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THE EFFECT OF VARIABLE FAT DIETS ON DEVELOPMENT OF DMBA- INDUCED MAMMARY TUMORS AND SERUM PROLACTIN LEVELS ROLE OF THE ADRENALS IN REGRESSION OF DMBA-INDUCED MAMMARY TUMORS DURING POSTPARTUM LACTATION BY Charles Frederic Aylsworth A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1979 ABSTRACT I. THE EFFECT OF VARIABLE FAT DIETS ON DEVELOPMENT OF DMBA-INDUCED MAMMARY TUMORS AND SERUM PRL LEVELS II. ROLE OF THE ADRENAL GLANDS IN REGRESSION OF DMBA- INDUCED MAMMARY TUMORS DURING POSTPARTUM LACTATION BY Charles Frederic Aylsworth l. The effects of variable fat diets fed to rats injected with DMBA on mammary tumor development and serum prolactin levels were studied. Rats fed the 20% high fat diet containing either predominately saturated or unsaturated fat showed significantly stimulated mammary tumor development with respect to tumor mass, tumor number, rate of growth, and latency period when compared with rats fed a 4.5% control fat diet. Basal serum prolactin levels were significantly elevated in only some of the blood sampled from rats fed high low fat diet showed little fat diets. Rats fed a 0.5% differences in tumor development or serum prolactin levels when compared with control animals. These results indicate consumption of a 20% high fat diet results in stimulated mammary tumor development whether they contain primarily saturated or unsaturated fat, and suggest that elevated serum prolactin levels may a mechanism through which represent, at least in part, this effect is manifested. The effects of bilateral adrenalectomy or estradiol 2. benzoate (EB) treatment (Lug/rat/day) were observed on growth of DMBA-induced mammary tumors during postpartum lactation. In the control and EB-treated postpartum lactating rats, the mammary tumors regressed to 50% of their average original diameter by day 25 postpartum. Adrenalectomy on day 3 postpartum prevented mammary tumor regression, and resulted in renewed mammary tumor growth. It is concluded that in rats adrenal cortical activity is primarily responsible for reduced mammary tumor growth during postpartum lactation. 3. The direct effect of glucocorticoid administration on the growth of DMBA—induced mammary tumors in vivo were studied. Female Sprague—Dawley rats with established DMBA-induced mammary tumors were given daily sc injections of dexamethasone either alone or in conjunction with haloperidol. Rats injected with HALO alone showed stimulated mammary tumor growth and elevated serum PRL levels when compared with controls. DEX when injected alone resulted in a significant regression of mammary tumors and reduced serum PRL levels. Simulta— neous injections of DEX and HALO caused a significant regression of mammary tumors and elevated serum PRL a synthetic levels. These results suggest that DEX, glucocorticoid, can directly inhibit mammary tumor growth in the presence of elevated serum PRL levels. ACKNOWLEDGMENTS to acknowledge and thank Dr. Joseph Meites for I wish his support and guidance during the course of the work involved in preparing this thesis. I would also like to acknowledge and thank Dr. Charles A. Hodson and Dr. Kenneth Pass for their help in formulating the research problems and their instruction and expertise in the laboratory techniques utilized in the experiments contained in this thesis. Thanks and recognition are due Dr. G.S. Kledzik and G. Berg for their measurement of serum corticosterone levels used in the lactation study. ii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . V LIST OF FIGURES . Vi INTRODUCTION. . . . . 1 REVIEW OF LITERATURE. . . . 3 DMBA-Induction of Mammary Tumors in the Rat 3 Hormonal Influences on DMBA—Induced Mammary Tumors. 5 Influence of Prolactin . . . . . . . . . . . . 5 Influence of Estrogen. . . 6 Influence of Adrenal Glucocorticoids . 8 Influence of Progesterone. . . . . . . . . . . . . 9 . . . . 10 Influence of Insulin . . . . . . . . . Fat Diets on Prolactin and Effects of High Mammary Tumor Induction . . 11 Tumor Induction and Development. . . . 11 Hormonal Effects of High Fat Diets 12 Implications on the Human Condition. . 14 Effects of Lactation on Growth of DMBA—Induced Mammary Tumors. . . . . . . . . . . . 15 Hormonal Responses to the Suckling Stimulus During Lactation . . . . . . . . . . 15 Effects of Lactation on Mammary Tumor Development and Growth . . . . . . . . . . . . . . 20 MATERIALS AND METHODS . . . . . . . . . 23 Research Animals. . . . . . . . . . . . . . . . . . . 23 Tumor Induction . . . . . . . . . . . . . . . . . . . 23 Tumor Measurements. . . . . . . . . . . . . . . . . . 24 Pregnancy Induction . . . . . . . . . . . . . . 24 Blood Sampling and Hormone Radioimmunoassays. . . . . 25 Hormone Treatments (Lactation Study). . . . 25 Diets (Variable Fat Diet— Tumor Development Study) . 26 . . . . . . . 26 O O O 0 Statistical Analysis. . . . . iii Page EXPERIMENTAL . . . . . . . . . 29 Effects of Variable Fat Diets on Development of DMBA—Induced Mammary Tumors. . . . . . . . . . . . 29 Objectives. . . . . . . . . . . . . . . . . . . 29 Procedure . . . . . . . . . . . . . . . . . . . 29 Results . . . . . . . . . . . . . . . . . . . . 30 Conclusions . . . . . . . . . . . . . . . . . . 36 Effects of Adrenalectomy and Estrogen Administration on the Regression of DMBA—Induced Mammary Tumors During Postpartum Lactation . . . . 38 Objectives. . . . . . . . . . . . . . . . . . . 38 Procedure . . . . . . . . . . . . . . . . . . . 39 Results . . . . . . . . . . . . . . . . . . . . 41 Conclusions . . . . . . . . . . . . . . . . . . 46 Effects of Dexamethasone and Haloperidol on Mammary Tumor Growth. . . . . . . . . . . . . . 49 Objectives. . . . . . . . . . . . . . . . . . . 49 Procedure . . . . . . . . . . . . . . . . . . . 49 Results . . . . . . . . . . . . . . . . . . . . 51 Conclusions . . . . . . . . . . . . . . . . . . 60 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 63 LIST OF REFERENCES . . . . . . . . . . . . . . . . . 68 iv LIST OF TABLES Table Page 1. Composition of Variable Fat Diets (percent of total diet) . . . . . . . . . . . . 28 2. Effects of Variable Fat Diets on Mammary Tumor Development in Rats . . . . . . . . . . . 33 3. Effects of Variable Fat Diets on Serum Prolactin (PRL) Levels. . . . . . . . . . . . . 34 4. Effects of Adrenalectomy (ADRENX) and Estradiol Benzoate (EB) on Average Tumor No. in Rats . . . . . . . . . . . . . . . . . . 43 5. Effects of Adrenalectomy (ADRENX) and Estradiol Benzoate (EB) on Serum Prolactin (PRL) in Lactating Female Rats Bearing DMBA-Induced Tumors . . . . . . . . . . . . . . 44 6. Effects of Dexamethasone (DEX) and Haloperidol (HALO) on Serum Prolactin (PRL) Levels in Rats . . . . . . . . . . . . . . . . . . . . 55 7. Effects of Dexamethasone (DEX) and Haloperidol (HALO) on Body Weight in Rats . . . . . . . . . 56 8. Effects of Dexamethasone (DEX) and Haloperidol (HALO) on Serum Prolactin (PRL) Levels in Rats . . . . . . . . . . . . . . . . . . . 58 9. Effects of Dexamethasone (DEX) and Haloperidol on Body Weight in Rats. . . . . . . . . . . . . 59 LIST OF FIGURES Figure Page 1. Effects of Variable Fat Diets on Mammary Tumor Development in Rats Injected With DMBA. . . 32 2. Effects of Variable Fat Diets on Body Weight in Rats Injected With DMBA. . . . . . . . . . . . 35 3. Effects of Adrenalectomy (ADRENX) and Estradiol Benzoate (EB) on Percent Change in Average Tumor Diameter During Lactation . . . . . . . . . 42 4. Effects of Adrenalectomy (ADRENX) and Estradiol Benzoate (EB) on Pup Weight Gain During Lactation . . . . . . . . . . . . . . . . . . . . 45 5. Effects of Dexamethasone (DEX) (300pg/rat/day) Administered Alone; Haloperidol (HALO) (0.5mg/kg/day) Administered Alone; and Dexamethasone Administered With Haloperidol (DEX + HALO) on Percent Change in Average Tumor Diameter. . . . . . . . . . . . . . . . . . 54 6. Effects of Dexamethasone (DEX) (SOpg/rat/day) Administered Alone; Haloperidol (HALO) (0.5 mg/kg/day) Administered Alone; and Dexamethasone Administered With Haloperidol (DEX + HALO) on Percent Change in Average Tumor Diameter. . . . . . . . . . . . . . 57 vi INTRODUCTION The 7,12—dimethy1benz(a)anthracene (DMBA) model of mammary tumor development and growth has long been the basis for much of the knowledge obtained from research and applied to therapy of human breast cancer. It has been well established that prolactin and estrogen are the two most important hormones involved with carcinogen—induced mammary tumorigenesis in rats. In general, physiologic and pharmacologic states which increase serum prolactin levels stimulate mammary tumor growth, and conditions that reduce serum prolactin levels reduce or inhibit mammary tumor growth. There are exceptions, however, such as during lactation when serum levels of prolactin are elevated as a result of the suckling stimulus. However, at this time, most mammary tumors have been determined to undergo a profound regression. This "paradoxical regression" of mammary tumors during postpartum lactation has been repeatedly observed, yet no adequate explanation has been demonstrated. Therefore, it was of interest to examine this phenomenon and attempt an explanation by focusing in on the elevated adrenalglucocorticoid and reduced estrogen secretions that occur concomitant with elevated serum prolactin during 1 lactation. Dietary factors have also been shown to profoundly influence carcinogen-induced mammary tumor development and growth. In particular, high fat diet consumption has been demonstrated to stimulate spontaneous and carcinogen— induced mammary tumor development in mice and rats. Both the type and relative amount of fat consumed in the diet seem to be important factors in the manifestation of this effect on mammary tumor development. In general, diets which contain primarily unsaturated fatty acids appear to stimulate mammary tumor development to a greater extent than diets containing equal amounts of saturated fatty acids. Mechanisms to explain this effect of high fat diets on mammary tumori— genesis have not been elucidated. In light of the large amount of fat consumption in societies that have correspond- ingly high incidence of breast cancer, compared to societies that have lower fat consumption; confirmation of this stimulatory effect of high fat diets, as well as a mechanism for such findings need to be investigated. It was for this reason that the effects of variable fat diets on mammary tumor develOpment and serum prolactin levels were examined. REVIEW OF LITERATURE DMBA—Induction of Mammary TumorsAin the Rat The carcinogenic prOperties of coal tar derivatives such as dimethylbenz(a)anthracene (DMBA) have been noted for a relatively long period of time. The first such Observation stemmed from the relatively widespread occurence of testicular carcinoma that developed in chimney-sweeps of nineteenth century Europe during the period of the Industrial Revolution. Since then potent carcinogens, such as 3—methylcholanthrene and 7,lZ—dimethylbenz(a)anthracene have been isolated. These chemical agents were initially determined to be highly carcinogenic in laboratory animals when skin cancers were produced as a result of their topical application to rabbit ears. DMBA is particularly effective in producing mammary tumors when administered subcutaneously, orally or intravenously to the Sprague—Dawley strain of rat. Huggins (1965) demonstrated that a single intravenous injection containing 5 mg of 7, 12— DMBA in a lipid emulsion administered to female rats 50 - 60 days old resulted in a 95-100% incidence of mammary adenocarcinomas within one to three months. When evaluating DMBA—induced mammary tumors in the rat as a model for studying human breast cancer one must consider the following criterion. Initially, the tumors should be induced with relatively high frequency in a reasonable amount of time and utilizing laboratory animals suitable for such study with respect to size, ease of handling, expense, etc. The tumors which develop should resemble human breast cancer histoloqically and in the manner in which they respond to drug and endocrine treatments. DMBA produces mammary adenocarcinomas in the rat primarily in the ductal epithelial portion of the mammary gland, which is also the primary site of human breast