactin binding to DMBA—induced mammary tumors were investigated. Isolated membranes from mammary tumors obtained from rats fed either a HF or a CF diet were measured for specific prolactin binding by a radioreceptor assay. No differences in specific prolactin binding was observed in rats fed either a high or low fat diet. Therefore, the effects of HF diet on mammary tumor development do not appear to depend on changes in prolactin binding. DEDICATION I dedicate this dissertation to my loving wife, Janie; who, along with our families, gave me the necessary encouragement, inspiration, and love in my research endeavors. ii ACKNOWLEDGMENTS I wish to thank the members of my guidance committee; Dr. James Bennett, Dr. Robert Pittman, Dr. H. Allen Tucker, and Dr. Clifford Welsch, for their valued assistance in formulating and defining the research problem considered here. I would especially like to acknowledge and thank Dr. Joseph Meites for teaching me the value of scholarly thought and imaginative research, directed toward a mean— ingful problem. iii LIST LIST INTR( LITE] Ge] TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . vii LIST OF FIGURES. . . . . . . . . . . . . viii INTRODUCTION. . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . 5 General Theories of Tumorigenesis . . . . . . 5 Tumor Progression Theory of Tumorigenesis. . . 5 The Berenblum Hypothesis . . . . . . . . 9 Somatic Mutation Theory. . . . . . . . . 12 Models for Studying Mammary Tumorigenesis . . . 14 Chemical Carcinogens. . . . . . . . . . l4 Transplantable Mammary Tumors. . . . . . . 18 Human Breast Cancer Cell Lines . . . . . . 19 Role of Hormones in Murine Mammary Tumorigenesis . 20 Neuroendocrine Relationships . . . '. . . . 20 Control of Anterior Pituitary Prolactin Secretion. . . . . . . . . . . . 23 Role of Prolactin in Murine Mammary Tumorigenesis . . . . . . . . . . . 27 Role of Estrogen in Murine Mammary Tumorigenesis . . . . . . . .. . .. . 30 Role of Insulin in Murine Mammary TumorigeneSis. 33 Role of Progesterone in Murine Mammary Tumorigenesis . . . . . . . . . . . 34 Role of Growth Hormone in Murine Mammary Tumorigenesis . . . .' _. . . f - - - 36 Role of Adrenal GlucocortiCOids in Murine Mammary Tumorigenesis. . . . . . . . . 37 iv Cha m RTTBDDTS EXP] E: Chapter Page Nutrition and Mammary Cancer . . . . . . . . 38 Effects of Dietary Fat on Mammary TumorigeneSis . 38 Tumorigenesis. . . . . . . . . . 51 Effects of Dietary Protein Intake on Mammary Tumorigenesis. . . . . . . . . . . 55 Effects of Vitamin A and Its Derivatives on Mammary Tumorigenesis . . . . . . . . . 58 Effects of Antioxidants on Mammary Tumorigenesis. 61 MATERIALS AND METHODS . . . . . . . . . . . 65 Research Animals . . . . . . . . . . . . 65 Tumor Induction . . . . . . . . . . . . 65 Tumor Measurements . . . . . . . . . . . 66 Blood Sampling and Radioimmunoassays . . . . . 66 Dietary Treatments . . . . . . . . . . . 68 Drug and Endocrine Manipulations. . . . . . . 68 Tissue Preparation and Prolactin Receptor Assay. . 70 Statistical Analysis. . . . . . . . . . . 72 EXPERIMENTAL Q o O O O O 0 O O O O O O O 73 Estimate of Caloric Intake in Rats Fed High Fat and Control Fat Diets. . . . . . . . . . 73 Objectives . . . . . . . . . . . . . 73 Procedure. . . . . . . . . . . . . . 73 Results . . . . . . . . . . . . . . 74 Conclusions . . . . . . . . . . . . . 76 Effects of High Fat Diet on Serum Prolactin LeVels During the Estrous Cycle in Female Rats . 76 Objectives . . . . . . . . . . . 33 Procedure. . . . . . . . . . . . . 78 Results . . . . . . . . 80 Conclusions . . . . . . . . . . . . . Effects of High Fat Diet on the Surge of Prolactin Induced by Estrogen and Progesterone in 81 Ovariectomized Female Rats . . . . . . . . Obj eCtiVGS o o o a o o o ‘ ‘ 33]]: Procedure. . . . . . - . - ~ ° ' ' ‘ . . . . 82 Results 0 a o o c o c c I P 84 Conclusions . . . . . . . . . . . . Chap Tm ,_,_. n DISCI LIST Chapter Role of Prolactin and Estrogen in Stimulation by High Fat Diet of Development of DMBA-Induced Mammary Tumors . . . . . . . Objectives . . . . . . . . . . Procedure. . . . . . . . . . . Results . . . . . . . . . . Conclusions . . . . . . . . . o o c Relation of Length of Treatment With High Fat Diet and Carcinogen Administration to Mammary Tumor Development . . . . . . . . . Objectives . . . . . . . . . . Procedure. . . . . . . . . . . Results . . . . . . . . . . . Conclusions Effects of High Fat Diet on Mammary Tumor Growth in Rats With Established DMBA—Induced Mammary Tumors. . . . . . Objectives . . . . . . . . . . Procedure. . . . . . . . . Results . . . . . . . . . . . Conclusions . . . . . . . . . The Effects of a High Fat Diet on Specific Binding in DMBA-Induced Mammary Tumors . Objectives . . . . . . Procedure. . . . . . . . . . . Results . . . . . . . . . . . Conclusions . . . . . . . . . . DISCUSSION. . . . . . . . . . . . LIST OF REFERENCES . . . . . Page 98 98 99 100 106 110 110 111 111 113 116 116 117 117 119 120 125 Tabl LIST OF TABLES Composition of Variable Fat Diets (% of Total Diet) 0 O O O I I I Q 0 O O C O 0 Effects of Dietary and Endocrine Treatments on Serum Prolactin Levels . . . . . . Effects of Semi-Synthetic Control Fat and High Fat Diet on Mammary Tumor Development . . . Effects of Duration of High Fat Diet Treatment on Mammary Tumor Development: Evidence for a Time Dose-Response Relationship . . . Effects of High Fat Dietary Treatments of Equal Duration on Mammary Tumor Development When Applied at Different Times During Early Mammary Tumorigenesis . . . . . . Effects of High Fat Diet on Growth of Established Mammary Tumors. . . . . . vii Page 69 89 90 102 104 108 115 Fig LIST OF FIGURES Figure Page 1. Food Consumption, Estimate of Caloric Intake and Body Weight Gains in Rats Fed 4.5% Control Fat and 20.0% High Fat Diets . . . 75 2. Effects of High Fat Diet on Serum Prolactin Levels During the Estrous Cycle of Female Sprague-Dawley Rats. . . . . . . . . 79 3. Effects of High Fat Diet on the Surge of Prolactin Induced by Estrogen and PrOgesterone in Ovariectomized Female Rats . . . . . 83 4. Effect of High Fat Diet on Mammary Tumor Development: Influence of Normal or Reduced Circulating Levels of Prolactin and Estrogen . . . . . . . . . . . . 88 5. Effects of High Fat Diet on Mammary Tumor DevelOpment: Influence of Replacement of Circulating Levels of Estrogen and Prolactin or Only Estrogen. . . . . . . . . . 92 6. Effects of High Fat Diet on Mammary Tumor Development: Influence of Elevated Prolactin With or Without Estrogen Replacement . . . 95 7. Effects of High Fat and Control Fat Semi— synthetic Diets on Mammary Tumor Development . . . . . . . . . . . 101 8. Effects of Duration of High Fat Diet Treatment on Mammary Tumor Development. . . . . . 103 9. Effects of High Fat Dietary Treatments of Equal Duration Administered at Different Times During Early Mammary TumorigeneSis. . . . 107 viii ——f Figure Page 10. Effects of High Fat Diet on the Growth of Established DMBA-Induced Mammary Tumor Growth in Rats . . . . . . 112 11. Effects of High Fat Diet on Mammary Tumor Development During Latter Stages of Mammary Tumorigenesis: Newly Palpable Tumors . . . 114 12. Effects of High Fat Diets on Specific Prolactin Binding to Mammary Tumor Membranes . . . . 118 ix INTRODUCTION It has been suggested that most cancers which occur in humans are causally related to environmental factors and con— ditions. In recent years, nutrition repeatedly has become one of the most intriguing and extensively examined environ— mental factors in cancer research. The incidence of mammary carcinomas among women has been related specifically to various nutritional components, particularly to dietary intake of protein and fat (Carroll, 1975). Approximately 50% of the cancers developing in women of Western cultures are believed to be related to nutritional factors (Wynder, 1976). Positive correlations have been established between per capita consumption of dietary fat and protein, and the incidence of breast cancer in women (Carroll, 1975). Generally, women belonging to Western cul- tures, wnose members consume greater quantities of dietary fat, exhibit a higher incidence of breast cancer than their counterparts in Eastern cultures who consume less dietary fat. Further support for the role of dietary factors in the etiology of human breast cancer is provided by epidemio- lOgical studies which reported that breast cancer mortalities increased among second generation Japanese migrants to the United States. A primary behavioral modification inherent in the assumption of a Western lifestyle by these individ— uals was the consumption of diets containing larger amounts of fat and protein. Epidemiological evidence has been substantiated by wide— spread reports that high fat diets stimulate mammary tumor development in many rodent mammary systems, including spon- taneous, transplantable and carcinogen-induced tumors. Ani- mals fed high fat diets develop a greater number of mammary tumors, more rapidly and with a higher incidence, than in similarly treated rats fed a lower fat diet (Carrol, 1975). The influence of dietary fat treatment on mammary tumor development does not appear to involve a general caloric effect, since the caloric intake of rats fed high and low fat diets were similar even though the diets dif- fered in caloric content (Gammal et a1., 1967). Tannenbaum (1945) demonstrated that when the caloric intake was equally reduced in mice fed high fat and low fat diets, the stimu— latory effect of the high fat diet was still observed. Although it has been conclusively demonstrated that the consumption of high fat diets can stimulate murine mammary tUmorigenesis, no adequate mechanism has been proposed to wholly account for these observed effects. Since most of the murine mammary tumor models are highly hormone dependent and responsive, particularly with regard to estrOgen and prolactin, many of the investigations concerned with eluci- dating the mechanisms by which high fat diets stimulate on an inl esi IQI mammary tumor development have primarily focused on the pos- sible mediation of these effects through the endocrine system. Prolactin, in particular, has been implicated in the mediation of the influence of dietary fat on mammary tumor development. Chan and Cohen (1974) observed that the differential effects between high and low fat diets on mammary tumor development could be completely eliminiated by chronic suppression of anterior pituitary prolactin secretion. Inhibition of the influence of estrogen by chronic administration of an anti— estrogen drug reduced mammary tumor development, but could not remove the differential effects of high and low fat diets on mammary tumor development. The role of prolactin in the stimulation of mammary tumorigenesis by dietary fat was further suggested by reports that high fat diets elevated circulating levels of prolactin at various times during the estrous cycle. These observations led to the View by Chan et a1. (1975) that the enhancement of mammary tumorigenesis by high fat diets was mediated indirectly via the hypothalamic-hypophyseal system, to increase pituitary pro- lactin secretion rather than by a direct action of dietary lipids on the mammary tissue. More recent reports, however, have determined that these putative effects of high fat diets on serum prolactin levels may not be valid or have minimal significance in the mediation of enhanced mammary tumorigenesis by high fat diets (Cave et a1., 1979; Ip et a1. 1980). Further investigation into the effects of dietary fat on anterior pituitary prolactin secretion, and other end gen the of th f0] Die 011‘ max aPl tun man mot re; ige the bui as; endocrine involvement in the stimulation of mammary tumori- genesis by dietary fat appears to be warranted. The Berenblum hypothesis, or the initiation-promotion theory of tumorigenesis, has been used to describe the effects of high fat diets on mammary tumor development. Carroll and Khor (1970) have reported that the type of diet consumed following, but not prior to, carcinogen administration, is important in its influence on mammary tumor development. Dietary fat does not appear to have an effect on the metab- olism, distribution, or uptake of the carcinogen by the mam- mary gland during initiation (Gammal et a1., 1968). It appears, therefore, that high fat diets stimulate mammary tumor development by acting during the promotional stage of mammary tumorigenesis, but additional aspects of tumor pro- motion by high fat diets need to be explored. Upon consideration of the research studies already reported on the influence of high fat diets on mammary tumor- igenesis, it was of interest to further assess the role of the endocrine system, with particular emphasis on prolactin, but also on estrogen, and to further evaluate the promotional aspects of high fat diets on mammary tumor development. [ah—.3 LITERATURE REVIEW General Theories of Tumorigenesis The observable phenomenon of tumorigenesis has spawned a multiplicity of research designs, provoking the advancement of various theories describing the processes involved in tumor development. As the development of most tumors appears to be mediated by similar factors, integration and synthesis of research observations have generated the formation and proposal of theories of tumorigenesis. Three of the most prominent and widely accepted theories of tumorigenesis will be considered here. These include the Tumor Progression Theory as proposed by Leslie Foulds, the Berenblum Hypothesis and the Somatic Mutation Theory originally advanced by Theodor Boveri. In the discussion which follows, the basic principles on which these theories are established and their relation to mammary tumorigenesis will be explored. Tumor Progression Theory of Tumorigenesis Tumor progression, as defined by Foulds (1969) asserts that tumors develop by the acquisition of sequential, herit- able, qualitative changes in one or more of the biological properties of the cells contained in the tumor. This tumor ————_’ I progression concept attempts to explain these qualitative changes that occur during the development of tumors. Sug- gesting that the process of tumor progression is a multistage phenomenon, Foulds classified such development as the initi- ation, intermediate, and advanced phases of tumorigenesis (Foulds, 1969). As an incipient neoplasia progresses through these phases of tumorigenesis, irreversible qualitative changes are produced whereby lesions with increasing degrees of neoplastic capacity may or may not be observed. Upon exposure to an initiator, cells in the area of exposure undergo a series of irreversible changes in their biological composition which may or may not be accompanied by histological or proliferative alterations, thus estab- lishing a potential for tumor development. In the region exposed to the initiating agent, a state of incipient neo- plasia is established possessing a finite capacity for neo- plastic development. The induced incipient neoplasia per- sists for the lifetime of the animal and may or may not at any time undergo the qualitative changes of tumor Progression. Lesions which develop from the region of incipient neoplasia during the initiation phase have been classified by Foulds as Group A lesions. This lesion-type represents either side effects of the initiating agent or dead-end hyperplasias which are not neoplastic by histo- lOgical examination and are nonparticipants in further neo- plastic development. Conversely, the region of incipient neoplasia has the potential to progress through other phases IIIIIIIIIIIIIIIIIIIIIE::::_________________——T 7 tumorigenesis, thereby evolving lesions with greater neo- plastic potentials. An analysis of Foulds' second, or intermediate, phase of tumorigenesis demonstrates a characteristic emergence of the earliest recognizable non-malignant, precancerous (Group B) lesions representing various degrees of neoplastic severity. Group B lesions may undergo one of four fates: complete regression to the original state of incipient neo- plasia, an extensive static existence without qualitative or quantitative changes, growth without qualitative changes in structure or neoplastic potential, or finally, qualitative progression into lesions having greater neoplastic capacity. Thus, during Foulds' intermediate stage, Group B lesions may be derived directly from an incipient neoplasia or from other Group B lesions having a lesser degree of neoplastic severity. Foulds' third phase of tumor progression is the advanced phase, characterized by the presence of clinically defined, malignant (Group C) lesions. Most Group C lesions have the capacity to be derived directly from the region of incipient neoplasia or indirectly from other Group B or Group C lesions with lesser degrees of neoplastic and malignant qualities. An exception is the highly malignant "Type C3" lesion in which no counterpart has been observed in humans and results only after extended tranplantation of tumors in experimental animals. Type C lesions appear to have sole derivation from 3 other Group C lesions exhibiting a lesser degree of ma malignancy, and thus are derived directly from the region of incipient neoplasia. Estrogen-induced and spontaneous mammary neoplasia occurring in high incidence strains of mice have provided a basis for understanding the early stages of mammary tumor progression. Each group of lesions defined by Foulds is observable in a single animal suggesting that progression occurs independently in different tumors within the same animal. Group A lesions in the estrogen-induced hyperplasia of the mouse mammary gland are produced by the side effects of estrogen and include changes in the mammary gland resemb- ling cystic disease or an inflammatory response as often observed with mastitis. However, no correlation has been established between the development of such cystic—type lesions and later developing advanced stage mammary tumors. Group B lesions in mouse mammary tumorigenesis are primarily hyperplastic nodules and plaques. It is not thought that these lesions represent consecutive stages along a single course of neoplastic development, but rather are alternate pathways through which the incipient neoplasm may proceed. Both hyperplastic nodules and plaques have the capacity to progress to lesions possessing higher degrees of mammary neoplastic potential. It is thought that hyperplastic nodules and plaques account for the majority, but not all, of the advanced phase, or Group C lesions. Group C lesions are composed of a wide variety of adenocarcinomas and adenoacanthomas which, in the mouse, are largely hormone independent and unresponsive to endocrine ablation or treatment. The Berenblum Hypothesis The Berenblum hypothesis, or the two—step mechaniSm of tumorigenesis, advances the premise that cancerigenesis is a multistep phenomenon. Berenblum hypothesized that tumorigenesis is composed of two discrete stages, initiation and promotion. The terms initiation and promotion were first suggested by Friedwald and Rous (1944) from studies involved with tumors arising from rabbit ear epidermis treated with the carcinogen, benzypyrene. Berenblum (1954, 1969) derived his hypothesis primarily by focusing upon the mouse integument tumor system (Berenblum & Shubik, 1947). Similar to the Tumor Progression Theory, initiation according to the Berenblum Hypothesis is a rapidly occur- ring process in which permanent changes in the cellular phenotype are induced in the tissue involved. These per- manent changes are most probably invoked by mutation-like alterations in the genome of the cell. Such a hypothesis is supported by the observations that a wide range of initiating agents have been shown to specifically bind covalently to nucleic acids, particularly DNA, in many tissues sensitive to their carcinogenic action (Brooks & Lawley, 1964; Goshman & Hadelberger, 1967; Prodi et a1., 1970). Depending on the type and dose of the initiator used, tumors can be produced IIIIIIIIIIIIIIIIIIII:::T______________——i 10 either by application of the initiator alone, as in the case of a large dose of a complete carcinogen, or by treatment with a subthreshold dose of an incomplete initiator and sub- sequently treated with a promoting agent. In the latter case, limited treatment with an initiator produces "dormant" tumor cells which require further stimulation by promoting agents to proliferate into tumors. Furthermore, according to the Berenblum hypothesis, a prolonged interval between the initiation and promotion events does not decrease the result- ing tumor yield. Berenblum and Shubik (1947) reported that , an interval up to 43 weeks between initiation with benz— pyrene and promotion with croton oil did not decrease the yield of skin tumors in mice. In contrast to initiation, promotion in tumorigenesis is a slow process in which repeated administration of rela- tively large doses of the promoting agents are usually required. Also, unlike initiation, the effects of promotion can largely be reversed when the application of the promoting agent is discontinued (Berenblum & Shubik, 1947). It has been demonstrated that promotion occurs only when the pro- moting agents are applied after, but not prior to, initiation. Unlike the mechanism of initiation, promotion does not induce permanent changes in the genome but rather influences the expression of pre-existing alterations induced by the initiator. It has been suggested by Berenblum (1974) that promoting agents may act by binding to various regulatory IIIIIIIIIIIIIIIIIIIII::::________________——T 11 proteins to exert their effect. Furthermore, promotors do not appear to act by simply inducing non—specific hyperplasia of the initiated tissue, but rather may induce specific hyperplasia and/or delayed maturation of altered undiffer- entiated stem cells. This concept is particularly plausible when considering the epidermal tumor models. Berenblum's classic example of initiation-promotion is based upon the benzpyrene-croton oil model using the mouse integument tumor system (Berenblum, 1941). In these studies, administration of subcarcinogenic doses of the initiating agent, benzpyrene, resulted in the development of few or no epidermal tumors. However, when the promoting agent, croton oil, was administered following the subthreshold treatment with benzpyrene, tumor incidence was increased and the latency period of tumor development was decreased, indicating enhanced tumorigenesis. When croton oil was given alone or prior to the subcarcinogenic dose of benzpyrene, few or no tumors developed. These results would indicate that croton oil alone has little or no carcinogenic activity and that the promoting activity of croton oil is only effective when applied after initiation. Although the concepts of the initiation-promotion hypothesis of mammary tumorigenesis were derived from, and are best applicable to the mouse integument and rabbit epidermis tumor systems, the hypothesis also appears to be relevant to many rat mammary tumor models. Hormones, par- ticularly estrogen and prolactin, have often been classified m.