Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland.

Mitochondrial biogenesis in the parenchymal cell of the mouse mammary gland appears to occur in two distinct phases: replication during cell proliferation, and maturation during cell differentiation. This study of the mitochondrial maturation phase in the mouse gland demonstrates a significant increase in organelle density on isopycnic sucrose gradient centrifugation during the transition from late pregnancy to day 8 of lactation. Differential fragility to high sucrose concentrations or changes in mitochondrial lipid composition do not satisfactorily explain the density increases. When organelle densities were assessed by centrifugation under iso-osmotic conditions with Ficoll gradients in 0.25 M sucrose, the mitochondria from pregnant glands were observed to be more dense than those from lactating glands. The two mitochondrial populations were also found to differ in their response to changes in sucrose concentration in the Ficoll gradients. When sucrose concentration was increased, the density of both pregnant and lactating gland mitochondria increased nonlinearly, the increase being greater with the lactating gland organelles. By use of mathematical models, the differing response was interpreted as a change in the density and osmotic activity of the mitochondrial internal compartment (inner membrane plus matrix space). We have proposed that the changes reflect a large expansion of the inner mitochondrial membrane and perhaps the matrix material during the transition into lactation in the differentiating parenchymal cell.

PIP: Developmental changes in mitochondria during pregnancy and the transition into lactation in the parenchymal cell of the mouse mammary gland were studied. There were 2 apparent distinct phases in mitochondrial biogenesis: replication during cell proliferation and maturation during cell differentiation. Isopycnic sucrose gradient centrifugation during the transition from late pregnancy to Day 8 of lactation revealed a marked increase in organelle density. This increase in organelle density could not be explained by differential fragility to high sucrose concentrations or changes in mitochondrial lipid composition. Mitochondria from pregnant glands were more dense than those from lactating glands as determined by centrifugation under iso-osmotic conditions with Ficoll gradients in .25 M sucrose. These mitochondrial populations also differed in their response to changes in the concentration of sucrose in the Ficoll gradients. The density of both pregnant and lactating gland mitochondria increased nonlinearly when the sucrose concentration was increased, with the increase in density being greater in the latter. The difference in response was mathematically interpreted to reflect a change in the density and osmotic activity of the mitochondrial internal compartment (inner membrane plus matrix space). It is proposed that the developmental changes observed reflect a large expansion of the inner mitochondrial membrane and possibly the matrix material during the transition into lactation in the differentiating parenchymal cell.

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure.

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.

PIP: The enzyme markers for mitochondrial inner and outer membranes throughout the pregnancy-lactation cycle in the mouse were compared. The ultrastructural changes of the organelle during the transitions were studied by electron microscopy. The activity of cytochrome oxidase in mouse mammary gland homogenates was found to increase 5- to 6-fold from late pregnancy to Day 8 of lactation, while that of monoamine oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased 5-fold. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. The pattern was the same in each preparation with little change until late pregnancy and then a 3- to 4-fold increase occurred and values reached a maximum by Day 8 of lactation. Electron micrographs of midpregnant and midlactating mammary parenchymal cells in situ were prepared, and changes in the mitochondrial morphology noted. The most significant change is the large increase in number of inner membrane cristae and an increase in matrix density in the lactating gland cell.

Direct action of estradiol on rat mammary tumors.

PIP: This study reports a direct action of 17-beta-estradiol on protein synthesis by 7, 12-dimethylbenz (alpha) anthracene (DMBA) induced rat mammary tumors. Sprague-Dawley female rats were given 20 mg of DMBA in sesame oil by gastric tube. Mammary tumors developed. When tumors reached 1.5 to 2 cm in diameter, animals were ovariectomized. At 4-7 days later animals were killed and tumors removed. Microscopic examination confirmed the tumors to be carcinoma of adenoid cystic variety with regressive changes. 14 tumors from 14 animals were found suitable for study. In vitro treatment with 17-beta-estradiol gave rise to a 7% increase in the rate of 3H-leucine incorporation, expressed as dpm/mg of TCA-insoluble protein. This increase was considered statistically significant (p.001). A large variation among different tumors was noted. Also results varied with differences in time after ovariectomy. Under the same incubation conditions the same percentage of increase in the rate of 3H-leucine incorporation had been observed in the study of the effect of estrogen on the uterus of ovariectomized rats. In other mammalian tissues studied those containing high levels of estrogen receptor were able to respond to direct stimulation of estrogen. It was concluded that estrogen directly stimulates protein synthesis in the mammary tumors. This supports the view that these tumors are estrogen responsive tissues.^ieng

