Biotransformation of estradiol by explant culture of human mammary tissue

In vivo experiments on strains of mice that differ in the risk of developing mammary cancer have demonstrated a correlation between the extent of 16 alpha-hydroxylation of estradiol and incidence of mammary cancer. The ability of human mammary terminal duct lobular unit (TDLU), the site of neoplastic transformation, to metabolize estradiol or to accumulate estradiol metabolites has not been unequivocally established. Using a newly developed human mammary TDLU explant culture system and a radiometric assay for estradiol metabolism, we compared the site-specific metabolism of estradiol by the 17-oxidation, 2-hydroxylation, and 16 alpha-hydroxylation pathways in noninvolved human mammary tissue. The relative extent of estradiol 16 alpha-hydroxylation was found to be increased in TDLU from patients in the luteal phase of the menstrual cycle in relation to either those from patients in the follicular phase or from postmenopausal subjects. This study demonstrates that TDLU can metabolize estradiol extrahepatically and that 16 alpha-hydroxylation in the target tissue is dependent on the phase of the menstrual cycle. Furthermore, the specific, risk-related increase in 16 alpha-hydroxylation suggests that intrinsic metabolic ability of the target tissue leading to the formation of 16 alpha-hydroxyestrone from estradiol may be a determinant in, or a marker for, the relative risk of developing mammary cancer.     

Effects of exogenous thyroxine on C-2 and C-16 alpha hydroxylations of estradiol in humans

Thyroid dysfunction in humans is known to alter the excretory pattern of estrogen metabolites, suggesting that thyroid hormone directly influences the oxidative metabolism of estradiol. We examined the extent to which a brief period of hyperthyroidism specifically affected estradiol hydroxylation at C-2 and C-16 alpha, the two primary and competing sites of estrogen oxidation, using an in vivo radiometric assay in healthy male volunteers. Hydroxylation at C-2 was increased by a 2-week course of thyroxine (4.3 micrograms/kg/d) from 29.9% +/- 2.6% to 35.9% +/- 3.1% (P = 0.04), while 16 alpha-hydroxylation was unchanged (10.3% +/- 0.8% versus 9.3% +/- 0.5%). The greater extent of oxidation at C-2 was evidenced by a twofold increase in the urinary excretion of 2-hydroxyestrone (2.88 +/- 0.32 versus 5.30 +/- 0.85 micrograms/g creatinine), while the excreted products of 16 alpha-hydroxylation were unchanged. At the same time, significant reductions in total cholesterol (173.8 +/- 7.9 versus 139.4 +/- 8.9 mg/dl), low-density lipoprotein cholesterol (110.0 +/- 5.3 versus 83.8 +/- 7.7 mg/dl), and apolipoprotein B (68.2 +/- 3.3 versus 53.1 +/- 3.6 mg/dl) were observed. Serum levels of estrone, estradiol, sex hormone-binding globulin, high-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, triglycerides, and apolipoprotein A-I were not significantly affected. This study adds to the growing evidence that catechol estrogen production in humans is more readily regulated than 16 alpha-hydroxylation, which is relatively refractory to treatment.     

Sensitivity and specificity of the bioassay of estrogenicity in mammary gland and seminal vesicles of male mice

Young intact (18 days old) and adult castrated males of CBA and C3H/Di mice were used for measuring the estrogenicity on the basis of growth response of mammary epithelial structures and the weight of seminal vesicles. It was demonstrated that heavier young males had disproportionally heavier seminal vesicles (sex steroid-responsive organs) than small animals at day 33 of age (that is on the day when experimental animals were killed and organs dissected). However, the weight of the spleen (sex steroid-nonresponsive organ) was proportionally related to body weight. To minimize variability in hormone responsiveness, all animals were weighed at the age of 18 days and only males weighing 8+/-1 g were used for hormone treatment. The percentage area of mammary fat pad occupiedby mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.01 microg x d(-1). The maximum effective dose of estradiol was 0.1 microg x d(-1) and dose 10 microg x d(-1) of estradiol decreased mammary size to control level (inverted-U-shaped dose-response curve). Progesterone alone stimulated mammary growth only in high doses (500 microg x d(-1) and higher) in young intact males, but had no effect on mammary growth in adult castrated animals. In young intact males, estradiol alone, or progesterone alone decreased the weight of seminal vesicles. No such inhibitory effect of these hormones was noted in adult castrated males. Progesterone acted synergistically with estradiol to produce higher mammary growth compared to that in males treated with estradiol alone. In the presence of progesterone seminal vesicles weight was decreased by estradiol given in such low doses as 0.001 microg x d(-1) of estradiol, which is 10 times lower than that effective in animals treated with estradiol alone. On the other hand, in the adult castrated males a combination of estradiol plus progesterone stimulated seminal vesicles weight. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate (a synthetic steroid exhibiting progestantial and estrogenic activities) and inhibited by both testosterone and cortisol. Estradiol, progesterone, norethindrone acetate, or testosterone did not affect spleen weight and size of mammary lymph nodes.However, cortisol significantly decreased not only spleen weights but also size of mammary lymph nodes. These results showthat simultaneous evaluation of mammary gland growth, seminal vesicles, and the spleen weight in the same animal is suitable for bioassay of estrogenicity as well as for detection of androgenic and antiandrogenic activities.     