‘ V( i 12 as promotors of carcinogen-induced mammary tumorigenesis. The role of hormones in mammary tumorigenesis will be explored in a subsequent section of this literature review. Promotion of mammary tumorigenesis has also been reported using the classical systemic promotor, phorbol (Armuth & Berenblum, 1974). In this study, rats treated twice weekly with intraperitoneal injections of phorbol for 10 weeks fol- lowing DMBA administration showed significantly augmented mammary tumor incidence when compared to rats treated with DMBA alone. However, in contrast to the effect of most promotors of skin tumorigenesis, administration of phorbol had no effect on the average latency period of mammary tumor development. Somatic Mutation Theory The permanent nature of the initiating event during tumorigenesis provided the premise upon which the Somatic Mutation Theory was evolved. Boveri (1914) suggested that alterations within the hereditary material of the cell (a "mutation" thereof) were responsible for the initiation of tumorigenesis. Credence to this hypothesis was not forth— coming for many years due to the failure to demonstrate that many carcinogenic substances could also cause mutations in various microbial and cell test systems. Maher et a1. (1968) and Miller and Miller (1966) reported that "activated forms" of non-mutagenic carcinogens, such as aminoacetyl-fluorine (AAF) were, in fact, able to cause mutations in bacterial IIIIIIIIIIIIIIIIIIIIIII-lllI---——_______I 13 test systems. Furthermore, it has been determined that most carcinogens are metabolized to an activated, mutagenic form in yiyg by cytochrome P450-dependent-microsomal-mixed- function oxidases which then act on the tissue to produce transformation. Subsequently, it has been shown that there is a positive correlation between the carcinogenicity and mutagenicity as determined by the Ames Salmonella/microsome test in which the enzymes required for metabolic activation of the carcinogens were provided (McCann et a1., 1975). Further evidence that somatic cell mutations may be responsible for the transformation of normal cells into cancer cells resulted from the observation that most carcinogenic compounds bind to DNA and could thereby induce tumorigenesis. Brooks and Lawley (1964) observed a correlation between the carcinogenicity of a compound in yiyg and its ability to bind to DNA. Binding to RNA and cellular protein, however, were not correlated to the carcinogenic potency of the substance. Finally, if one assumes that somatic cellular mutations are the primary factors involved in the induction of cancer, then those cells deficient in enzymes required for their DNA repair mechanisms should have a higher incidence of trans- formation than cells possessing normal DNA repair mechanisms. Cells deficient in DNA repair enzymes have an increased susceptibility to transformation by chemical carcinogens and ultraviolet radiation (Maher et a1., 1976; Maher et a1., 1977). Individuals with deficiencies in DNA repair mech- anism, as in xeroderma pigmentosium patients, have an —7— 14 extremely high incidence of skin cancer when exposed to ultraviolet radiation. Therefore, the relationship between the cells' ability to repair errors in DNA and cancer inci- dence, suggests that mutations cause tumorigenesis. The relationship between the somatic mutation theory and mammary tumorigenesis has been based primarily on the observation that many carcinogens shown to induce mammary tumors in rats also bind selectively to DNA in mammary cells and can transform mammary cells in YEEEQ (Janss et a1., 1972; Dao, 1970). Models for Studying Mammary Tumorigenesis The selection of a model for the study of human breast cancerigenesis should consider the following criteria. Initially, tumor induction should proceed with a relatively high frequency in a reasonable period of time utilizing laboratory animals suitable for such study with respect to size, ease of handling, and expense. Developing tumors should resemble human breast cancer both morphologically and in biological characteristics relating to local inva- siveness and metastasis to distant sites. Chemical Carcinogens Aminofluorenes Chemically induced mammary tumors have provided much information contributing to an understanding of the endo- crine effects on mammary tumorigenesis and growth. The IIIIIIIIIIIIIIIIIIIIIllllllll---———_____I 15 first chemicals to be successfully used in the induction of mammary tumors in experimental animals were the amino- fluorenes. Wilson et a1. (1941) first reported that 2- aminofluorene could initiate tumors in mammary tissue when administered in the diet. Bielschowsky (1944) showed that female Wistar rats fed aminoacetylfluorene (AAF) developed mammary tumors, primarily adenocarcinomas. Also reported was the observation of reduced incidence of AAF-induced mammary tumors in ovariectomized female or intact male rats. These observations suggest that the development of AAF- i induced mammary tumors is dependent on an ovarian influence. However, ovariectomy had little effect on the growth of established AAF-induced mammary tumors (Bielschowsky, 1944). Symeonidis (1954) reported that the susceptibility to carcinogenesis by AAF was greatest in immature female rats, and decreased with age. Polycyclic Aromatic Hydrocarbons By far, the most commonly cited class of chemical carcinogens that has been used in the study of mammary tumorigenesis are the polycyclic aromatic hydrocarbons. Shay et a1. (1949) found that repeated intragastric adminis— trations of 3-methy1cholanthrene (MC) resulted in the develop- ment of mammary adenocarcinomas in female Wistar rats. Shay et a1. (1952) also reported that mammary tumors could be induced in male rats and that the number and type of tumors that developed was largely influenced by hormone treatment. —7 16 Huggins et a1. (1958) reported that most of the carcinomas induced by MC were hormone sensitive tumors observing that ovariectomy resulted in a delay in tumor induction, whereas hypophysectomy completely prevented tumor induction. This suggests that both ovarian and pituitary factors influenced mammary tumor development induced by MC. Ovariectomy also was reported to cause rapid regression of established MC- ,induced mammary tumors. Seven-twelve Dimethylbenz(a)anthracene (DMBA) has become the most widely used carcinogen for the induction of mammary tumors in experimental animals in the last 20 years. Huggins et a1. (1959) reported that administration of DMBA induced largely hormone dependent mammary tumors with a relatively short latency period. Huggins et a1. (1961) further showed that a single intragastric administration of 20 mg of DMBA was as effective in inducing mammary tumorigenesis as repeated doses of MC. The optimum age for induction of mammary tumorigenesis by DMBA has been observed to occur between 50 and 65 days of age, after which time the mammary gland becomes relatively unresponsive to the carcinogen (Huggins et a1., 1961). Huggins (1965) also showed that a single intravenous injection of smaller doses of DMBA into rats 50 to 60 days of age results in the development of multiple mammary adenocarcinomas in Sprague-Dawley rats. Young et a1. (1963) reported that the first tumors to develop after administration of DMBA were largely adenocarcinomas which histologically resembled human breast cancer in that .____l —7—' 17 they were derived primarily from the ductal epithelial portion of the mammary gland. The hormone responsiveness of DMBA—induced mammary tumors also appears to be similar to human breast cancer. However, one important deficiency of the DMBA-induced mammary tumor is its inability to locally invade surrounding tissues and metastasize to dis- tant sites as often observed in human breast cancer (Young et a1., 1963). Polycyclic aromatic hydrocarbons by themselves are pre— carcinogens and like many other types of carcinogens require enzymatic activation. Metabolism of polycyclic aromatic hydrocarbons to ultimate carcinogens is by arylhydrocarbon hydroxylase (AHH). AHH is a cytochrome P450 dependent microsomal mixed function oxidase. AHH oxidizes polycyclic aromatic hydrocarbons to epoxides, which are thought to represent the active forms of these carcinogens. Nitrosamines Nitrosamines, such as N—methyl—N-nitrosourea (NMU) have been reported to be potent mammary gland carcinogens in BUF/n and Sprague-Dawley rats (Guillino et a1., 1975; Rose et a1., 1980). All of the mammary tumors induced by 3- monthly intravenous injections of NMU were adenocarcinomas and papillary carcinomas, most of which appeared to be hor- mone responsive (Guillino et a1., 1975). Recently, McCormick et a1. (1981) have shown that a single injection of NMU induces a high incidence of mammary tumors in Ira. IIIIIIIIIIIIIIIIIIIIIII::::______________——r 18 Sprague-Dawley rats in a dose-responsive fashion. However, the most important feature of the NMU-induced mammary tumor is its ability to metastasize to some small degree to bone, spleen, and lung tissue (Guillino et a1., 1975). Upon such consideration, the NMU—induced mammary tumor may prove to be a better model for the study of human breast cancer than other carcinogen—induced mammary tumor models which do not induce metastatic tumors. However, the degree of metastasis does not approach that occurring in human breast cancer. Transplantable Mammary Tumors Many transplantable mammary tumor cell lines have been developed. A major advantage to using the transplantable mammary tumor cell line is that much of the heterogeneity seen with the carcinogen—induced mammary tumors is eliminated. This is especially important for many biochemical investi- gations since primary carcinogen-induced mammary tumors often have diverse biological characteristics. Two of the most widely used transplantable tumors are the MTW9 and the R3230AC. The MTW9 transplantable mammary tumor was developed in Wistar/Furth rats bearing a somatomammotropic pituitary tumor (MtT), injected with a sub—carcinogenic dose (10 mg) of 3-methy1cholanthrene, and perpetuated in female rats bearing MtT (Kim & Furth, 1960). The MTW9 requires sus- tained elevated serum prolactin levels and physiological concentrations of ovarian steroids for growth. Elevation of —7 19 serum prolactin by either co—implantation of MtT, pituitary isografts to the kidney capsule, or by injection with periphenozine, have been reported to be required for develop- ment and growth of MTW9 (Kim & Furth, 1960; MacLeod et a1., 1964; Hollander & Diamond, 1978). Ovariectomy at the time of MTW9 transplantation, or after MTW9 has been established, inhibits development and growth of the tumor (MacLeod et a1., 1964; Kim & Furth, 1960). The R3230AC transplantable mammary adenocarcinoma was derived from a spontaneous mammary tumor in the Fisher rat (Hilf et a1., 1967). R3230AC is an autonomous tumor, grow- ing equally well in both intact or castrated, male or female rats. Unlike most carcinogen—induced and transplantable mammary tumors, exogenously administered estrogen and/or prolactin inhibited the growth of R3230AC in yiyg. Such treatment also appears to stimulate the differentiation of the tumor into a secretory tissue as indicated by the pres- ence of milk proteins such as casein and d-lactalbumin (Hilf et a1., 1967; Turkington & Riddle, 1969). Human Breast Cancer Cell Lines The cell line MCF—7, derived by Soule et a1. (1973) from a pleural effusion in a post-menopausal woman with metastatic breast cancer, is the most universally employed human breast cancer cell line. The MCF-7, unlike many other derived human breast cancer cell lines, has been well charac- terized as containing human mammary epithelial cells, using —7 20 morphological, chromososomal, and biochemical criteria. It has been reported to contain receptors for glucocorticoids, estrogens, progestins. androgens, and insulin, lending further support to its mammary epithelial derivation. These parallels in morphology are also associated with the hormone responsiveness of the cell line. Recently, other human breast cancer cell lines (the ZR-75 cell lines) have been derived from malignant effusions (Monaco & Lippman, 1978). These, like the MCF-7, have been characterized as derived from human mammary epithelial cells and are similarly hormone-responsive. Role of Hormones in Murine Mammary Tumorigenesis Neuroendocrine Relationships Treatments which influence neuroendocrine function dramatically affect murine mammary tumorigenesis. Drugs which act on the central nervous system, particularly the hypothalamus, have long been shown to influence murine mam— mary tumor development and growth. Lacassagne and Duplan (1959) first showed that tranquilizers, such as reserpine could hasten the development of spontaneous mammary tumors in C3H mice. Reserpine, a catecholamine storage inhibitor and depletor, also stimulated the growth of DMBA-induced mammary tumors in rats (Welsch & Meites, 1970). Hypo— thalamic dopamine activity is inversely correlated with mammary tumorigenesis and growth. Inhibition of dopamine activity by dopamine receptor blockers, such as haloperidol —7 21 and pimozide, also stimulated DMBA—induced mammary tumor growth (Quadri et a1., 1973; Hodson et a1., 1978). Other antidopaminergic drugs, such as perphenazine, sulpiride, and methyl-dopa, also stimulated mammary tumor development and growth (Pearson et a1., 1969; Pass & Meites, 1977; Quadri et a1., 1973). Drugs which increase hypothalamic dopamine activity inhibit mammary tumor development and growth. L—DOPA, the immediate precursor of dopamine inhibited growth of DMBA- induced mammary tumors (Quadri et a1., 1973). Dopamine agonists, such as various ergot alkaloids, piribedil, and iproniazid, suppressed growth of established carcinogen- induced mammary tumors (Cassell et a1., 1971; Hodson et a1., 1978; Nagasawa & Meites, 1970). An increase in hypo- thalamic dopaminergic activity by ergot alkoloids such as bromoergocryptine (CB-154) and ergocornine also inhibited mammary tumor development in mice and rats (Welsch & Gribler, 1973; Nagasawa & Morii, 1981. Treatments with drugs which influence hypothalamic norepinephrine and serotonin activity also affect mammary tumor growth in rats. Hodson et a1. (1978) reported that a decrease in brain serotonin activity by parachlorophenyl- alanine (PCPA) inhibited growth of DMBA-induced mammary tumors. Administration of the d-adrenergic agonist, clonidine, also inhibited DMBA-induced mammary tumor growth, suggesting that increased noradrenergic activity IIIIIIIIIIIIIIIII:::__________________——7 22 may have a suppressive effect on mammary tumor growth (Hodson et a1., 1978). Hypothalamic lesions also affect mammary tumorigenesis. Lesions of the median eminence area of the hypothalamus enhanced DMBA-induced mammary tumor growth (Welsch et a1., 1969; Klaiber et a1., 1969), whereas lesions of the preoptic area of the hypothalamus and the amygdala caused significant regression of these tumors (Welsch et a1., 1969). Median eminence lesions also stimulated spontaneous mammary tumor development in rats (Welsch et a1., 1970). However, median eminence lesions placed prior to carcinogen administration inhibited mammary tumor development (Klaiber et a1., 1969). Biuni and Montemurro (1971) also reported that lesions of the anterior and medial hypothalamic areas stimulated spon— taneous mammary tumor development in C302F female mice. Lesions of the posterior hypothalamus had no effect on mam- mary tumor development. Hypothalamic releasing factors such as TRH also stimu— lated mammary tumor growth by increasing serum prolactin levels (Chen et a1., 1977). Suppression of endogenous opioid activity with opiate antagonists, such as naloxone or naltrexone, inhibited mammary tumor growth as a result of inhibition of anterior pituitary prolactin secretion (Aylsworth et a1., 1979). 