cancer development (Huggins at al., 1959). This is opposed to lobulo-alveolar mammary adenocarcinomas that occur in many types of mouse mammary tumors. Finally, the hormonal responsiveness of the DMBA—induced mammary tumors in the rat somewhat resembles that of human breast cancer. DMBA—induced mammary tumors do not metastisize, however, which is an important deviation from the situation in human breast cancer (Young et 31., 1963). In general, it appears that the rat DMBA—induced mammary tumor represents a workable model for studying human breast cancer. Recent discovery and initial use of nitrosamines, such as N—nitrosomethylurea, have shown that such compounds may be even more desirable than DMBA as a carcinogen since they appear to produce metastatic mammary tumors in the rat (Guillino at al., 1975). i 5 Hormonal Influences on DMBA—Induced Mammary Tumors Influence_gf Prolactin In general, treatments that increase prolactin (and produce hyperprolactinemia) stimulate mammary tumor growth (Welsch and Nagasawa, 1977). Meites (1972) found that the presence of prolactin was required for the development and growth of DMBA tumors in the rat. The increase of serum prolactin induced by grafting pituitaries under the kidney Cepsule,stimulated DMBA-induced mammary tumor growth (Welsch gt gl., 1968). Surgical manipulations which elevate serum prolactin levels, such as lesion of the median emminence, stalk section and adrenalectomy, also stimulate mammary tumor growth (Clemens g3 g1., 1968; Chen g3 g1., 1976). Drugs, such as reserpine, haloperidol, perphenazine and others that increase serum prolactin also stimulate mammary tumor growth (Welsch and Meites, 1970; Quadri g: g1., 1973). Physiological conditions, including pregnancy (McCormick and Moon, 1965) and pseudOpregnancy (Dao, 1959), which also cause hyperprolactinemia, stimulate mammary tumor growth. One exception to the general principle that elevated prolactin levels stimulate mammary tumor growth is post— partum lactation. At this time, when prolactin levels are elevated due to the suckling stimulus mammary tumors regress (McCormick and Moon, 1965). This is the basis of one of the problems studied in this thesis. Conditions and treatments that decrease serum prolactin levels (produce hypoprolactinemia) generally inhibit development and growth of mammary tumors (Welsch and Nagasawa, 1977). Pargyline, L-dopa, and ergot alkaloids such as ergocornine, decrease serum prolactin and inhibit mammary tumor growth (Quadri, 1973; Nagasawa and Meites, 1970). Treatments such as hypophysectomy and restricted food intake, which remove or decrease anterior pituitary hormones in general, inhibit growth of carcinogen—induced mammary tumors in the rat (Kim and Furth, 1960; Tannenbaum, 1959). Hyperprolactinemia prior to carcinogenic treatment decreases the incidence of developing mammary tumors (Kledzik g3 g1., 1974). The rationale for this phenomenon is not entirely clear, but it is hypothesized that a hyperprolactinemic state produces a predominantly lobulo- alveolar growth of the mammary gland which renders the mammary tissue refractory to carcinogen treatment (Welsch, 1977). As previously stated, carcinogens such as DMBA produce tumors predominantly in the ductal epithelial tissue of the relatively undeveloped mammary gland in the rat (Huggins g3 g1., 1959). Influence of Estrogen High doses of estrogen have been demonstrated to inhibit development and growth of DMBA—induced mammary tumors in the rat (Meites and Sigouris, 1953). However, estrogen is required for induction of mammary tumors by DMBA since ovariectomy inhibits development of such tumors (Dao, 1962). Talwalker g3 g1. (1964) observed that while estrogen replacement in ovariectomized rats reversed the inhibition of ovariectomy on mammary tumor development, prolactin and growth hormone together could promote mammary tumor develop- ment in the absence of ovaries. This suggests that pituitary hormones are more important than estrogen for development of mammary tumors in rats. Estrogen also has been demonstrated to exert complex effects on growth of established DMBA—induced mammary tumors. Huggins gt g1. (1959) observed that ovariectomy induced regression of established carcinogen-induced mammary tumors, an effect that could be reversed by replacement with estrogen. Low and moderate doses of estrogen admin— istered to intact, tumor bearing rats have been shown to stimulate mammary tumor growth (Huggins g3 g1 ., 1965). However, in the absence of prolactin (as a result of hypophysectomy) such stimulation or maintenance of tumor growth by estrogen is not evident (Meites, 1972). During pregnancy, mammary tumor growth is stimulated (McCormick, 1965) presumably due to the combination of increased ovarian steroid secretion and placental prolactin secretion (Friesen, 1973). Estrogen also has been demonstrated to increase serum prolactin levels (Meites and Nicoll, 1966), which may be one of the means by which estrogen influences mammary tumor growth. However, large doses of estrogen have been shown to inhibit mammary tumor growth, but simultaneously stim- ulate prolactin release (Meites and Sigouris, 1953). High levels of estrogen may act peripherally to inhibit mammary tumor growth. Kledzik g3 g1. (1976) observed that large doses of estrogen decreased prolactin receptors in DMBA- induced mammary tumors, thereby preventing the elevated serum prolactin from stimulating tumor growth. Therefore, it appears that estrogen, although it may not be absolutely essential as indicated in experimental regimens, is important under most physiological conditions for mammary tumor de— velopment and growth by acting directly on the mammary tumor and indirectly through its influence on prolactin secretion. Influence of Adrenal Glucocorticoids Adrenal glucocorticoid hormones appear to inhibit mammary tumor growth in rats by acting directly on the tumor tissue and indirectly by inhibiting prolactin secretion. Dexamethasone, a synthetic glucocorticoid, has been shown to decrease basal serum prolactin levels (Schwinn g3 g1., 1976 and Harms g3 g1., 1975). Euker g3 g1. (1975) and Harms g3 g1. (1975) demonstrated that dexamethasone reduced the stress—induced increase in serum prolactin caused by exposure to either anesthesia. TRH (thryotrOpin releasing hormone)—induced increases in prolactin are blocked by chronic administration of dexamethasone (Schwinn g3 g1., 1976). 9 Chen gt "1. (1976) showed that removal of glucocorti- coids by adrenalectomy stimulated mammary tumor growth and increased serum prolactin levels. Replacement with gluco— corticoids inhibited mammary tumor growth and reduced serum prolactin to control levels (Chen g; gl., 1976). Hilf g3 g1. (1962) showed that exogenously administered corticosterone inhibited growth and development of transplantable mammary tumors in the rat. Influence of Progesterone Progesterone is generally thought to have a stimulatory effect on the development of carcinogen induced mammary tumors. Huggins g; g1. (1962) demonstrated that progesterone treatment 1 month after DMBA administration shortened the latency period of tumor development, stimulated growth of existing tumors, and increased the number of tumors that developed. Huggins g3 g1. (1962) showed that pregnancy induced after DMBA administration had similar effects as that of progesterone treatment during the same time period. This study indicated that the increased progesterone secretion during pregnancy is primarily responsible for the observed increase in mammary tumor development and growth. Jabara (1967) studied the effects of progesterone treatment just prior to and following DMBA administration, and obtained results similar to Huggins gt g1. (1972). Tumor incidence and tumor number were increased in groups given progesterone. The latency period was also reduced in 10 progesterone treated rats. Jabara (1967) also observed that exogenous progesterone had little or no effect on the growth of established mammary tumors. McCormick g5 g1. (1965) demonstrated that pregnancy stimulated the growth of established carcinogen—induced mammary tumors. However, it was not determined whether the elevated progesterone or the presence of placental lactogen was the main contributor to the stimulated tumor growth. Influence of Insulin Insulin appears to have an important effect on the growth of carcinogen—induced mammary tumors. Heuson and Legrow (1972) observed that removal of insulin by alloxan—induced di— iabetes resulted in regression of 90% of the mammary tumors present. Cohen and Hilf (1974) obtained similar re— sults when 60% of the mammary tumors present regressed when rats were made diabetic by an injection of streptozotocin. The effect of chemical—induced diabetes on mammary tumors was reversed by insulin replacement. Further research on the effect of diabetes on mammary tumors by Smith g3 g1. (1977), determined that streptozotocin induced diabetes reduced prolactin receptors in the regressing mammary tumors, suggesting a possible explanation for the observed regression of mammary tumors in these rats. In intact rats, administra- tion of insulin has not been demonstrated to influence mammary tumor development or growth. 11 Effects of High Fat Diets on Prolactin and Mammary Tumor Induction Tumor Induction and Development High fat diets long have been shown to influence tumors. Tannenbaum (1942) reported that high fat diets ingested by mice increased the incidence of spontaneous breast carcinomas and induced skin tumors, while decreasing the latency period of development of these tumors. High fat diets also increase the incidence and shorten the latency period of carcinogen—induced (DMBA) mammary tumors in rats (Gammal et al., 1967). The type of fat used appears to be significant for increasing tumors in animals fed high fat diets. Unsaturated fats appear to stimulate tumorigenesis to a greater degree than saturated fat. Gammal gt gt. (1967) demonstrated that high fat diets containing corn oil, consisting mainly of unsaturated fatty acids, stimulated 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 ten different fats and oils in their diets and determined that, in general, high fat diets that contained primarily unsaturated fats stimulated tumori- genesis to a greater extent than high fat diets containing primarily saturated fats. Hopkins gt gt. (1976) also determined that diets containing unsaturated fats increased tumor incidence and shortened the latency period of mammary 12 tumors. High fat diets appear to exert their effects at the promotional stage of mammary carcinogenesis. Carroll and Khor (1970) demonstrated that the type of diet ingested by a rat after carcinogen administration produced a greater influence on tumorigenesis than the type of diet ingested prior to DMBA administration. Their summary suggests that high fat diets consumed by rats after DMBA administration stimulated mammary tumorigenesis regardless of whether high or low fat diets were supplied prior to the injection of DMBA. Although high fat diets have a high caloric content, their effects on tumorigenesis appear to be independent of caloric intake. Tannenbaum (1945) demonstrated that a calorically restricted diet inhibited tumor formation in mice. However, at any level of caloric restriction, a high fat diet was less inhibitory than a low fat diet on tumor induction. It thus appears that the tumor enhancing effect of high fat diets is due primarily to a specific action of fat rather than to a general caloric effect. Hormonal Effects of High Fat Diets Since hormones, especially prolactin and estrogen, play predominant roles in controlling mammary tumorigenesis, the effects of high fat diets may be mediated wholly or in part by hormonal mechanisms. Chan and Cohen (1974) used hormone antagonists in an attempt to explain how high fat diets enhance mammary tumorigenesis in rats. Nafoxidine, 13 (1— 2- p—3,4—dihydro-6-methoxy—2—phenyl—l—l—napthyl phenoxyethyl -pyrrolidine hydrochloride) an extrogen antagonist, inhibited tumor development but did not remove the differential effects of low and high fat diets. How— ever, CB-154, (2-bromo—deergocryptine methanesulfate), an anti-prolactin agent completely suppressed the formation of all palpable tumors when a 5 mg DMBA dose