Mammary nodules in dogs during four years' treatment with megestrol acetate or chlormadinone acetate.

PIP: A 7 year study of megestrol and chlormadinone in female dogs is in progress. This report characterized histopathologically 60 mammary nodules during the first 4 years of the study. 100 purebred female beagles, 6-12 months of age, were randomly assigned to 5 equal groups. One group was used as a control. Oral doses were .01, .10, and .25 mg/kg/day of megestrol acetate in coconut oil in capsules and of chlormadinone acetate .25 mg/kg/day in lactose tablets. These doses were 1, 10, and 25 times the projected dose of megestrol for humans and about 25 times the human dose of chlormadinone. After 2 years 4 dogs from each group were necropsied. One high-dose megestrol-treated and 1 chlormadinone-treated dog had benign mixed mammary tumors. Palpable nodules were first observed at 16 months in the chlormadinone-treated dogs, at 18 months in dogs given the high dose megestrol and at 27 months in the dogs treated with middle-dose megestrol. Transitory nodules were found in 4 control dogs after 21 months and in low dose megestrol-treated dogs at 26 months. Of 38 grossly detected nodules evaluated microscopically from the megestrol-treated dogs 27 were nodular hyperplasia, 5 were benign mixed mammary tumors, 3 were ductal dialatations, 1 was a lymph node, 1 was fat necrosis and 1 was the umbilicus. Of 22 nodules from the chlormadinone-treated dogs 12 were nodular hyperplasia, 4 benign mixed mammary tumors, 1 chondromucoid degeneration and 1 adenocarcinoma with widespread metastases. 3 nodules were lymph nodes and 1 other had no mammary tissue. Involutions, regression and sclerosis of many areas of nodular hyperplasia were evident at 4 years. Thus of the 60 nodules evaluated during the first 4 years of the study 50 were non-neoplastic and 10 were neoplastic. It is considered that the 1 adenocarcinoma may have been spontaneous and not a treatment-related neoplasm. A precursor stage through nodular hyperplasia apparently did not occur.^ieng

Oral contraceptive use and fibrocystic breast disease with special reference to its histopathology.

The relationship between use of oral contraceptives and fibrocystic breast disease was examined in a hospital-based case-control study undertaken in New Haven, Connecticut, from 1977 to 1979. Particular emphasis was placed on the extent of epithelial atypia and other histopathologic characteristics found in the biopsy specimens from the cases. Women who had ever used oral contraceptives were at a somewhat decreased risk for fibrocystic disease as a whole. Cases with high atypia and controls had similar patterns of oral contraceptive use, whereas cases with low and intermediate atypia had less oral contraceptive use than controls. Cases with intermediate atypia reported the lowest oral contraceptive use. Subjects with biopsy specimens exhibiting gross cysts, microscopic cysts, or papillomatosis were about 50% less likely to have used oral contraceptives than controls.

Effect of oral contraceptives on the mammary glands of Rhesus monkeys: a preliminary report.

PIP: To determine the relationship of oral contraceptive use and breast cancer, 96 rhesus monkeys were administered either Enovid-E (2.5 mg norethynodrel and .1 mg mestranol) or Ovulen (1 mg ethynodiol diacetate and .1 mg mestranol) cyclically for 5 years at doses of 1, 10 and 50 times the human dose. The animals' progress was compared with a control group of 32 monkeys. General physical and mammary gland examinations were conducted before treatment and monthly thereafter. During the 5 year study period none of the treated animals demonstrated clinical evidence of mammary gland lesions, no deaths from breast malignancy occurred, and no palpable breast nodules were found.^ieng

Role of prolactin in rat mammary carcinogenesis: detection of carcinogenicity of low-dose carcinogens and of persisting dormant cancer cells.