23 Control of Anterior Pituitary Prolactin Secretion Most of the above mentioned effects of hypothalamic lesions and drug treatment on mammary tumor development and growth are mediated by their action on pituitary prolactin secretion. Secretion of prolactin by the mammalian anterior pituitary is under a tonic inhibitory influence from the hypothalamus. Thus, separation of the anterior pituitary from this inhibitory influence by stalk section or trans- plantation of anterior pituitaries to ectopic sites resulted in enhanced prolactin secretion and elevated serum prolactin levels (Meites & Nicoll, 1966). Destructive lesions of the median eminence area of the hypothalamus also elevated serum prolactin levels (Meites et a1., 1963). Disruption of com- munication between the hypothalamus and anterior pituitary via the hypophysial portal vessels removes the predominantly inhibitory effect of the hypothalamus on secretion of pro- lactin by the anterior pituitary. Talwalker et a1. (1963) and Pasteels (1963) were first to demonstrate that the hypothalamus contains a factor that inhibited anterior pituitary secretion. Acid hypothalamic extracts were shown to suppress anterior pituitary prolactin secretion in yitgg. Pharmacological and physiological conditions, such as the suckling stimulus, estradiol, reserpine, and perphenazine, all known to elevate serum prolactin levels, were shown to decrease hypothalamic PIF activity (Ratner & Meites, 1964; Ratner et a1., 1965; Danon et a1., 1963). Evidence for a 24 prolactin releasing factor (PRF) also was demonstrated by Meites et a1., (1960) and Mishkinsky et a1. (1968). The chemical structures of these putative releasing and release inhibiting factors have not been identified. Hypothalamic neurotransmitters also are involved in the control of anterior pituitary prolactin secretion. These substances act either directly on the pituitary or indirectly by influencing the putative PIF and PRF peptides. Dopamine (DA) exerts an inhibitory effect on anterior pituitary pro- lactin secretion. Pharmacological agents that increase hypothalamic dopaminergic activity depress serum prolactin levels. Thus, dopamine agonists such as various ergot alkoloids, apomorphine, and piribedil, reduced serum pro- lactin levels (Nagasawa & Meites, 1970; Mueller et a1., 1976). Conversely, drugs which decrease hypothalamic dopaminergic activity, such as reserpine, chlorpromazine, and haloperidol, increased serum prolactin levels (Lu et a1., 1970; Dickerman et a1., 1973). Dopamine directly inhibited prolactin release from incubated anterior pitui— taries in yitrg (MacLeod, 1976). There is considerable eVidence that dopamine is the major physiological PIF (MacLeod, 1976; Greibrokk et a1., 1975). shaar and clemens (1974) reported that removal of catecholamines from hypo- thalamic extracts completely eliminated all prolactin inhibiting activity of these extracts in vitro. However, When dopamine receptors were blocked by haloperidol, pro- lactin secretion was still inhibited in a ——i 25 hypothalamic-pituitary co—incubation system, suggesting that inhibitory factors other than dopamine are present in the hypothalamus (Ojeda et a1., 1974). The role of norepinephrine (NE) in the control of anterior pituitary prolactin secretion is not clear. Injections of DOPS, a precursor of NE, increased prolactin release (Donoso et a1., 1971). Inhibition of NE synthesis by disulfram decreased prolactin release (Meites & Clemens, 1972). These observations suggest that the noradrenergic system may exert a stimulatory influence on prolactin secretion. However, other noradrenergic agonists, such as clonadine, depressed serum prolactin levels (Meites et a1., 1977; Hodson et a1., 1978). NE also directly inhibited prolactin release in yitrg (MacLeod, 1976). Serotonin influences anterior pituitary prolactin secretion ig vivo, presumably by its effect on hypothalamic PIF or PRF release. Injection of the serotonergic pre- cursors, tryptophan or 5 hydroxytryptophan, or serotonergic agonists, such as quipazine, elevated serum prolactin levels (Meites & Clemens, 1972; Meites et a1., 1977). Con- versely, serotonin antagonists, such as methysergide or PCPA, blocked the suckling-induced rise in prolactin in lactating rats (Kordon et a1., 1973; Blake et a1., 1973). Furthermore, the suckling induced rise in prolactin is associated with increased hypothalamic serotonergic activity (Kordon et a1., 1973). Thus, present evidence suggests that serotonin exerts a stimulatory effect on anterior pituitary secretion of prolactin. et at IIIIIIIIIIIIIIIIIE:::7________________—7 26 Acetylcholine reduced serum prolactin levels (Grandison et a1., 1974), an effect thought to be mediated through the adrenergic system (Grandison & Meites, 1976). However, a physiological role for acetylcholine on the control of pro- lactin secretion has not been established. More recently, the endogenous opiate peptides have been shown to influence anterior pituitary prolactin secretion. B-endorphin, leucine— and methionine-enkephalin, and other opiates increased serum prolactin levels (Meites et a1., 1979). Conversely opiate antagonists, such as naloxone, reduced serum prolactin levels (Meites et a1., 1979). The opiates reduced dopamine and increased serotonin activity in the hypothalamus (Meites et a1., 1979), actions that would increase prolactin secretion. Pituitary target tissue hormones also have an important influence on serum prolactin levels. Estrogen was shown repeatedly to increase serum prolactin levels, and can act directly on the anterior pituitary to stimulate prolactin secretion (Meites et a1., 1972). However, there also is evidence that the effects of estrogen on anterior pituitary prolactin secretion are mediated in part via hypothalamic mechanisms involving a decrease in PIF (DA) activity and/or an increase in PRF activity (Meites, 1979). Adrenal glucocorticoids decreased, whereas adrenal- ectomy increased serum prolactin levels (Chen et a1., 1976). Inhibition of prolactin secretion by glucocorticoids appears to be mediated by a direct action on the anterior —7—7 27 pituitary (Leung et a1., 1980). Thyroid hormones directly stimulate secretion of prolactin by the anterior pituitary in yitrg. Nicoll and Meites (1963) showed that addition of either thyroxine or triiodothyronine to anterior pituitary organ cultures increased prolactin release. However, radio— immunoassay of prolactin in the blood of hypo- or hyper- thyroid rats showed no significant change (Chen et a1., 1976). Role of Prolactin in Murine Mammary Tumorigenesis By influencing the control of anterior pituitary pro— lactin secretion, mammary tumor development and growth are dramatically affected. In general, treatments and physio- logical conditions that elevate serum prolactin stimulate mammary tumorigenesis, whereas treatments and physiological conditions that reduce serum prolactin inhibit mammary tumor- igenesis (Welsch & Nagasawa, 1977). Boyne et a1. (1973) and Hawkins et a1. (1976) reported that higher mammary tumor incidence was associated with strains of rats which have comparatively greater basal serum prolactin levels. Hyper- prolactinemia induced by grafted pituitaries or median eminence lesions enhanced spontaneous mammary tumor develop- ment in rats (Welsch et al., 1970a; Welsch et al., 1970b). Loeb and Kirtz (1937) showed that subcutaneous pituitary grafts increased mammary tumor incidence in mice. Hyper- prolactinemia produced in rats after carcinogen adminis- tration stimulated mammary tumor development, whereas 28 hyperprolactinemia induced prior to carcinogen adminis- tration inhibited mammary tumorigenesis (Klaiber et a1., 1969; Clemens et a1., 1968). Also, administration of reserpine or haloperidol to evelate serum prolactin prior to DMBA treatment, decreased mammary tumorigenesis (Welsch & Meites, 1970; Kledzik et a1., 1974). It appears that hyperprolactinemia stimulates growth of normal mammary tissue and development of a predominantly lobulo—alveolar system, rendering the mammary gland refractory to the action of the carcinogen (Welsch & Nagasawa, 1977). Hypophysectomy reduced the incidence of mammary tumors in mice (Korteweg & Thomas, 1946). Reduction of serum pro- lactin with ergot drugs or L-DOPA either prior to or after carcinogen treatment reduced mammary tumor development in rats (Clemens & Shaar, 1972; Kledzik et a1., 1974). In a recent report by Nagasawa and Morii (1981) temporary sup- pression of serum prolactin with CB-154 between weeks 4 and 11 of life suppressed subsequent spontaneous mammary tumor development in rats. Neuroendocrine treatments that elevate serum prolactin levels also stimulate growth of murine mammary tumors. These include median eminence lesions (Welsch et a1., 1969), pituitary homografts (Welsch et a1., 1968; Harada, 1976), and various dopamine antagonists (Quadri et a1., 1973; Pearson et a1., 1969; Hodson et a1., 1978; Pass & Meites, 1977). Physiological conditions which elevate serum prolactin levels, such as pregnancy (McCormick & Moon, 1965) and IIIIIIIIIIIIIIIIII:::T_______________——' 29 pesudopregnancy (Dao, 1959), also stimulated mammary tumor growth. However, during lactation, when serum prolactin levels are increased, most mammary tumors regressed (McCormick & Moon, 1965), probably because of enhanced adrenal glucocorticoid secretion (Aylsworth et a1., 1979). Elevated serum prolactin levels appear to be able to sustain mammary tumor growth, at least for a limited time period, even in the absence of estrogen. Welsch et a1. (1969) showed that hyperprolactinemia induced by median eminence lesions could maintain established mammary tumor growth for a limited period of time in the absence of ovarian influence. Administration of large doses of prolactin to ovariectomized rats also stimulated mammary tumor growth (Nagasawa & Yanai, 1970). Manni et al. (1977) reported that elevation of serum prolactin levels by perphenazine could support and restore DMBA-induced mammary tumor growth even if the influence of estrogen was removed by ovari- ectomy or Tamoxifen adminstration. However, Sinha et a1. (1973) reported that the effects of hyperprolactinemia on mammary tumor growth in the absence of ovarian influences were transitory, and that sustained mammary tumor growth required replacement with ovarian steroids. Prolactin can act directly on carcinogen-induced mammary tumor tissue in vitro to stimulate growth as measured by incorporation of 3H-thymidine into DNA (Lee et a1., 1975; Welsch & Rivera, 1972). However, estrogen combined with prolactin produced w] —:—— 30 a marked synergistic effect on in yitrg mammary tumor growth when compared with treatment using either hormone alone. Suppression of serum prolactin causes rapid regression of most established hormone—dependent mammary tumors. Hypophysectomy caused regression of most mammary tumors in the rat (Clifton & Sridharan, 1975), and upon replacement of prolactin these tumors resumed growth. Dopamine agonists such as pargyline, L-DOPA, and ergot alkoloids, reduced serum prolactin levels and inhibited rat mammary tumor growth (Quadri et a1., 1973; Nagasawa & Meites, 1970). Involvement of prolactin in the growth of human breast cancer is not as clear as in animal tumor models. Hypo- physectomy has been reported to cause regression of breast tumors in humans (Kennedy et a1., 1956; Pearson & Ray, 1960; Van Gilder & Goldenberg, 1975). However, regression of breast cancer in w0men by suppression of serum prolactin with L-DOPA and ergot drugs has rarely been reported (McGuire, 1975; Schultz et a1., 1973; Heuson et a1., 1972). Role of Estrogen in Murine Mammary Tumorigenesis Estrogen has a profound influence on mammary tumori- genesis. Removal of estrogen influence by ovariectomy or by use of anti-estrogen drugs, such as Tamoxifen, prior to or shortly after carcinogen administration inhibited mammary tumorigenesis (Dao, 1962; Jordon, 1976). Talwalker et a1. (1964) showed that replacement of estrogen following ovari- ectomy partially reversed the inhibitory effect of e1 p] dl Cl —7—' 31 ovariectomy on mammary tumorigenesis. Administration of low or moderate doses of estrogen increased mammary tumor incidence in mice and rats (Bern & Nandi, 1961; Dao, 1962). Since estrogen increases serum prolactin levels (Meites & Nicoll, 1966), it has been suggested that such a stimulatory effect is mediated, at least in part, through its effects on prolactin secretion (Furth, 1973). Brooks & Welsch (1974), and Welsch et a1. (1977) have shown that while chronic treatment with estrogen increased the incidence of mammary hyperplasias and tumor development in C3H mice, concurrent reduction of serum prolactin levels with CB-154 blocked the stimulatory effects of estrogen on mammary tumorigenesis. Estrogen also influences the growth of established carcinogen-induced tumors. Removal of estrogen by ovari— ectomy or Tamoxifen resulted in a rapid regression of most established carcinogen-induced mammary tumors (Huggins et a1., 1959; Jordan & Jaspar, 1976). Replacement with estrogen reversed regression of mammary tumors induced by ovari- ectomy. Administration of low or moderate doses of estrogen to intact DMBA-induced tumor bearing rats also stimulated mammary tumor growth (Huggins et a1., 1962). However, estrogen has no effect on mammary tumor growth in the absence of the pituitary (Sterental et a1., 1963). Large doses of estrogen inhibited mammary tumor growth and induced tumor regression (Huggins et a1., 1962). These inhibitory effects of large doses of estrogen can be reversed by concurrent administration of large doses of prolactin i 32 (Meites et a1., 1971). Large concentrations of estrogen also can inhibit the action of prolactin in XAEEQ (Welsch & Rivera, 1972), an effect that can be reversed by increasing the amount of prolactin added to the culture media (Chan et a1., 1976). Since there is no evidence that large doses of estrogen inhibit pituitary prolactin secretion, it is likely that estrogen acts on the tumor tissue to antagonize the action of prolactin. Evidence of such an inhibitory effect of pharmacological doses of estrogen on mammary tumor growth was reported by Kledzik et a1. (1976). These investigators showed that large doses of estrogen reduced specific prolactin binding to mammary tumor membranes and thereby inhibited the peripheral action of prolactin on the tumor. Estrogen stimulated incorporation of 3H-leucine into protein in cultures of DMBAvinduced tumor tissue (Lee et a1., 1975). However, Welsch and Rivera (1972) reported that estrogen over a wide range of concentrations was either without effect or inhibited in vitro DNA synthesis. Estro- gen may not be essential for carcinogen-induced murine mam- mary tumorigenesis under some experimental conditions, but it does appear to have an important stimulatory role in mammary tumor development and growth under most physio- lOgical conditions, by acting directly on the mammary tumor tissue and indirectly through its influence on anterior pituitary prolactin secretion. p1 Min man all inl Leg bee of Col 33 Role of Insulin in Murine Mammary Tumorigenesis Insulin appears to have an important effect on murine mammary tumorigenesis and growth. Removal of insulin by alloxan—induced diabetes shortly after DMBA administration inhibited mammary tumor development in rats (Heuson & Legros, 1972). Induction of diabetes by alloxan in rats bearing DMBA—induced mammary tumors caused regression of 90% of the tumors present (Heuson & Legros, 1972). Similarly, Cohen and Hilf (1974) reported that streptozotocin-induced diabetes also caused regression of 60% of the DMBA-induced mammary tumors present, and that replacement with insulin reversed the regression. Administration of relatively large doses of insulin to intact rats stimulated DMBA-induced mammary tumor growth (Heuson et a1., 1972). However, the effects of insulin removal or administration on mammary tumor growth depends on the tumor model used. Growth of the R3230AC transplantable, autonomous, but hormone responsive mammary adenocarcinoma, was inhibited by administration of insulin, whereas growth was enhanced in diabetic rats (Cohen & Hilf, 1975). Removal of insulin also blocked the growth of MCF-7 human breast cancer cells transplanted into diabetic athymic nude mice (Shafie, 1980). Insulin receptors have been identified in both rat mammary tumors and in human breast cancers (Hilf et a1., 1978; Holdaway and Friesen, 1977). Recently, Shafie and Hilf (1981) found a positive correlation between insulin bin mam I5]? 34 binding and the magnitude of the biological response of the mammary tumor tissue to insulin ig yiggg. The presence of insulin is required by most DMBA-induced mammary tumors to proliferate in culture (Heuson et a1., 1966). Heuson and Legros (1971) also showed that insulin could stimulate DNA synthesis in most DMBA—induced mammary tumors in yitrg. Insulin also increased DNA synthesis in human breast cancer tissue i5 yitrg (Welsch et a1., 1976). It has been suggested that the effect of insulin on mammary tumor growth may be mediated through its effect on prolactin receptors. In support of this concept, Smith et a1. (1977) showed that removal of insulin by streptozotocin- induced diabetes decreased prolactin receptors in the regres— sing, insulin-dependent tumors. This would suggest that insulin deprivation can render the tumors less responsive to circulating prolactin levels and may thereby cause regression of these tumors. Role of Progesterone in Murine Mammary Tumorigenesis Progesterone is generally thought to have a stimulatory effect on murine mammary tumorigenesis. Huggins et a1. (1962) showed that treatment with progesterone 1 month after DMBA administration shortened the latency period of mammary tumor appearance and increased the number of developing tumors. Jabara (1967) similarly showed that progesterone treatment just prior to and following DMBA treatment enhanced mammary tumor development. Pregnancy induced shortly after 196 be] mar tei pr) llld 35 DMBA also enhanced mammary tumorigenesis (Huggins et a1., 1962). Increased progesterone secretion during pregnancy is believed to be primarily responsible for stimulation of mammary tumor development. However, progesterone adminis- tered 20 days prior to and 20 days following DMBA adminis- tration inhibited mammary tumorigenesis (Kledzik et a1., 1974). Thus, as with prolactin, increased progesterone prior to carcinogen administration inhibits, whereas increased progesterone after carcinogen administration stimulates mammary tumorigenesis. The effect of progesterone on the growth of existing mammary tumors is not clear. Huggins et a1. (1962) reported that progesterone treatment stimulated growth of estab- lished DMBA—induced mammary tumors. Jabara (1967) however, showed that exogenous progesterone had little or no effect on growth of established mammary tumors. Pregnancy stimu- lated DMBA—induced mammary tumor growth, but whether increased progesterone secretion or some other pregnancy hormone such as placental lactogen was primarily responsible for the enhanced growth, has not been determined (Huggins et a1., 1962; McCormick & Moon, 1965). Other tumor models, such as the transplantable rat mammary carcinoma MTW9 and the pregnancy dependent TPD MT—4 mouse mammary tumor appear to be more progesterone dependent and responsive than many of the carcinogen-induced mammary tumor models (Diamond et a1., 1980; Matsezawa & Yamamoto, 1975). Progesterone also —7—' 36 appears to be of primary importance in promoting mammary tumorigenesis in dogs (Briggs, 1977). Role of Growth Hormone in Murine Mammary Tumorigenesis The effects of growth hormone (GH) in murine mammary tumorigenesis and growth are largely permissive or supple— mentary to other endocrine influences. Talwalker et a1. (1964) reported that GH and prolactin were somewhat more effective than prolactin alone in promoting carcinogen— induced mammary tumorigenesis and growth in ovariectomized rats. Administration of GH was shown to have either no effect or a slight stimulatory effect on the growth of established carcinogen-induced mammary tumors in intact rats (Nagasawa & Yanai, 1970; Li & Yang, 1974). Injections of GH overcame the inhibitory effects of protein deficiency on mammary tumor growth, but were without effect on mammary tumor growth in rats fed normal protein rations (Welsch & Meites, 1978). Median eminence lesions which suppress GH, but increase prolactin secretion by the anterior pituitary, caused rapid growth of mammary tumors. Iturri and Welsch (1976) showed that GH caused a slight stimulation of carcinogen-induced rat mammary tumors in 23539. However, these effects were much less than the stimu— latory effects of prolactin. Also, Welsch et a1. (1978) found that GH was ineffective in stimulating incorporation 3 Of H-thymidine into DNA in organ cultures of human breast Ill. 37 tumors. Thus, GH does not appear to have an important role in mammary tumor growth. Role of Adrenal Glucocorticoids in Murine Mammary Tumorigenesis Large doses of adrenal glucocorticoids inhibit mammary tumor growth ig yiyg and in yitrg. The inhibitory effects of glucocorticoids on mammary tumor growth in yiyg may be by a direct action on mammary tumor tissue, or by an indirect mechanism through inhibition of anterior pituitary prolactin secretion (Schwinn et a1., 1976). Chen et a1. (1976) showed that adrenalectomy stimulated mammary tumor growth and ele- vated serum prolactin levels, whereas glucocorticoid replacement inhibited mammary tumor growth and reduced serum prolactin levels. Hilf et a1. (1965) noted that exo— genous administration of glucocorticoids to intact rats inhibited the growth of transplantable mammary tumors. Brennan (1973) and Hayward (1970) have shown that adminis- tration of glucocorticoids decreased mammary tumor growth in women. Elevated adrenal glucocorticoid secretion also appears to be involved in regression of established mammary tumors during post-partum lactation, since adrenalectomy after parturition blocked the regression and restored mam— mary tumor growth equal to that of non-lactating intact controls (Aylsworth et a1., 1979). Aylsworth et a1. (1979) showed that the synthetic glucocorticoid, dexamethasone, inhibited established DMBA-induced mammary tumor growth even in the presence of elevated serum prolactin levels, SU IIIIIIIIIIIIIIIIIII[:::T______________——' 38 suggesting that glucocorticoids may act directly on mammary tumor tissue to inhibit growth. Glucocorticoids appear to be necessary for prolactin to maximally stimulate DNA synthesis in yitrg (Lewis & Hallowes, 1974). However, high concentrations of gluco- corticoids inhibited growth of DMBA-induced rat mammary tumors and human breast cancer cells in yitgg (Koyama et a1., 1972; Osborne et a1., 1979). Therefore, glucocorticoids may have a permissive role in mammary tumorigenesis, but in high doses are inhibitory to this process. Nutrition and Mammary Cancer Effects of Dietary Fat on Mammary Tumorigenesis A nutritional factor related to mammary tumors that has generated much interest in recent years is dietary fat. One of the first reports that linked increased fat consump- tion to enhanced mammary tumor development was the classic study by Tannenbaum (1942) who showed that high fat diets fed to C H and DBA mice resulted in an increased incidence 3 of spontaneous mammary tumors. Diets which contained high levels of olive or corn oil also increased the incidence of spontaneously developing mammary fibroadenomas in Sprague- Dawley rats (Benson et a1., 1956; Davis et a1., 1956). Dunning et a1. (1949), Engel and Copeland (1951) and Gammal et a1. (1967) showed that diets containing high levels of fat (corn oil) enhanced development of mammary tumors 39 induced by diethylstilbesterol (DES), AAF, and DMBA. More