_—__7 A THE EFFECTS OF HORMONES AND DRUGS ON THE GROWTH OF CARCINOGEN» INDUCED MAMMARY TUMORS IN RATS Thesis for‘the Degree of 'M‘. S. MICHIGAN STATE UNIVERSITY JAMES. L. CLARK 1972 9 e - w . HERA R’ 1’ ~3 Michigan State University “East! magma BY " HOAE & SONS' 890K BINDERY THE. LIBRARY amosns smusroq. magma; ABSTRACT THE EFFECTS OF HORMONES AND DRUGS ON THE GROWTH or CARCINOGEN-INDUCED MAMMARY TUMORS IN RATS by James L. Clark The effects of hormones and drugs on carcinogen-induced mammary tumors in rats were investigated in order to test further the current theory that prolactin plays a major role in promotion of mammary tumor growth. Some of the drugs used are known to alter catecholamine content of the hypothalamus and also to alter prolactin secretion. All agents employed in these studies influenced prolactin secretion and thereby caused inhibition or stimulation of mammary tumor growth. 1. Estradiol benzoate (EB) administered daily in doses of 20 ug for 20 days completely inhibited growth of mammary tumors induced by'the carcinogen 7,l2-dimethylbenzanthracene (DMBA), whereas mammary tumors in control rats increased by about 50% in diameter. When 1 mg of exogenous prolactin was administered simultaneously with 20 ug of EB, the inhib- itory effect of EB was overcome completely and mammary tumor diameters increased about 50%. The inhibitory action of EB upon mammary tumor growth appears to be exerted via inhibition of the peripheral action of prolactin on the mammary gland. Administration of exogenous prolactin (1 mg) overcomes this inhibition by "flooding" the mammary tissue with prolactin. James L. Clark 2. Ovine prolactin (1 mg) injected daily for 3 weeks significantly increased growth of mammary tumors. Androgen treatment for 3 weeks (5 mg testosterone propionate for 1 week, followed by 5 mg of 11B-hydroxy-l7-methy1testosterone for 2 weeks) had no significant effect mammary tumor growth. L-dopa (10 mg) for 3 weeks did not affect mammary tumor growth. Both Iproniazid (15 mg daily, reduced to 5 mg) and Pargyline (10 mg daily, reduced to 5 mg) prevented an increase in the growth of DMBA-induced mammary tumors. The dose of L-dopa may not have been sufficient to reduce prolactin secretion long enough to influence mammary tumor growth. The androgens are believed to have been given in insufficient doses to inhibit growth of mammary tumors. 3. Daily injections of haloperidol (150 ug) for 3 weeks enhanced growth of DMBA-induced mammary tumors. Lysergic acid diethylamide (LSD) (6 ug) and ergonovine (3.6 mg) tended to inhibit mammary tumor growth while Pargyline (6 mg) completely inhibited mammary tumor growth. Haloperidol has been reported to decrease hypothalamic catecholamines, thereby increasing prolactin release from the anterior pituitary gland. Pargyline interferes with prolactin release by increasing hypothalamic catecholamine and prolactin inhibiting factor (PIP) contents. LSD and ergonovine are believed to act directly on the anterior pituitary gland to inhibit prolactin secretion. This investigation supports the theory that prolactin is the major influence in promotion of mammary tumor growth in rats. THE EFFECTS OF HORMONES AND DRUGS ON THE GROWTH OF CARCINOGEN-INDUCED MAMMARY TUMORS IN RATS BY James L. Clark A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1972 Dedicated to Tina, Tom, and Tip ACKNOWLEDGMENTS During the course of this work the author received encouragement and assistance from many people, all deserving thanks and mention in the fruit of my labors. Foremost are the people who helped me and whom I assisted in the three experiments covered in this thesis. Eldon E. Cassell, John Lu, and Kaleem Quadri all worked patiently with me in the respective experiments. Their knowledge and presence was vital to the work. I am grateful to all three. My thanks are extended to Dr. Sam Dickerman for his ever-present guidance and example as a researcher. Marie Gelato was a source of assistance and my chief inspiration in this and other research. Others to whom I offer my thanks are Dr. Wolfgang Wuttke, Dr. Elias Dickerman, Dr. Steve Clark, Jim Dibbet, Ken Kortright. Thanks to Pam Rashid for typing the tables herein, and thanks to Amylou Davis for her guiding me through departmental procedures. Lastly, I want to thank the members of my guidance committee: Dr. Tom Jenkins, Department of Anatomy; Dr. W. D. Collings, Associate Chairman, Depart- ment of Physiology; and especially Dr. E. M. Convey, Department of Dairy, for his understanding, patience, and guidance throughout the course of my graduate work at Michigan State University; most importantly, I thank Dr. Joseph Meites, Department of Physiology, my major professor, for his direction of my graduate work. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . Hypothalamic control of prolactin secretion . . . . The role of catecholamines . . . . . . . . . . . . . Direct effects of hormones and drugs . . . . . . . . Short loop feedback . . . . . . . . . . . . . . . Mammary tumor induction, development, growth, and inhibition . . . . . . . . . . . . . . . . . . . . MATERIALS AND mmODS O O O O O O O O O O O O O O O O O O EXPERIENTAL O O 0 O O O O O O O O O O O O O O O O O O O 0 Experiment I. Effects of Estradiol Benzoate and Prolactin on Growth of Carcinogen-induced Mammary Tumors . . . . . . . . . . . . . . . . . . . . . . Experiment II. Effects on Mammary Tumor Growth of Prolactin, Testosterone, and Drugs that Alter Hypothalamic Catecholamines . . . . . . . . Experiment III. Effects on Mammary Tumor Growth of Ergots, Haloperidol, and Pargyline . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . SUWY AND CONCLUS I ONS I O I O O O O O O O O O O O O O O BIBLIOGMPHY O O O O I I I O O O O O O O O O O O O O O O 0 iv Page vi w \Dxlnbw 10 16 17 17 21 27 31 36 38 Table II. III. LIST OF TABLES Page Effects of Estradiol Benzoate and Prolactin on Growth of Carcinogen-induced Mammary Tumors . . . . . . . 19 Effects on Mammary Tumor Growth of Prolactin, Testosterone, and Drugs that Alter Hypothalamic Catecholamines . . . . . . . . . . . . . . . . . . . . . . 23 Effects on Mammary Tumor Growth of Ergots, Haloperidol, and Pargyline . . . . . . . . . . . . . . . . 29 Figure II. III. IV. LIST OF FIGURES Page Effects of Estradiol Benzoate and Prolactin on Growth of Carcinogen—induced Mammary Tumors . . . . . . . 20 Effects on Mammary Tumor Growth of Prolactin, Testosterone, and Drugs that Alter Hypothalamic Catecholamines . . . . . . . . . . . . . . . . . . . . . . 24 Effects on Mammary Tumor Growth of Prolactin, Testosterone, and Drugs that Alter Hypothalamic Catecholamines . . . . . . . . . . . . . . . . . . . . . . 26 Effects on Mammary Thmor Growth of Ergots, Haloperidol, and Pargyline . . .,. . . . . . . . . . . . . 