PIP: Rats used were W/Fu strain with a reported incidence of spontaneous mammary tumors (MTs) of 17%. Most of these have been benign fibroadenomas in older females. X-ray or fast neutron irradiations were given in subthreshold doses, some as low as 10 rads. Also, low doses (150 mg) of a chemical carcinogen (N-nitroso-N-butylurea; NBU) prolactin (MtH). The prolactin was provided by grafting a syngeneic mammatropic pituitary tumor (MtT.W95 or MtT.W98). Tumors developing at the implantation site were chormophobe adenomas. The latent period of tumor induction decreased after the 4th generation of animal passage of the tumors. In some rats, ovariectomy was done 1 week before X-irradiation. No MT was found in 18 untreated controls during 1 year of observation. No MT developed in 32 rats having only the grafts for 9-12 months. Total-body-X-irradiation with 25 or 50 rads alone elicited no MT for 1 year. Among 27 rats exposed to 200 rads of X-rays 2 developed MT fibroadenomas after 5 and 7 months. The supplemental administration of prolactin greatly increased and accelerated the occurrence of MTs in irradiated rats. Most of these were adenocarcinomas. Ovariectomized rats had fewer MTs than intact females. A correlation was found between dose and MT incidence. The persistence of chemically transformed MT cells was shown by the addition of prolactin several months after NBU therapy. MTs developed in 8 of 10 rats within 3 months after the MTT graft became palpable. All but 1 of these were adenocarcinomas. Apparently there had been no repair or elimination of damaged cells in the dormant state. It is suggested that progressive growth of MTs may depend on the hormonal milieu of the host.^ieng

Oral contraceptives as related to cancer and benign lesions of the breast.

We conducted a case-control study to search for any relationship between use of oral contraceptives and development of breast cancer or benign breast disease. Women less than 50 years old with these diseases were matched with 2 controls by age, race, religion, and hospital. Home interviews elicited information on oral contraceptive use and other host and environmental factors. The study population comprised 1,770 women, including 452 with breast cancer and 446 with benign breast disease. The relative risk of developing cancer or benign disease was measured by matched set and summary chi-square analyses. Although the relative risk of developing breast cancer among "ever-users" of oral contraceptives was 1.1, the risk among women using oral contraceptives for 2-4 years was 1.9 (significantly increased). This risk estimate reached 2.5 for the 2- to 4-year users if they were still taking oral contraceptives when entered into study. Moreover, prior biopsy for benign breast disease increased the cancer risk among long-term users by as much as 11-fold. The relative risk of breast cancer did not vary by age, interval since first use, earliest year of use, or interval since last use. These results could be interpreted to indicate that oral contraceptives did not induce breast cancer but may have accelerated the growth rate of preexisting breast cancer. The relative risk of developing benign breast disease among ever-users of oral contraceptives was 0.8 (significantly reduced); it decreased with longer duration of use until it reached 0.2 for women who took these hormones 8 years or more. The relative risk of benign breast was not affected by earliest year of use or interval since last use. We concluded that oral contraceptives reduced the incidence of benign breast disease, but that use of steroid hormones is ill-advised for women with already established benign breast disease.