was given to high fat and low fat—fed rats. CB-154 also abolished the differential effect of high fat diets on mammary tumor induction in rats given 10 mg DMBA. Thus it appears that enhancement of mam— mary tumor development by high fat diets may be mediated in part through increases in circulating levels of prolactin. How— ever, no measurements of hormones were made in this report to support such a theory. Chan and Cohen (1975) advanced a hormonal hypothesis to explain the effect of a high fat diet on mammary tumorigenesis. The basis of their hypothesis was that the ratio of prolactin to estrogen (PROLACTIN:ESTROGEN) is important in controlling mammary tumor development and growth rather than the absolute values of the circulating hormone levels. It appears then, that an increase in the PROLACTIN:ESTROGEN ratio results in increased mammary tumor growth and development. Chan and Cohen (1975) believe then that chronic high fat diets increase the PROLACTIN:ESTROGEN ratio which then leads to enhancement of growth and develOpment of normal and neoplastic mammary growth. Chan, Didato and Cohen (1975) reported evidence that —T— 14 apparently lent support to their hypothesis, by observing that high fat diets increased serum prolactin levels during the proestrus-estrus stage of the estrous cycle of the rat. They also found that high fat diets had no effect on serum prolactin levels during the metestrus-diestrus stage of the cycle. More recently, Cave gt gt. (1979) observed that a high fat diet had no effect on serum prolactin levels although it increased mammary tumorigenesis in carcinogen-treated rats. Implications on the Human Condition Such observations of the effects of high fat diets on rat carcinogenesis may provide important insight into the explanation for the marked differences in the incidence of breast cancer in women of Western and Eastern cultures. Postmenopausal women in the U.S.A. show approximately a 4-fold greater incidence of breast cancer when compared to Japanese postmenopausal women (Segi gt 31., 1969). One appearent difference between the two groups is the type of diet consumed. In general, American women consume a relative- ly high fat diet as compared to Japanese women (Carroll, 1975). Carroll (1975) reported that a high correlation exists between the total fat consumption and death rate from breast cancer. The mechanism by which high fat consumption in human subjects stimulates breast tumorigenesis remains to be determined. ————i 15 Effects of Lactation on Growth of DMBA-Induced Mammary Tumors Hormonal Responses to the Suckling Stimulus During Lactatipn The control of release of hormones necessary for lactation is mediated by a nervous reflex initiated by stimu- lation of the nerve endings within the nipples by the suckling stimulus, and is integrated through neural cir— cuits in the hypothalamus (anrs gt gt., 1956). Edwardson and anrs (1967) demonstrated that a quantitative relationship exists between the volume of milk products and the intensity of the suckling stimulus. They concluded that decreasing the innervation from the nipples reduces the intensity of nervous stimulation reaching the hypothalamo- hypophysial system, and thereby causes a reduction in the pituitary hormones that affect lactation. Myers, gt gt. (1975), demonstrated that a quantitative relationship exists between the suckling stimulus and serum prolactin levels, by showing that elevated prolactin levels during lactation are directly proportional to litter size, which was used as an index of the intensity of the suckling stimulus. According to Harris (1948) hypothalamic neurohumors are released into the hypothalamo—hypophysial portal vessels in response to suckling or to other types of neuronal activity and thereby control the release of anterior pituitary hormones. Prolactin, which is the primary anterior 16 pituitary hormone concerned with lactation, is tonically inhibited by the neurohormone "prolactin—inhibiting fac— tor" (PIF) (Talwalker gt gt., 1963) which some have identified as dopamine. Ratner and Meites (1964) demon— strated that hypothalamic extracts from suckled rats had no capacity to inhibit PRL release from pituitaries of control cycling rats incubated ta ytttg. This would appear to be a result of a decrease in PIF synthesis in the hypothalamus which is caused by the suckling stimulus. Minaguchi and Meites (1967) showed that the suckling stimulus decreased the content of PIF in the hypothalamus and thereby increased the synthesis and release of PRL from control pituitaries incubated in vitro. In summary, the suckling stimulus decreases PIF activity in the hypothalamus via a neural-hypo— thalamic reflex, thereby reducing the inhibitory influ- ence of the hypothalamus on pituitary PRL release. Stern and Voogt (1973/74) also compared prolactin levels in suckled lactating and cycling diestrus rats, and showed that suckling increased serum prolactin levels. The suckling stimulus in rats during lactation affects the gonadotropins(LH and FSH) in a manner opposite to that of prolactin. Minaguchi and Meites (1967) showed that suckling caused a reduction in leuteinizing hormone releasing hormone (LHRH) content in the hypo- thalamus. This was demonstrated by a reduced effective- ness of hypothalamic extracts from suckled 17 rats to release LH from pituitaries of control cycling rats. Also, pituitary LH concentrations were significantly decreased in suckled lactating rats. Lu gt gt. (1967b) reported that serum Lh levels were depressed in postpartum lactating mother rats. Lu gt gt. (1976a) also reported that postpartum lactating rats showed a reduced LH response to exogenous LHRH administration. Elevated serum prolactin levels during postpartum lactation appear to be partly responsible for this reduction in LH at this time and appear to work via a hypothalamic mechanism (Lu gt gt., 1976b; Clemens gt gt., 1969). The ovaries also appear to have a role in the inhibi— tion of LH release from the pituitary during postpartum lactation. Hodson gt gt. (1978) showed that the positive feedback of estrogen and progesterone on LH release and the response of the pituitary to LHRH stimulation are reduced in lactating rats, suggesting that the ovarian steroids may act at both the hypothalamic and pituitary levels to decrease LH secretion during postpartum lactation. Suckling has also been shown to inhibit the postcastration rise in LH (McCann gt gt., 1961;Smith gt gt., 1977) and to depress basal levels of LH (Smith gt gt., 1977). FSH appears to be much less affected by the suckling stimulus during lactation than does LH. Circulating FSH levels during postpartum lactation remained at values similar to basal cycling levels (Smith gt gt., 1977). However, suckling did appear to inhibit the postcastration rise in FSH in lactating ovarectomized female rats (Smith l8 gt gt., 1977). Lu gt gt. (1976a) also found that suckling can inhibit FSH release from the pituitary. Such inhibitory effects of suckling on basal gonadotropin levels are not evident in nursing women (Soria gt gt., 1976). Growth hormone (GH) is also involved in lactation and appears to be released in response to the suckling stimulus. Grosvenor (1964) showed that injections of growth hormone improved lactation in rats deprived of spinal cord connec— tions between the mammary gland and the CNS. This would suggest that the rat may normally release GH in response to the suckling stimulus. In the same study, Grosvenor found that pituitary GH concentration was reduced following a thirty minute acute suckling period which suggests that a considerable amount of GH was discharged in response to suckling. Also Chen gt gt. (1974) showed that serum GH is increased 30 minutes after suckling in rats. Adrenalcorticotropin (ACTH) secretion also is in— creased in response to suckling. Poulton and Reece (1957) showed by indirect measurement that the pituitary—adrenal cortex axis is stimulated during lactation in the rat. They demonstrated that adrenal cholesterol concentration is reduced during lactation which reflects an increased adrenal cortical activity at this time. Poulton and Reece used the ascorbic acid depletion bioassay for ACTH to measure the decrease in pituitary ACTH concentration following suckling. Together, these observations indicate that the pituitary—adrenal cortex axis is stimulated at this time. ’f— 19 Voogt gt gt. (1969) observed that an acute suckling period (i.e. a 12 hour non-suckling followed by % to 3 hours of suckling) resulted in increased plasma ACTH levels in the lactating rat. Adrenal glucocorticoid secretion also appears to be increased in the lactating rat in response to suckling. Voogt gt gt. (1969) observed an increase in total serum cor— ticosterone levels in the lactating rat following an acute suckling period of B or 3 hours. Stern and Voogt (1973-74) also demonstrated that total plasma corticosterone levels were increased following a similar acute suckling period when compared with normal cycling levels of corticosterone. However, when the lactating rats were with their litters continually, no significant increase in total plasma corti— costerone was observed when compared with basal cycling levels of corticosterone. Increased glucocorticoid activity in the lactating rat also may be produced by a decrease in corticosteriod binding globulin (CBG) levels. CBG is a plasma globular protein that binds glucocorticoids, thereby rendering them unavail— able for action at the cellular level (Westphal, 1971). Gala and Westphal (1965) observed a marked decrease in CBG activity in lactating rats and suggested that this had a possible role in initiation of lactation in the postpartum rat. Further studies by Gala and Westphal (1966) suggested that TSH may be the anterior pituitary factor that mediates the control of CBG synthesis. It appears then that an —i— 20 increase in glucocorticoid secretion from the adrenal glands caused by stimulation of the ACTH—adrenal axis due to suckling, combined with a decrease in CBG synthesis, results in a marked increase in free glucocorticoid hormones available for action on the mammary gland. Ovarian steriod secretion also appears to be influ— enced during lactation, both by changes in anterior pitui— tary secretion and by the ovaries entering a period during which they are temporarily refractory to gonadotropin stim~ ulation. Smith and Neill (1977) showed that estradiol secre— tion is depressed in ovariectomized levels in lactating rats (Smith and Neill, 1977). Zarate gt gt. (1972) also observed estrogen secretion to be depressed in postpartum women. Such an inhibition of estrogen secretion appears to be due to ovarian refractoriness to gonadotropin stimula- tion as well as to decreased gonadotropin secretion from the pituitary, since exogenous administration of gonadotropins failed to elevate estrOgen levels during postpartum lacta— tion in women (Zarate gt gt., 1972). Progesterone secretion appears to be elevated during postpartum lactation in rats, because of the high circulating levels of prolactin at this time (Smith and Neill, 1977). Effects of Lactation on Mammary Tumor Development and Growth Lactation has been shown to inhibit the induction and development of carcinogen—induced mammary tumors and cause regression of existing, growing mammary tumors. Dao et al. 21 (1960) reported that lactating rats appeared to be unre— sponsive to the action of carcinogenic agents such as 3—methylcholanthrene. The hyperprolactinemic state during lactation may stimulate lobuloalveolar growth in the mammary gland at the time of carcinogen administration, and since carcinoqen-induced mammary tumors arise predominately from the ductal epithelium, such a preponderance of the lobulo— alveolar elements of the mammary gland may prevent the carcin- Ogen from inducing mammary tumorigenesis (Welsch and Nagasawa, 1977). Lactation also has been shown to cause regression of existing carcinogen—induced mammary tumors in rats (McCormick and Moon, 1965, 1967a, 1967b; McCormick, 1972) and women (McGuire, 1972), despite high serum prolactin levels. However, an adequate explanation for this paradoxical regression has yet to be advanced and is the basis for one of the research projects in this thesis. One possible explanation for this phenomenon is that estrogen secretion is reduced during postpartum lactation (Labhsetwar and Watson, 1974; Smith and Neill, 1977). Since estrogen is necessary for maximal mammary tumor growth (Meites, 1972), reductions in estrogen secretion during lactation could contribute to the observed tumor regression. Another possible explanation for postpartum regression of mammary tumors is the elevated free glucocorticoid levels resulting from a reduction of corticosteroid binding globulin and an increase in corticosterone secretion from .f... __ 22 the adrenal glands. Administration of adrenal glucocorti- coids has been shown to decrease mammary tumor growth in non- lactating rats (Hilf gt gt., 1965; Chen gt gt., 1976) and in women (Brennan, 1973; Hayward, 1970), whereas adrenal- ectomy results in increased tumor growth and elevated serum prolactin concentrations in rats (Chen gt gt., 1976). Adrenal glucocorticoids also may directly inhibit mammary tumor growth. Further work to examine the role of adrenal secretions and ovarian steriod secretion during lactation is necessary to elucidate the reason for the paradoxical regression of mammary tumors during postpartum lactation. MATERIALS AND METHODS Research Animals Animals used in the variable fat diet study were female Sprague-Dawley obtained from Spartan Research Animals Haslett, Michigan. Animals used in the lactation study were obtained from Harlan Research Animals, Indiana— polis, Indiana. The animals were housed one to a cage in metal suspension cages in the variable fat diet study. In 1 the lactation study lactating females and their litters were isolated in plastic cages. All animals were main— tained in a temperature controlled (25¢ 1°C) room on a 14 hour light (5:00A.M.