30 vi INTRODUCTION Less than 20 years ago it was demonstrated that the hypothalamus exerted a regulatory influence on anterior pituitary function (Harris, 1955). Today it is known that the hypothalamus produces hypophysio- tropic hormones which control the release and perhaps synthesis of the six hormones of the anterior lobe of the pituitary gland (Ganong, 1966; Meites, 1970; Burgus and Guillemin, 1970). Thus, cortiocotropin releasing factor (CRF) from the hypothalamus stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary (Saffran and Schally, 1955). In a similar manner, luteinizing hormone releasing factor (LRF) stimulates luteinizing hormone (LH) release (McCann gt_31,, 1960), thyrotropin releasing factor (TRF) stimulates thyroid stimulating hormone (TSH) release (Guillemin gt_al,, 1962), prolactin inhibiting factor (PIF) inhibits prolactin release (Meites gt_213, 1961; Pasteels, 1962), growth hormone releasing factor (GRF) stimulates growth hormone (GH) release (Deuben and Meites, 1964), and follicle-stimulating hormone releasing factor (FRF) stimulates follicle-stimulating hormone (FSH) release (Igarashi and McCann, 1964; Mittler and Meites, 1964). These hypophysiotropic agents travel via axons from various nuclei in the hypothalamus to the storage terminals in the median eminence (Kobayashi and Matsui, 1969; Sulman, 1970). It is here that the secretory granules containing these factors enter the portal circu- lation destined for the cells of the anterior pituitary gland. The special nature of the blood vessel walls in this region enables the granules to enter the circulation easily (Clementi and Ceccarelli, 1970). This is the "final common pathway" to the adenohypophysis. Many drugs and hormones cause an increase or decrease in pro- 1actin secretion via the hypothalamus. Such agents may act by altering the contents of catecholamines and PIF in the hypothalamus. Drugs and hormones may also have a direct effect upon pituitary prolactin secre- tion. It was the objective of the present investigation to determine the effects on growth of carcinogen-induced mamary tumors in rats of some drugs and hormones that previously have been demonstrated to alter prolactin secretion. LITERATURE REVIEW Hypothalamic control of prolactin secretion During the 1950's the work of several researchers presented in. yiyg_evidence for an inhibitory influence of the hypothalamus on ante- rior pituitary prolactin secretion (Desclin, 1950, 1956; Everett, 1954, 1956; Sanders and Rennels, 1957; Alloiteau, 1958; Boot st 21,, 1959). Later, Pasteels (l961a,b) and Meites gt_a1, (1961) showed an inhibition of prolactin secretion by the hypothalamus ig_yi££g, Finally, a series of thorough and convincing experiments (Talwalker g£_21,, 1963) left little doubt regarding the validity of the concept of a specific "prolactin-inhibiting factor" (PIF) of hypothalamic origin. Other laboratories have confirmed this work (Danon g£_§l,, 1963; Gala and Reece, 1964), and today it is firmly entrenched as a scientific fact. PIF is produced in the hypothalamus and is carried by way of the hypophysial portal circulation to the anterior pituitary gland where it exerts an inhibitory control over pituitary prolactin secretion. When the anterior pituitary gland can escape hypothalamic control, the secretion of prolactin is increased (Meites §t_al,, 1963). Hypothalamic involvement is lost after specific median eminence lesions, pituitary stalk section, some central nervous system-depressant drugs, pituitary transplants, or ig_!i§£g_culture. In all cases, after hypo- thalamic control is removed, prolactin secretion is increased. As a result of the elevated prolactin concentrations, changes may occur in the mammary glands, ovaries, uterus, and vagina (Sulman, 1970). The role of catecholamines The hypothalamus contains a high content of norepinephrine and dopamine (Vogt, 1954; Laverty and Sharman, 1965). These two catechola- mines are present in areas of the hypothalamus which have influence over the secretory activity of the anterior pituitary gland. Nerve terminal concentrations of norepinephrine are highest in the supraoptic, para- ventricular, dorsomedial, and periventricular nuclei (Fuxe, 1965), while DA neuron cell bodies are concentrated in the arcuate and periven- tricular nuclei with their terminals in the median eminence (Fuxe 22.2}:' 1967). Evidence implicating these catecholamines as participants in control of anterior pituitary function has been accumulating for the last several decades. Everett (1964) has published a review which presents evidence supporting the possible involvement of the autonomic nervous system in regulation of gonadotropin secretions. Changes in hypothalamic input cause fluctuations in catechola- mine content. For example, lesions in the tegmentum of the mesencepha- lon, lateral hypothalamic area, or subthalamus lead to the disappearance of hypothalamic catecholamine terminals (Dahlstrom.gt_al,, 1964; Anden st 21., 1965, 1966a,b). Stefano and Donoso (1967) showed by fluoromet- ric procedures that changes in the functional state of the pituitary- gonad axis tend to affect noradrenergic neurons in the anterior hypo- thalamus. Norepinephrine concentrations appeared to be maximal during proestrus and minimal at estrus. After castration norepinephrine concentrations increased, but simultaneous treatment with estrogen and progesterone led to a reduction in norepinephrine concentrations in the hypothalamus. Dopamine fluctuations occurred at the same time and were found to be inversely related to the changes in norepinephrine (Donoso and Stefano, 1967; Donoso §E_§l,, 1969). Other workers claim that a-methyl-para-tyrosine or other agents inhibiting tyrosine hydrox- ylase, an enzyme important in norepinephrine synthesis, lead to the disappearance of the effects of gonadectomy (WUrtman, 1970). Reis and wurtman (1968) demonstrated that brain norepinephrine content undergoes diurnal variations. In addition, stress, hypophysectomy, pregnancy, and lactation have been associated with changes in catecholamine content in the hypothalamus (Fuxe and Hokfelt, 1969). Coppola (1969) reported an age-related increase in catecholamine pool size and turnover rate; the increase is accelerated after puberty. His work showed that an absence of ovarian steroids (for example, after oopherectomy) was followed by a marked increase in pool size and turnover rate. Steroid replacement therapy lowered catecholamine content. From these experiments he con- cluded that there was a strong indication of a reciprocal relationship between circulating gonadal steroid concentrations and hypothalamic catecholamine release and/or synthesis. In order to elucidate the possible involvement of catecholamines in the hypothalamus-pituitary axis, drugs have been used to alter hypo- thalamic catecholamine content. Drugs that increase hypothalamic cate- cholamines tend to decrease serum prolactin concentrations and drugs that depress hypothalamic catecholamines tend to increase serum pro- lactin