recent evidence has shown that high fat diets stimulated mammary tumorigenesis in many different carcinogen-induced and transplantable tumors in both mice and rats (Hillyard & Abraham, 1979; Carroll & Khor, 1975). Contributing to the increased interest in studying the effects of dietary lipids on breast carcinogenesis is the strong positive cor— relation found between breast cancer incidence and dietary fat consumption among women (Carroll & Khor, 1975). Initiation Versus Promotional Action of Dietary Fat on Mammary Tumorigenesis Since mammary adipose tissue has been proposed to act as a depot or storage site for administered hydrocarbon- type carcinogens (Dao & Sunderland, 1959), it was suggested that the effects of high fat diets may be mediated through the adipose surrounding the susceptable mammary gland. This hypothesis to explain stimulation of mammary tumorigenesis by high fat dietary intake appears unacceptable, since most reports have shown that the level of fat intake after car- cinogen administration is more important in determining mammary tumor development than the level of dietary fat intake before carcinogen administration (Carroll & Khor, 1970; Hopkins et a1., 1976). In contrast, 1p (1980) showed that a delay of initiation of high fat diet treatment by up to 20 weeks after DMBA still resulted in enhanced mammary tumor development. Carroll and Khor (1975) reported that a 40 high fat diet treatment initiated 1-2 weeks after DMBA enhanced mammary tumorigenesis. However, in contrast to Ip (1980), if they delayed the dietary treatment by 4 weeks after DMBA, little or no enhancement of mammary tumorigenesis was observed. Furthermore, Gammal et a1. (1968) showed that 3H—DMBA by the mammary gland and its uptake and clearance of fat pad is not altered to any appreciable degree by dietary fat consumption, and concluded that the effects of dietary lipids on mammary tumorigenesis is not likely to be mediated by such a mechanism. However, Janss et a1. (1972) showed that DMBA binds to DNA in mammary parenchymal cells and can persist there for up to 2 weeks after DMBA administration. It has been suggested therefore, that high fat diets may still have a direct effect on carcinogen activity at the mammary gland even after most of the DMBA has been cleared from the circulation. Since Berenblum (1969) has defined a promoter as an agent that augments tumor induction when administered after completion of the initiating action, but not when administered before initiation, most of the evidence would indicate that high fat diets act as promoters in mammary tumorigenesis rather than as initiators. Influence of Type of Dietary Fat The type of fat consumed in high fat diets has a sig~ nificant effect on the tumor enhancing ability of the diet. Gammal et al. (1967) demonstrated that high fat diets con- taining corn oil, which consists mainly of unsaturated fatty 41 acids, stimulated mammary tumorigenesis to a greater degree than a similar high fat diet that contained coconut oil which consists mainly of saturated fatty acids. Carroll and Khor (1971) used 10 different fats and oils in their high fat diets, and determined that in general, high fat diets that contained primarily unsaturated fatty acids stimulated mammary tumorigenesis to a greater extent than equally high fat diets that contained primarily saturated fatty acids. Hopkins and Carroll (1979) used high fat diets containing different amounts of unsaturated and satu— rated fatty acids to show that a certain amount of unsatu- rated fat, as well as high levels of dietary fat, are required to enhance mammary tumorigenesis. They reported that a diet containing 3% sunflower oil (which contains primarily unsaturated fatty acids) and 17% tallow or coco- nut oil (which contain primarily saturated fatty acids) was just as effective in stimulating mammary tumor development as a 20% sunflower oil diet, and was much more effective in stimulating mammary tumorigenesis than diets containing either 3% sunflower oil or 17% tallow or coconut oil. Recent evidence has implicated linoleic acid as the most important unsaturated fatty acid involved in stimu- lation of mammary tumorigenesis by high fat diets. Hillyard and Abraham (1979) suggested that linoleic acid is mainly responsible for stimulating mammary tumor growth by showing that 0.1% linoleic acid added to a fat free diet was as effective in stimulating mammary tumor growth as a 15% corn 42 oil diet. These results appear to contradict the report of Hopkins and Carroll (1979), that both certain amounts of unsaturated fatty acids and high levels of dietary fat were required to enhance tumorigenesis. Tinsley et a1. (1981) used multiple regression statistical tests to isolate the effects of single fatty acids on mammary tumor development. They reported that increasing levels of linoleic acid in the diet was most strongly associated with increased mammary tumorigenesis (i.e., linoleic acid had the highest regression coefficient of all fatty acids tested). Further indication that linoleic acid is of primary importance in stimulating mammary tumorigenesis and growth was shown by Wicha et a1. (1979). These investigators showed that normal and neoplastic mammary cell growth could be stimulated in yigrg by addition of unsaturated fatty acids, especially linoleic acid, and inhibited by addition of saturated fatty acids. Proposed Mechanisms by Which High Fat Diets Stimulate Mammary Tumorigenesis General caloric effect of high fat diets. Since most of the experimental diets used to investigate the effects of dietary lipids on mammary tumorigenesis have a higher caloric content than the comparable control or low fat diets, it has been suggested that these differences may be the means by which high fat diets stimulate mammary tumor devel- opment. However, Tannenbaum (1945) showed that even though 43 caloric restriction inhibited mammary tumorigenesis in mice, at any specific degree of caloric restriction, high fat diets were less inhibitory than similarly restricted diets that contained lower amounts of fat. Thus, the effects of high fat diets on mammary tumorigenesis appears to be due to the specific action of the fat rather than increased caloric intake. Furthermore, Gammal et a1. (1967) and other inves— tigators showed that rats fed a low fat diet consume greater amounts of the diet than similar rats fed high fat diets, resulting in similar caloric consumption of the rats fed low and high fat diets. Also, these and other investigators have reported that the growth rates and body weight gains of rats fed diets containing different levels of fat were similar. Therefore, a general caloric effect of high fat diets is not thought to be the mechanism by which high fat diets stimulate mammary tumorigenesis. Mechanisms involving the endocrine system. Since estrogen and prolactin are two of the most important hor- mones involved in murine mammary tumorigenesis (Meites, 1972), most of the investigations of endocrine involvement in the effects of dietary fat on mammary tumorigenesis have focused on these hormones. Chan and Cohen (1974) were first to implicate prolactin in stimulation of mammary tumori- genesis by high fat diets. They reported that when serum prolactin levels were chronically decreased by CB-154 (bromo- ergocryptine), mammary tumor development was decreased and the the rec hig de( 44 the differential effects of high fat and low fat diets on mammary tumorigenesis were eliminated. Also reported was the observation that when Nafoxidine, a specific estrogen receptor blocker, was chronically administered to rats fed high fat and low fat diets, mammary tumor development was decreased, but the differential effects of the diets were not eliminated. These findings led to the suggestion that the enhancement of mammary tumorigenesis by high fat diets was mediated through alterations in circulating levels of pro- lactin, but not estrogens. Further evidence in support of this hypothesis was provided by Chan et a1. (1975, 1977), who reported that serum prolactin levels during the after- noon of the proestrous-estrous stage of the estrous cycle was elevated in rats fed high fat diets, and suggested that the enhancement of mammary tumor development by high fat diets are mediated indirectly by increased prolactin secretion via the hypothalamic-pituitary system rather than by a direct action of the fat on the mammary gland itself. One must note however, the small sample size and large variability present in the prolactin values reported by these authors. Conversely, Cave et a1. (1979) found no consistent elevation of serum prolactin levels in rats fed high fat diets and no increase in prolactin synthesis in 23339 in pituitaries from rats fed high fat diets. Thus, the effects of dietary fat on serum prolactin levels is not clear and is a problem which is addressed in the experi— mental section of this thesis. lac die lac re} 45 Ip et al. (1980) attempted to clarify the role of pro- lactin in the promotion of mammary tumorigenesis by high fat diets using median eminence lesions to elevate serum pro- lactin levels in rats fed high and low fat diets. Ip reported that although serum prolactin levels were similar in both the high fat and low fat median eminence lesioned rats, tumor incidence in the rats fed a high fat diet was still greater than in the rats fed a low fat diet. The authors concluded that although prolactin may partially mediate the effects of high fat diets on mammary tumori— genesis, other factors must be involved. High fat diets also stimulated growth of the hormone responsive transplantable mammary adenocarcinoma R3230AC (Hillyard & Abraham, 1979) which is inhibited by elevated serum prolactin levels (Hilf et a1., 1971). This would imply that an increase in serum prolactin levels as hypothesized by Chan et a1. (1975) is not involved in mediating the effects of high fat diets on the growth of this type of mammary tumor. Chan et a1. (1977) also reported small increases in circulating estrogen levels at certain stages of the estrous cycle in rats fed high fat diets, but do not attribute this effect to stimulation of mammary tumorigenesis by high fat diets. Furthermore, Carroll and Khor (1975) reported that 3H-estradiol distribution and clearance from the mammary gland is not different in rats that were fed high fat and control fat diets. Thus, it would appear that estrogen secretion and metabolism are not appreciably altered by high IIIIIIIIIIIIIIIII3::7_________________——7 46 dietary fat and do not provide an adequate explanation for the stimulatory effects of high fat diets on mammary tumorigenesis. It is possible that the mammary gland and incipient mammary tumor tissue of rats on high fat diets may be more sensitive to circulating levels of prolactin and estrogen and could thereby show enhanced tumorigenesis. Mammary glands from rats fed high fat diets showed more histological evidence of hypertrophy and secretory activity in response to implant DES than rats fed a lower fat diet (Dunning et a1., 1949). This stimulated response of the mammary glands in rats fed high fat diets was also correlated with increased incidence of mammary tumors. Recently, Cave et a1. (1981) reported that mammary tumors from rats fed a high fat diet showed an increase in lactogenic hormone binding activity when compared to mammary tumors from rats fed a low fat diet. Thus, mammary glands and tumors from rats fed high fat diets may be more responsive to normal circulating levels of estrogen and prolactin, and thereby cause enhanced mammary tumorigenesis. Recent reports have also implicated the possible involvement of prostaglandins in the mediation of the effects of high fat diets on mammary tumorigenesis. Inhibition of prostaglandin synthesis by administration of indomethecin prevented the growth promoting effects of high unsaturated fat diet consumption on a transplantable mammary tumor in BALB/c mice (Hillyard & Abraham, 1979). —7—7 47 Mechanisms involving the immune system. The immune system plays an integral role in tumorigenesis (Chirigos, 1977), and has been implicated in mediating the effects of high fat diets on mammary tumorigenesis. There is ample evidence that polyunsaturated fatty acids are capable of suppressing immune function. Offner and Glauson (1974) showed that polyunsaturated fatty acids can inhibit antigen— induced proliferation of lymphocytes in yitrg. Kollmorgen et a1. (1979) reported that the conconavalin A-induced blastogenesis of spleen lymphocytes from rats fed a high fat diet was inhibited as compared to spleen lymphocytes from rats fed low fat diets. Also, nonspecific stimulation of the immune system inhibited stimulation of mammary tumorigenesis by high fat diets (Kollmorgen et a1., 1979). Polyunsaturated fatty acids increased the survival of skin allografts in rodents (Ring et a1., 1974) and are an effect— ive immunosuppressive therapeutic agent following renal transplantation in humans (Uldell et a1., 1975). PGEl and PGE2 inhibited immune responses in lymphocyte test systems (Plecia et a1., 1975). Since polyunsaturated fatty acids such as linoleic acid are precursors of prostaglandins, mediation of the immunosuppressive effects of high unsatu- rated fat diets by prostaglandins is possible. As mentioned above, inhibition of prostaglandin synthesis by indomethecin blocks the stimulation of mammary tumorigenesis by high fat diets (Hillyard & Abraham, 1979). Thus, high fat diets containing large amounts of polyunsaturated fatty acids, may the tun 48 themselves or via prostaglandins, suppress cell mediated immune response systems and thereby enhance mammary tumorigenesis. Direct mechanisms by which high fat diets stimulate mammary tumorigenesis. In addition to indirect mechanisms involving the endocrine and immune systems, direct effects of high fat diets on the mammary gland and tumor tissue may explain enhanced mammary tumorigenesis by high fat diets. Tannenbaum (1945) postulated a direct metabolic stimulation of potential tumor cells by dietary fat. Dunning et a1. (1949) showed that high fat diets may cause an increased sensitization of mammary glands and tumors to administered DES. Gammal et a1. (1967) showed that the composition of mammary tissue reflects the type of fat that is consumed in the diet. Thus, rats which are fed diets containing high levels of corn oil show increased levels of unsaturated fatty acids in their mammary tissue, particularly linoleic acid, the principle fatty acid of corn oil. Membranes with such altered fatty acid composition have different biophysi— cal characteristics than membranes containing lesser amounts Of polyunsaturated fatty acids. DeKruyff et a1. (1973) showed an increased equilibrium flux of l4C-erythritol across the membranes of Acholephasma Laidlawaii cells grown on linoleic acid enriched media. Thus, the passage of erythritol across these membranes appeared to be enhanced by incorporation of linoleic acid into the membranes. It is p0: m01 49 possible that the passage of other substances such as hor- mones or nutrients into mammary gland or tumor cells may also be enhanced by incorporation of polyunsaturated fatty acids into these membranes of animals fed high fat diets and thereby provide more favorable conditions for mammary tumors to develop and grow. Polyunsaturated fatty acids are converted to lipid peroxides by normal free radical reactions involved with fatty acid metabolism. Since lipid peroxidation has been associated with carcinogenesis (Shamberger et a1., 1973), the possibility that high fat diets exert their effects on mam— mary tumorigenesis through such a mechanism has been pro— posed recently. King et a1. (1979) reported that anti- oxidants such as butylhydroxytoluene (BHT) decreased the stimulation of mammary tumor development by saturated and unsaturated high fat diets. High fat diets which were deficient in selenium, an antioxidant, caused further stimu- lation of mammary tumorigenesis when compared to high fat diets containing normal amounts of selenium (Ip & Sinha, 1981). Chan and Dao (1981), and Carroll (1975) reported that rats fed a non-purified laboratory rat chow showed decreased DMBA-induced carcinogenesis when compared to puri— fied diets containing equal amounts of fat. It is worthy of note that antioxidants such as BHT are routinely added to commercially prepared non-purified rat chow, and may therefore account for the difference observed. IIIIIIIIIIIIIIIII::::________________——T 50 Relation to the Human Condition Experimental evidence from animal mammary tumor studies investigating the effects of dietary fat on mammary tumori- genesis provides important insight in an attempt to explain international and intercultural differences in breast can- cer incidence. In general, Western countries and cultures that consume greater amounts of fat in their diets have a higher incidence of breast cancer than Eastern countries and cultures which consume smaller amounts of dietary fat (Carroll & Khor, 1975). Epidemiological studies by Lea (1966) and others have shown a high positive correlation between dietary fat intake and mortality from breast cancer. Carroll (1975) also demonstrated a strong positive corre— lation (r = +0.935) between total dietary fat intake and age- adjusted breast cancer mortality. However, when the intake of vegetable fat was considered alone, little or no cor- relation with breast cancer mortality was seen. Further- more, adipose tissue from Americans was shown to contain less linoleic acid than adipose tissue from Japanese (Insull et a1., 1969). Thus, the importance of unsaturated fatty acids as seen in experimental mammary tumor models may not be relevant when considering human breast cancer. Buell (1973) reported that the incidence of breast cancer increased by five times among Japanese women who immigrated to the United States when compared with breast cancer incidence in native Japanese women. Also Hems (1978) found that changes in breast cancer rates in 20 countries wer anc‘ ta] wil lll 1237!?!) 51 were positively correlated with similar changes in total fat and animal protein intake. Increases in breast cancer mor— tality in Japan over the past 20 years have been associated with a marked shift toward a Western lifestyle and an increase in dietary fat in Japan (Reddy et a1., 1980). Therefore, although such epidemiological studies cannot define causative relationships between dietary fat and human breast cancer, the strong correlations between dietary fat consumption and incidence of human breast cancer are relevant to experimental animal mammary tumor studies. Elucidation of the mechanisms by which high fat diets stimulate mammary tumorigenesis in animal models may also provide further understanding of the etiology of human breast cancer. Effects of Caloric Restriction on Mammary Tumorigenesis Caloric restriction is another aspect of nutrition that has been shown to have a profound effect on mammary tumori- genesis. In general, restriction of food intake increases the latency period of development and decreases the incidence and growth rates of many types of spontaneous, transplanted and carcinogen-induced cancers in mice and rats (White, 1961). Tannenbaum (1940) reported that diets which were restricted to one-half the normal caloric amounts decreased spontaneous mammary tumor incidence in DBA mice. A prolongation of the latency period required for spontaneous mammary tumors to appear was also observed in food restricted animals (Tannen- baum, 1942). Huseby et a1. (1945), reported a decrease in IIIIIIIIIIIIIIIEIII:—____________________——T 52 mammary tumor incidence with a 33% caloric restriction in C3H mice. Dunning et a1. (1949) showed that caloric restriction reduced the development of diethylstilbestrol (DES)-induced mammary tumor development. Conversely, C H 3 mice that were made obese by treatment with thioglucose developed spontaneous mammary tumors with a shorter latency period than non—treated controls (Waxler et a1., 1953). Clayson (1975), using data from the work of Tannenbaum (1940), has shown that an increase in the incidence of spon- taneous mammary tumorigenesis in C3H and DBA mice is posi- tively correlated with an increase in caloric intake. Also, a correlation between body weight and tumorigenesis in mice with varying degrees of caloric restriction has been estab- lished (Tannenbaum & Silverstone, 1953). However, such a correlation could not be established between body weight and incidence of spontaneous mammary tumors in mice fed full rations. Recently, Tucker (1979) reported that the develop- ment of spontaneous mammary tumors, as well as other types of tumors, were inhibited in rats and mice treated by restricting their food intake by 20% over 2 years. Also noted in this study was an increase in the latency period of tumor appearance. The 20% caloric restriction also increased the longevity of the restricted animals when compared with the full-fed control animals. Sylvester et a1. (1981) showed that restriction of food intake by 50% from 1 week prior to DMBA administration to 30 days following DMBA administration permanently inhibited mammary tumorigenesis even though the rat the in) of mi ma 53 rats were returned to full-fed rations for the remainder of the experiment. Caloric restriction has also been noted to have an inhibitory effect on the growth of established mammary tumors. Tannenbaum (1942) demonstrated an inhibitory effect of caloric restriction on spontaneous mammary tumors in DBA mice. Tarnowski et a1. (1955) showed that the growth of many types of transplantable mammary carcinomas could be inhibited in a dose related manner by caloric restriction. Welsch and Meites (1978) and Leung et a1. (1980) reported that caloric restriction inhibited the growth of established DMBA-induced mammary tumors. Finally, the death rate from breast cancer in humans was demonstrated to be positively correlated with caloric intake (Carroll & Khor, 1975). However, when compared to the correlation seen for high fat diets (r = +0.935), the correlation between caloric intake and mortality from human breast cancer is not as strong (I = +0.737). The inhibitory effects of caloric restriction on mam- mary tumorigenesis appear to be mediated not through the initiation, but the promotional stage of mammary tumori- genesis. Tannenbaum (1942) showed that spontaneous mammary tumorigenesis culd be inhibited when caloric restriction was initiated at 2, 5, or 9 months of age. The diversity of different types of tumors that can be affected by caloric restriction (White, 1961) suggests that a common mechanism is involved. Carroll (1975) suggested simply that the lack of tal< ger su} (C.