—7:00P.M.)-10 hour dark lighting regiment. Animals were fed ad libitum on a diet of tap water and rat chow (Ralston Purina Co., St. Louis, MO) except when variable fat diets were used (see below). Tumor Induction Mammary tumors were induced in all animals by the method of Huggins gt gt. (1965). Virgin female Sprague- Dawley rats, 55—60 days of age, were given a single intra- venous (i.v.) injection of lml lipid emulsion containing 5mg of 7,12—dimethy1benz(a)anthracene (DMBA), while under light ether anesthesia. Tumors appeared l to 3 months from 23 24 DMBA injection in both studies. Tumor Measurements Tumors were palpated and measured, and body weights were recorded at weekly intervals in the variable fat diet study beginning 1 month after administration of DMBA. Palpable mammary tumors were measured with a vernier caliper and the two largest perpendicular diameters were recorded. Weekly measurements for each tumor were averaged and summed for each rat, and expressed as "the sum of the average tumor diameter per rat." In the lactation-tumor study tumors were palpated and measured at weekly intervals following the onset of pregnancy until parturition. During lactation, tumors were measured at 2 to 5 day intervals since the changes in tumor sizes appeared to be quite dramatic. Tumor growth or regression was expressed as the percent change in average tumor diameter when compared to initial measurements (i.e., at parturition, prior to initi— ation of the respective treatments.) Tumor measurements in the dexamethasone-halOperidol study were taken at weekly intervals for a 3—week period upon the initiation of the drug treatment. In this study tumor growth or regression was expressed as the percent change in average tumor diameter when compared with pretreatment measurements. Pregnancy Induction As tumors began to develop approximately six weeks following DMBA treatment, one male Sprague-Dawley rat was 25 placed in a cage housing four females with developing mam— mary tumors and allowed to mate at will. Daily vaginal smears were taken from each female each morning to determine the presence of sperm in the vagina. If sperm was present, the rats were assumed to be impregnated and were isolated in separate cages. They were placed into an appropriate treat- ment group and allowed to bear their young. Following parturition, the litters were adjusted to a uniform number, and treatment was initiated three days postpartum. Btood figmpling and Hormone Radtgimmunoassayg Serial blood samples were taken at weekly intervals during lactation and at monthly intervals for the variable fat diet—tumor development study. All samples were taken between 10:00 A.M. and 12:00 noon. Serum was extracted by centrifugation and assayed for prolactin by the method of Niswinder gt gt. (1969). Total plasma corticosterone was measured at Endocrine Sciences, Tarzana, California, by competitive binding radioimmunoassay. Hormone Treatments (Lactation Study) Estradiol benzoate was administered daily by subcutaneous injections to one group of lactating rats at a dose of l‘pg/ rat. Control and adrenalectomized rats in the same study were injected daily with equivolumes of corn oil vehicle. Glucocorticoid influence on tumor growth during 26 lactation was removed by bilateral adrenalectomy. Dexamethasone was administered subcutaneously at a relatively high dose of 300 pg/rat and a relatively low dose of Sng/rat per day. Corn oil and a 5% ethanol solution were vehicles used to deliver the dexamethasone in these experiments. Haloperidol was administered daily by subcutaneous injections at a dose of 0.5 mg/kg per day. Corn 011 and 0.89% saline were vehicles used to deliver haloperidol in the dexamethasone-haloperidol study. Diets (Variable Fat Diet—Tumor Development Study) The composition of the diets employed in this study are shown in Table 1. Corn oil and animal lard were used as sources of unsaturated and saturated fatty acids, respec- tively. Diets were prepared weekly with the proportion of ingredients added on a percentage-weight basis. Rats were fed ad libitum and fresh food was provided every two days. Casein, cellulose, salt, mineral mixture and vitamin supple— ments were obtained from U.S. Biochemical Corporation, Cleveland, Ohio. Sucrose, corn oil and lard were purchased from local sources. Statistical Analysis Statistical differences between hormone levels in each study were determined by use of analysis of variance (ANOVA) and Student Neuman Keuls (S—N—K) statistical tests. 27 Differences in tumor number and latency period of development also were tested for significance utilizing the S-N—K analysis. Statistical differences in tumor development and growth in the variable fat study were analyzed by analysis of covariance. 28 .wad: peep mo m1 comm 0o. coped mmB coHumoHMprow §H> mo macho Hm m if; ll 1 m m cm o.me mm Anyway new team m m om o.ma mm AHflo shoot ham team a A m.e m.mm mm cam Honucoo a A m.o m.eo mm owe 30a iiifllflll i mwudfiflz pamm mmodsqmu p§\fio BOO mmonosm fimwmo #6585. 3pr H38 mo £50.89 muwHQ pom mHQmHHQ/ mo coflemoagoo . H maflme EFFECTS OF VARIABLE FAT DIETS ON DEVELOPMENT OF DMBA-INDUCED MAMMARY TUMORS AND SERUM PROLACTIN LEVELS Objectiveg Although the stimulatory effects of high fat diets on DMBA—induced mammary tumorigenesis have been definitely demonstrated (Carroll, 1975), no adequate explanation for the mechanism(s) involved has been forthcoming. Since some inves— tigators have suggested that hormonal mechanisms, specifically prolactin, are involved (Chan and Cohen, 1974; Chan gt gt., 1975) it was of interest to determine the effects of variable fat diets on basal serum prolactin levels as well as attempt to confirm previous findings on the stimulatory effect of high fat diets on mammary tumor development. Procedure Female Sprague—Dawley rats, 55—60 days of age were in- jected with 5 mg 7, lZ-dimethylbenz(a)anthracene via the tail vein and placed on one of the four variable fat diets (see Table 1). Approximately four weeks after DMBA administration, the rats were palpated for presence of tumors. Tumor growth and body weights were measured and recorded at weekly intervals. Three blood samples were taken at 29 30 monthly intervals in the morning, beginning approximately one month after the beginning of the dietary treatment. Blood was sampled via orbital sinus puncture under light ether anesthesia. Treatment was suspended following three months of dietary treatment, at which time the rats were fed a diet consisting of control Purina lab blocks. After two months on the normal diets a final blood sample was taken. Mammary tumor development was approximated by summing the average tumor diameters for each rat and expressed as the means of the sum of the average tumor diameter per rat. Statistical differences in mammary tumor development among treatment groups were determined by analysis of covariance. Mammary tumor numbers were recorded in each rat and expressed as the mean tumor number per rat. Mean latency period was determined for each group by averaging the number of days that elapsed between DMBA treatment and appearance of the tumor for each tumor palpated. Statistical differences in average tumor number and latency period were determined by analysis of variance and S-N-K analyses. Prolactin was measured in serum by the method of Niswinder, et al., (1969). Statistical differences between treatment groups were determined by analysis of variance and S-N-K analyses. Results Figure 1 shows the effects of variable fat diets on mammary tumor development in rats injected with DMBA. Both IIIIIIIIIIIIIIIII::::_________________——T 31 the high fat diet containing primarily saturated fatty acids (lard) and the high fat diet containing primarily unsaturated fatty acids (corn oil) resulted in increased mammary tumor development. The low fat diet fed to DMBA-treated rats resulted in a marginally significant (p<0.05) suppression of tumor development when compared with controls. The effects of variable fat diets on percent tumor induction, average tumor number and average latency period of tumor development are listed on Table 2. Both the unsatu- rated high fat diet (corn oil) and the saturated high fat diet (lard) resulted in an increase in percent of tumor induction when compared with control fat diets. Both types of high fat diets also significantly (p<0.05) reduced the mean latency period of tumor development when compared with controls. Finally both high fat diets resulted in an increase in average tumor number when compared with controls. The low fat diet had no significant effect on latency period, percent tumor induction or average tumor number when compared with the control fat diet. The effects of variable fat diets on basal serum prolactin levels are presented in Table 3. Unsaturated high fat diets (corn oil) were associated with increased basal serum prolactin levels at the first sampling time period, approximately one month after initiation of the diet treatment and DMBA injection. In the subsequent sampling time periods, during diet treatment, prolactin was elevated but the differences were not significant. High fat diets containing primarily saturabai fat (lard) significantly elevated serum prolactin 32 110 ".0 .o‘HFkorn oil) 0...... 100 ;F. A It, DUO...OOOQOOO'IOOOOOO‘OOOOOO H F ('0 I'd ) 380 i, = I. “no [3 I! [i so ’ Tumor Diameter: 0- O f o :‘ < 3.0 3 29 a 2 W a O 5 6 7 8 9 10 H 12 13 week: after OM B A , _ I Figure 1. Effects of Variable Fat Diets on Mammary Tumor Development in Rats Injected With DMBA. CF Represents 4.5% Control Fat Diet; LF Represents 0.5% Low Fat Diet; HF (corn oil) and HF (lard) Represent 20.0% High Fat Diets. mHoHEOo fies 8.3960 533 3.8..» 0 when pcmmmwmmm n .z.m.m a cams .m 3 a. 11 oo.H H m.am we woos 0m.o « o.o o Aeneas one roam oa.H H o.mm mo woos us.o H m.e ma Iago shoot use nose o.e « N.oo as wm.mo o.o H o.m m can 30a no.m 4 H.oo om wo.oo am.o a a.e o pan Houpcoo Bowed 50933 mug» cofloscfi . oz HOSE. mobs ugmwfi. mmmumfim mo . oz H083. ”:80me montage. mo .02 mama cum ucQEOHo>mo H959 g co 39.5 umm managing wo Bomwmm . N wanna. 34 “#ch Honucoo co coondm mums mums Hmpmw mcuEQe N :93 mam—Ewm mo mHoHEoo fig 8% 5:3 8.on o 02-mom fl Mg a “gamuu mo coflmflflfi Burma 9303 .m o.~ « o.mm om.vH « m.eo v.m « s.em om.v a m.om o Aeneas umm amen m.m H o.em m.o « H.m~ s.o H m.om oe.o « m.Hm Ha Iago auooo pom seem m.oH « o.om N.m « o.mH H.s a H.mm m.H n m.HH o one son o.o « v.e~ m.v « m.oH m.o « m.oH av.m a o.mH o new Houuaoo ammll mm m Mm mama Adimcv magma 5E gnaw wo .02 g maw>mu Adamo cauomaoum ezuwm no mumao has wanmeum> mo mpowmmm . m manna. 