concentrations (Coppola 25_31,, 1965, 1966; Van Maanen and Smelick, 1968; MacLeod gt_21,, 1969). Kamberi g£_al, (1971) reported that infusion of epinephrine into the third ventricle had no effect on prolactin release except at pharmacological levels; however, low doses of dopamine were effective in inhibiting prolactin release, presumably by increasing the release of PIF. An intracarotid injection of dopamine or epinephrine was followed by a decrease in anterior pituitary pro- lactin content but no change in the serum prolactin concentration (Lu §£;21,, 1970). A single intraperitoneal injection of L-dihydroxy- phenylalanine (L-dopa) was effective in significantly reducing the serum prolactin concentration (Lu and Meites, 1971; Wedig and Gay, 1970). Norepinephrine infused into the third ventricle inhibited PIF activity in the hypothalamus (Mittler and Meites, 1967), thereby increasing prolactin release. These four agents, dopamine, norepine- phrine, epinephrine, and L-dopa presumably raise brain catecholamine content directly. Other drugs are believed to inhibit catecholamine degradation and, thus, lead to an increase of hypothalamic catecholamines. The monoamine oxidase inhibitors Iproniazid, Pargyline, and Lilly compound 15461 have all been found to reduce serum prolactin concentrations (Lu and Meites, 1971). On the other hand, reserpine (Coppola gt_213, 1965; Ratner g5 21,, 1965; MacLeod gt_al,, 1969; Lu gt_al,, 1970), chlorpromazine (Lu SEHElrr 1970), haloperidol (Janssen, 1967; Abuzzahab, 1971; Dickerman gt_31,, in press) and related neuroleptic drugs cause a decrease in hypothalamic catecholamines in the hypothalamus and a significant increase in prolactin concentrations in the serum. Blockers of catecholamine-synthetic enzymes also cause an increase in serum prolactin concentrations. Some of these are armethyl-para-tyrosine, a-methyl-meta-tyrosine, and a-methyl-dopa (Fuxe and Hokfelt, 1969; Glowinski, 1970; Lu g£_al,, 1970; Lu and Meites, 1971). Thus, in summary, the hypothalamus exerts control over the secretory activity of the anterior pituitary gland and is known to contain a high content of catecholamines. Changes in the reproductive cycle produce changes in hypothalamic catecholamine content. Drugs able to increase or decrease catecholamine content of the hypothalamus are available. An increase in hypothalamic catecholamines results in increased PIF activity and decreased prolactin release, whereas a decrease in hypothalamic catecholamines results in decreased PIF activ- ity and increased prolactin release. There is a strong indication that circulating gonadal steroids play an important role in the catecholamine fluctuations in the hypothalamus. Direct effects of hormones and drugs A number of hormones and drugs are capable of acting directly upon the adenohypophysis to influence prolactin release (see Meites 22_ 31., 1963). Of the gonadal, thyroid, and adrenal cortical hormones capable of stimulating prolactin release, estrogen appears to be the most notable. Employing the use of a highly sensitive radioimmunoassay, Chen and Meites (1970) treated different groups of ovariectomized rats with low, intermediate, or high doses of estradiol benzoate and found that all doses increased the serum and pituitary concentrations of prolactin. Estradiol benzoate implants in the median eminence also increase both serum and pituitary prolactin concentrations (Nagasawa g£_31,, 1969). Removal of endogenous estrogen by ovariectomy and adrenalectomy is followed by a decrease in prolactin concentrations (Pearson st 21,, 1969). Estrogen or a combination of estrogen and progesterone have been shown to increase serum prolactin concentrations indirectly by decreasing PIF in the hypothalamus (Ratner and Meites, 1964; Minaguchi and Meites, 1967). In addition to this indirect effect, estrogen also exerts a direct effect upon the anterior pituitary gland to enhance the secretion of prolactin (Nicoll and Meites, 1962; Ratner 3.3 a. , 1963) . In contrast, certain ergot drugs are known to decrease the serum prolactin concentrations. Nagasawa and Meites (1970) showed that ergo- cornine decreased serum and pituitary prolactin concentrations. Further work had indicated that ergocornine probably acts directly upon the anterior pituitary gland (Malven and Hoge, 1971; Lu §E_21,, 1971) and also indirectly by increasing the PIF content of the hypothalamus (Wuttke gtnal., 1971) to inhibit prolactin secretion. Zeilmaker and Carlsen (1962) administered ergocornine to lactating rats, resulting in a temporary inhibition of milk production. Ergocryptine, ergonovine, and several other ergot alkaloids have recently been found to reduce serum prolactin concentrations (Meites gt_213, unpublished). Quadri and Meites (1971) demonstrated the effectiveness of lysergic acid diethylamide (LSD) in preventing the prolactin peak in the rat during the afternoon of proestrus. It is evident from the preceding that a number of agents normally present in the mammalian system interact to influence the synthesis and release of prolactin. In addition, there are a number of synthetic drugs capable of directly affecting the internal concen- tration of prolactin. By carefully experimenting with these hormones and drugs, endocrinologists hope to learn more about the physiology of the pituitary and hypothalamus and to be able to correct malfunctions. Short loop feedback Meites and Sgouris (1953) first suggested that prolactin might feed back on the pituitary to inhibit prolactin secretion. Subsequent work by MacLeod gt_§1, (1966) and Chen gt_al, (1967) showed that prolactin-secreting pituitary tumor transplants led to a decrease in hypothalamic PIF activity. This suggested that the feedback mechanisms acted via the hypothalamus. Later work by Clemens and Meites (1967, 1968) revealed that median eminence implants of prolactin also signif— icantly decreased serum and pituitary prolactin concentrations as a result of increasing PIF in the hypothalamus. Transplantation of two or more anterior pituitaries or daily administration of exogenous pro- lactin to intact rats resulted in a decreased concentration of prolactin in the i§_§itu_pituitary (Welsch g£_§1,, 1968; Sinha and Tucker, 1968). Finally, using the sensitive prolactin radioimmunoassay, Voogt and ~Meites (1971) found that prolactin implants in the median eminence of pseudopregnant rats caused a decrease in the concentration of prolactin in the pituitary, termination of pseudopregnancy in most rats within three days, and failure of prolactin to reach normal concentrations in the serum in the subsequent estrus. The efforts of these various workers indicate that prolactin, indeed, has a profound inhibitory influence on its own secretion. 