PIP: A 3-year case-control study of 1770 women under age 49 from the San Francisco Bay area, who were admitted to area hospitals with newly diagnosed breast cancer or with benign noninflammatory breast lesions, is reported. There were 452 with breast cancer, 446 with benign breast disease, 433 with other medical conditions, and 439 with other surgical conditions. Detailed information was obtained concerning each subject's contraceptive practices, menstrual and obstetric experiences, hormone use, medical and surgical history, and family history of breast cancer. Those without breast disease were selected as suitable controls concerning risk factors. In all instances the risk of breast disease was compared between women who had or were using oral contraceptives and others. Current users of oral contraceptives were 16% of patients with breast cancer and 13% of control patients. Contraceptive users were 9% of patients with benign breast disease. 1/2 of those using oral contraceptives had done so for 2 years or less. Only 15% of those who used these drugs had done so for over 6 years. Relative risk for all age groups of developing breast cancer was estimated to be increased by 10% among oral contraceptive users. The risk was less in older patients. With duration of oral contraceptive use, risk was significant only for the 2-4 year users in whom it was increased twofold (p less than .01). Relative risks were the same with the different dose levels of contraceptive compounds. Among ever-users, cancer risk was much greater (six to elevenfold) for women with prior history of biopsy for benign breast disease, particularly among those who had used oral contraceptives more than 6 years. Other contraceptive measures are recommended for such women. Observation for long-term effects should include study of type-specific benign disease as related to use of the hormones and later development of cancer. It may be that use of oral contraceptives accelerated the growth rate of preexisting subclinical malignant lesions and that after 2 years these lesions reach the level of clinical recognition, all being diagnosed within the next 2 years.

Oral contraceptive use and fibrocystic breast disease of different histologic classifications.

The relationship between oral contraceptive (OC) use and occurrence of fibrocystic breast disease (FBD) of different histologic classifications was evaluated with data from a cohort study. Biopsy specimens from 232 women with FBD were classified into different atypia categories. In 96 matched pairs of OC users and nonusers, atypia scores were lower in users than in nonusers. Women without breast diseases (500 OC users and 500 nonusers) were sampled from the original cohort to form a two-stage "anamorphic" study with the 232 cases of FBD. The previously shown inverse association between OC use and FBD occurrence was present and increased with increased length of OC use. However, the "protective effect" of OC use did not vary for different histologic classifications of FBD. The findings from both paired and anamorphic analyses of the data are not consistent with the hypothesis that the use of OC is associated with decreased frequency only of FBD with minimal epithelial atypia.

Potent inhibitory effect of a new antiestrogen (RU 16117) on the growth of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

At the daily dose of 24 mug for a period of 4 weeks, RU 16117 (11alpha-methoxyethinyl estradiol), a new antiestrogen, led to 65% reduction of the number of already established dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats. Not only the tumor number but also the tumor size was reduced by RU 16117 in a manner similar to that seen after ovariectomy. The absence of an inhibitory effect of doses of 0.1 to 12.5 mug 17beta-estradiol (E2) per day, a dose-range which covers the low estrogenic activity of the RU 16117 doses used, suggested that the inhibitory effect of RU 16117 was not due to its estrogenic activity. Decreased levels of receptors for E2, progesterone, and prolactin were found in the tumors remaining after ovariectomy; treatment with the dose of RU 16117 sufficient to inhibit tumor growth (24 mug) had a similar inhibitory effect on the levels of E2 and prolactin receptors. These data suggested that a reduction of hormone receptor levels in the tumor tissue could be a mechanism by which RU 16117 acts as a potent inhibitor of the growth of DMBA-induced mammary carcinoma.

PIP: The new antiestrogen RU 16117, at doses of 8 or 24 mcg daily, had been shown to completely prevent the development of rat mammary cancer when given from the day after 7,12-dimethylbenz(a)anthracene (DMBA) administration. This study was undertaken to investigate the effect of this compound on the growth of DMBA-induced tumors which had already developed in Sprague-Dawley rats. The effect was compared with that of castration. Levels of receptors for 17beta-estradiol (E2), progesterone, and prolactin (PRL) were correlated with the response. At about 3 months after DMBA administration animals with palpable tumors were selected. The rats were then treated daily for 4 weeks with .1, .5, 2.5, or 12.5 mcg E2 or with 2, 8, or 24 mcg RU 16117 injected in .1 ml of 1% gelatin in .9% NaCl. Controls were injected with the vehicle alone. For comparison, a group of rats were ovariectomized. After 4 weeks' treatment rats were killed, blood collected, and a cytosol was prepared from tumor tissues. Binding assays and radioimmunoassays were done. 8 and 24 mcg doses of RU 16117 led to 45 and 65% inhibition of tumor number, respectively, and tumor size was markedly reduced. Lower doses had less effect. Ovariectomy had an effect similar to that of 24 mcg RU 16117. E2 doses did not change the number or size of tumors. Decreased levels of receptors for E2, progesterone, and PRL were found in the tumors remaining after ovariectomy. The 24 mcg dose of RU 16117 had a similar effect on levels of E2 and PRL receptors. It was considered likely that RU 16117 exerts its inhibitory activity at both the hypothalamic-pituitary and tumor levels.