35 C F .. 300 g ’ H F(|ard) E “0.; L F a o . . v .o"“-v'::--""'° " WW “\ H F (corn on I) h ’a.«?o: OOIOOQQO..'........ c ...'.O'W I I’..'. ' 2 250 I... C ' If...“ m a... . “..”ooom~” “0". ; MWOOM. >. D O n 20C) 4 5 6 7 8 9 1O 1 1 1 2 13 weeks after start of diet trootment Figure 2. Effects of Variable Diets on Body Weight in Rats Injected With DMBA. CF Represents 4.5% Control Fat Diet; LF Represents 0.5% Low Fat Diet, HF (corn oil) and HF (lard) Represent 20.0% High Fat Diets. 36 levels at the first and third sampling times, approximately one and three months following initiation of the diet treatment, respectively. At the second sampling time (two months after initiation -of diet treatment) prolactin levels were again elevated but not significantly above controls. The low fat diet had no significant effect on basal serum prolactin levels when compared with controls at any of the sampling times. At the final sampling time, two months after variable fat diet treatment was suspended, serum prolactin levels were similar in all groups. The effects of the variable fat diets on body weight are shown in Figure 2. Rats fed the low fat diet had significant- ly lower body weights when compared with controls at all time periods except at week 4. Both high fat diets resulted in Siguficantly decreased body weight at weeks 8,9,10 and 13 after initiation of diet treatment when compared with controls. Conclusions The results from this study indicate that, contrary to Carroll and Khor (1971), both saturated and unsaturated types of high fat diets were equally effective in stimulating mam- mary tumor development in rats treated with DMBA when tumor development was expressed as percent tumor induction, latency period, or by tumor diameters and numbers. Low fat diets, however, appeared to have little or no effect on mammary tumor development when measured by these parameters. 37 Because high fat diets fed to rats resulted in small, inconsistent elevations in basal serum prolactin levels, it is only possible to conclude that these effects of high fat diets on prolactin may explain only part of the mechanism(s) by which high fat diets stimulate tumor development. The high caloric content of the high fat diets alone probably did not contribute to the increased tumor develop- ment, since these rats did not gain more body weight than controls. In fact, rats fed the high fat diets showed significantly lower body weights at the final four time periods when compared with controls. However, more of the fat in the high fat diet may have been utilized for tumor development. Also, body composition may have been altered by the high fat diets, and perhaps resulted in altered metabolism of the hormones (estrogen and prolactin) most involved in mammary tumor development. EFFECTS OF ADRENALECTOMY AND ESTROGEN ADMINISTRATION ON THE REGRESSION OF DMBA—INDUCED MAMMARY TUMORS DURING POSTPARTUM LACTATION IN RATS Objectives The paradoxical regression of mammary tumors in rats (Dao and Sunderland, 1959; McCormick and Moon, 1969) and human subjects (McGuire, 1965) during post—partum lactation despite high serum prolactin (PRL) levels has been well documented, but no adequate explanation for this phenomenon has been advanced. Elevated serum PRL levels normally stimu— late mammary tumor growth in rats (Meites, 1972; Welsch and Nagasawa, 1977). During pregnancy in rats there is increased growth of existing mammary tumors (Dao and Sunderland, 1959; McCormick and Moon, 1969) in the presence of elevated serum levels of placental PRL (Friesen, 1969), but during lactation when serum levels of pituitary PRL are increased, most mammary tumors regress. One possible explanation for this regression during postpartum lactation is that estrogen secretion is reduced during this period (Smith and Neill, 1977). Since estrogen is necessary for mammary tumor growth (Meites, 1972), a re— duction in estrogen secretion during lactation could 38 39 contribute to the observed tumor regression. Free glucocorti- coid levels also are elevated during lactation as a result of the reduction in corticosteriod-binding globulins (Gala and Westphal, 1965) and the suckling stimulus (Voogt gt gt., 1969). Administration of adrenal glucocorticoids have been shown to decrease mammary tumor growth in rats (Hilf gt gt., 1965) and in humans (Brennan, 1973), whereas adrenalectomy resulted in increased tumor growth and elevated serum PRL secretion in rats (Chen gt gt., 1976). In addition to their effects on PRL secretion, the glucocorticoids also may directly inhibit mammary tumor growth. The objective of this study was to elucidate the possible role of estrogen and the adrenals on regression of mammary tumors during postpartum lactation in rats. Procedure Virgin female Sprague—Dawley rats, 55 days of age, were each given an intravenous injection of 1 ml lipid emulsion containing 5 mg of 7,l2—dimethylbenz(a)anthracene (DMBA). The DMBA was kindly supplied by Dr. Paul Shurr of the Upjohn Co., Kalamazoo, Mi. When the tumors first began to appear, approx— imately 6 weeks after DMBA administration 4 females per cage were placed with 1 male and allowed to mate at will. The presence of sperm in the vagina, as determined by daily vaginal smears, was considered to be a positive indicator of pregnancy. Impregnated females were placed in separate cages and allowed to bear their young. 40 Three days after parturition, lactating rats containing one or more tumors at least 1 cm in average tumor diameter were divided into 3 groups and treated as follows: Group 1, intact controls were given daily sc injections of 0.1ml corn oil vehicle; Group 2 were given daily so injections of 1 ug estra- diol benzoate (EB) in 0.1ml corn oil; Group 3 were bilaterally adrenalectomized and given daily injections of 0.1ml corn oil. All adrenalectomized rats were given 0.9% NaCl in their drink— ing water and their diet was supplemented with sugar cubes. A fourth group of non—lactating, tumor bearing rats served as a second control group. Tumor measurements and body weights were observed at 2-4 day intervals from the day of parturition to day 25 postpartum. Average tumor diameters for each rat were determined by using the mean of the 2 largest diameters of each tumor as measured with vernier calipers. Blood samples were taken on the morning of days 3 (pretreatment), 8, l4, and 20 postpartum by orbital sinus puncture while the rats were under light ether anesthesia. Serum was separated by centrifugation and stored at -200C until assayed for PRL and corticosterone by radioimmunoassay. Serum PRL was assayed by a standard procedure (Niswinder gt gt., 1969) and corticosterone by a standard method employed at Endocrine Sciences, Tarzana, CA (by Gary Berg and Gary Kledzik). Statistical differences in serum PRL and corticosterone levels and tumor measurements between control and treatment groups were tested by Student—Newman-Keuls Test. A difference of P<0.05 was considered to be significant. 41 Results Figure 3 shows the effect of the different treatments on mammary tumor growth during lactation. After 25 days of lac— tation, average tumor diameter was reduced by almost 50% in both the untreated lactating controls and the EB treated lac- tating rats, as compared to initial measurements at the time of parturition. In the adrenalectomized lactating group, average tumor diameter increased nearly 40% over prepartum measurements. The growth rate of tumors in the adrenalecto- mized lactating rats between days 5 and 15 postpartum was similar to intact non-lactating control rats. The effects of the different treatments on average tumor number are shown in Table 4. Rats in the intact lactating group showed no significant change in average tumor number during lactation. By contrast, the EB treated lactating rats showed a significant decrease in average tumor number by the end of the 21 day treatment period. A significant increase in average tumor number was observed in the adrenalectomized lactating group. The effects of the various treatments on serum PRL levels are shown in Table 5. When compared with the non- lactating controls, serum PRL levels were elevated in all lactating groups as a result of the suckling stimulus. Serum PRL levels in the adrenalectomized group were higher than in the intact control lactating group during the first 2 weeks of lactation, but returned to control levels by the 3rd week of treatment. Serum PRL levels in the EB treated rats decreased Percent Change in Ave. Tumor Diameter Figure 3 . 42 + 50 .eeeeeeeeeeeeeeeeeee Non- 'OC'. con "0' +4 0 ...o°°. I, —_ lac t.+ Adrenx + 3 O ..e°.. ,’ II + 2 0 “.0. I, + 1 o I' e. I O ’0' I, e. ’ I/ :‘K I - 2 O ‘2‘ \ \l \ -30 2A ’0‘.~uuuuu,~’ ..‘u.,"~' 4 O \ oom‘ clam-m- ----- Lac t. 0 E B '50 \\\ /‘~~-,”;b--- lact. Control Part ’ 5 1O 15 2O 25 tx Days Postpartum Effects of Adrenalectomy (ADRENX) and Estradiol Benzoate (EB) on Percent Change in Average Tumor Diameter During Lactation. 43 .0 m8 fies» 8% 5E; Hodvm O .o sad Hafiz aoummeoo ewes mo.ovm Q .mmflfimmz w as.o H m.o no.o a o.o o.o H e.m o.o « N.m A.o « e.o A xzmmo< + .pomq ow.o H A.m o.o H A.m s.o H s.m o.o a H.« o.o a e.a o mm + .uomq N.H H o.e v.H a H.o N.H a A.m e.H « o.e e.H H v.4 s maonucoo .uomq ....... In no.o m o.m o.o « v.m nunluluui mo.o H A.o o .ncoo .pomqucoz Hm sea ea sac m sea a sac o sad mumm newsumwne umm\.oz noese mo .02 BE 5. .oz H0859 madame/4 co EM: 382% Hoflpmfimm pan szmmofl @Upowamcwfi mo muommwm . v magma. 44 .mHoHEOo mcflBDMH guns 8% coca Ho.ovmo .mHOHquo .ubdalco: fins pogo secs 80.0va .m.m H Emma m Emma... + Home 28... N am odofl « moo 9&2. a SS Dao H mom A QNNH « oom boo in omm 051% a mod nAoH r” oom A mm + .83 how H mmm nAm H omm boo a o8 home A woe A $828 .83 Ad H Toe To “ mam mo H HAN «ma 4 4.8 m .ucoo $63.82 358% on >8 3 s8 o N8 m N8 3mm penthouse SERVE £969 Jam :5me mo .02 whose BBEHIEE madame 3mm £96m 93363 on ammo 5838.1 spam no as BSNcwm Hoaomfimm new 35% genomes no Boohm . m manna Figure 4 . Average Pup Weight (gm) 45 60 [CONTROL I I [I 50 I [I [I f.“ ’I 3: IADRINX / [I so / . ‘1, / .0eee""‘ ’/ y... .o..‘ 2 O / I.I""'".’” 1....“ (1.3” ‘° 49.11;” I Part . 5 1O 15 20 25 tx Day: Postpartum Effects of Adrenalectany (ADRENX) and Estradiol Benzoate (EB) on Pup Weight Gain During lactation. 46 during the initial sampling period, but returned to control levels during the final two sampling periods. Bilateral adrenalectomy reduced serum corticosterone to nearly undetectable levels. However, there were no signifi-~ cant differences in the serum corticosterone levels of lacta- ting as compared to non—lactating controls. Both EB treat— ment and adrenalectomy reduced lactation efficiency as indicated by the decreased growth rate of pups when compared with the control lactating group (Figure 4). Conclusions This study demonstrates that adrenalectomy during post— partum lactation completely prevented regression of mammary tumors in rats, and resulted in tumor growth equal to that of non—lactating, intact controls. These observations suggest that the adrenals inhibit mammary tumor growth during post— partum lactation. Serum PRL levels in the adrenalectomized rats were higher than in intact lactating rats for the first two weeks after parturition. This increase in PRL probably contributed to the reversal of the mammary tumor regression induced by adrenalectomy. Chen gt gt., (1976) reported that adrenalectomy of non-lactating tumor-bearing rats resulted in enhanced mammary tumor growth and elevated serum PRL concentrations, whereas, cortisol administration produced opposite effects on mammary tumor growth and serum PRL. Removal of any direct inhibitory effect by adrenal glucocorticoids on mammary tumor tissue also may have *7— 47 contributed to tumor regression. Glucocorticoids such as corticosterone in the rat, may directly inhibit growth of mammary tumors, and reverse any stimulatory effect exerted by elevated PRL levels during lactation. This may be similar to the effects of administration of high doses of estrogen to induce mammary tumor regression, which at the same time ele- vated serum PRL concentration (Kledzik, gt gt., 1976). The inhibitory action of high doses of estrogen on mammary tumor growth has been shown to be exerted