10 Mammary tumor induction, development, growth, and inhibition The induction of mammary tumors in laboratory animals may be carried out by any of several methods. In mice mammary cancer may be elicited by treatment with estrogens or prolactin, or by pituitary transplants (Muhlbock and Boot, 1967; Boot, 1969). In rats the methods include estrogen treatment (Noble and Collip, 1941; Noble and Cutts, 1959; Huggins, 1965), exposure to radiation (Hamilton SE.2$:!.19543 Huggins and Fukunishi, 1963), and administration of aromatic chemical carcinogens--the last being the most effective and convenient means of producing experimental mammary tumors. These aromatic carcinogens include 2-acetaminofluorene (Wilson gt 31., 1941), methylcholanthrene (Shay gt_al,, 1949), and dimethylbenzanthracene (Bachmann gt_al,, 1938). Mammary adenocarcinoma induced by 7,12-dimethylbenzanthracene (DMBA) appears to be hormone-dependent (Pearson st 21,, 1969), and Huggins (1965) demonstrated a quick and simple method for the induction of mammary cancer of this type in 100% of the rats treated. Thus, research- ers now have an experimental situation closely resembling hormone- dependent human breast cancer. Much of the original work leading to an understanding of the hormonal relationships associated with breast cancer in humans has come from experimental surgical ablation. Thus, ovariectomy (Beatson, 1896), adrenalectomy (Fekete gt_31,, 1941), orchidectomy (Farrow and Adair, 1942) , and hypophysectomy (Luft and Olivecrona, 1953) were recomended as measures to be taken in cases of advanced breast cancer in humans. Along with hormone and drug treatments, these surgical procedures became weapons exhibiting varying degrees of effectiveness in the struggle against cancer. 11 Oopherectomy, in may cases, leads to a reduction in the size of carcinogen-induced mammary tumors, whereas estrogen treatment in ovari- ectomized rats previously treated with a carcinogen promotes growth of the tumors (Huggins gt_313, 1959). When rats are first ovariectomized and then treated with a carcinogen, no tumors develop (Huggins g£_gl,, 1961; Dao, 1962; Talwalker gt_g1,, 1964). However, if these rats are treated with estrogen or receive transplanted ovaries simultaneous with or shortly after administration of the carcinogen, tumors will appear although the incidence is lower than in intact rats treated with the carcinogen alone (Dao, 1962; Talwalker £5 31,, 1964). These findings indicate that estrogen appears to be necessary for the development of mammary cancer in rats. After hypophysectomy carcinogen-induced mammary tumors regress (Kim and Furth, 1960; Kim 32 21,, 1960; Sterental gt_al,, 1963). When, however, prolactin was administered to such rats, the tumors resumed growth rapidly, and many new tumors appeared (Pearson g£_21,, 1969; Furth, 1961). Cessation of prolactin treatment resulted in a rapid regression of the tumors again. Estrogen administration to humans (Pearson and Ray, 1959) and rats (Sterental gt_§1,, 1963) could not produce an exacerbation of tumor growth after hypophysectomy. Such evidence strongly suggests that rat mammary cancer induced by DMBA is dependent on prolactin secretion from the anterior pituitary gland. MacLeod §t_gl, (1964) found that transplanted mammary tumors failed to grow in rats previously ovariectomized, even in the presence of a tranSplanted mammosomatotropic tumor. Daily treatments of estro- gen and progesterone from the time of ovariectomy allowed growth of the 12 tumors. In a similar experiment Murota and Hollander (1971) delayed the estrogen and progesterone treatment for two months. Prolactin concentrations in the serum increased and the mammosomatotropic tumor continued to grow, but the transplanted mammary tumor failed to grow. However, one month after the onset of estrogen and progesterone treat- ment another mammary tumor was transplanted, and this time the tumor survived and began to grow promptly. These studies appear to indicate a requirement for ovarian stimulation of the mammary gland in order for mammary cancer to develop. Recent work suggests that prolactin may play a major role in the development of mammary tumors, while estrogen is necessary to prepare the mammary gland for the action of prolactin. Talwalker g£_21, (1964) found that ovariectomized rats treated with DMBA did not develop mammary tumors. When, in addition, estrogen or a combination of prolactin and growth hormone were administered, some tumors developed, although not in 100% of the cases as with intact rats injected with DMBA. By experimentally increasing serum prolactin concentrations, Clemens st 31. (1968) observed maintenance of tumor growth even after removal of the ovaries. The same researchers found similar results when they removed both ovaries and adrenals while inducing elevation of the serum pro- lactin concentrations (Welsch §t_§1,, 1969). Despite the fact that in both cases the tumors could be maintained only temporarily, the results indicate that prolactin plays an important role in tumor growth. At any rate it is evident that both prolactin and estrogen are required for the development of carcinogen-induced mammary tumors. Apparently prolactin and growth hormone are able to promote tumor development in the mammary 13 gland without the ovaries, but as mentioned previously estrogens were unable to promote manmary cancer in the absence of the pituitary gland. Experimental manipulations on pituitary prolactin secretion should exert a profound influence upon mammary cancers. Indeed, Welsch st al. (1970a) found a marked increase in the incidence of spontaneous mamary tumors in rats bearing multiple pituitary grafts. Bilateral lesions of the median eminence evoked similar results (Clemens 32 21,, 1968; Welsch gt_313, 1969, 1970b). Both experimental situations are known to increase the serum prolactin concentration. In addition, it has been reported that certain drugs known to enhance circulating pro- lactin concentrations can promote development of DMBAeinduced mammary tumors. Both perphenazine (Pearson gt_gl,, 1969) and reserpine (Welsch and Meites, 1968) yield such a response. These findings constitute further evidence for the major role of prolactin in promotion of DMBA- induced mammary tumors in rats. On the other hand, certain drugs that inhibit prolactin secre- tion have been demonstrated to decrease growth and development of mam- mary tumors. Two ergot drugs which were found to suppress mammary cancer are ergocornine (Nagasawa and Meites, 1970; Yanai and Nagasawa, 1970; Cassell 22.2lrv 1971) and ergocryptine (Heuson £2 21,, 1970; Cassell gt_al,, 1971). The role of estrogen is of great importance in the interrela- tionships of endocrine organs and their influence upon the mammary gland and the development of tumors. Estrogen administration in high, intermediate, or low doses causes an increase in serum and pituitary prolactin concentrations, while ovariectomy and adrenalectomy are 14 followed by a decrease in serum prolactin concentrations in the rat. Thus, tumor growth