A new virus in a spontaneous mammary tumor of a rhesus monkey.

PIP: This study investigated the ultrastructure and development of virus particles detected in a spontaneous mammary tumor of an 8 year old rhesus monkey. During a 3-month period of tumor growth, several biopsies were performed. Thin section electron microscopy of the tumor tissue revealed 2 types of particles: 1) an intracytoplasmic, electron-dense, ring-shaped particle, and 2) an extracellular particle with an outer unit membrane and a central dense nucleoid. The intracytoplasmic development and virus maturation by a process of budding at the level of the cell membrane was reconstructed.^ieng

Increased incidence of mammary tumors in the female rat grafted with multiple pituitaries.

PIP: This study evaluates the role of pituitary homografts on mammary tumorigenesis in the female Sprague-Dawley rat. 5 pituitaries were grafted unilaterally over the inguinal, abdominal, and thoracic region of the mammary gland, and 2 pituitaries were grafted underneath the kidney capsule of each rat. Number and percentage of rats with mammary tumors 9 months after grafting were: Group 1, 13/45 (30%), Group II, 9/12 (75%), and Group III, 8/13 (61%). Since pituitary homografts secrete relatively large amounts of prolactin and small amounts of other pituitary hormones, the results indicate that an additional source of prolactin significantly enhances mammary tumorigenesis in the female rat.^ieng

Anovulation and increased androgenic activity as breast cancer risk in women with fibrocystic disease of the breast.

The urine of 26 otherwise healthy women with fibrocystic disease of the breast was assayed by gas chromatography for testosterone and androstanediol (5alpha-androstane-3alpha, 17beta-diol), the major metabolite of dihydrotestosterone. The mean values for both androgens were significantly higher than in 18 normal women in the same age range. Sixteen of the 26 fibrocystic disease patients also had endometrial hyperplasia. Since the endometrial specimen was obtained in the premenstrual period, the presence of hyperplasia proved that the menstrual cycle in over two-thirds of the fibrocystic disease patients was nonovulatory.

PIP: Urine of 26 otherwise healthy women with fibrocystic breast disease (FCD) was assayed by gas chromatography for testosterone (Tes) and androstanediol (Ans), the major metabolite of dihydrotestosterone. The 26 patients under study had been subjected 3-5 months previously to biopsies of breast lumps, diagnosed as FCD. The histological features varied from simple cystic formations with moderate epithelial proliferation in 11 women (Group 1) to pronounced intraductal epithelial hyperplasia accompanied by epithelial cell atypia in 15 women (Group 2). The urinary Tes and Ans difference in FCD patients and controls (18 normal women) was significant at the level of p .01 for both androgens. In controls the mean excretion levels of Tes and Ans were 6.5 and 35 mcg/24 hours, respectively. In FCD patients, the mean Tes and Ans values were 17.4 and 68.5 mcg/24 hours, respectively. Group 2 presented a higher urinary Tes level than patients in Group 1, but the difference was not significant. The Ans level of Group 1 patients was significantly above normal (p .01) and near significantly higher (p .08) than that of the Group 2 patients; whereas the Ans level of Group 2 patients did not differ significantly from the normal value. Endometrial specimens showed that 16/26 FCD patients had endometrial hyperplasia. Since the endometrial specimen was obtained in the premenstrual period (Days 20-22), the presence of hyperplasia proved that the menstrual cycle in over two-thirds of the FCD patients was nonovulatory.