- W be in 96 54 of availability of nutrients required for tumorigenesis to take place accounted for the inhibition of mammary tumori— genesis by caloric restriction. Caloric restriction also suppressed the secretion of most anterior pituitary hormones (Campbell et a1., 1977) and induced a state of pseudo- hypophysectomy (Mulinos et a1., 1940). It has therefore been suggested that an endocrine mechanism may be involved in mediation of the effects of caloric restriction on tumori— genesis. Huseby et a1. (1945) showed that the normal mammary gland development of C3H mice was inhibited by caloric restriction. Also noted in this study were changes in the structure and function of the ovaries and uteri analogous to changes associated with hypophysectomy. Such observations led to the inference by Huseby (1945) that the apparent decrease in hormonal stimulation of the mammary gland may be associated with the inhibition of mammary tumorigenesis by decreased food intake. More direct evidence for an endocrine involvement in the inhibition of mammary tumorigenesis was provided by Sylvester et a1. (1981). These investigators showed that supplementation with estrogen or a combination of estrogen, prolactin, and growth hormone, reversed many of the effects of caloric restriction on DMBA-induced mammary tumori— genesis. Also, Welsch and Meites (1978) and Leung et a1. (1980) showed that the inhibition of established DMBA-induced mammary tumor growth caused by caloric restriction was associated with reduced serum prolactin and estrogen levels, and not imh ger 1i: 55 and could be blocked by daily injections of haloperidol which elevated serum prolactin levels. Boutwell (1948) noted that adrenal hypersecretion may contribute to the inhibitory effect of caloric restriction on mammary tumori- genesis. Adrenal glucocorticoids inhibit growth of estab- lished mammary tumors (Aylsworth et a1., 1979; Hilf et a1., 1965), yet an inhibitory role for adrenal glucocorticoids on mammary tumor development has not been established. Effects of Dietary Protein Intake on Mammary Tumorigenesis The effects of dietary protein on mammary tumori- genesis are not as definitive as the effects of other dietary components such as caloric restriction or dietary fat, and thereby make construction of generalizations difficult. Tannenbaum and Silverstone (1949) reported that spontaneous mammary tumorigenesis was stimulated in mice fed diets con- taining levels of protein either greater than (45% protein) or less than (9% protein) the control level (18% protein), even though the caloric intake was regulated in each group. Conversely, White and Andervont (1949) found that diets which were severely restricted in protein content (4% casein) and deficient in cysteine prevented formation of spontaneous mammary tumors in C3H mice. However, such treatment also caused a reduction in body weight gains (similar to caloric restriction), and aberrations in estrous cycles. Engel and Copeland (1952) demonstrated an inverse relationship between dietary protein intake and induced 56 mammary tumorigenesis observing that high protein levels in the diet were associated with reduced mammary tumor development. Furthermore, Gilbert et a1. (1958) reported that rats fed diets containing high levels of casein (77%) had a lower incidence of spontaneously developing mammary fibroadenomas when compared with rats fed diets containing 12-15% protein. Shay et al. (1964) showed that a semi— synthetic 64% casein diet increased mammary tumor develop— ment when compared to a commercially prepared diet containing 27% protein. However, the many differences that exist between the semi—synthetic and commercially prepared diets preclude assigning any definitive conclusions concerning the effects of high protein diets on mammary tumorigenesis. More recently, Clinton et a1. (1979) showed that reduced g protein intake prior to DMBA administration stimulated mammary tumor development, whereas similar treatment after DMBA administration had no effect. Conversely, increased protein intake before DMBA administration reduced mammary tumorigenesis, but had no effect when administered after DMBA treatment. These results suggest that dietary protein can influence the initiation phase, but not the promotional phase of mammary tumorigenesis. Because of the inconsistent evidence attempting to ascertain the effect of dietary pro- tein on mammary tumor development, more extensive investi- gation is needed. Evidence from examining the effect of reduced dietary protein intake on the growth of established mammary tumors 57 is more consistent. White and Belkin (1945) have shown that transplanted adenocarcinomas grow at a reduced rate in severely protein deficient mice when compared to control mice consuming normal amounts of protein. Also the growth of established DMBA—induced mammary tumors was inhibited when rats were fed a protein deficient (6% protein) diet (Welsch & Meites, 1978). The incidence of breast cancer in women appears to be associated with intake of protein in the diet. Carroll , and Khor (1975) found a strong positive correlation between breast cancer mortality and animal protein consumption. However, no correlation was observed with vegetable protein . consumption and death due to breast cancer. In an epidemio- logical study controlled for height, weight, and age of menarche, Gray et a1. (1979) also positively correlated | breast cancer incidence and consumption of animal protein. The effects of dietary protein on mammary tumor develop- ment may involve an altered metabolism of carcinogens induced by changed in dietary protein intake. Clinton et a1. (1979) have shown that ary1-hydrocarbon-hydroxylase (AHH) activity at the time of DMBA administration is elevated when protein intake is increased. AHH is the principle mixed function oxidase involved in the conversion of polycyclic aromatic hydrocarbon-type carcinogens to more hydrophillic and readily excretable compounds. Thus, the attenuated mammary tumorigenesis seen with high protein consumption prior to carcinogen administration may be a result of reduced car cat mec of 5111 58 carcinogenic response of DMBA due to a more rapid detoxifi- cation caused by increased AHH activity. An endocrine mechanism has also been suggested to explain the inhibition of growth of established mammary tumors resulting from con- sumption of a protein deficient diet. Administration of growth hormone overcame the inhibition of mammary tumor growth in protein deficient rats bearing DMBA-induced mam- mary tumors (Welsch & Meites, 1978). These investigators suggest that the effect of growth hormone on mammary tumor growth is probably mediated indirectly through conservation of body proteins rather than direct stimulation of mammary tumors. Effects of Vitamin A and Its Derivatives on Mammary Tumorigenesis Another nutritional component influencing mammary tumor- igenesis that has generated interest in recent years has been Vitamin A and associated retinoid compounds. The prin— ciple function of Vitamin A is maintenance of the integrity of epithelial tissues. Additionally, Vitamin A and its derivatives may participate in the stabilization and main- tenance of permeability characteristics of cellular and intracellular membranes (Harper, 1979). A deficiency in Vitamin A resulted in the replacement of certain secretory epithelium with a keratinized epithelium similar to that induced by some carcinogens (Harris et a1., 1972). co ea tr in (I) IIIIIIIIIIIIIIIIII::::________________——T 59 Recent reports suggest that administration of retinoic compounds inhibits murine mammary tumorigenesis. However, earlier work by Brandis and Anton (1966) showed that adminis- tration of retinoid compounds enhanced mammary tumorigenesis induced by cytoxen. Also, Schmahl et a1. (1972) reported that retinyl palmitate had no effect on DMBA-induced mammary tumorigenesis. More recent investigations by Moon and his co-workers (1976) have shown conclusively that exogenous administration of retinyl derivatives such as retinyl acetate can inhibit mammary tumorigenesis. Daily administration of 2.5 mg of retinyl acetate beginning 7 days after treatment with DMBA decreased both the incidence and the number of developing benign and malignant tumors (Moon et a1., 1976). Retinyl acetate also inhibited mammary tumorigenesis induced by N-methyl-N—nitrosucrea (NMU), (Moon et a1., 1977). Rettara et a1. (1975) reported that growth of transplantable mammary adenocarcinomas in mice could be inhibited by retinyl acetate. Most recently, Welsch et a1. (1980) reported that suppression of serum prolactin levels by CB-154 potentiated the inhibition of tumorigenesis induced by retinyl acetate. Retinoid compounds appear to inhibit mammary tumori- genesis both at the initiation and promotional phases. McCormick et a1. (1980) found that short-term treatment with retinyl acetate from 2 weeks before to 1 week after DMBA was nearly as effective in permanently inhibiting mammary tumorigenesis as treatment with retinyl acetate for up to 30 weeks after DMBA. This would suggest that retinyl ac< t0 60 acetate can act in the initiation and promotional phases to inhibit mammary tumor development. The mechanism by which retinyl compounds inhibit mammary tumorigenesis is not clear. Because the major biological function of Vitamin A is to induce proper differentiation of epithelial tissues, it has been suggested that retinoids may act by reversing carcinogen-induced anaplasia. However, Thompson et a1. (1979) reported that short—term treatment with retinyl acetate after carcinogen treatment temporarily suppressed mammary tumor development and continuous adminis- tration of retinyl acetate was required to inhibit mammary tumorigenesis induced by NMU. These results suggest that retinyl acetate inhibits the progression of mammary tumor development instead of reversing the carcinogen—induced anaplasia. Hill and Shih (1974) demonstrated that retinoic acid inhibits liver mixed function oxidases that are responsible for the activation of many polycyclic aromatic hydrocarbons such as DMBA, benzo(a)pyrene, and methylcholanthrene, thereby reducing the initiation capacity of these compounds. Since the uptake and binding of many carcinogens such as DMBA and NMU to cellular proteins and DNA are completed within 2 weeks of administration (Janss et a1., 1972), such a pro- cess would probably not be affected by administration of retinyl acetate after this time (Moon et a1., 1976). There- fore, while inhibition of mixed function oxidases could explain the decrease in initiation of mammary tumorigenesis, a1 (11 61 it would not explain the inhibition of mammary tumor develop- ment by retinoids during the promotional stage. The toxic effects of large doses of retinyl compounds do not appear to totally account for their inhibitory effects on mammary tumorigenesis. No changes in body weight gains or in liver function and structure were associated with suppression of mammary tumorigenesis by retinyl acetate (Moon et a1., 1976, 1977). Also, no changes in estrous cycle activity in rats treated with retinyl acetate is necessary for inhibition of mammary tumorigenesis to occur (Moon et a1., 1976). There- fore, it would not appear that the effects of retinyl acetate are mediated by any major alteration in endocrine or repro- ductive function. However, more careful examination of the effects of retinyl compounds on the endocrine system is required to properly assess the role of hormones in the inhibition of mammary tumorigenesis by retinoids. There is evidence that retinoic acid may inhibit mammary tumorigenesis via the immune system by stimulation of thymus-derived killer cell induction (Lotan & Dennert, 1979). Finally, retinoid binding proteins have been identified in NMU- induced mammary tumors, suggesting that retinyl compounds may act directly at the mammary cell to inhibit tumor devel- opment and growth (Mehta & Moon, 1979). Efifigcts of Antioxidants on Mammary W Antioxidants such as butylated hydroxanisole (BHA), butylated hydroxytoluene (BHT) and alpha tocopherol L—_ 62 (Vitamin E) are among the most widely used compounds added to commercially prepared foods consumed by both humans and laboratory animals. Because consumption of these anti- oxidants are frequently encountered in the human diet, these compounds have been of interest, particularly in relation to their possible role in carcinogenesis (Wattenberg, 1978). Antioxidants inhibit a wide variety of chemically induced neoplasms (Wattenberg, 1978). Wattenberg (1972) reported that DMBA-induced mammary tumorigenesis could be inhibited by addition of either BHA or BHT to the diets consumed by rats. Furthermore, diets supplemented with BHT resulted in a decrease in the incidence of mammary tumors induced by AAF. Harman (1969) reported that alpha— tocopheral suppressed mammary tumorigenesis induced by DBA. However, Wattenberg (1972) reported no such effect of alpha— tocopheral in DMBA-induced mammary tumorigenesis. Antioxidants are thought to exert their inhibitory effects on mammary tumorigenesis via their effects on the mixed function oxidases involved in the metabolism of carcinogenic compounds. BHT increased the detoxification of AAF by enhancing its conjugation to glucoronic acid and excretion (Granthram et a1., 1972), thereby lowering the availability of the carcinogen for activation reactions. BHA altered the metabolism of benzo(a)pyrene by mixed function oxidases such that there was a decrease in the epoxidation and increase in the detoxification reactions. Such actions of BHT and BHA could account for their inl Cd] 0X 63 inhibitory effect on the induction of mammary tumors by carcinogens. Selenium is also involved in the endogenous anti- oxidation reactions and affects mammary tumorigenesis. Selenium administered in the drinking water of C3H mice over a 15—month period decreased the incidence of spontaneous mammary tumors from 83% to 10% (Schrauzer & Ishmed, 1974). Harr et a1. (1973) showed that selenium deficient diets stimulated mammary tumor development when fed to AAF- treated rats and that this effect could be reversed by selen- ium supplementation. Medina and Shepart (1980) reported that selenium inhibited spontaneous mammary tumorigenesis in BALE/cf C H (MuMTV-positive) mice without alterations in 3 normal reproductive function or growth processes. Ip and Sinha (1981) reported that selenium deficient, high poly- unsaturated fat diets enhanced DMBA—induced mammary tumori- genesis to a greater extent than high polyunsaturated fat diets which contained normal levels of selenium. In humans, increased incidence of breast cancer has been associated with geographic locations in which selenium is known to be lacking (Schrauzer & Ishmed, 1974). The biological function of selenium important to its role in mammary tumorigenesis is its effect on glutathione peroxidase activity (Griffin, 1979). Selenium itself is not an antioxidant, but rather functions indirectly as an antioxidant through the selenium containing enzyme gluta- thione peroxidase. Glutathione peroxidase converts PO' ge th an p1 ti 64 potentially harmful hydrogen peroxide and hydroperoxides generated from lipid metabolism to water. Thus, removal of the peroxides by glutathione peroxidase prevents oxidation and subsequent damage to various cellular components and proteins by the peroxides. Selenium also appears to have an effect on mixed function oxidases which metabolize many types of carcinogens. Marshall et a1. (1979) and Ip and Sinha (1981) reported that selenium may impede activation and/or accentuate detoxification of AAF and DMBA. Medina and Shepard (1980) reported that selenium had little or no effect on pre-neoplastic mammary tumor outgrowth lines main— tained in BALB/c (MuMTV-negative) mice or on the growth of transplanted primary mammary tumors. These results suggest that selenium may act through either inhibition of the carcinogen-induced transformation of normal mammary tissue or through inhibition of the promotion of previously transformed cells. MATERIALS AND METHODS Research Animals Animals used in studies described here were female Sprague-Dawley rats obtained from Harlan Research Animals, Indianapolis, IN, and Charles River Research Animals, Wilmington, MA. Animals were housed in metal suspension cages and maintained in a temperature controlled (251°C) room on a 14 hour light (0500-1900 hr)/10 hour dark illumina- tion regimen. All animals were fed ad libitgm a diet of tap water and either laboratory rat chow in pelleted form (Ralston Purina Co., St. Louis, MO), or a semi-synthetic variable fat diet as specified. Tumor Induction Mammary tumors were induced in animals by the method of Huggins (1965). Virgin female Sprague-Dawley rats, 55-60 days of age, were given a 1 ml lipid emulsion con- taining 5 mg of 7,12—dimethylbenz(a)anthracene (DMBA) by tail vein injection under light ether anesthesia. The DMBA emulsion was kindly provided by the Upjohn Co., Kalamazoo, MI. Most tumors became palpable 1-3 months after DMBA injection. 65 66 Tumor Measurements Tumors were palpated and measured at weekly intervals, beginning 1 month after administration of DMBA. Palpable mammary tumors were measured with a vernier caliper and the two largest perpendicular diameters were recorded and averaged. Weekly tumor measurements were totaled for each rat, and expressed as "the sum of the average tumor diameter per rat" for each treatment group. In addition, mammary tumor development was assessed by the average latency period of tumor appearance, which was calculated for all tumors in each treatment group. This value represents the period of time (in weeks) between DMBA administration and the initial appearance of tumors as determined by palpation. In the studies examining the effects of high fat diets on the growth of established mammary tumors, tumors were palpated and measured immediately prior to and at weekly intervals after initiation of treatments. Tumor growth or regression was expressed as the percent change in average tumor diameter for each rat when compared to the initial tumor measurements. Tumors developing after the initiation of treatments were also measured and recorded. Blood Sampling and Radioimmunoassays Blood was sampled in the tumor induction and growth experiments by the orbital sinus puncture technique, using light ether anesthesia. All blood samples were taken between 1000 and 1200 hr to minimize variability in serum prolactin levels. 