may be affected by variations of the estrogen titer in the blood. Nagasawa gt_al, (1969) found that estrogen implanted in the median eminence of rats with DMBA-induced mammary tumors increased serum and pituitary prolactin concentrations and growth of mammary tumors. In another experiment, Welsch g£_§13 (1969) reported that ovariectomy at the time of median eminence lesion was followed by an initial stimulation of mammary tumor development with a subsequent regression. The stimulation is presumably due to the elevated prolactin concentrations in the serum associated with the median eminence lesion; however, regression of the tumors suggests that removal of the ovaries is responsible for the latter phenomenon. Thus, it appears that, although prolactin may be the major hormone promoting mammary tumor growth, estrogen may affect the growth and development of mamary tumors in rats. Estrogen has been used for years in humans to keep breast cancer in check (Haddow, 1935; Landau £2 21,, 1962). Meites (in press) found that high doses of estrogen (20 ug) caused mammary tumor regression in rats despite the fact that prolactin concentrations were elevated. Male sex hormones have also been discovered to be effective against mammary cancers (Lacassagne, 1939; Loeser, 1941; Council on Pharmacy and Chemistry, 1951). Others have shown that an estrogen-progesterone combination (Huggins and Yang, 1962; Landau g£_31,, 1962) or potent synthetic estrogens (Haddow st 31,, 1944; Nathanson, 1946) may lead to regression of breast cancer. 15 Although high or low serum prolactin concentrations may be a major influence on the promotion or regression of mammary tumors in rats, there are other factors which may play a less significant role but exert considerable influence upon the induction, growth, and development of mammary cancer. Therefore, the purpose of the exper- imental work in this thesis was to attempt to elucidate some of the factors involved in control of mammary tumor growth in rats, and hopefully, to provide some new approaches for treating human breast cancer . MATERIALS AND METHODS All animals used in these experiments were virgin female Sprague- Dawley rats obtained from Spartan Research Animals, Inc., Haslett, Mich. l The animals were maintained on a regular lighting schedule of 14 hours if of light per day. Temperature was held constant at 24 11° C, and food (Wayne Lab Blox, Allied Mills, Chicago, Ill.) and tap water were given a§_libitum. In addition, dietary supplements of carrots and oranges were supplied weekly. At 55 days of age intact rats were administered a single intra- venous injection into the tail vein of a lipid emulsion containing 5 mg of 7,12-dimethy1benzanthracene (DMBA) (The Upjohn Co., Kalamazoo, Mich.) after the method of Huggins (1965). Palpable mammary tumors were present within 1—3 months after DMBA administration in 100% of the animals. The rats were then divided into groups of equal size with efforts for uniformity of tumor incidence and mean tumor diameter among the groups. With the rats placed under light ether anasthesia, total tumor number per rat, the largest diameter per tumor as measured by calipers, and body weights in grams were recorded at the beginning of each experiment. These measurements continued at regular intervals after the onset of treatment. Further details of the treatments are given under each separate experiment. All data were analyzed by comparing regression lines through an analysis of covariance. 16 EXPERIMENTAL Experiment I: Effects of Estradiol Benzoate and Prolactin on Growth of Carcinogen-induced Mammary Tumors Objectives The purpose of the first experiment was to learn more about the mechanism whereby large doses of estrogen inhibit mammary tumor develop- ment. Prolactin and estrogen can promote mamary tumor development and growth. Small doses of estrogen can promote mammary tumor development and growth, whereas large doses are known to inhibit mammary tumor growth. Large doses of estrogen are commonly used in the treatment of breast cancer in women. Likewise, such large doses of estrogen inhibit mammary adenocarcinoma in rats and other experimental animals. Never- theless, estrogen at high, intermediate, or low doses increases serum prolactin concentrations and has never been observed to decrease the prolactin concentration in the serum. Therefore, large doses of estro- gen do not appear to inhibit mammary tumor growth by inhibiting prolac- tin secretion. Thus, the objective of this investigation was to determine how large doses of estrogen could inhibit mammary tumor growth in rats. 17 18 Procedure All treatments were administered via subcutaneous injection daily for 20 days. Treatments were as follows: GROUP 1: 0.2 ml of corn oil (controls) GROUP 2: 20 ug of estradiol benzoate (Nutritional Biochemicals Corp., Cleveland, Ohio) in 0.2 ml of corn oil GROUP 3: 20 ug of estradiol benzoate in 0.2 ml of corn oil + 1 mg of ovine prolactin (NIH-P-S-8, 28 IU/mg, National Pituitary Agency, NIH) in 0.2 m1 of 0.9% saline Largest tumor diameter, number of tumors per rat, and body weights in grams were measured every 5 days during the 20 days of treatment. Results Mean tumor diameter of the control group increased from 702t12 mm to 107:t16 mm and mean number of tumors per rat increased from 2.4:tO.4 to 3.4:tO.7 during the 20 days of treatment (Table I). The group receiving estradiol benzoate exhibited a decrease in mean tumor diameter from 131 £21 mm to 126 £30 mm which was significantly different (p‘<.01) from the control group. Likewise, the mean tumor incidence decreased from 2.52:0.4 to 1.7:to.3 and this was also significantly different (p‘<.01) from the control group (see Figure I). The group administered estradiol benzoate and prolactin showed an increase in mean tumor diameter from 95 i 13 mm to 142 i 16 um and in mean tumor incidence from 2.9:t0.3 to 3.22:0.4. Neither was different from the control group. It appears that the ovine prolactin overcomes the effect of the estradiol benzoate in inhibiting mammary tumor growth. 19 $.OHN.mV Am.OHH.mV Am.OH¢.mv 3.0M H H 0H H NvH m H HMH bH H. mNH m m 0mm Ohm AOHV GHUOMHOHm + mm +| \D O O H M +I SHONE: Am.OHm.Hv Am.OHw.NV Am. om H mNH hm mNH mm H hMH m NO +l 4-! 98 3.0 H mé H3 3. H 5 8m «2 8: mm +I CHOHvHHV $.0Hm.mv Am.o.flm.mv Am.OHm.NV Av.0Hv.NV H N. H H m H 3 m4 H om 2 H 8 m H m «H H 2. mmm mom a: 303:8 mane om mane ma when S 23c m H020 133 HHHHHEH Hume Ho .8: 9292.259 THHH Ham Ham mnosa. «0 .oz game Game .93 Bow. zdmz .m.m H 25 $9328 moses zmmz muofiPH. mung: pmoochncmmocHoumu mo susouu so cHuUMHoum use 35 masonsmm HOHpmuumm mo muoommm .H 01.69 I40 - I20 - 3 o o 0 M10 In mm 0 O 40 - 20- Figure I. 20 ”t" E B + "OIOCfin «- .oul -l.-I-..... .o '- e a.... .I" O..-(" ' . 