Prolactin binding to mammary gland, 7,12-dimethylbenz(a)-anthracene-induced mammary tumors, and liver in rats.

Specific binding of radioactively labeled prolactin was determined in membrane preparations from mammary glands and livers of rats during pregnancy and lactation. Prolactin binding to mammary gland increased throughout late pregnancy and early lactation, reached a maximum on Day 11 of lactation, and then declined. Maximum prolactin binding to liver membrane preparations was observed during late pregnancy and declined throughout lactation. Estradiol benzoate (20 mug/day), administered on Days 5 to 10 of lactation, reduced prolactin binding to mammary gland by 55%, increased binding to liver 2-fold, and reduced litter weight gain by 25%. Prolactin binding to 7,12-dimethylbenz(a)anthracene-induced mammary tumors was 3 times higher than that observed in lactating mammary gland. Administration of prolactin enhanced tumor growth but decreased specific prolactin binding to tumors. Lergotrile mesylate inhibited and estradiol benzoate (2 mug/day) enhanced tumor growth, but neither treatment affected prolactin binding to tumor membrane preparations. In contrast, higher doses of estradiol benzoate (20 mug/day) inhibited tumor growth and reduced prolactin binding. Prolactin binding varied widely within all groups of mammary tumors and was not clearly related to growth response or to altered circulating estrogen and/or prolactin levels. Hormone dependence in this animal tumor model is complex and may not be predicted on the basis of prolactin-binding capacity alone.

PIP: Experiments designed to study the changes in prolactin (PRL) binding to rat mammary tissue and liver during pregnancy and lactation, to study the relationship between PRL binding and growth in 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and to measure PRL binding in DMBA tumors, lactating mammary gland and liver following administration of pharmacological doses of estrogen are described. Specific binding of radioactive PRL binding to mammary gland increased throughout late pregnancy and early lactation, reached a maximum on Day 11 of lactation and then declined. Maximum binding to liver membrane preparations was observed during late pregnancy and declined throughout lactation. The administration of 20 mcg estradiol benzoate (EB/day on Days 5-10 of lactation, reduced PRL binding to mammary gland by 55%, increased binding to liver 2-fold and reduced litter weight gain by 25%. PRL binding to DMBA-induced mammary tumor was 3 times higher than that observed in lactating mammary gland. PRL administration enhanced tumor growth but decresed specific PRL binding to tumors. Lergotrile mesylate inhibited and 2 mcg EB enhanced tumor growth, but neither treatment affected PRL binding to tumor membrane preparations. However, 20 mcg EB inhibited tumor growth and reduced PRL binding. PRL binding varied widely within all groups of mammary tumors and was not clearly related to growth response or to altered circulating estrogen and/or PRL levels. It is concluded that hormone dependence in the rat tumor model is complex and may not be predicted on the basis of PRL-binding capacity alone.

Clinical toxicology of estrogens.

PIP: This paper reviews both minor and major adverse reactions caused by estrogenic substances (natural and synthetic, steroidal and nonsteroidal) of which diethylstilbestrol is the prototype of nonsteroidal synthetic estrogen. Minor side effects include nausea, breast tenderness, and excessive cervical secretions (most common), headache, and water and salt retention (less common and often eradicated by lowering estrogen dosage). Vertigo, yeast infections, depression, and photosensitivity are other minor effects. Major side effects are discussed in some detail. Major effects include those on the endocrine system (e.g., feminization in boys and men and precocious puberty in girls); breast tumors; endometrial carcinoma; ovarian tumors; hypertension; thromboembolism; blood clotting excesses; various metabolic effects (including lipid metabolism and carbohydrate metabolism alterations); liver changes (bile alterations and neoplasms); porphyria; melanoma; and effects on a fetus in situ during maternal estrogen administration. In general, lowering doses of estrogen should help eradicate or alleviate most of these effects.^ieng

17 beta-Hydroxysteroid dehydrogenase activity in human breast epithelial cell and fibroblast cultures.