67 In the studies examining the effects of high fat diets on serum prolactin levels, blood was sampled via an implanted right atrial cannula. This allowed for frequent sampling of blood from unanesthetized, undisturbed rats. Silastic cannulas (0.025 inches I.D.; 0.047 inches O.D.) were inserted into the right external jugular vein, and threaded through the superior vena cava into the right atrium. The free end of the cannula was threaded subcutaneously across the back of the neck and exited l-2 cm caudal from the base of the skull. After the cannula was secured in place, it was flushed with approximately 0.2 m1 of sterile heparinized saline (100 IU/ml) and closed with a knot at the free end i of the cannula. Cannulated rats were then injected with 0.2 ml Longicil to prevent infection and placed in individual wire cages. At least 2 hours prior to blood collection, the free end of the cannula was out immediately proximal to the closure knot and a Silastic extension tube was attached to the cannula and extended outside the cage. Cannulated rats with attached extension tubes could move freely in their cages and were free to consume food and water. Serum from collected blood was separated by centrifuga- tion and stored at -20°C until assayed for prolactin, using the radioimmunoassay method developed by Niswender et a1. (1969). pr be WE 68 Dietary Treatments The composition of the semi-synthetic diets used in the following experiments are shown on Table l. Diets were prepared 1 to 2 times per week, or as required, with the proportion of ingredients added on a percentage-weight basis. Prepared diets were stored at 4°C until fed. Rats were fed ad libitum with fresh food provided at least every two days. The casein, cellulose, salt mixture and vitamin supplement were obtained from 0.8. Biochemical Corp., Cleveland, OH. Sucrose and corn oil were purchased from local sources. Drug and Endocrine Manipulations In studies examining the effects of high fat diets on mammary tumor development and growth, serum estrogen and prolactin levels were controlled by using the following combinations of endocrine and drug treatments: bilateral ovariectomy without hormone replacement to reduce circulating levels of estrogen and prolactin; bilateral ovariectomy followed by administration of estradiol benzoate (EB) at a dose of 2 ug/rat/day to replace basal levels of estrogen and serum prolactin; bilateral ovariectomy followed by injections of EB and 2-bromo-a-ergocryptine (CB—154) at a dose of 0.2 mg/kg/day to replace physiological levels of estrogen while maintaining reduced serum prolactin levels; bilateral ovariectomy followed by administration of haloperi- dol (0.5 mg/kg/day) to elevate serum prolactin levels while 69 .musuxHE pomp mo EmumoHHx zoom on pooch mm: GOADmoawHuuom CAEMDH> kuou mo manna occlmuqozem m m o.o~ o.ma mm Imam. use none 4 a m.a m.mm mm Iwm.ac has Houucoo mwusuxaz mmoHsH Hao wmonoom cflwmmo ucwEumoHB unm taco cnou .Ibwaa Hence no my woman has mannaum> no cOAuam0dsoout.H magma mai fol sex le\ tr< we: 70 maintaining reduced estrogen levels; bilateral ovariectomy followed by injections of haloperidol and EB to elevate serum prolactin levels in the presence of physiological levels of estrogen. Sham operated rats were used as con- trols in which normal cyclic levels of estrogen and prolactin were present. The BB and CB-154 were suspended in a solution of 0.85% physiological saline containing 0.3% ethanol. Halo- peridol was dissolved in 0.3% tartaric acid. All drugs were administered by subcutaneous injection into the back of the animal. Sham operated and ovariectomized control rats received injections of vehicle alone. The effects of high fat diets on induced prolactin surges also were examined. In this study, proestrus-like surges were induced by administration of EB and progesterone into long—term ovariectomized rats. Six weeks after bilateral ovariectomy, animals were injected with EB at a dose of 20 ug/rat; 72 hours later, the animals were injected with progesterone at a dose of 5 mg/rat. The resulting surge of prolactin was observed 4-5 hours following progesterone administration. Tissue Preparation and Prolactin Receptor Assay Rats bearing multiple DMBA-induced mammary tumors were killed by decapitation at the end of the experiment. All identifiable mammary tumors were immediately excised, wrapped in aluminum foil, and frozen on dry ice. Mammary —:—— ’ 71 tumor tissues were stored at —50°C until assayed for pro- lactin binding activity. All mammary tumors were homogenized in 0.3 M sucrose for 45 sec. in a Waring Blender with a Special microcup attachment. The homogenate was centrifuged at 11,000 rpm for 20 min and the pellet was discarded. The supernatant was centrifuged at 40,000 rpm for 60 min to obtain the particulate membrane pellet. This pellet was resuspended in a Tris buffer (0.025M Tris, pH 7.6 and 10 mM CaClZ). Protein concentrations for each membrane preparation was determined by the method of Lowry et al. (1951). All samples were then diluted to uniformity with the Tris buffer so that 500 ug of membrane protein were present in 100 pl of membrane preparation. Ovine prolactin was iodinated by the lactoperoxidase- 125 glucose oxidase method of Tower et al. (1977). I-labeled prolactin was diluted in Tris buffer to give approximately 70,000 cpm/lOO ul. Individual tumor samples were assayed in quadriplicate. Total binding of ovine prolactin was 125 determined in tubes containing 100 ul of I—labeled pro- lactin, 300 pl of membrane preparation containing 1.5 mg protein and 100 pl of Tris buffer. Parallel incubation to determine non-specific ovine prolactin binding were per- formed using the same reactants, except 100 pl of excess unlabeled ovine prolactin (l ug/lOO ul) replaced the 100 pl of Tris buffer diluent. In all tubes, the total incubation Volume was 0.5 ml. All assay tubes were incubated for 2 days at 40°C after which 3 ml Tris buffer were added to 72 terminate the incubation, and the tubes were centrifuged at 16,000 rpm for 30 min. The resulting pellets were counted for l min in a Nuclear-Chicago gamma counter. Specific binding was determined for each sample by subtracting the cpm bound in the presence of excess unlabeled ovine pro— lactin (i.e., non-specific binding cpm) from the cpm bound in the absence of ovine prolactin (i.e., total binding cpm) and was expressed as a percentage of total radioactive label used in each incubation. Statistical Analysis Significant differences in hormone levels between treatment groups were determined by using either students‘ "t" test or by analysis of variance (ANOVA) and the Student- Newman-Keuls' (S-N-K) statistical tests, when appropriate. Differences in body weight, tumor diameters, tumor number, and latency period of mammary tumor development were tested for significance by ANOVA and S-N-K analyses. A difference of p<0.05 was considered to be significant. IQ me i} [W EXPERIMENTAL Estimate of Caloric Intake in Rats Fe d High Fat and Control Fat Diets Objectives Nutritional effects have been shown to influence murine mammary tumorigenesis, particularly with respect to caloric intake (Tennenbaum, 1959). Since the semi-synthetic diets used in this and subsequent experiments differed in their caloric content, it is conceivable that the stimulation of mammary tumor development by high fat diets may be due to differences in caloric intake between rats fed high and control fat diets. The purpose of this experiment was to estimate the caloric intake of rats fed a 20% high fat diet and a 4.5% control fat diet, and to relate any observed differences in caloric intake to possible involvement in Stimulation of mammary tumor development by high fat diets. Procedure \ Fifty female Sprague-Dawley rats approximately 250 grams in body weight were fed ad libitum either a 4.5% control fat or a 20% high fat diet. Over a 4 week treatment period, daily food consumption was measured by subtracting the amount 73 of pr ra su ca we BC 74 of food remaining each day from the amount of food given the previous day. The daily caloric intake for control fat fed rats was calculated by multiplying the grams of food con- sumed by each rat by the caloric content of each diet. The caloric contents for the control fat and the high fat diets were 3.795 Kcal/gram and 4.480 Kcal/gram, respectively. Body weights were measured and recorded at weekly intervals. Results Figure 1 shows the food consumption and caloric intake of rats fed control fat and high fat diets during the 4 week treatment period. As shown after 4 days on their respective diets, rats fed the 20% high fat diet consistently consumed less of their diet than similar rats fed the 4.5% control fat diet. After the fourth day of dietary treatment, rats fed the 20% high fat diet consumed an average of between 1 and 5 grams less of their diet per day than rats fed the 4.5% control fat diet. Caloric intake, calculated for rats fed either diet, demonstrated that beginning 4 days after initiation of dietary treatment, there were no significant differences in caloric intake between rats fed the high fat and control fat diets. Also shown on Figure l are the average body weights measured during the 4 week treatment period. No differences in body weight were observed between rats fed the 4.5% con- trol and the 20% high fat diets. Fig Figure l. 75 (A) O O 275 f — cr b“ 250 null-nu H, Body Weight (grams) 1 00 "’utfl'um 75 (kcol) 50 Caloric Intake per rat 30 20 Diet Consumed per rat (grams) 0 5 10 15 20 25 Days After Initiation of Diet Treatment Food Consumption, Estimate of Caloric Intake and Body Weight Gains in Rats Fed 4.5% Control Fat and 20.0% High Fat Diets. Vertical bars represent S.E.M. 76 Conclusions These results indicate that rats have the ability to regulate their caloric intake whether they are fed a high fat or a control fat diet. After a 4 day period of adjust- ment to their dietary regimen, rats fed the high fat diet consumed less of their diet and maintained a daily caloric intake similar to rats fed the control fat diet. The ability of rats to control their caloric intake is further indicated by the observation that over a 4 week dietary treatment period, no significant difference in body weight was observed in rats fed the high fat diet compared to rats fed the con- trol fat diet. Therefore, the enhanced development of mammary tumors by consumption of a high fat diet cannot be explained on the basis that the high fat diet had a higher caloric content than rat diets containing normal amounts of fat. Effects of High Fat Diet on Serum Prolactin Levels During the Estrous Cycle in Female Rats Objectives Prolactin has been reported to have a major facilitatory role in murine mammary tumorigenesis. Therefore, elevation Of serum prolactin levels by high dietary fat may be one mechanism by which high fat diets enhance mammary tumor development. The effects of high fat diets on serum pro- lactin levels during the estrous cycle have not been thoroughly investigated. Chan et al. (1975) claimed that cor lex €81 77 consumption of high fat diets elevated serum prolactin levels in rats during the proestrus-estrus stages of the estrous cycle, and suggested that such an effect may account for stimulation of mammary tumor development by high fat diets. However, other investigators have not been able to confirm such an increase in serum prolactin levels by high fat diets (Cave et a1., 1979). Furthermore, since the rat is more active and consumes most of its daily food ration at night, any acute or short-term effects of high dietary fat on serum prolactin may not be observed from blood sampled only during the day. Also, by using indwelling right atrial cannulas, blood may be sampled from conscious, undisturbed rats, and this had not been done previously. This would allow for more accurate physiological measurements of serum prolactin levels in blood sampled from rats treated with different dietary fat treatments, and would not be influenced by drug-induced anesthesia or physiological stress. The purpose of this study was to observe, in detail, the effects of high fat diet consumption on serum prolactin levels during the entire estrous cycle of cycling female rats, and to attempt to relate these effects to the stimulation of mammary tumor development by high fat diets. Procedure Sixty mature female Sprague-Dawley rats were fed either a 20% high fat or a 4.5% control fat diet for 4 weeks, during which time estrous cycling patterns were monitored by daily collection of vaginal smears. After 4 weeks, 24 ho 1’10 78 normally cycling rats (12 from each group) were implanted with indwelling right atrial cannulas and maintained on their respective dietary regimen. Beginning the day after cannulation, and continuing for the next 4 days serial blood samples were taken at 4 hour intervals during each stage of the estrous cycle. Additional blood samples were taken at hourly intervals between 1600 and 1900 hr during the after- noon of proestrus. Serum prolactin was measured by radioimmunoassay according to the method of Niswender et a1. (1969). Differ— ences in serum prolactin levels between dietary treatment groups at each time sampled during the estrous cycle were tested for statistical significance by students' "t" test. Differences were considered to be statistically significant if p<0.05. Results The effects of dietary fat on serum prolactin levels are shown in Figure 2. Both control fat and high fat diet rats showed normal serum prolactin levels throughout the estrous cycle and a typical surge of prolectin during the afternoon of proestrus. Basal serum prolactin levels for both control fat and high fat fed rats ranged from 20 to 60 ng/ml throughout the cycle. Prolactin levels during the afternoon of proestrus were elevated to between 300 and 400 ng/ml in both treatment groups. Additionally, a smaller surge of prolactin was observed during the afternoon of 79 400 ................ “F 300 200 PRL ("G/ml) 15 120 80 4O — 0500 1 700 0500 1700 0500 1700 0500 1 700 0100 DIESTRUS PROESTRUS ESTRUS METESTRUS Figure 2. Effects of High Fat Diet on Serum Prolactin Levels During the Estrous Cycle of Female Sprague—Dawley Rats. CF represents 4.5% control fat diet. HF represents 20.0% high fat diet. Vertical bars represent S.E.M. n=10 es s} (1; ml [—h In 80 estrus. No significant difference in serum prolactin at any time during any stage of the estrous cycle was detected between rats fed either the 4.5% control fat or the 20% high fat diet. Also, the estrous cycling behavior was not altered by consumption of the high fat diet during the 4 week treatment period. A total of 20 out of 28 rats fed the 4.5% control fat diet and 19 out of 29 rats fed the 20% high fat diet showed consistent 4-5 day estrous cycles, as indicated by daily vaginal smears. Additionally, in both dietary treat- ment groups, 10 out of 12 rats implanted with indwelling cannulas maintained their normal cycling pattern during the following 4 days of blood sampling. Conclusions These results show that consumption of a high fat diet does not significantly influence serum prolactin at any time during the estrous cycle. In contrast to results reported by Chan et a1. (1975), no elevation of serum prolactin levels during the afternoons of estrus or proestrus were observed in rats fed high fat diets when compared to rats fed control fat diets. Chan et a1. (1975) claimed that high fat diets stimulate serum prolactin levels during the afternoons of proestrus and estrus. However, the data reported by these investigators combined serum prolactin values during proestrus and estrus, and showed a large amount of variability. The prolactin values obtained in the present experiment are believed to be more precise and to re di —:—— 81 represent a more meaningful assessment of the effects of dietary fat on serum prolactin levels. Therefore, stimula- tion of mammary tumor development by a high fat diet does not appear to be mediated through elevation of serum pro- lactin levels. Effects of High Fat Diet on the Surge of Prolactin Induced by Estrogen and Progesterone in Ovariectomized Female Rats Objectives To clarify further the effects of high fat diets on serum prolactin levels, the effects of dietary fat on induced surges of prolactin were investigated. It has been shown that surges in prolactin and gonadotropins resembling those occurring just prior to ovulation, can be induced in long- term ovariectomized rats treated with estrogen and progesterone (Freeman et a1., 1976; Huang et al., 1980). The purpose of this experiment was to compare the surges of prolactin induced by estrogen and progesterone in ovariectomized rats fed a 4.5% control fat and a 20% high fat diet. Procedure Twenty female Sprague—Dawley rats, 200—225 grams in body weight were bilaterally ovariectomized. Two weeks after ovariectomy, rats were placed on either a 4.5% control fat or a 20% high fat diet. Six weeks after ovariectomy, or 4 weeks after initiation of the dietary treatment, all rats were implanted with indwelling right atrial cannulas —:—— 82 and injected with 20 ug of estradiol benzoate (EB); 72 hours later, all rats were injected with progesterone at a dose of 5 mg per rat. Blood was sampled via the indwelling cannula immediately prior to (1100 hrs) and following progesterone administration, at 1400, 1600, 1700, 1800, 1900, and 2200 hrs. Serum prolactin was measured by radioimmunoassay according to the method of Niswender et al. (1969). Sta- tistical significance was tested between the two dietary treatment groups using the students' "t" test and a level of significance of p<0.05. Results The effects of dietary fat on serum prolactin levels in EB-progesterone treated ovariectomized rats are summarized on Figure 3. As shown, a surge in prolactin was induced. Prior to progesterone treatment, basal serum prolectin levels in both control fat and high fat fed rats were approximately 30 ng/ml. Five hours after injection of progesterone, serum prolactin levels were observed to peak at nearly 400 ng/ml in both the control fat and high fat treatment groups. Sub- sequently, serum/prolactin levels declined to nearly 100 ng/ml at 2200 hrs, 11 hours after progesterone treatment. At no time were serum prolactin levels significantly differ- ent between rats fed either the 20% high fat or the 4.5% control fat diet. 83 500 400 Illllllllllllll ”F “l 300 (ngfino 200 100 1100 1300 1500 1700 1900 2100 2300 prog TUNE Figure 3. Effects of High Fat Diet on the Surge of Prolactin Induced by Estrogen and Progesterone in Ovariect- omized Female Rats. CF represents 4.5% control fat diet. HF represents 20.0% high fat diet. Vertical bars represent S.E.M. PROG: indicates progesterone injection (5 mg per rat). 