1" ---.I.llIIII.a E. _/ Controls / 1” I I I ” ’ ” '1 J I O 5 IO 15 20 Time in Day: Effects of Estradiol Benzoate and Prolactin on Growth of Carcinogen-induced Mammary Tumors. MTD -mean tumor diameter. Treatments were: 20 ug of estradiol benzoate (EB) alone or with 1 mg of ovine prolactin; controls received corn oil. 21 Experiment II: Effects on Mammary Tumor Growth of Prolactin, Testosterone, and Drugs that Alter Hypothalamic Catecholamines Objectives This investigation was undertaken in order to explain the effects of experimentally increasing serum prolactin concentration, serum testos- terone concentration, or brain catecholamdne content on mammary tumor growth. Prolactin stimulates mammary tumor growth. Testosterone can directly inhibit mammary tumor growth, while increasing the catechola- mine content in the hypothalamus inhibits prolactin secretion. Procedure All treatments were administered via subcutaneous injection daily for 3 weeks. They were as follows: GROUP 1: 0.3 ml of 0.9% saline (controls) GROUP 2: 10 mg of Iproniazid phosphate (Hoffman-LaRoche, Inc., Nutley, N.J.) in 0.3 m1 of 0.9% saline GROUP 3: 15 mg of Pargyline hydrochloride (Abbott Laboratories, North Chicago, Ill.) in 0.3 ml of 0.9% saline GROUP 4: 10 mg of Levodopa dihydroxyphenylalanine (L-dopa) (Nutritional Biochemicals Corp., Cleveland, Ohio) in 0.34 ml final volume of a combination of 0.5 N HCl brought to pH 6.5 by 0.5 N NaOH GROUP 5: 5 mg of testosterone propionate (Nutritional Bio- chemicals Corp., Cleveland, Ohio) in 0.2 ml corn oil GROUP 6: 1 mg of ovine prolactin (NIH-P-S-8, 28 IU/mg, National Pituitary Agency, NIH) in 0.2 ml of 0.9% saline GROUP 7: 5 mg of testosterone propionate in 0.2 m1 of corn oil + 1 mg of ovine prolactin in 0.2 ml of 0.9% saline 22 After one week of treatment an androgen with more anabolic potency was used to replace testosterone propionate. Five mg of llB-hydroxy-17- methyltestosterone in 0.2 ml of corn oil was substituted for the duration of the experiment. Measurements of body weights in grams, mammary tumor diameters in mm, and mammary tumor incidence were recorded every 7 days. Treat- ments lasted for a period of 3 weeks. Results Mean tumor diameter of the control group increased from 35::11 mm to 59 :22 mm and mean tumor incidence increased from 3.1 i 1.1 to 3.8 i 1.4 during the 3 weeks of treatment (Table II). The prolactin group exhibited an increase in both mean tumor diameter from 40:t12 mm to 118:122 mm and the mean number of tumors per rat from 3.0:t1.0 to 8.6:t1.5. When compared to the control group, the mean tumor diameter and mean tumor incidence of the prolactin group showed a significant (p‘<.001) increase (see Figure II). On the other hand, Pargyline, with an initial mean tumor diameter of 45ztl3 mm and a final mean tumor diameter of 27::9 mm, and Iproniazid, with an initial mean tumor diameter of 37:tl6 mm and a final mean tumor diameter of 39::9 mm, were both significantly different (p‘<.01) from the control group in their effect on mean tumor diameter. Although the rats in the Iproniazid group showed no change in mean tumor incidence which was 3.8:tl.1 at the beginning of the experiment and 4.0:tl.l at the end, the Pargyline group exhibited a decrease in mean tumor incidence from the original 3.4:to.8 to 2.3:t0.6 which was sig- nificant (p‘<.01) when compared to the controls. 23 8.0Hm.8 8.03.8 8.30.8 8.0312 0 H R S H mm «H H mm «A H 3 mmm mmm 5 00383.1 3.30.3 8. HHmé 3 8.30.3 2.30.2 0 H mm 0 H 00 S H 00 3 H am 0.00 05. 2: 033083 8.35.8 8.30.2 8.HH~.2 8.30.8 8 H 8 3 H am 0H H mm 2 H 3 «an 8.0. 8v «downs 8. 0H5 8 8.30.8 8.0H0.2 8.0H0.8 0H H 08 0H H 8 0H H 00 S H 0e 08 man E 03038.1 0 509608 8.0H0.3 8.0H~.3 8.02:3 8.35.8 S H 0m 2 H S 3 H mm 0 H d. 400 80H 8e 80805 8.HH8.8 8.HHO.8 GAHWE 8.30.8 mm H 8: on H 08 ma H 8 3 H 00 man 8N E 03030.8 8.38.8 8.HH0.2 8.332 S.HHH.8 «H 0m 3 H mm 3 H 8 S H mm 000 mmm 8V 205.80 333 0 meme: m x33 H H300 303 HmHHHcH 888 no .on azmzaamma A.m.mHuum Ham 3955. mo .oz 0005 853 .93 woom zmmz .m.mH 055 $9823 moron. zcmz mocHEMHonooumo ogHmzuém HoUHd umfi moons one .ocououmoumoa .cHDOMHoum mo nuzouo H0039 whose—oz so muoommm . HH OHQMB 24 120. // Prolactin Ioo _ /‘ / .x E 80. x”/ I E /". 5 60 . ’./' . a Control 2 4O _ 2’ “\“ _ \Q‘ Iproniazid 20 . ~ Pargyline 0 . . ‘ (’ '2 ‘3 Weeks Figure II. Effects on Mammary Tumor Growth of Prolactin, Testosterone, and Drugs that Alter Hypothalamic Catecholamines. MTD:-mean tumor diameter. Treatments were: 1 mg ovine prolactin, 15 mg Iproniazid (reduced to 5 mg). 10 mg Pargyline (reduced to 5 mg), controls received saline. 25 L-dopa and androgen treatment were ineffective in inhibiting mammary tumor growth. Mean tumor diameters and mean tumor incidences increased from 43i17 mm and 3.0:1.2 to 61i21 mm and 3.7i1.5, respectively, for L-dopa and from 4lzt9 mm and 3.7:t0.8 tumors per rat to 582t19 mm and 4.01:0.8 tumors per rat for the androgen-treated group (see Figure III). An increase in mean tumor diameter from 402t19 mm to 692t10 mm and an increase in the mean number of tumors per rat from 3.0:t0.6 to 3.72:0.7 during the 3 weeks of treatment for the group treated with androgen and prolactin together were not different from the increase in the same measurements in the control group. Likewise, there is no difference between this group and the group treated with androgen alone. However, when the group treated with androgen and prolactin is compared to the group receiving prolactin alone there is a significant inhibition (p‘<.01) of mammary tumor growth in the former. This indicates that the androgen llB-hydroxy-l7-methyltestosterone can overcome the mammary tumor growth-stimulating effects of ovine prolactin. In spite of their respective effects upon prolactin secretion and the mammary glands, eropa and the androgen did not inhibit mammary tumor growth. This may be explained by the facts that L—dopa acts rapidly but has a short duration, while the androgen is not as potent as those used clinically. Both Iproniazid and Pargyline caused hyperactivity in the rats. At the end of the first week the dose of Iproniazid was decreased by half, and by the end of the second week both drugs were reduced to 5 mg daily per rat. Despite the reduction in dose, animals in both groups lost weight and several died. 7O 60 4O 30 M10 in mm. 20 'IO Figure III. 26 Androgen ,’°""' + Prolactin ./ l-d , - — ‘ ’ Control Androgen Effects on Mammary Tumor Growth of Prolactin, Testosterone, and Drugs that Alter Hypothalamic Catecholamines. MTD==mean tumor diameter. Treatments were: 5 mg of androgen alone or with 1 mg of ovine prolactin, 10 mg L-dopa, controls received saline. 