at the tumor tissue level, resulting in a decrease in specific PRL receptors in the mammary tumors, thereby preventing PRL from exerting its normal growth stimulating effect (Kledzik, gt gt., 1976). Whether glucocorticoids can similarly reduce PRL receptors in mammary tumor tissue remains to be demonstrated. Serum corticosterone levels in the intact lactating rats were similar to those of the non—lactating controls. This is in agreement with a previous report by Stern and Vooqt (1973- 74), who compared total serum corticosterone levels in non— lactating rats with suckled lactating rats. However, they also found that after an acute period of suckling, total serum corticosterone levels were significantly elevated above non— lactating control values. In the present study, blood was not collected after acute periods of suckling, but randomly, and any elevations in serum corticosterone immediately after suckling may have been missed. Although the total plasma corticosterone concentrations were not different between lactating and non—lactating control _ 48 animals, it is possible that the glucocorticoid was biologi- cally more active in the lactating rats. Gala and Westphal (1965) reported that the plasma protein that binds corticos- terone (transcortin) is decreased during lactation, thereby remkring corticosterone more active than in non—lactating rats. Treatment with the dose of EB given did not prevent mammary tumor regression during lactation, nor consistantly affect serum PRL levels when compared with untreated lactating controls. Other doses of estrogen may need to be tested to conclusively determine whether the low estrogen secretion during postpartum lactation in rats (Smith and Neil, 1977) has any effect on mammary tumor regression. Reduced milk secretion, as indicated by decreased pup weight gain in the EB and adrenalectomized rats, is not believed to be responsible to any extent for the increased mammary tumor growth after adrenalectomy, or for the decrease in mammary tumor growth observed in the estrogen treated rats. The present observations suggest that the adrenal glands during postpartum lactation in rats are mainly responsible for the regression of mammary tumors. Whether regression of breast cancer in women during postpartum lactation also is related to adrenal cortical function remains to be demonstrated but there is evidence that glucocorticoid secretion is increased during lactation in women (Lyons, gt gt., 1958). EFFECTS OF DEXAMETHASONE AND HALOPERIDOL ON MAMMARY TUMOR GROWTH Objectives Since the paradoxical regression of most DMBA—induced mammary tumors during postpartum lactation appears to be largely due to an adrenal influence, it was of interest to observe the direct influence of glucocorticoids on mammary tumor growth when serum prolactin levels are elevated. In this study an "artificial lactation” hormone state was induced in rats bearing mammary tumors by using dexamethasone, a synthetic glucocorticoid, in conjunction with haloperidol, a dOpamine receptor blocker which elevates serum prolactin levels. In postpartum lactation in rats, there is evidence that prolactin and ACTH-glucocorticoid release are elevated by the suckling stimulus (Stern and Voogt, 1973/74). The objective of this study was to observe the effect of exogenous glucocorticoid administration on mammary tumor growth when serum prolactin levels were elevated, to further clarify the role of the adrenal glucocorticoid influence on mammary tumor regression during postpartum lactation. Procedure Virgin female Sprague-Dawley rats 55 days old were given a single intravenous injection of 1 ml lipid emulsion 49 50 containing 5 mg of 7,12—dimethylbenz(a)anthracene (DMBA). Approximately 8 weeks later, when each rat had at least one tumor measuring more than 1 cm in average tumor diameter, the rats were divided into their respective treatment groups. In the first experiment the groups were divided as follows: Group 1, controls, received two 0.1 ml sc injections of corn oil vehicle once daily for the duration of the experiment; Group 2 was given daily sc injections of 300 pg dexamethasone (DEX) and a second daily injection of 0.1 ml corn oil vehicle; Group 3 received daily sc injections of haloperidol (HALO) at a dose of 0.5 mg/kg suspended in 0.1 m1 corn oil and a second daily injection of 0.1 ml corn oil vehicle; Group 4 received daily so injections consisting of 3001ng dexamethasone in 0.1 ml corn oil in addition to haloperidol at a daily dose of 0.5 mg/kg contained in 0.1 ml corn oil. Therefore each rat was given two injections daily in a total volume of 0.2 ml. In the second experiment the treatment groups were similarly divided, with the exception that a lower dose (Solug/rat) of dexamethasone and different injection vehicles were used. The dexamethasone was suspended in a 5% ethanol solution and the haloperidol was suspended in 0.89% saline. The same injection schedule was used as in the previous experiment, so that each rat in every group was injected twice daily in a total volume of 0.2 ml. The duration of treatments in both experiments was 3 weeks. Tumor measurements and body weights were observed and recorded at weekly ; - . L-) I-- .. ..l‘. ‘.- b 1-. 7‘ r‘ .':‘.:'.*:' ricer”- 51 intervals for 3 weeks in both experiments. Average tumor diameters for each tumor were determined by using the mean of the two largest diameters as measured with vernier calipers. Tumor growth was expressed as percent change in average tumor diameter when compared with the initial measurements made prior to the initiation of treatment. Blood samples were collected in the first experiment by orbital sinus puncture in the morning between 1000 and 1100 hr, 1 and 2 weeks following the initiation of the treatment, and by decapitation at the conclusion of the experiment. In the second experiment blood was sampled by orbital sinus puncture 10 days following the start of the treatments and again by decapitation at termination of the experiment. All blood was sampled 1 hour after daily injec- tions of the respective treatment groups. Serum was sepa- rated by centrifugation and was stored at —200 until assayed for PRL by a standard method (Niswinder gt gt. 1969). Statistical differences in tumor growth, body weight and serum prolactin levels between the control and treatment groups were determined utilizing analysis of variance (ANOVA) and Student Newman Keuls (S-N—K) statistical tests. A difference of p<0.05 was considered to be significant. Results Figure 5 shows the effects of the various treatments in the first experiment on tumor growth expressed as percent change in average tumor diameter. Dexamethasone (DEX) 52 (300 ug/rat) administered alone as well as with haloperidol (DEX + HALO) produced significant regressions in tumors at the end of 2 and 3 weeks of treatment. Dexamethasone alone reduced average tumor size by over 40% by the termination of the experiment, and dexamethasone given with haloperidol reduced average tumor size by 25% when compared with pre- treatment measurements. Haloperidol (HALO) administered alone significantly stimulated tumor growth at 2 and 3 weeks of treatment, increasing tumor size by 45% when compared with initial measurements. In the control group average tumor diameters were increased by 20% by the conclusion of the 3 week experiment. The effects of the different treatments in the first experiment on serum PRL levels are shown in Table 6. Halo- peridol (0.5 mg/kg) administered alone significantly (p<0.01) elevated serum PRL at each of the sampling times when compared with controls. In addition dexamethasone and haloperidol given together significantly (p<0.01) elevated serum PRL levels above the group given haloperidol alone. Daily administration of dexamethasone (300,ug/rat) reduced serum PRL below controls at all sampling times, significantly (p 0.0% at the first two weeks of treatment. The effects of dexamethasone (300/Mg/rat) and haloperidol treatment on body weight are shown on Table 7. Dexamethasone administered alone and dexamethasone given with haloperidol reduced body weight by 15.1% and 17.5% respectively when compared with initial measurements. Significant (p(0.05) 53 reductions in body weight were seen at 3 weeks of treatment in both the rats given dexamethasone alone and in the rats given dexamethasone and haloperidol as compared with controls. Haloperidol decreased body weight insignificantly by 1.9% when compared with initial measurements but there were no significant differences when compared to controls at any time. Rats injected with corn oil vehicle showed a slight in- significant reduction of 4.8% in body weight by termination of the experiment. The results of the second experiment of this study in which dexamethasone was given at a dose of 50 pg/rat with and without haloperidol are shown in Figure 6 and Tables 8 and 9. As in the first experiment, dexamethasone (DEX) given alone and dexamethasone administered with haloperidol (DEX + HALO) resulted in significant reductions in average tumor diameter. Dexamethasone alone significantly reduced tumor size by week 1 of treatment (p<0.05) when compared with controls. Dexamethasone alone decreased tumor size by nearly 60% at the termination of treatment when compared with pretreatment measurements. Similarly dexamethasone given with haloperidol reduced tumor size at weeks 2 and 3 of treatment (p<0.05 and p<0.01, respectively) when compared with controls. Dexamethasone and haloperidol administered together decreased tumor size by nearly 40% by the end of the treatment when compared with pretreatment values. Haloperidol (HALO) given alone stimulated tumor growth by over 80% when compared with initial measurements, but there were no IN AVE. TUMOR DIAMETER A N a 0| 0 O O O 8 O % CHANGE n o 40 54 HALO CONTROL DEX‘HALO Figure 5. I 2 3 Week of Treatment Effects of Dexamethasone (DEX) (300ug/rat/day) Administered Alone; Haloperidol (HALO) (0.5 mg/kg/day) Administered Alone; and Dexamethasone Administered With Haloperidol (DEX + HALO) on Percent Change in Average Tumor Diameter. ,- oqam m> mo.ovm m maonucoo m> Ho.ovm o maonueoo m> mo.ovm U .m.m H smash .m xmos co coflumuflmmomp >9 pan m can H mmeB co mowflccomp ouduocsm macaw Hmuflnuo %Q poademm vocab upcmEummnu mo GOHDMHDHCH Adamo mxmmz .m 5 a. m.o.oo.Ao A o.mmm m.o.oo.om H m.Aoo w.o.om.om « o.vo a ones + xma o.oo.HH H o.mNH o.oA.Hm A H.oom o.oA.om A o.aom o Asmo\ox\m5m.oo oaam o.m A m.o 6H.m H o.A ee.o a m.m o Asmo\umn\mz\oomo xmo A.m n o.oa o.m H o.om hm.o a o.ma m Honpcoo maoagm> m. m. 1m. when .02 ucoeummne M AHEchV maw>mq 4mm Ebnmm whom ca mao>oq Adamo cauomaonm esnmm co Aoqamo Hooflnwdoamm new Axmno mcommepmsmxoo no movemem .0 dance 56 acmemnsmmme Hmouflce LDAB pmnmmeoo ucmAmB mpon CH meMSU pcmommm p maouucoo .m> mo.va o .m.m H CMOZO pcmEumoHu mo mxmmz m m.AH| om.m M m.mmm 0m.m H m.ANN om.v H o.mmm v.m H m.mmm m qum + xmo o.H- m.oH A e.oAN o.o H H.oAm A.a A H.oAm o.m a o.mom o Asmo\ox\ma m.oo came H.mHI oH.o H A.Hmm oo.v H m.mmm oa.o H m.mmm m.m H m.mAm m Ammp\umw\mdoomv xmo m.v| H.NH H o.oAm m.m M «.mom m.w H A.MAN am.m H w.mmm m Honucou oaoecw> nomcmco w m. m. .Mw .eauwmm mums ucmEumoHB Amequv pamflwz xpom ommnm>¢ Mo .02 mumm ca beefing soom co Aoqamv Hooflnmaonm was Axmav maommeuoemxmo no mpownmm .h OHQMB 1(N) 80 60 4O 20 20 4O °/ocuANoE IN AVE. rumon DIAMETER Figure 6 . 57 HALO CONTROL it . -DEX . HALO it lIIIIIIIIIIIIIIII-IIII-IIIIIIII-IIIIII 1 2 3 Week of treatment J. Effects of Dexamethasone (DEX) (Sng/rat/day) Administered Alone; Haloperidol (HALO) (0. 5 mg/kg/day) Administered Alone: and Dexamethasone Administered With Haloperidol (DEX + HALO) on Percent Change in Average Tumor Diameter. 58 Qqam .m> mo.oCm o mHoHEoo .m.