9g di In 84 Conclusions These results indicate that ovariectomized rats fed diets containing either control or high levels of fat show similar prolactin surges induced by EB and progesterone injections. They support previous observations that high fat diets do not stimulate serum prolactin during the pre- ovulatory surge on the afternoon of proestrus. Thus, they strengthen the conclusion that the stimulatory effects of high fat diets on mammary tumor development are not mediated via elevation of serum prolactin levels. Role of Prolactin and Estrogen in Stimulation by High Fat Diet of Development of DMBA- Induced Mammary Tumors Objectives The stimulatory effects of high fat diets on mammary tumor development in rats are well established (Carroll, 1975; Aylsworth, 1979). However, the mechanism(s) involved are not clear. Some investigators believe that enhancement of the pre-ovulatory surge of prolactin on the afternoon of proestrus is the mechanism through which such stimulatory effects of high dietary fat are mediate (Chan et al., 1975). However, consideration of the observations previously pre- sented in this thesis, as well as other published reports (Cave et al., 1978), makes it apparent that high fat diets do not influence serum prolactin levels significantly at any stage of the estrous cycle in the rat. Therefore, the ———f 85 stimulatory effects of high fat diets on mammary tumor development are mediated by other mechanisms. Chan and Cohen (1975) also suggested that prolactin may exercise a greater influence on high fat stimulation of mammary tumor development than estrogen, since administration of the anti—prolactin drug, (CB—154), could abolish the stimulatory effects of high fat diets on mammary tumori- genesis, whereas administration of an anti-estrogen drug, Nafoxidine, could not do so. In the present study, the influence of estrogen and prolactin upon the stimulatory action of high dietary fat during mammary tumor development was examined. The objective of this study was to determine whether high fat diets could effectively stimulate mammary tumorigenesis in rats whose levels of estrogen and prolactin were maintained at controlled levels. Attempts were made to determine the optimal require- ments for estrogen and prolactin to permit high fat diets to exert their stimulating effects on mammary tumorigenesis, and to evaluate the relative importance of estrogen as against prolactin in this process. Procedure Two hundred forty female Sprague-Dawley rats, 55 days Of age, were injected with DMBA as described previously (See p. 67). Five days after DMBA administration, animals were either bilaterally or sham ovariectomized. Beginning 10 days after DMBA administration, and continuing for the —7—i 86 duration of the experiment, rats were fed either a 20% high fat, or a 4.5% control fat, semi—synthetic diet. Ten days after DMBA administration, reduced levels of estrogen and/or prolactin were selectively replaced in ovariectomized rats, as follows: reduced levels of both estrogen and prolactin were maintained in ovariectomized rats; physiological levels of estrogen were injected while reduced serum prolactin levels were maintained, by administering both estradiol benzoate (EB, 2 ug/rat/day) and bromoergocryptine (CB-154, 0.2 mg/kg/day); physiological levels of estrogen and basal levels of prolactin were maintained in ovariectomized rats, by injection of EB (2 ug/rat/day); serum prolactin levels were elevated and serum estrogen levels were reduced by injection of haloperidol (0.5 mg/kg/day) to ovariectomized rats; serum prolactin levels were elevated and physiological levels of estrogen were maintained by administration of haloperidol and EB. Sham operated rats were used as controls, in which normal cyclic levels of estrogen and prolactin were present. Developing mammary tumors in each rat were palpated and measured at 7 to 10 day intervals, beginning 4 weeks after DMBA administration. Also measured were average tumor number, percent tumor induction, and average latency period for tumor development. Blood was collected 3, 9, and 16 Weeks after DMBA injection, and was radioimmunoassayed for prolactin to insure that the effects of the administered drugs on serum prolactin levels were similar in rats fed high and low fat diets. E 87 Results The effects of high fat diets on DMBA-induced mammary tumor development, as influenced by normal and reduced circulating levels of estrogen and prolactin, are presented on Figure 4 and Table 2. It was observed that mammary tumor development in rats on a high fat diet was stimulated when compared to similarly treated rats fed a control fat diet. Beginning 7 weeks after DMBA administration and continuing throughout the experiment, sham-operated rats fed the high fat diet showed a significantly greater accumulation of mammary tumor mass than sham-operated control fat diet rats. Sham-operated rats fed high fat diets also showed an increase in average tumor number and percent tumor induction, and a decreased average latency period when compared to sham- operated rats fed a control fat diet (Table 2). Reduction of both serum prolactin and estrogen levels by ovariectomy resulted in near complete suppression of mammary tumor development in both control fat and high fat diet rats (Figure 4). Average tumor number and percent tumor induction were similarly reduced in ovariectomized rats fed either a high fat or a control fat diet. Table 3 shows the effects of sham and bilateral ovariec- tomy on serum prolactin levels. Serum prolactin levels were reduced in ovariectomized rats fed either a control or a high fat diet. Moreover, at no time did serum prolectin levels differ significantly between rats fed either a con- trol or a high fat diet. Therefore, in rats with normal per lot (cm) Diameters Iumor Sum at Ave. Figure 4. dd“ O-‘N’w O‘NUAUO‘VNO 88 SHAM CONTROL T c F O ,t ‘ ‘. OVEX CONTROL Weeks after 0M BA Effect of High Fat Diet on Mammary Tumor Develop- ment: Influence of Normal or Reduced Circulating Levels of Prolactin and Estrogen. A. Animals were sham ovariectomized (SHAM) 5 days after DMBA administration. B. Animals were bilaterally ovariectomized (OVEX) 5 days after DMBA administration. CF represents 4.5% control fat diet; HF represents 20.0% high fat diet. indicates p<0.05 vs similarly treated rats fed control fat diets. 89 mdumum .muossu mo Hogans Hanan ou map oumaumoummmcfl COmHHmmEoo Haven . mo 0 3 . o.ov .umHo Haw Houucoo m emu mpmu owuwwuu saumHHsHm :HH3 emummEOo a n m n .E.m.m H Qmwzm gpfldan,unu N.“uW."..u. ”lfllfiinuu u.u u u n H iiliilrillliiliilliii iliIIIii1Iilllliiiiliiiiilililiii nHv.o H Hv.~H mm Hm.mm nos.o H mH.o ma mm oqo av.o H Hm.vH v6 Ho.mn om.o H Hm.m oH no o~o.a H mo.nH ma mm.mm mm.o H oo.H pH mm one: + xm>o vm.~ H m~.0H A Ho.Hm na.o H mm.o mH no Av.o H oo.oH oo H>.oo oo.H H oo.v ma a: . n mm+xm>o vm o H Hm.ma mm av.mw ~v.o H mk.H ma mo emHmo+ amm.o H MH.NH on Ho.~m HH.H H em.m «H m: . i + xm>o mm o + Ho.MH Hm wo.on oo.H H mm.m 5H no mm onH.H H nH.~H n HN.HH MH.o H mH.o sH mm 00.0 H oo.oH H Hk.o oo.o H 60.0 ma mo HoHuqooixm>o an.o H mm.m mnH $0.00H nom.H H mm.m mH m: mk~.o H mn.m mm H~.mm mam.o H mm.m EH mo HoHucooiemgm Ax3v wheeze :oHuUSpCH umm nod wumm ucmeumwue ucwaummua moaned mocmumq mo .02 scene w .02 Hesse mo .02 mumuoflo mawu00©sm moans cum: Hmuoe ommuw>< .wwumum wcfluooccm pououa< >2 poocozawsH mm acoEQan>oo “0559 xumeemz :0 umwo umm nmflm mo muumwwmll.m wands L 9O lli .2.m.m H QMQZQ .pcmaummnu mmzo Hmpmm mxmmzm iiiiiiiiliiiiiiiiiui IIIiiiIii1ililiiiiiiiiiiiiiiiiiiiii m.mm H m.mvv m.Hm H H.osa o.m~ H m.omm mm m.~v H H.mmv m.m~ H m.mma m.MH H m.w~m mo oqo m.oH H «.mmm H.mH H H.m- H.m H e.ma~ m: H.m H m.wm~ H.HH H m.km~ m.m H m.mm~ mo oqam + xm>o H.o H «.mH m.H H m.m v.0 H o.OH mm H.H H ~.w m.H H m.HH m.o H H.m mo HmHmo+mm+xm>o H.0H H H.4H m.m H m.m~ Hm.m H H.mm mm N.o H m.mm H.m H m.Hm m.m H H.mm mo mm + xm>o m.m H n.o~ o.m H H.~N o.m H H.mH mm m.m H m.o~ m.m H o.mH m.~ H a.m~ so Honucoo xm>o m.» H k.mm H.mH H m.om H.HH H e.mm mm H.@ H s.mm H.OH H m.mv no.e H H.Hm so HOHucoo swam wa m Mm ucwEumwuB ucmeummue wumumflo wCHHOOpcm AHE\ocv :Huomaoum Esumm .me>oH :HuomHoHa ssHmm co mucosummue HCHHooecm new sumHmHo Ho mHomHHmiu.m mHnma 91 circulating levels of estrogen or prolactin, high fat diets stimulated mammary tumorigenesis, whereas reduction of circulating levels of both estrogen and prolactin by ovariec- tomy suppressed mammary tumorigenesis equally in rats fed low or high fat diets. The effects of replacement with estrogen after ovariec- tomy on mammary tumor development in rats fed control or high fat diets are shown on Figure 5 and Table 2. Replace— ment of estrogen by daily injections of BB in ovariectomized rats resulted in mammary tumor development approximately equal to that of sham operated rats fed a control fat diet, with respect to accumulation of tumor mass (Figure 5), average tumor number, and percent tumor induction (Table 2). However, such treatment appeared to delay the appearance of palpable tumors as indicated by an increased average latency period in ovariectomized EB-treated rats as compared to sham operated control rats. Moreover, no significant difference in mammary tumor development was observed in ovariectomized EB-treated rats fed either a high fat or a control fat diet, as indicated by accumulation of tumor mass (Figure 5) or average tumor number (Table 2). However, the average latency period was reduced, and percent tumor induction was in- creased in high fat, ovariectomized, EB-treated rats when compared to similarly treated rats fed a control fat diet (92.9% vs 70.6%). Examination of Table 3 reveals that serum prolactin levels in ovariectomized rats treated with 2 pg Of EB per day were approximately the same as sham operated to! (cm) Diameter; pet Ave. 1 umor Sum of Figure 5. b 92 OVEX 658 HF CF OVEX 9 EB t CB—154 HF CF Weeks cite! DMBA Effects of High Fat Diet on Mammary Tumor Develop— ment: Influence of Replacement of Circulating Levels of Estrogen and Prolactin or Only Estrogen. A. Replacement of serum estrogen and prolactin levels in ovariectomized (OVEX) rats by daily administration of estradiol benzoate (EB). B. Replacement of serum estrogen levels in ovari- ectomized (OVEX) rats while maintaining reduced prolactin levels by daily administra- tion of estradiol benzoate (EB) and bromo- ergocryptine (CB-154). CF represents 4.5% control fat diet; HF represents 20.0% high fat diet. i 93 control rats, 90 minutes following injection of EB. No significant differences in serum prolactin levels were observed in rats fed high or control fat diets. The effects of replacement of physiological levels of estrogen and reduced levels of prolactin on mammary tumor development in rats fed control and high fat diets are shown on Figure 5 and Table 2. Treatment of ovariectomzed rats with EB and CB-154, to replace physiological levels of estrogen while maintaining reduced serum prolactin levels, resulted in suppression of mammary tumor development when compared to sham-operated control fat diet rats. No signi- ficant differences in mammary tumorigenesis were observed in ovariectomized rats treated with EB and CB—154 and fed either control or high fat diets, as indicated by accumula- tion of tumor mass (Figure 5), percent tumor induction, or average latency period (Table 2). However, an increase in average tumor number was observed in ovariectomized EB and CB-154 treated rats fed a high fat diet, as compared to similarly treated rats fed a control fat diet. Table 3 shows that serum prolactin levels were reduced in ovariectomized EB and CB-154 treated rats when compared with sham—operated control rats. Also, no significant differences in serum prolactin levels were observed in con- trol and high fat diet fed ovariectomized rats treated with EB and CB—154. Effects of elevated serum prolactin levels, without replacement of physiological levels of estrogen, on mammary IIIIIIIIIIIIIIIIIII-Illll---————____l 94 tumor development in ovariectomized rats fed high and con- trol fat diets are shown on Figure 6 and Table 2. Elevation of serum prolactin levels through daily administration of haloperidol, in the absence of normal circulating levels of estrogen, resulted in markedly suppressed mammary tumor development in both control and high fat fed rats. Accumula— tion of tumor mass, average tumor number and percent tumor induction were reduced in ovariectomized-haloperidol treated rats when compared to sham-operated control rats. No differ- ences in mammary tumor development were observed in ovari— ectomized rats treated with haloperidol and fed either a control or high fat diet. Serum prolactin levels were substantially elevated in ovariectomized rats treated with haloperidol when compared to sham-operated control rats (Table 3). No difference in serum prolactin levels was observed in ovariectomized— haloperidol treated rats fed either a high or a control fat diet. The effects of elevated serum prolactin levels and replacement with physiological levels of estrogen on mammary tumor development in ovariectomized rats fed control and high fat diets are shown on Figure 6 and Table 2. At 11 weeks following DMBA administration, and throughout the experiment, ovariectomized rats fed the high fat diet and treated with haloperidol and EB accumulated a significantly greater tumor mass when compared to similarly treated rats fed the control fat diet (Figure 6). Upon termination of 95 OVEXOEB v HALO 9 HF 8 7 6 5 v 5 CF E s 4 O a 3 3 . 5 '5 2 3 E 3 p- > C 1.5 3'. HF 3 f3 51.0 1 {’5 ‘ “ “u". 1 CF 05 0 III-I-quuuu"..% "10"" l Figure 6. ..'.-o 9 11 13 15 17 19 21 23 Weeks after DMBA Effects of High Fat Diet on Mammary Tumor Develop— ment: Influence of Elevated Prolactin With or Without Estrogen Replacement. A. Circulating prolactin levels were elevated in ovariectomized (OVEX) rats by daily admin- istration of haloperidol (HALO). BI Circulating levels of estrogen were replaced and prolactin levels were elevated in ovari- ectomized (OVEX) rats by daily administration of estradiol benzoate (EB) and haloperidol (HALO). CF represents 4.5% control fat fiet; HF represents 20.0% high fat diet. ()indicates p<0.05 vs similarly treated rats fed control fat diets. 96 the experiment, high fat diet ovariectomized rats treated with haloperidol and EB showed an increase in average tumor number, and percent tumor induction, but a shorter latency period for tumor palpation when compared to similarly treated rats fed a control fat diet (Table 2). Serum prolactin levels were elevated in ovariectomized rats treated with EB and haloperidol (Table 3). No signi- ficant differences in serum prolactin levels were observed in ovariectomized EB and haloperidol treated rats fed either a control or high fat diet. Conclusions These results show that high fat diets can stimulate the development of DMBA-induced mammary tumors in the presence of controlled blood concentrations of prolactin and estrogen. Although serum estrogen and prolactin levels were controlled in ovariectomized rats by daily injections of haloperidol and estrogen, high fat diets stimulated all aspects of mammary tumor development when compared to similarly treated rats fed a control fat diet. High fat diets also enhanced some aspects of mammary tumor development in ovariectomized rats with varying amounts of hormone replacement. Therefore, it appears that high fat diets can stimulate mammary tumor development by a mechanism independent of alterations of estrogen or prolactin secretion. These data suggest that high fat diets stimulate mammary tumor development by another mechanism, possibly by an increase 97 in the biological responsiveness of the incipient mammary tumor tissue to circulating levels of estrogen and prolactin. Even though mammary tumors in both high fat and control fat diet rats were exposed to similar circulating hormone levels, development of tumor tissue in rats fed high fat diets was enhanced. These results demonstrate that stimulation of mammary tumor tissue by high fat diets required near normal circula- ting estrogen and prolactin levels. Only when mammary tumor development was maximally stimulated, as observed in sham- operated controls and in ovariectomized rats injected with haloperidol and EB, were all parameters of mammary tumor development significantly increased by high fat diets. This indicates that the presence of adequate circulating levels of both estrogen and prolactin are required for enhancement of mammary tumor develOpment by high fat diets. This con— tradicts the report by Chan and Cohen (1975) that stimulation of mammary tumorigenesis by high fat diets occurred despite inhibition of the peripheral action of estrogen by the antiestrogen, nafoxidine. These results also contradict their view that the stimulating effects of high fat diets on mammary tumor development are mediated via an increase in prolactin secretion. 98 Relation of Length of Treatment With High Fat Diet and Carcinogen Administration to Mammary Tumor Development Objectives High fat diets appear to stimulate mammary tumor develop- ment during the promotional stage of tumorigenesis since their effects are observed only when administered after, but not before carcinogen treatment (Carroll & Khor, 1970; Hopkins et al., 1976). Other promotional characteristics of high fat diets on mammary tumorigenesis, however, have not been described. One of the properties of promoting agents is their dose-response influence on tumorigenesis. An increase in dose or duration of treatment with a promoting agent enhances the response of potential tumor tissue. The effects of promoting agents are largely reversible, since removal of promoting influences decrease their stimulatory effects. A delay in administration of a promoting agent after carcinogen induction does not decrease tumor develop- ment. Therefore, administrations of a similar dose of a promoting agent at different times during the developmental phase of tumorigenesis does not alter tumor responsiveness. The objective of this study was to assess some of the properties of dietary fat treatment as a promoting agent on mammary tumor development. This investigation attempted to determine whether high fat dietary treatments of equal time duration influenced mammary tumor development similarly when administered at different periods of carcinogen administra- tion. This study also determined whether high fat diets 99 could stimulate mammary tumor development in a dose-response fashion, where dose represents time duration of high fat diet treatment. In addition, the influence of consumption of a semi-synthetic diet on mammary tumor development was observed in rats fed either a semi-synthetic or a commerci— ally prepared diet containing equalivalent amounts of fat. Procedure Female Sprague—Dawley rats, 55 days of age, were injected with DMBA as previously described (Huggins, 1965). Two days following DMBA administration, rats were placed on one of the following dietary regimens: a 4.5% control fat diet for the duration of the experiment (0-16 weeks); a 20% high fat diet for the duration of the experiment (0-16 weeks); a 20% high fat diet for one week after DMBA administration, followed by a 4.5% control fat diet (0—1 week); a 20% high fat diet for 3 weeks after DMBA administration, followed by a 4.5% control fat diet (0-3 weeks); a 20% high fat diet for 6 weeks after DMBA administration, followed by a 4.5% con- trol fat diet (0-6 weeks); a 20% high fat diet for 3 weeks beginning 2 weeks following DMBA administration, preceeded by and followed by a 4.5% control fat diet (2-5 weeks); a 20% high fat diet for 3 weeks beginning 4 weeks after DMBA administration, preceeded by and followed by a 4.5% control fat diet (4—7 weeks). An additional control group was fed a commercially prepared diet in pelleted form (laboratory rat chow) containing approximately 4.5% fat for the entire 100 experiment. Developing mammary tumors were palpated and measured at weekly intervals beginning 4 weeks after DMBA administration. Mammary tumor development was assessed in each treatment group by examination of accumulation of tumor mass, expressed as the sum of the average tumor diameter per rat, the average tumor number, the percent tumor induction, and the average latency period for tumor development. Results The results of treatment with a semi-synthetic 4.5% control fat diet, a 20% high fat diet, and a commercially prepared pelleted diet containing approximately 4.5% fat, are shown on Figure 7 and Table 4. The high fat diet stimulated most aspects of mammary tumor development, con— firming previously described results. No significant differ— ences in mammary tumor development were observed between rats fed a 4.5% control fat diet or a commercially prepared diet containing approximately the same amount of fat. Accumula- tion of mammary tumor mass Was not significantly different during the 16 week treatment in rats fed the control fat diet or the laboratory rat chow (Figure 7). Furthermore, no significant differences in average tumor number or mean tumor latency period were observed (Table 4). The effects of different durations of treatment with a high fat diet on DMBA—induced mammary tumor development are shown on Figure 8 and Table 5. Treatment with the high fat diet for one week after DMBA treatment had no effect on Figure 7. 101 10.0 Sum of Ave. Tumor Diameter per rat (cm) Weeks after DMBA Effects of High Fat and Control Fat Semi-synthetic Diets on Mammary Tumor Development. CF repre— sents 4.5% control fat semi-synthetic diet; HF represents 20.0% high fat semi-synthetic diet; LB represents commerically prepared laboratory rat chow in pelleted form. ()indicates p<0.05 vs similarly treated rats fed control fat diets. .SH 1 8 mo 0H emedsoo 5:3 3.0de .E.m.m H meZm 102 bov.o H vw.m omH wm.wn bmo.a H mm.o mm Ava I 0 V mm mm.o H mm.oH mm wo.mm mv.o H vo.m em Ava 1 ov mu mmm.o H mm.HH wv ww.mo www.o H oo.m vm Ama 1 ov mg Amxzv muOESB coHuosch Ham Ham mumm chfiumoue UOHHOQ xocoumq mo .02 HOEJB w .02 HOESB mo .02 wnmuwfio H0658 cmmz Hmuoe ommuw>< .chEdon>wQ HOESB xumEEcz co uoHo Ham 20H: new pom Houucoo oeumcucxmiflewm mo muowwwm11.v wanna 103 “10 9.0 .5 115(0-16) 11¢“ A i “\“‘. \g 8.0 *e"‘ 3 7.0 , HF(O-6) O. ~¢~ ”’ *s‘ I I. :s‘ ;” $0- 6 o *.“e‘ fi” E ! ’l ...eeee HF(0'3) a 5 e, .oeoooe'. .- t I ..e O 5 o :5. [2.000 3 4.0 151/: ,; HF(0-1) Q, 5 ' {if c F(0-16) a g 3.0 > < 3 2.0 E 3 V) to 4 6 8 1O 12 14 16 Weeks after DMBA Figure 8. Effects of Duration of High Fat Diet Treatment on Mammary Tumor Development. HF represents 20.0% high diet; CF represents 4.5% control fat diet; ( ) denote period of dietary treatment expressed in weeks. *indicates p<0.05 vs similarly treated rats fed control fat diet. i .ucmEuwwuu humumflp mo OOHde mmuocwp m000£b00H00 ca mumbesz . 