27 Experiment III. Effects on Mammarerumor Growth of Ergots, Haloperidol, and Pargyline Objectives The purpose of the final experiment was to test the effects on mammary tumor growth of the two ergot drugs ergonovine and lysergic acid I _1 diethylamide (LSD) and the tranquilizing drug haloperidol, which inhib- its-hypothalamic catecholamine activity. A reduced dose of Pargyline was tried with the aim of inhibiting mammary tumor growth without caus- ing a loss of body weight or a hyperactivity in the animals. Procedure Rats in each group were administered a single, daily injection subcutaneously as listed below: GROUP 1: 0.3 ml of corn oil (controls) GROUP 2: 150 ug of haloperidol (McNeill Laboratories, Inc., Fort Washington, Penn.) in 0.3 m1 of corn oil emulsion GROUP 3: 1.5 ug the lst week, 3.0 ug the 2nd week, 6.0 pg the 3rd week of LSD (courtesy of Dr. T. M. Brody, Chairman of the Department of Pharmacology, Michigan State University, East Lansing, Mich.) in 0.3 ml of a saline-corn oil mixture (1:1) GROUP 4: 1.8 mg the lst and 2nd weeks, 3.6 mg the 3rd week of ergonovine (Eli Lilly and Co., Indianapolis, Ind.) in 0.3 ml of 0.9% saline GROUP 5: 6.0 mg of Pargyline hydrochloride (Abbott Laboratories, North Chicago, Ill.) in 0.3 ml of 0.9% saline Treatments lasted for a period of 3 weeks and measurements of the largest tumor diameter, tumor incidence, and body weights in grams were recorded every 7 days. 28 Results Mean tumor diameter of the control group increased from Slzt 16 mm to 822t24 mm and mean tumor incidence from 3.6:tl.l to 6.0:t1.3 during the 3 weeks of treatment (Table III).‘ The haloperidol group exhibited an increase in both tumor diameter from.29:t10 mm to l3l:t 31 mm and the mean number of tumors per rat from 2.7:t0.9 to 8.5:tl.3. When compared to the control group, the mean tumor diameter and the mean tumor incidence of the haloperiodol group showed a significant increase (p<<.01) (see Figure IV). On the other hand, Pargyline, with an initial mean tumor diameter of 65:t20 mm and a final mean tumor diameter of 43::17 nun significantly inhibited mammary tumor growth (p‘<.01). Likewise, there was a significant decrease (p‘<.01) in the mean number of tumors per rat from 4.3:t0.9 to 3.3:t0.7 when compared to the control group. Mean tumor diameters and mean tumor incidences increased in the groups receiving ergonovine, from 552t18 mm to 68::20 mm and 3.9:t1.2 to 4.6: 1.3, respectively, and LSD, from 49 :18 mm to 59¢ 16 m and 4.02:1.2 to 4.6:t1.3, but neither group exhibited a significant differ- ence when compared to the control group. However, the final dose of LSD (6 ug) appears to have an effect on the mammary tumors; perhaps this dose would inhibit mamary tumor growth if administered for 3 weeks. Rats did not appear to be harmed by the reduced amount of Pargyline although they still lost weight. None of the other groups exhibited any unusual reactions to treatment. The animals receiving LSD seemed generally more docile than the rats in the other groups. 29 843.8 8.30.8 8.35.3 8.0Haé 8 H H3 5 H 0a 2 H mm 0H H 0m 8m mam 8V HonHHmmonm 8.02.8 8.03:8 8.0Hm.$ 8.0Hm.3 S H 2. S H me 3 H 0m on H mm mmm 8m 80 8283a 8.30.3 8.38.3 8.38.3 8.HH0.3 3 H 0m 8 H 8 0m H 0m 3 H 00 mom Ea E 28.: 03.31530 oHoe UHmHom>A 8.30.3 8.30.3 2.30.3 8.30.2 0H H we 3 H 80 00 H 8 3 H mm 8m mom E maH>oaomHm 8.30.8 8.30.3 8.30.3 3.38.8 H H 8 S H 80 3 H mm 3 H 3. 08 mam 8e 385000 mMOOB m m¥003 N #003 H umeO HmGHm HMHpHGH Amug MO .OZV azmzaamme A.m.m Ham Ham muossa «0 .oz amaze lasso .9: seem zmmz .m.m Assv mmemzmoo mozoe zmmz ocHHhmumm one .HooHuomonm .muomum mo cusouw Hosea unseen: co muoommm .HHH pome 120 100 IITDInInnu h 0 G O O O N O Figure IV. 30 Hal0perido| Effects on Mammary Tumor Growth of Ergots, Haloperidol, and Pargyline. MTD:-mean tumor diameter. Treatments were: 150 pg haloperidol, 1.5-6.0 ug LSD, 1.8-3.6 mg ergonovine, and 6 mg Pargyline; controls received corn oil. DISCUSSION Prolactin appears to be the most important hormone related to the promotion of mammary tumor growth in rats (Furth, 1961, 1962; Meites and Nicoll, 1966; Pearson 2E_213, 1969; Welsch gt_§l:, 1970a,b). This research involved attempts to alter carcinogen-induced mammary tumor growth in rats by indirect manipulation of prolactin concentration through alteration of hypothalamic control of prolactin secretion. The DMBA-induced mammary tumor and human breast cancer resemble each other in two respects--their common origin in ductal tissue and their respon- siveness to hormone treatment. The types of drugs employed in these experiments have all been shown to affect circulating prolactin concen- trations, while the steroid hormones also have direct effects upon the mammary glands. An attempt was made to determine the mechanism by which large doses of estrogen inhibit mammary cancer growth. The results of this work indicate that large doses of estrogen interfere with the peripheral action of prolactin on mammary tumor growth. Large doses of estrogen have been used by clinicians and researchers to inhibit breast cancer in women and experimental animals. However, it was found that all doses stimulate prolactin release from the anterior pituitary gland (Chen and Meites, 1970). Therefore, the mechanism of action of large doses of estrogen in inhibiting mammary tumor growth cannot be explained by the inhibition of prolactin secretion. Meites and Sgouris (1954) found 31 32 that ovarian steroids appeared to prevent prolactin from exerting its full influence upon the mammary gland. The results of the first exper- iment suggest that this may be the mechanism by which estradiol benzoate inhibits mamary tumor growth. Nagasawa and Meites (unpublished), using several dose levels of estradiol benzoate, found that daily injections of a dose of 20 mg was most effective in inhibiting mammary tumor growth in rats. The results of Experiment I show that this dose of estradiol benzoate was effective in preventing the increase of mean tumor diameter and mean number of tumors per rat found in the control group. Increasing the amount of prolactin in the serum by daily injections of exogenous NIH ovine pro- lactin (1 mg) overcame the inhibition by estradiol benzoate, and mean tumor diameter and mean tumor incidence were no different from those of the control group. These findings suggest that large doses of estrogen interfere with the peripheral action of prolactin on the mammary tissue and, thereby, inhibit growth of the mammary adenocarcinoma. Further research is required in order to determine the biochemical mechanisms involved in this interaction between estrogen and prolactin. Androgens have also been used in the treatment of mammary cancer with effectiveness in 20% or more of the cases (Rosoff, 1960; Calabresi and Parks, 1970). In Experiment II testosterone propionate was found to be ineffective during the first week of treatment and a more potent anabolic androgen, llB-hydroxy-l7-methyltestosterone, was substituted. However, this hormone also failed to inhibit mammary tumor growth, although when given at the same time as prolactin, llB-hydroxy-17- methyltestosterone prevented the significant increase in mean tumor 33 diameter found in the group receiving prolactin alone. Since clinical studies indicate that improvements in the status of patients with mam- mary cancers are very slow with androgen treatment, it would appear that the time span of this experiment was not sufficient for the effects of the androgen treatment to be manifested. However, the principal reason for the ineffectiveness of these two androgens is probably that they were not potent enough in androgenic activity. In rats given prolactin there can be no doubt that there was a significant stimulatory effect upon the growth of mammary tumors.' There was nearly a threefold increase in both mean tumor diameter and tumor incidence in three weeks. In addition, the effects of haloperidol in Experiment III are equally marked.