>. Ho. 9m p mHoBcoo .m> mo.ovm o .m.m 1 com: + n Hm man no coflmwflmoomp .3 new OH hump co wagofimmp ofifiocum msfim HSHQHO \E ponEmm pooHn “ugmmifl mo coflmflflfi Hmpmm. m no .m m.o.oA.mm H A.HHN m.o.oa.mm a o.mom o ones + xmo o.oA.oH A N.oo o.oo.om « N.oom o Asmoxoxxoeim.oo ones A.H A A.oH om.m A e.om o Ammo\pmn\m:omv xmo N.o A H.om ha.oH A o.oo o Honucoo maoasm> IHIN. truduH. mums . oz pgmofl. HEAR: mHm>QH Hmm EPHMm mpmm ca mHm>mq Aémv CHHAOMHOHQ gm CO AQHANZV HOUHHQQOHMZ mug AXWOV OQOmMfiQhMXOD MO mflnvmwwm . w mHQmB 59 omom .fl gmmz Q Hcoaumonp mo Mock m m.mH- oH H Hom oH H mom oH H mAm o H mom o OHHH + xmo m.o+ HH H How mH H oom oH H oom AH H How o Asma\HmH\oe m.oo QHHH A.MH: HH H omm NH H Hom oH H oom oH H mom o Asmo\HmH\oo omo xmo m.o+ om H mom om H Aom om H oom now H oom o HOHHcoo mHOHgm> omcmco w m m mH .HEHon mpmu .oz HcmEumoHe $533 HnJmHmz wpom ommum>¢ mumm CH uanmz Npom co AOHHmo HooHHmmonm ocm Axmao mcommnumsmxmo Ho muomHHm .m OHQMB 60 significant differences when compared with the control group. In this experiment vehicle injected control animals showed increased average tumor diameter of over 40% by week 3 of treatment when compared with initial values. Table 8 shows the effects of the various treatments on serum prolactin levels sampled 10 days and 21 days after initiation of treatment. The results in this experiment were similar to those found in the first experiment. Dexa— methasone significantly (p<0.05) reduced serum prolactin levels at both sampling times when compared with controls. On the other hand, haloperidol administered alone or with dexamethasone increased serum prolactin levels significantly (p<0.05) above controls at both sampling times. Again, as in the first experiment, dexamethasone administered with halo- peridol increased serum prolactin above that in rats given haloperidol alone at both sampling times. The effects of the various treatments in this experiment on body weight are shown in Table 9. Again dexamethasone alone and dexamethasone given with haloperidol reduced average body weight (by 13.7% and 15.2%) when compared with initial values, respectively. However, there were no significant differences in body weight between any of the treatment groups at any observed times. Conclusions In the previous study it was determined that the adrenal glands play an important role in the regression of DMBA—induced mammary tumors during postpartum lactation. 61 The present study indicates that glucocorticoids such as dexamethasone are capable of inhibiting mammary tumor growth and producing regression of such tumors even when circulating prolactin levels are elevated. In the first experiment, the significant reductions in body weight pro- duced by the high levels of dexamethasone administered (300 pg/ rat/day) may have contributed to the regression of the tumors. Welsch and Meites gt gt., (1978) showed that caloric restric— tion induced by decreased food intake inhibited tumor growth while at the same time reducing body weight. Such inhi- bition of tumor growth is possibly due to decreases in circulating levels of prolactin and/or estroqen. In the second experiment, no significant reductions in body weight were observed in animals treated with the lower dose of dexamethasone (50 ug/rat) and therefore did not contribute to the regression of tumors observed. The effect of dexamethasone on serum prolactin levels confirms previous findings of Schwinn gt gt., (1975) and Harms gt gt., (1975) that dexamethasone reduces basal serum prolactin levels in the rat. This decrease in serum prolactin in rats treated with dexamethasone may have contributed to the tumor regression observed in that group. But in the group treated with dexamethasone together with haloperidol, serum prolactin levels were elevated even above values in rats treated with haloperidol alone. This indicates that dexa— methasone may act directly on the mammary tumor to cause tumor regression or via other mechanisms. 62 The exact mechanisms by which glucocorticoids cause mammary tumor regression, even in the presence of elevated serum prolactin levels, is yet to be determined. Most of the influence of glucocorticoids on mammary tumors may be exerted directly on the tumor. Glucocorticoids may act like high doses of estrogen to decrease the number of pro- lactin binding sites on the plasma membranes of mammary tumors Kledzik gt gt., 1976). Such a reduction in prolactin binding sites would reduce the effectiveness of elevated serum prolactin levels to stimulate mammary tumor growth. Glucocorticoids also may act to reduce receptor sites for other hormones that may be associated with mammary tumor growth such as insulin, growth hormone, estrogen, etc. Osborne gt gt., (1979) showed that dexamethasone directly inhibits the growth of human breast cancer cells, in vitro, apparently by antagonizing the action of insulin. Dexamethasone and the endogenous glucocorticoids may also act through their own intercellular receptor sites to exert their inhibitory action on mammary tumor growth. Further studies need to be done to determine the precise mechanism by which glucocorticoids act on mammary tumors. DISCUSSION In most physiologic and pharmacologic hyperprolactin— emic states, DMBA-induced mammary tumors respond with accelerated growth. However, in the hyperprolactinemics examined in this thesis such a correlation was not always apparent. In the lactation and dexamethasone studies, elevated prolactin levels due to the suckling stimulus or to administration of haloperidol with dexamethasone were associated with marked regression of mammary tumors. On the other hand, in the variable fat diet study, enhanced mammary tumorigenesis was not consistantly correlated with elevated serum prolactin levels. It can be concluded from the postpartum lactation study that the adrenal glands appear to be a major inhibitory factor in regression of mammary tumors during lactation. Decreased estrogen secretion during postpartum lactation apparently does not play an important role in regression of mammary tumors at this time. Since gluco— corticoids secreted from the adrenals are important for induction and maintenance of lactation, and adrenal cortical secrection is elevated during postpartum lactation in response to the suckling stimulus (Voogt gt gt., 1969), it would appear that these hormones are responsible for the 63 64 observed postpartum regression of mammary tumors. The dexamethasone—haloperidol study demonstrates that dexa- methasone, a synthetic glucocorticoid, is able to cause regression of mammary tumors even in the presence of ele— vated serum prolactin caused by administration of haloperidol. This suggests endogenous adrenal glucocorti- coids such as corticosterone in the rat may exert their inhibitory effects directly on the mammary tumor. Elevated serum glucocorticoid levels may cause a decrease in prolactin receptors similar to that produced by high estrogen levels as was observed by Kledzik gt gt., (1976). Such a decrease in prolactin receptors would reduce the ability of the circulated prolactin to exert its stimulatory effect on the mammary tumor. Furthermore, glucocorticoids themselves may have a direct inhibitory action on the mammary tumor mediated through their own intracellular receptor. Glucocorticoid receptors have been identified in normal as well as in neoplastic mammary tissue and such tumor inhibitory effects may be achieved through such receptors. Osborne gt gt., (1979), have reported that dexamethasone directly inhibits the growth of human breast cancer cells in vitro presumably antagonizing the action of insulin. Further study to establish the inhibitory role of glucocorticoids needs to be done. First, restoration of normal tumor regression during postpartum lactation by replacement of the glucocorticoid influence removed by 65 adrenalectomy would provide further convincing evidence that elevated adrenal glucocorticoid secretion during postpartum lactation is primarily responsible for the regression of mammary tumors during this state. Also studies designed to determine the effects of glucocorticoids on prolactin receptors within the mammary tumors would pro- vide a mechanism by which these substances exert their inhibitory effect. Finally studies on the direct effect of glucocorticoids on tumor growth dynamics 1g vitro would also provide evidence on the mechanisms by which gluco— corticoids inhibit mammary tumor growth. The results from the variable fat diet study confirm some previous findings by Gammal gt gt., (1967) and Carroll gt gt., (1971) that 20% high fat diets stimulate mammary tumorigenesis in rats. However, the data indicate that, contrary to the findings of Carroll and Khor (1971), diets containing saturated fats are equally effective in stimulating mammary tumor develOpment as diets containing equal concentrations of unsaturated fats. It also is apparent that a 0.5% low fat diet has little or no effect on tumor development. Further studies are necessary to determine if high fat diets have similar stimulatory effects on the induction and growth stages of mammary tumorigenesis, as in the developmental stage. Further work also is necessary to determine the effects of high fat diets on the neuroendocrine and endocrine systems, to provide a more complete explanation of the if 66 mechanism by which high fat diets stimulate mammary tumor development. The small, inconsistent elevation of serum prolactin levels observed in the high fat diet groups, probably does not completely account for such a dramatic stimulatory effect on mammary tumorigenesis. The prolactin values were highly variable at most of the times sampled. This seems to stem from the fact that the rats did not consume their respective diets at the same time. Therefore any acute or short term effects of the high fat diets on prolactin might have been overlooked. It would be helpful to synchronize feeding by removing the food for a short time and then replacing it to acutely feed the animals. The effects of high fat diets on the fluctuations of hormones, especially prolactin, during the estrous cycle in rats, also needs to be examined to determine if such diets can affect hormone values at specific times during the cycle. The effects of high fat diets on other mammoqenic hormones also needs to be examined. Estrogen, which with prolactin represents the other important hormone involved in mammary tumorigenesis, may play a role in the stimulatory effects of high fat diets. Therefore, changes in estrogen clearance and/or secretion which may result in increased serum levels of estrogen, may further clarify the mechanisms by which high fat diets stimulate mammary tumor development. Increased fat intake also could result in increased fat content, especially surrounding the mammary gland, which 67 may localize estrogens as well as other steroid hormones, and thereby increase the effectiveness of circulating steroid hormones on mammary tumor development. High fat diets also may have acute effects on insulin and other metabolic hormones that could influence mammary tumorigenesis. Since insulin and growth hormone appear to have synergistic or permissive effects on mammary tumor growth and development, these hormones may play some role in the high fat diet stimulation of mammary tumorigenesis. Finally, there has recently been strong evidence that high fat diets may have a direct stimulatory effect on tumor growth. Wicha gt gt., (1979) showed that lipids may have a direct effect on normal and neoplastic mammary epithelial cell growth. In this study, unsaturated fatty acids stimulated the growth of normal and neoplastic tissue tg ytttg. Increased fat intake, then, may "sensitize" the mammary gland and/or tumor to normal or basal hormone levels. Prolactin receptors might be increased, or a change in cell membrane fluidity of the mammary gland may result in increased sensitivity. Work both on the direct effects on the mammary tumor cells as well as on the systemic and central effects of high fat diets needs to be pursued in order to clarify the precise mechanisms for stimulation of mammary tumorigenesis by high fat diets. LIST OF REFERENCES Brennan, J.J., 1973. Corticosteroids in the Treatment of Solid Tumors. Medical Clinics of North America 57: 1225—1239. Carroll, K.K., 1975. Experimental Evidence of Dietary Factors and Hormone-dependent Cancers. Cancer Res. 35: 3374-83. Carroll, K.K. and H.T. Khor, 1970. Effects of Dietary Fat and Dose Level on 7,12—Dimethylbenz(a)anthracene on Mammary Tumor Induction in Rats. Cancer Res. 30: 2260-2264. Carroll, K.K. and H.T. Khor, 1971. Effects of Level and Type of Dietary Fat on Incidence of Mammary Tumor Induced in Female Sprague—Dawley Rats by 7,12-Dimethyl- benz(a)anthracene. Lipids. 