Primary cultures of human breast cells prepared from surgical specimens of reduction mammoplasty were used to study the activity of the enzyme 17 beta-hydroxysteroid dehydrogenase (E2DH) which converts estradiol (E2) into its less active metabolite estrone. This study was performed in both epithelial and stromal cells separated, after collagenase digestion of the tissue, on a Percoll gradient, and then cultured as monolayers in Ham's F 10 medium supplemented differently for epithelial cells and fibroblasts. E2DH activity was strikingly higher in epithelial cells than in fibroblasts, since after [3H]E2 incubation (2 nM), 600 fmol/micrograms DNA were metabolized to estrone in epithelial cells after 1 h, whereas an equivalent amount was hardly obtained in fibroblast cultures after 24 h. The affinity and capacity of E2DH were greater in epithelial cells with apparent Michaelis-Menten constant (Km) = 0.6 +/- 0.1 microM and maximum velocity (Vmax) = 250 to 360 pmol/micrograms DNA/h, whereas they were 10 +/- 1 microM and 50 to 70 pmol/micrograms DNA/h, respectively, in fibroblast cultures. Moreover, the E2DH activity was 2 to 5 times higher in epithelial cells cultured in the presence of the progestin medroxyprogesterone acetate, whereas it remained unchanged in fibroblasts cultured under the same conditions. This increase in E2DH activity was dose dependent from 10(-10) to 10(-7) M medroxyprogesterone acetate and inhibited by both actinomycin D and cycloheximide. This system of differential breast cell culture appears to be a fruitful tool for the study of the hormone dependence of normal breast growth and differentiation. Due to the presence of E2DH, epithelial cells are more apt to undergo and to moderate E2 action. Moreover, epithelial cells are a possible site of progesterone modulation of E2DH activity. Therefore, E2DH could be a good marker both for epithelial cells and their hormone dependence.

Grwoth hormone, prolactin, and cortisol in dogs developing mammary nodules and an acromegaly-like appearance during treatment with medroxyprogesterone acetate.

PIP: Concentrations of (GH) growth hormone, (PRI) prolactin, cortisol, progesterone, and (MPA) medroxyprogesterone acetate were determined by RIA in blood sera collected from beagle bitches 17 months after initiating treatment with MPA (75 mg/kg.3 months; n = 12) MPA vehicle (controls; n = 12), or progesterone implants which produced physiological levels of progesterone (13.8 + or - 2.1 ng/ml; n = 12). In the MPA-treated bitches, mean MPA levels were 104 + or - 6 ng/ml, mean GH levels were elevated (9.5 + or - 3.0 vs. 0.4 + or - 0.1 ng/ml; P 0.01); mean PRL levels were unchanged (13.7 + or - 2.8 vs. 12.6 + or - 1.2 ng/ml); and mean cortisol levels were suppressed (1.7 + or - 0.2 vs. 13.7 + or - 1.4 ng/ml; P 0.01) in comparison to those in control animals. None of these parameters was significantly affected by progesterone treatment. External signs of an acromegaly-like condition and large mammary gland nodules (diameters, 5 mm) were noted in, and limited to, 9 bitches with elevated ( 2.5 ng/ml) GH levels (12.8 + or - 3.0 ng/ml). These were 8 MPA-treated bitches which developed the acromegal-like condition during treatment and 1 progesterone-treated bitch which appeared acromegalic before treatment and in which the condition was considered to have developed spontaneously. The data suggest that the acromegaly-like changes and large mammary nodules in dogs administered the contraceptive progestin MPA occurred as a result of MPA-induced elevations in GH. The results do not preclude the possibility that the MPA-induced suppression of cortisol and/or the direct action of MPA on the mammary glands also contributed to mammary nodule formation. MPA-treated dogs may also provide a unique experimental model for studying chronic elevations in endogenous GH levels and for testing compounds for their ability to suppress GH levels.^ieng

Estradiol binding to nuclear receptors in human breast cancer tissue (MCF-7 cell line) and in dimethylbenz(a)anthracene-induced mammary carcinoma.