~ Haloperidol has been found to stimulate an increase in serum prolactin concentration of greater than elevenfold in rats (Dickerman gt_gl,, in press). It was shown to deplete the hypothalamus of catecholamine and PIF activities. Thus, sufficient increase of circulating prolactin concentrations, either by injectionuof prolactin or indirectly by administering haloperidol to rats with DMBA-induced mammary tumors, promoted a powerful stimulation of mammary tumor growth. These findings lend strong credence to the. concept that prolactin plays a major role in stimulating growth of carcinogen-induced mammary tumors in rats. The remaining drugs in Experiment II, L-dopa, Iproniazid, and Pargyline, were employed in efforts to reduce the circulating concen- tration of prolactin and, in so doing, to prevent promotion of mammary tumor growth by prolactin. These three drugs are believed to increase the content of brain catecholamines, and they have been found to 34 decrease serum prolactin concentrations in rats (Lu and Meites, 1971). Both Iproniazid and Pargyline, inhibitors of catecholamine degradation, inhibited mammary tumor growth. L-dopa, a precursor in the biosynthesis of dOpamine and norepinephrine, failed to inhibit mammary tumor growth in this experiment. Lu and Meites (1971) injected a similar dose of L-dopa intraperitoneally and found that it reduced serum prolactin concentrations for up to two hours after injection. It is possible, therefore, that more frequent treatment with L-dopa would have inhib- ited mammary tumor growth. The results of injecting two agents, Iproniazid and Pargyline, known to inhibit prolactin secretion into rats with DMBA-induced mammary tumors tend to support the concept used as the basis for this work. Neither of the ergot drugs, ergonovine and LSD, inhibited mammary tumor growth. Ergot derivatives inhibit prolactin secretion. Quadri and Meites (1971) found that LSD inhibits the prolactin peak during proestrus. The fact that these two drugs were not effective in the inhibition of mammary tumor growth may be explained by the fact that the doses used were not high enough. The dose of ergonovine was doubled late in the second week of treatment and LSD doses were doubled at the end of weeks one and two. Both drugs appeared to be more effective in preventing mammary tumor growth during the third week of treatment. Previously, two other ergot drugs, ergocornine and ergocryptine, were shown to be effective in inhibiting mammary tumor growth in rats (Cassell £2.2l3' 1971; Nagasawa and Meites, 1970). After termination of the treatments the growth of mammary tumors in the control group continued at the same rate. Although not recorded 35 in the present data,-the trend for tumors whose growth had been inhibited by drugs earlier showed an increase in growth subsequent to termination of treatment. Those whose growth was previously stimulated (i.e., the prolactin and haloperidol groups) subsequently exhibited a decrease in the mammary tumor growth rate. A similar observation has been made by others (Cassell, 1971; Quadri and Meites, unpublished). SUMMARY AND CONCLUSIONS The effects of hormones and drugs on carcinogen-induced mammary tumors in rats were investigated in order to test further the current hypothesis that prolactin plays a major role in the promotion of mammary tumor growth. Some of the drugs used are known to alter catecholamine content of the hypothalamus and also to alter the secretion of prolac- tin. All agents employed in these experiments influenced prolactin and thereby caused inhibition or stimulation of mammary tumor growth. 1. EB administered daily in doses of 20 ug for 20 days com- pletely inhibited growth of mammary tumors induced by DMBA, whereas mammary tumors in the control group increased by about 50% in mean tumor diameter. When 1 mg of ovine prolactin was administered simultaneous to 20 ug of EB, the inhibitory effect of EB was overcome completely and mammary tumor diameters increased about 50%. The inhibitory action of EB upon mammary tumor growth appears to be exerted via inhibition of the peripheral action of prolactin on the manmary gland. Administration of exogenous prolactin (1 mg) overcomes this inhibition by "flooding" the mammary tissue with prolactin. 2. Ovine prolactin (1 mg) injected daily for 3 weeks signifi- cantly increased growth of mammary tumors. Androgen treatment (5 mg of testosterone propionate for 1 week, followed by 5 mg of llB-hydroxy-l7- methyltestosterone for 2 weeks) had no effect upon mammary tumor growth. L-dopa (10 mg) for 3 weeks had no effect upon mammary tumor growth. 36 37 Both Iproniazid (15 mg daily, reduced to 5 mg) and Pargyline (10 mg daily, reduced to 5 mg) significantly inhibited mammary tumor growth. The dose of L—dopa may not have been sufficient to reduce prolactin secretion long enough to influence mammary tumor growth. The androgens were given in insufficient doses to inhibit growth of mammary tumors. 3. Daily injections of haloperidol (150 pg) for 3 weeks enhanced growth of DMBA-induced mammary tumors significantly. LSD (6 pg) and ergonovine (3.6 mg) appeared to have an inhibitory effect on mammary tumor growth, while Pargyline (6 mg) completely inhibited mammary tumor growth. Haloperidol has been reported to decrease hypothalamic cate- cholamines, thereby increasing prolactin release from the anterior pituitary gland. Pargyline and Iproniazid interfere with prolactin release by increasing hypothalamic catecholamine and prolactin inhib- iting factor (PIF) contents. LSD and ergonovine are believed to act directly on the anterior pituitary gland to inhibit prolactin secretion. This investigation supports the theory that prolactin is the major influence in the promotion of mammary tumor growth in rats. BIBLIOGRAPHY Abuzzahab, F.S., Sr. 1971. Effects of haloperidol and amantadine on rat brain catecholamines and behavior. Fed. Proc. 30: 381. Alloiteau, J.J. 1958. Sur la nature du controle de la fonction luteo- trophe de 1'hypophyse anterieure par 1'hypotha1amus chez 1a ratte. C.R. Acad. Sci. (Paris) 247: 1047-1049. Anden, N.E., A. Dahlstrom, K. Fuxe, and K. Larsson. 1965. 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S E R A R m L V H S R E w UN TATE m s 1mm HI 3 INIHIHIH 3