6: 415-420. Cave, W.T., J.T. Dunn, and R.M. MacLeod, 1979. Effects of Iodine Deficiency and Diet Modification on Hormone Dependent Cancer in Rats. Cancer Res. 39: 729—733. Chan, P.C. and L.A. Cohen, 1974. Effects of Dietary Fat, Antiestrogen and Antiprolactin on the Development of Mammary Tumors in Rats. J.Nat.Cancer Inst. 52: 25-30. Chan, P.C. and L.A. Cohen, 1975. Dietary Fat and Growth Promotion of Rat Mammary Tumors. Cancer Res. 35: 3384—3386. Chan, P.C., F. Didato, and L.A. Cohen, 1975. High Dietary Fat Elevation of Rat Serum Prolactin and Mammary Cancer. Proc. Soc. Exp. Biol. Med. 149: 133-135. Chen, H.J., C.J. Bradley, and J. Meites, 1976. Stimulation of Carcinogen-induced Mammary Tumor Growth in Rats by Adrenalectomy. Cancer Res. 36: 1414-1417. Chen, H.J., G.P. Muellar, and J. Meites, 1974. Effects of L—Dopa and Somatostatin on Suckling-Induced Release of Prolactin and GH. Endocrine Research Communications. 1: 283—291. 68 69 Clemens, J.A., M. Sar, and J. Meites, 1969. Inhibition of Lactation and Luteal Function in Postpartum Rats by Hypothalamic Implantation of Prolactin. Endocrinology. 84: 868-872. Clemens, J.A., C.W. Welsch, and J. Meites, 1968. Effects of Hypothalamic Lesions on Incidence and Growth of Mammary Tumors in Carcinogen—treated Rats. Proc. Soc. Expt. Biol. Med. 127: 969—972. Cohen, N.D. and R. Hilf, 1974. Influence of Insulin on Growth and Metabolism of 7,12—Dimethylbenz(a)anthracene— induced Mammary Tumors. Cancer Res. 34: 3245-3252. Dao, T.L., 1962. The Role of Ovarian Hormones in Initiating the Induction of Mammary Cancer in Rats by Polynuclear Hydrocarbons. Cancer Res. 22: 973—981. Dao, T.L., F.G. Bock, and M.J. Greiner, 1960. Mammary Carcinogenesis by 3—Methylcholanthrene II. Inhibitory Effect of Pregnancy and Lactation on Tumor Induction. J. Nat. Cancer Inst. 25: 991-1003. Dao, T.L., and H. Sunderland, 1959. Mammary Carcinogenesis by 3—methylcholanthrene. I. Hormonal Aspects of Tumor Induction and Growth. J. Nat. Cancer Inst. 23: 567—585. anrs, J.T., and R.M. Baddeley, 1956. Neural Pathways in Lactation. J. Anat. 90: 161—171. Edwardson, J.A. and J.T. anrs, 1967. Neural Factors in the Maintenance of Lactation in the Rat. J. Endocrinology. 38: 51—59. Euker, J.S., J. Meites, and G.D. Riegle, 1975. Effects of Acute Stress on Serum LH and Prolactin in Intact Castrate, and Dexamethasone—Treated Male Rats. Endocrinology. 96: 85-91. Friesen, H.G., 1973. Placental Protein and Polypeptide Hormones. tg: Handbook of Physiology, Sect.7, Vol. II, Part 2. R.O. Greep and E.B. Astwood (eds.). Am. Physiol. Soc., Washington, D.C. 294—309. Gala, R.R. and U. Westphal, 1965. Corticosteroid— Binding Globulin in the Rat: Possible Role in the Initiation of Lactation. Endocrinology 76: 1079-1089. Gala, R.R. and U.Westphal, 1966. Influence of Anterior Pituitary Hormones on the Corticosteroid—Binding Globulin in the Rat. Endocrinology 79: 55-68. 70 Gammal, E.B., K.K. Carroll, and E.R. Plunkett, 1967. Effects of Dietary Fat on Mammary Carcinogenesis by 7,12—Dimethylbenz(a)anthracene in Rats. Cancer Res. 27: 1737-1742. Grosvenor, C.E., 1964. Effect of Suckling Upon Pituitary Growth Hormone (STH) Concentration in the Lactating Rat. The Physiologist. 7: 150. Guillino, P.M., H.M. Pettigrew, and F.H. Grantham, 1975. N—Nitromethylurea as Mammary Gland Carcinogen in Rats. J. Nat. Cancer Inst. 54: 401—408. Harms, P.G., P. Langlier, and S.M. McCann, 1975. Modification of Stress—Induced Prolactin Release by Dexamethasone or Adrenalectomy. Endocrinology. 96: 475-479. Harris, G.W., 1948. Neural Control of the Pituitary Gland. Physiol. Rev. 28: 139-179. Hayward, J., 1970. Hormones and Human Breast Cancer, Springer-Verlag, Inc. New York. Heuson, G.J. and N. Legros, 1972. Growth of 7,12— Dimethylbenz(a)anthracene—induced Mammary Carcinoma in Rats Subjected to Alloxan Diabetes and Food Restriction. Cancer Res. 32: 226—232. Hilf, R., I. Michel, C.Bell, J.J. Freeman, and A. Borman, 1965. Biochemical and Morphological Properties of a New Lactating Mammary Tumor Line in the Rat. Cancer Res. 25: 286—299. Hodson, C.A., J.W. Simpkins, and J.Meites, 1978. Inhibition of Luteinizing Hormone Release and Lutein— izing Hormone Releasing Hormone Action by the Ovaries of Postpartum Lactating Rats. Endocrinology. 102: 832-836. Hopkins, G.J., C.E. West, and G.C. Hard, 1976. Effect of Dietary Fats on the Incidence of 7,12—Dimethylbenz(a)— anthracene—Induced Tumors in Rats. Lipids. 11: 328—333. Huggins, C., 1965. Two Principles of Endocrine Therapy of Cancers: Hormone Deprival and Hormone Interference. Cancer Res. 25: 1163-1167. Huggins, C., G. Briziarelli, and H. Sutton, 1959. Rapid Induction of Mammary Carcinoma in the Rat and the Influence of Hormones on Tumors, J. Expt. Med. 109: 25—42. 71 Huggins, C., R.C. Moon, and S. Morh, 1962. Extinction of Experimental Mammary Cancer I. Estradiol and Progesterone. Proc. Nat. Acad. Sci. USA. 48: 379-386. Jabara, A.G., 1967. Effects of Progesterone on 9,10—DMBA—Induced Mammary Tumors in Sprague—Dawley Rats. Br. J. Cancer. 21: 418—429. Kim, U. and J. Furth, 1960. Relation of Mammary Tumors to Mammaotropes. II. Hormone Responsiveness of 3-Methylcholanthrene Induced Mammary Carcinomas. Proc. Soc. Exp. Biol. Med. 103: 643-645. Kledzik, G.S., C.J. Bradley, S. Marshall, G.A. Campbell, and J. Meites, 1976. Effects of High Doses of Estrogen on Prolactin-binding Activity and Growth of Carcinogen—Induced Mammary Cancers in Rats. Cancer Res. 36: 2953-2956. Kledzik, G.S., C.J. Bradley, and J. Meites, 1974. Reduction of Carcinogen—Induced Mammary Cancer Incidence in Rats by Early Treatment with Hormones or Drugs. Cancer Res. 34: 2753—2956. Labhsetwar, A.P. and D.J. Watson, 1974. Temporal Relationship Between Secretory Patterns of Gonado- tropins, Estrogens, Progestins and Prostiglandin-F in Periparturent Rats. Biol. of Reprod. 10: 103—110. Lu, K.H., H.T. Chen, L. Grandison, H.H. Huang and J. Meites, 1976a. Reduced Luteinizing Hormone Release by Synthetic Luteinizing Hormone- Releasing Hormone (LHRH) in Postpartum Lactating Rats. Endocrinology 98: 1235—1240. Lu, K.H., H.T. Chen, H.H. Huang, L. Grandison, S. Marshall, and H. Meites, 1976b. Relation Between Prolactin and Gonadotrophin Secretion in Postpartum Lactating Rats. J. Endocrinology 68: 241—250. McCann, S.M., T. Graves, and S. Teleisnik, 1961. The Effect of Lactation on Plasma LH. Endocrinology 68: 873—874. McCormick, G.M., 1972. The Effect of Varying the Length of the Nursing Period on the Postpartum Growth of Chemically Induced Rat Mammary Tumors. Cancer Egg. 32: 1574—76. 72 MCCOImiCk, G.M. and R.C. Moon, 1965. Effect of Pregnancy and Lactation on Growth of Mammary Tumours Induced by 7,12—Dimethylbenz(a)anthracene. Brit. J. Cancer 19: 160-166. McCormick, G.M. and R.C. Moon, 1967a. Effect of Nursing and Litter Size on Growth of 7,12—Dimethylbenz(a)- anthracene-induced Rat Mammary Tumors. Br. J. Cancer 21:21: 586—591. McCormick, G.M. and R.C. Moon, 1967b. Hormones Influencing Postpartum Growth of 7,12-Dimethylbenz(a)— anthracene-Induced Rat Mammary Tumors. Cancer Res. 27: 626-631. McGuire, W.L., 1975. Prolactin and Breast Cancer. tg: Prolactin and Human Reproduction. P.G. Crosignani and C. Robyn (eds.). Adademic Press, London, p. 143. Meites, J., 1972. Relation of Prolactin and Estrogen to Mammary Tumorigenesis in the Rat. J. Nat. Cancer Inst. 48: 1217—1224. Meites, J., and C.S. Nicoll, 1976. Adenhypopysis: Prolactin. Ann. Rev. Physiology 28: 57—88. Meites, J. and J.T. Sigouris, 1953. Can the Ovarian Hormones Inhibit the Mammary Response to Prolactin? Endocrinology 53: 17—23. Minaguchi, H. and J. Meites, 1967. Effects of Suckling on Hypothalamic LH-releasing Factor and Prolactin— inhibiting Factor and on Pituitary LH and Prolactin. Endocrinology 80: 603—607. Myers, M.M., V.H. Denenberg, E. Thoman, W.R. Holloway and D.R. Bowerman, 1975. Effects of Litter Size on Plasma Corticosterone and Prolactin Response to Ether Stress in the Lactating Rat. Neuroendocrin— ology 19: 54—58. Nagasawa, H. and J. Meites, 1970. Suppression by Ergo- cornine and Iproniazid of Carcinogen-Induced Mammary Tumors in Rats: Effects on Serum and Pituitary Prolactin Levels. Proc. Soc. Expt. Biol. Med. 135: 469—472. Niswender, G.D., C.L. Chen, A.R. Midgley, Jr., J. Meites and S. Ellis, 1969. Radioimmunoassay for Rat Prolactin. Proc. Soc. Exp. Biol. Med. 130: 793—797. 73 Osborne, C.K., M.E. Monaco, C.R. Kahn, K. Huff, D. Bronzert, and M.E. Lippman, 1979. Direct Inhibition of Growth and Antagonism of Insulin ction by Glucocorticoids in Human Breast Cancer Cells in Culture. Cancer Res. 39: 2422-2428. Poulton, E.R. and R.P. Reece, 1957. The Activity of the Pituitary-Adrenal Cortex Axis During Pregnancy and Lactation. Endocrinology 61: 217-225. Quadri, S.K. J.L. Clark, and J. Meites, 1973. Effects of LSD, Pargyline, and Haloperidol on Mammary Tumor Growth in Rats. Proc. Soc. Expt. Biol. Med. 142: 22—26. Ratner, A. and J. Meites, 1964. Depletion of Prolactin— inhibiting Activity of Rat Hypothalamus by Estradiol or Suckling Stimulus. Endocrinology 75: 377-382. Schwinn, G., A. von aur Muhlen, and U. Warnecke, 1976. Effects of Dexamethasone on Thyrotropin and Prolac— tin Plasma Levels in Rats. Acta Endocrinologica 82: 486—91. Segi, M., M. Kurihara, and J. Matsuyama, 1969. Cancer Mortality for Selected Sites in 24 Countries No. 5 (1964-1965). Sundai Japan Department of Public Health, Tohoku University School of Medicine. Smith, M.S. and J.D. Neill, 1977. Inhibition of Gonadotropin Secretion During Lactation in the Rat: Relative Contribution of Suckling and Ovarian Steroids. Biology of Reproduction 17: 255-261. Smith, R.D., R. Hilf, and E.E. Senior, 1977. Prolactin Binding to 7,12-Dimethylbenz(a)anthracene— induced Mammary Tumors in Diabetic Rats. Cancer Res. 37: 4070-4074. Soria, J., A. Zarate, E.S. Canales, and H. Villalobos, 1976. Effect of Suckling on Serum Follicle— Stimulating Hormone and Luteinizing Hormone in Nursing Women. Neuroendocrinology 20: 43—46. Stern, J.M. and J.L. Voogt, 1973/74. Comparison of Plasma Corticosterone and Prolactin Levels in Cycling and Lactating Rats. Neuroendocrinology 13: 173-181. Talwaker, P.K., J. Meites, and H. Mizuno, 1964. Mammary Tumor Induction by Estrogen or Anterior Pituitary Hormones in Ovariectomized Rats Given 7,lZ-Dimethyl—l,2—Benzanthracene. Proc. Soc. Expt. Biol. Med. 116: 531—534. 74 Talwalker, P.K., A. Ratner and J. Meites, 1963. In Vitro Inhibition of Pituitary Prolactin Synthesis and Release by Hypothalamic Extract. Amer. J. Physiol. 205: 213-218. Tannenbaum, A., 1942. The Genesis and Growth of Tumors III. Effects of a High Fat Diet. Cancer Res. 2: 468-475. Tannenbaum, A., 1945. The Dependence of Tumor Formation on the Composition of the Calorie—Restricted Diet As Well As On the Degree of Restriction. Cancer Eng. 5: 616-625. Tannenbaum, A., 1959. Nutrition and Cancer. In: The Pathophysiology of Cancer. R. Honburger (ed.) Hoeber-Harper, New York, pp. 517-562. Voogt, J.L., M. Sar, and J. Meites, 1969. Influence of Cycling, Pregnancy, Labor and Suckling on Corti— costerone-ACTH Levels. Am. J Physiol. 216: 655-658. Welsch, C.W., J.A. Clemens, and J. Meites, 1968. Effects of Multiple Pituitary Homografts or Progesterone. on 7,12—Dimethylbenz(a)anthracene-induced Mammary Tumors in Rats. J. Nat. Cancer Inst. 41:465—471. Welsch, E.W. and J. Meites, 1970. Effects of Reserpine on Development of 7,12—Dimethylbenz(a)anthracene— induced Mammary Tumors in Female Rats. Experientia 26: 1133-1134. Welsch C.W. and J. Meites, 1978. Prolactin and Mammary Cancerigenesis. In: Progress in Cancer Research and Therapy, Vol. 9. R.K. Sharma and W.E. Criss(eds.) Raven Press, New York, pp. 71—92. Welsch, C.W., and H. Nagasawa, 1977. Prolactin and Murine Mammary Tumorigenesis: A Review. Cancer Res. 37: 951-963. Westphal, U., 1971. Steroid Protein Interactions. Springer—Verlag, New York, pp. 259-266. Wicha, M.S., L.A. Liotta, and W.R. Kidwell, 1979. Effects if Free Fatty Acids on the Growth of Normal Neoplastic Rat Mammary Epithelial Cells. Cancer Res. 39: 426-435. Young, S., D.M. Cowan, and L.E. Sutherland, 1963. The Histology of Induced Mammary Tumors in Rats. J. Pathol. Bacteriol. 85: 331—340. Zarate, A., E.S. Canales, J. Soria, F. Ruiz and C. Mac Ovarian Refractoriness During Lacta- Gregor, 1972. tion in Women. Am. J. Obstet. Gynec. 112:1130-1132. —' [_ — Iii — my ~ ' Lift; ~:_:_»__'r— ___.__.-. —; , l . . i! Ill\llUlllHHfl)“lllllllWNHIIWIHHHIIHH“HUMHI