PIP: The presence or absence of estrogen receptors in the nuclei of human breast tumor may be a useful tool in determining whether the tumor will or will not respond to endocrine therapy. This paper describes an assay which measures both unoccupied and occupied nuclear receptors in human breast cancer tumors. The assay was predicated on the fact that at low salt concentration, the nuclear receptor is bound to chromatin particles and can be separated from the soluble components containing proteolytic acitivity. Nuclear estradiol receptors were measured in human breast cancer tissue (MCF-7 cell line) and in DMBA (dimethylbenz(a)anthracene-induced rat mammary carcinomas) tumors. Complete translocation of the cytoplasmic receptor in the MCF-7 cells was observed compared to only 35-50% of the cytoplasmic receptors seen in the nucleus of the DMBA tumor after estradiol injection. The study also showed 6 pmol/mg DNA for total unoccupied nuclei and cytoplasmic estrogen receptors, and 25% of it in the nucleus; this finding differed from Zava et al's finding of 2 pmol/mg DNA and 75% in the nucleus, probably because of differing methodology or use of a later passage of cell line. 29 out of the 34 tumors with cytoplasmic receptors were found to contain unoccupied nuclear receptors, indicating that free nuclear receptors are not exceptions. The assay used in this study is currently being used to determine the translocative ability of the cytoplasmic receptors in human breast carcinomas.^ieng

The influence of prolactin secretion on human lactation.

The elevation of endogenous prolactin secretion using thyrotopin releasing hormone was associated with significant increases in mammary milk production in postpartum women. More-over, a specific effect was seen on the percent fat composition which has been shown to rise as much as 228% over pretest conditions. As in the bovine, administration of high doses of estrogen is associated with mammary breast development and the sudden removal of this stimulus if accompanied by nipple stimulation is associated with non-puerperal lactation. The inhibitory effects of estrogen on the mammary cellular response to circulating prolactin has been deduced from studies in pregnant and parturient women by measuing the TRH-induced prolactin response. These studies support a relationship between prolactin and sex steroids on the initiation and maintenance of human lactation.

PIP: The relationship between the secretion of pituitary prolactin (hPRL) and the initiation and maintenance of human lactation was investigated. HPRL secretion was stimulated by suckling and the change was especially significant beyond the 8th postpartum day when basal hPRL concentrations return to prepregnancy levels. Intravenous thyrotropin releasing hormone (TRH) further elevated hPRL and was associated with significant increases in mammary milk production in postpartum women. Milk fat concentrations were also seen to rise, as much as 228% over pretest conditions. TRH but not suckling increased plasma pituitary thyrotropin (hTSH) concentrations. Administration of estrogens led to decreased secretion of LH and FSH, indicating a potentiating effect on the hypothalamus and/or pituitary gland. Estrogens also were associated with mammary breast development; removal of this stimulus if accompanied by nipple stimulation led to nonpuerperal lactation.

Oral contraceptive use influences resting breast proliferation.

The controversy that surrounds oral contraceptive use and breast cancer risk has arisen from epidemiologic studies, yet the direct effect of such use on breast tissue remains undefined. Breast epithelial proliferation was assessed by 3H-thymidine labeling of normal lobular units dissected from benign biopsies of 347 females aged 14 to 48 years. Factors shown to influence this response included cycle phase, time since menarche (breast age), and parity status. Multivariate analysis allowing for these influences was used to compare activity of natural cycles and those artificially regulated by oral contraceptives (OC). The increased activity in nulliparous OC users was highly significant (P less than .005). Comparing the effect of differences in OC type, whether combined, triphasic, progestin only, or according to estrogen or progestin content, showed a heterogeneity in response that was significant (P less than .01). Examined specifically, the formulation of OC according to progestin content did not have a significant influence, although progestin-only OC was most active, while the influence of increasing estrogen content was significant (P less than .05). However, emphasis is given to acknowledging the multiple factors and interactive processes responsible for breast epithelial stimulation when considering strategies of intervention.