0 .AmH I ov mo 0H poncmfioo cuss mo.ovm a .z.m.m H 00020 104 nov.o H vo.m omH wm.mn nNo.H H mm.m mm Ara I ov mm nav.o H mm.m mHH wo.mh nam.o H ow.v mm Am I ov mm nhm.o H 0H.m mm wm.Hm Qmm.o H mm.v mm Am I 0. mm mm.o H wo.HH mm wo.m> ov.o H mm.m vm AH I ow mm me.o H mw.oa mu wo.mh 0m¢.o H vo.m vm oAmH I ow m0 IIIIIIIIIIIIIIIIIIIIIIIII .mx3v wHOEdB COHHOSOCH Ham Hem mumm uswEHmmHB cofluwm museumq mo .02 HOESB w .02 HOEDB mo .02 mumHMHQ H0859 can: Hmuoe ommu0>< .dHnmcoHHmawm uncommwmImmoo wEHB m HOH 00cmpH>m "Heme IdoH0>0o H0859 >umEEmz co ucwsumone HwHo Ham :on Ho coHHmnso mo muomwwmII.m magma 105 mammary tumorigenesis. No differences in accumulation of tumor mass (Figure 8), percent tumor induction, average tumor number, or average tumor latency period were observed between rats fed the high fat diet for one week after DMBA administration and rats fed the control fat diet for the entire treatment period. Examination of mammary tumor development in rats fed a high fat diet for a longer period of time suggests that a dose—response relationship exists for the stimulation of mammary tumorigenesis by high fat diets. With increasing duration of treatment with high dietary fat, from 0, 3, 6, to 16 weeks, enhanced mammary tumor development was observed as indicated by accumulation of tumor mass (Figure 8), average tumor number and average tumor latency period (Table 5). No differences in percent mammary tumor incidence were observed in rats fed a high fat diet for different lengths of time. Data presented in Figure 8 also suggest that withdrawal of the high fat diet and subsequent control low fat diet treatment, resulted in a partial reversal of the stimulatory effects on mammary tumor development by the high fat diet. Mammary tumor development between 4 and 10 weeks following DMBA administration progressed at nearly identical rates in groups of rats treated with a high fat diet for the entire experiment (0-16 weeks), for only 6 weeks after DMBA adminis— tration (0-6 weeks), or for only 3 weeks after DMBA (0-3 weeks). When the high fat dietary treatment was withdrawn 3 or 6 weeks after DMBA administration, subsequent mammary 106 tumor development was diminished when compared to rats main- tained on the high fat diet for a longer period. These results suggest that the early stimulatory effect of a high fat diet on mammary tumor development ceased after the removal of the high fat dietary treatment. The effects of a high fat diet fed to rats for equal time durations but at different periods during early mammary tumorigenesis are shown in Figure 9 and Table 6. These data show that a delay in administration of high fat dietary treatment does not significantly alter development of mammary tumors. In general, rats fed the high fat diet from 0 to 3 weeks, from 2 to 5 weeks, or from 4 to 7 weeks after DMBA, showed similar accumulation of mammary tumor mass (Figure 9) and average tumor number (Table 6). However, mammary tumors developed more rapidly in rats fed the high fat diet from 0 to 3 weeks after DMBA treatment, as indicated by a signi- ficantly reduced mean tumor latency period observed in these rats as compared with rats fed from 2 to 5 weeks and from 4 to 7 weeks after DMBA treatment. No significant differences in body weight were observed in any treatment group throughout the experiment. Conclusions These data suggest that a high fat diet acts as a promotor in the classical sense, as defined by Berenblum (1954), to enhance mammary tumorigenesis. A dose—response relationship is suggested in which increasing duration of 107 101) 9 o HF (0415) f‘ “e E e“. u .3 2’ 8.0 O . I. s a Q 7.0 ¢¢¢ 3 oc“ 3 6.0 , HF (4-7) ,5 y“ 1.. HF (>3) 0 pom}! _.-I HF(2-5) _ 5 O ..--' I" ".I'. ‘E’ I .-"' : e‘ I .J. 3 5 : I.- .- 4.0 i .0. I’- g : : CF (0-16) 2 2 3.0 ‘- o 2.0 E 3 10 1-0 o 4 6 8 10 12 14 16 Weeks after DMBA Effects of High Fat Dietary Treatments of Equal Duration Administered at Different Times During Early Mammary Tumorigenesis. HF represents 20.0% high fat diet; CF represents 4.5% control fat diet. ( ) denote period of dietary treatment. Figure 9. 108 .Hcoeummuu xumume mo toHumd mouocet mummnuswnmm 0H wnonfiszo .Ama I 0. m0 0H Umummfioo 00:3 mo.ovmQ eSQMOm H cmmzm n36 H $5 0.: H23 0184 H 38 3 GH I 3 mm mad H 3.3 HNH «93 age H EH am i I 3 mm 35 H 3:3 2: Horse hHmd H 3.1V mm 3. I .2 mm 23.0 H 35 mm 3.8 Emma H mm; mm 8 I 8 mm mN.o H mm.oa Mb mo.mh mv.o H vo.m «N OA®H I ow ho Amx3v mHOEdB coHuooch pom Hod mumm Hcmfipmmna cOHH0m Naccented mo .02 HOESB w .02 HOEdB Ho .02 xumumflm Hesse cow: Hmuoe mmmuw>< .mHmwcmeHoEDB wumEEmz xaumm mCHuso moEHe ucmuwwwHo um thHdd< con: chfidoH0>0o Meade zumeemz :0 :oHumuso stvm mo mucmenmwue mumuoHo Ham :mHm mo muowwwmII.m manna 109 high fat dietary treatment (i.e., dose) resulted in in- creased mammary tumor development (response). Administration of a high fat dietary treatment for equal durations of time, but at different periods during early tumorigenesis, did not significantly alter the sub- sequent development of mammary tumors. Thus, the actions of a high fat dietary treatment, like classical promotors, are not dependent upon some critical period during mammary tumor development, but are equally effective in promoting mammary tumor development regardless of when they are administered. These results support findings by Ip (1980) that when the administration of high fat dietary treatment was delayed for up to 20 weeks after DMBA administration, high fat diets were still capable of enhancing mammary tumor development. These observations conflict with those of Carroll and Khor (1975) who reported that a delay in the initiation of dietary treatment until 4 weeks after DMBA administration resulted in little or no stimulation of mammary tumor development. The effects of high fat diets on mammary tumor develop- ment appear to be largely reversible, since cessation of high fat diet treatment resulted in less mammary tumor development when compared with rats continually maintained on the high fat diet. Therefore, these dietary effects are not permanently induced modifications, but rather are dependent upon continuous high fat administration. —7— 110 This study also demonstrates that the consumption of a semi-synthetic diet does not alter mammary tumor develop- ment, since no significant differences in mammary tumorigene— sis were observed between rats fed a commercially prepared laboratory chow and a semi-synthetic diet with similar composition. These results appear to contradict the report by Chan and Dao (1980) that commercially prepared “non- purified" diets reduced mammary tumor development when com- pared to similar semi-synthetic diets. Effects of High Fat Diet on Mammary Tumor Growth in Rats With Established DMBA-Induced Mammary Tumors Objectives Much of the research concerned with the effects of high fat dietary treatment on mammary tumor development has been concerned primarily with the initial developmental stages of mammary tumorigenesis. These stimulatory effects of high fat diets on development of carcinogen-induced mammary tumors are well documented and are further confirmed by the data presented in this thesis. Wicha et al. (1978) noted that polyunsaturated fatty acids can stimulate the growth of normal mammary gland cells and mammary tumor cells in vitro, but this observation has not been extended to include 3g vivo conditions. The purpose of this study was to deter- mine whether the stimulatory effects of a high fat dietary treatment shown during the developmental stages of mammary 111 tumorigenesis could also be observed in its established stages of growth. Procedure Virgin female Sprague—Dawley rats, 55 days of age, were injected with DMBA, as described previously (Huggins, 1965). Approximately 12 weeks after DMBA administration, rats bearing multiple mammary tumors greater than 1 cm in average tumor diameter were placed on either a 20% high fat or a 4.5% control fat diet. Pretreatment measurements of average tumor diameter were recorded and treatment groups were adjusted, so that each group was similar with respect to average tumor diameter, average tumor number, and average body weight. Mammary tumor growth was monitored by weekly tumor measurements, and results were expressed as percent change in average tumor diameter. In addition, newly developing tumors were palpated and measured at weekly intervals. Blood was sampled by oribtal sinus puncture in the morning between 1000 and 1100 hr, 2 weeks after initiation of dietary treatment, and by decapitation upon termination of the experiment. Separated serum was measured for prolactin by radioimmunoassay (Niswender et al., 1969). Results Figure 10 shows the effects of a high fat dietary treatment on the growth of established mammary tumors, 112 80 a u a 8 o o o 3 0 Percent Change in Average Tumor Diameter 8 0 1 2 3 4 5 Weeks After Initiation of Diet Treatment Figure 10. Effects of High Fat Diet on the Growth of Established DMBA-Induced Mammary Tumor Growth in Rats. CF represents 4.5% control fat diet; HF represents 20.0% high fat diet. (Dindi- cates p<0.05 vs similarly treated rats fed control fat diet. 113 expressed as percent change in average tumor diameter. Mammary tumor growth in rats fed the high fat diet was markedly stimulated when compared to rats fed the control low fat diet. During the 4 and 5 week periods after initiation of dietary treatment, mammary tumor size was significantly greater in rats fed the high fat diet when compared with rats given the control low fat diet regimen. After 5 weeks of treatment, average tumor size was increased by approximately 70% in rats fed the high fat diet, whereas average tumor size was increased by approximately 25% in rats given the low fat dietary treatment. The high fat diet also enhanced growth of newly palpable mammary tumors during the 5 week treatment period, as shown in Figure 11 and in Table 7. Following treatment, rats fed the high fat diet showed a significant increase in the number of newly developing mammary tumors when compared to rats fed the control fat diet. No effects on body weight or in serum prolactin levels were observed during the 5 week treatment period (Table 7). Conclusions Results from this study show that a high fat diet can stimulate growth of previously established DMBA—induced mammary tumors, thereby lending support to the observations of Wicha et a1. (1978) that polyunsaturated fatty acids stimulate mammary tumor growth i3 yigrg. In the experiment presented here, consumption of a polyunsaturated high fat diet stimulated mammary tumor growth 1g vivo. Sum of Average Tumor Diameter per rat Figure 11. 114 NEWLY PAL PABLE TUMORS c’I-Ilr' CF 1 2 3 4 5 Weeks After Initiation of Dietary Treatment Effects of High Fat Diet on Mammary Tumor Development During Latter Stages of Mammary Tumorigenesis: Newly Palpable Tumors. CF represents 4.5% control fat diet; HF represents 20.0% high fat diet. (bindicates p<0.05 vs similarly treated rats fed control fat diet. 115 .mo m> mo.ovdo . . . I :00: E m m + n .ucmEHmwuH xumuwao wo COHHmHHHcH Houmm mwmom m.OH H H.vmm 0mw.o H m.w m.m H n.mm N.m H H.0v ma mm m.oa H H.mmm Hm.o H N.m o.m H w.Hm o.v H w.mm ma mu AmEdum. Ham Hon mm mva mumm ucweumwua uzmfloz .oz HOESB IIIIIIIIIIIIIIIIIIIIIIII mo .02 humuwflo zoom Hmcflm momum>< Hmcflm AHE\ch cHuomHond Eduwm .wHOESB xuoEEdz cozmHHnmumm mo zuzouu :0 Hoflo Dom cmflm mo muOdmwMII.n manna 116 The high fat diet also stimulated development and growth of newly palpable mammary tumors when administered for 12 weeks after DMBA treatment. These observations con- firm the study by Ip (1980), which determined that a delay of up to 20 weeks in administration of high fat dietary treatment following DMBA induction resulted in enhanced mammary tumor development. The concept that high fat diets act as promoting agents to enhance mammary tumorigenesis also is supported by these findings, since by definition, pro- motors stimulate tumor development even when treatment has been delayed after carcinogen administration. The Effects of a High Fat Diet on Specific Prolactin Binding in DMBA—Induced Mammary Tumors Objectives It is apparent from studies previously discussed that a high fat diet does not stimulate mammary tumor development by an increase in serum prolactin levels. Furthermore, it has been determined that high fat diets are capable of stimulating mammary tumorigenesis when circulating estrogen and prolactin are maintained at constant levels, thus not excluding the possibility that high fat diets may sensitize the incipient mammary tumor tissue to hormone action. High fat diets may exert a direct effect on mammary tumor development by increasing prolactin receptor activity Within the mammary tumor. This would result in an increase in the activity of normal circulatory levels of prolactin, 117 thereby stimulating mammary tumor development and growth. The purpose of this study was to measure the prolactin binding capacity of mammary tumors obtained from rats fed high fat and low fat diets, and to correlate these results with any observed effects on mammary tumor development. Procedure Female Sprague-Dawley rats were injected with DMBA according to the previously defined procedure and placed on either a 4.5% control fat or a 20% high fat diet. At 10 to 12 weeks following DMBA administration, rats bearing multiple mammary tumors 1—2 cm in average tumor diameter were sacri- ficed and their tumors were excised and frozen. After a sufficient number of tumors had been collected from both groups, membranes from these tumors were prepared and assayed for prolactin binding capacity according to the radioreceptor assay procedure described previously. Results The effects of a high fat diet on specific prolactin binding to mammary tumor membranes are shown on Figure 12. No significant differences in specific prolactin binding were observed in mammary tumor membranes obtained from rats fed either the control fat dietary regimen or the high fat regimen. 118 10 8 (15) (23) A b -l e: a. o I n or F ‘0- u — .E = '0 = .5 = n 4 _.-:= .9 = 3'. = 5 = 8 == 0, 2 = 4.57-CF 20%HF Figure 12. Effects of High Fat Diets on Specific Prolactin Binding to Mammary Tumor Membranes. ( ) denote number of samples in each group. 119 Conclusions These results indicate that high fat diets do not influence prolactin binding to membranes of mammary tumors, and therefore cannot provide an explanation for the increase in development of carcinogen-induced mammary tumors observed in rats fed a high fat diet. This appears to contradict a recent report by Cave et al. (1981) observing that lactogenic hormone binding is increased in rats fed high fat diets. Differences in the method of assessing specific prolactin binding, however, may account for the discrepancy between these two studies. DISCUSSION Many mechanisms have been proposed in an attempt to explain how high fat diets stimulate mammary tumorigenesis. However, to date, no conclusive mechanism has been established to wholly account for the stimulatory effects of high fat diets on mammary tumor development. The research reviewed in this thesis has primarily focused upon investigation of the involvement of the endocrine system, specifically estrogen and prolactin, in the enhancement of mammary tumori- genesis by dietary fat. The most extensively investigated and widely accepted premise describing the stimulatory role of dietary lipids in mammary tumor development has involved the endocrine system, particularly anterior pituitary prolactin secretion. The development of this premise by Chan et al. (1975) was based upon observations that suppression of serum prolactin levels could block the stimulatory effect of high fat diets on mammary tumorigenesis. Furthermore, these investigators reported that high fat diets elevated serum prolactin levels during selective periods of the estrous cycle. These observations led to the conclusion that the effects of high 120 121 fat diets on mammary tumor development are mediated indirectly through the hypothalamic-hypophyseal system, resulting in the elevation of serum prolactin levels. Conclusions drawn from the data presented in this thesis, however, show that the stimulation of mammary tumor development by high fat diets does not involve an alteration in anterior pituitary prolactin secretion as has been pro- posed by Chan et al. (1975). Serum prolactin levels during the estrous cycle were not influenced by dietary fat. Furthermore, when circulating levels of prolactin and estrogen were controlled by endocrine and drug manipulations, high fat diets continued to stimulate mammary tumor develop- ment. These results suggest that high fat diets may stimu- late mammary tumor development by a direct mechanism, possibly through involvement of a sensitization of the incipient mammary tumor tissue to circulating levels of estrogen and prolactin. Support for the above suggestion was provided by Dunning et a1. (1949) who noted that mammary glands from rats fed high fat diets showed an enhanced proliferative and secretory response to implanted diethylstilbesterol. Furthermore, Cave et al. (1981) showed that the binding of lactogenic hormone was increased in mammary tumors from rats fed high fat diets, thereby rendering the tumor tissue more respon- sive to circulating levels of prolactin and growth hormone. However, the observations made in this thesis could not con- firm this report by Cave et al. (1981) since it was determined 122 that there was no change in specific prolactin binding to mammary tumor membranes from rats fed control fat and high fat diets. Consumption of high fat diets alters whole body com— position, resulting in an increase in the proportion of fat. An increase in the size of the mammary fat pad may increase the localization of lipid soluble substances, including estrogens, in the mammary gland. This effect also may enhance the aromatic conversion of steroids to estrogens in the fat pad surrounding the incipient mammary tumor. Both effects would tend to potentiate the estrogenic influence on developing mammary tumors to rats fed a high fat diet. There also is other evidence that the stimulatory effects of high fat diets on mammary tumor development may not involve an endocrine mechanism. The growth of the R3230AC transplantable rat mammary tumor carcinoma is stimulated by high fat diets (Hillyard & Abraham, 1979) and inhibited when serum prolactin levels are elevated (Hilf et a1., 1967). If the stimulation of mammary tumor growth by high fat diets involved either an elevation of serum prolactin levels (as proposed by Chan et al., 1975), or a sensitization of the tumor tissue to circulating levels of prolactin, then the growth of this tumor could expect to be inhibited rather than stimulated by high fat diets. Early work by Tannenbaum (1942) demonstrated that the development and growth of hormone independent mammary tumors in mice were stimulated by high fat diets, lending further support IIIIIIIIIIIIIIIIIllllllll---——_______, 123 to the view that hormones are not involved in this phenom— enon. The development and growth of many non—endocrine related tumors also have been shown to be enhanced by high fat diets. These observations would suggest that a common mechanism may be involved in the promotion of tumorigenesis that does not include the endocrine system. Also, the distribution and clearance of estrogen from the mammary gland does not differ in rats fed high fat and low fat diets (Carroll & Khor, 1975). Non-endocrine related mechanisms for the stimulation of mammary tumor development were not investigated in this thesis. The fatty acid composition of cell membrane phospholipids of mammary gland and tumor tissue is modified by the amount and type of fat consumed by the animal. Such modifications in the cell membrane, by incorporation of large amounts of polyunsaturated fatty acids, may cause alterations in membrane permeability, protein involvement in the trans- port of nutrients, the adenyl cyclase system, and hormone- receptor interactions which may ultimately be manifested in enhanced tumor development. Additionally, the peroxidation of polyunsaturated fatty acids during lipid metabolism has been implicated in the stimulation of mammary tumor development by high fat diets. During normal lipid metabolism, polyunsaturated fatty acids are converted to various lipid peroxides, which have been demonstrated to have many deleterious effects on the structure and function of various cellular components when 124 present in high concentrations. It is possible that con- sumption of high fat diets containing large amounts of polyunsaturated acids increases the production of lipid peroxides which then may act to promote mammary carcino- genesis. Therefore, it can be concluded that the effects of high fat diets on mammary tumorigenesis do not involve an eleva- tion in serum prolactin levels, but are probably mediated by a direct mechanism on the mammary tumor tissue. This direct action of lipids on the incipient mammary tumor tissue may include sensitization of the tissue to hormones, or non— endocrine related phenomenon in which the biophysical nature or the metabolism of the neoplastic cell is changed, resulting in promotion of mammary tumorigenesis. LIST OF REFERENCES LIST OF REFERENCES Armuth, V., & I. Berenblum. Promotion of mammary carcino- genesis and leukemogenic action of phorbol in virgin female Wistar rats. Cancer Research 34: 2704-2707, 1974. Aylsworth, C. F. I. The effect of variable fat diets on development of DMBA—induced mammary tumors and serum prolactin levels. II. Role of the adrenals in regres- sion of DMBA-incuded mammary tumors during postpartum lactation. Thesis for the Masters Degree. Michigan State University, 1979. Aylsworth, C. F., C. A. Hodson, G. Berg, G. Kledzik, & J. Meites. Role of adrenals and estrogen in regression of mammary tumors during post-partum lactation in the rat. Cancer Research 32: 2436-2439, 1979. 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