Sensitivity and specificity of bioassay of estrogenicity on mammary gland and uterus of female mice

Young intact (18 days of age) and adult ovariectomized (OV-X, ovariectomized between 21 to 24 days of age) C3H/Di mice were used to measure the estrogenicity on the basis of the growth response of mammary epithelial structures and weight of the uterus. The percentage area of the mammary fat pad occupied by mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.001 microg.d(-1). The maximum effective dose of estradiol was 0.01 microg.d(-1) and the dose 10 microg.d(-1) of estradiol decreased mammary size to control levels (inverted-U-shaped dose-response curve). Progesterone alone progressively stimulated mammary growth in young intact females from dose 125 microg.d(-1), in adult OV-X animals from dose 1000 microg.d(-1). Both in young intact and adult OV-X animals, uterine weight progressively increased during estradiol treatment. Progesterone alone had no effect on uterine weight in young intact animals; in adult OV-X animals, uterine weight was increased starting from dose 250 microg.d(-1). Progesterone acted synergistically with estradiol to produce higher mammary growth than that in females treated with estradiol alone. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate and inhibited by cortisol in both young intact and adult OV-X animals. Testosterone inhibited estradiol plus progesterone stimulated growth of mammary gland only in OV-X animals, but stimulated uterine weights in both young intact and adult OV-X animals. Spleen weight and size of mammary lymph nodes were not affected by estradiol, progesterone, norethindrone acetate or testosterone, but were decreased by cortisol. Cortisol also decreased the percent area of the mammary fat pad occupied by mammary epithelial structures, but had no effect on weight of the uterus. These results show that bioassay of estrogenicity in females is not specific. Mammary and uterine growth is stimulated not only by estrogens but also by progesterone and testosterone, respectively.     

Nafoxidine administered to newborn female GR mice arrests the development of their mammary glands

Mice of the GR strain develop many hormone-dependent mammary tumors in response to estrogen and progesterone stimulation. Since this strain is so sensitive to steroid hormones, we administered a single dose of the antiestrogen Nafoxidine to female GR mice within 24 hours after their birth. This treatment arrested the development of their mammary glands and when the mice were adults, 10 weeks old, they did not cycle normally but were in a state of persistent estrus. Whole mounts of mammary glands from Nafoxidine-treated mice revealed cystic areas within some ducts and bulbous swellings at the ends of others. No hyperplastic alveolar nodules (HAN) were identified in the glands. In contrast, a single dose of 17 beta estradiol administered within 24 h after birth, resulted in a highly branched gland displaying typical end buds, a few alveoli and more HAN than were observed in glands of control adult mice of the same strain. Thus Nafoxidine treatment not only arrested the development of the mammary glands in female GR mice (causing them to appear "masculinized") but it also produced abnormalities within the glands.     

The effect of doxorubicin and retinoids on proliferation, necrosis and apoptosis in MCF-7 breast cancer cells

Doxorubicin (Adriamycin) is the most active drug in the treatment of breast cancer. The aim of this study was to investigate the interaction of doxorubicin and retinoids in the inhibition of proliferation of hormone sensitive (ER+) human breast cancer cell line MCF-7 and to find out whether this combination can result in the enhancement of its therapeutic effect. As a comparison we also used estradiol and tamoxifen. We also made an attempt to elucidate the effect of these compounds on the stimulation of the apoptotic pathway in breast cancer cells. Cell proliferation in a 24-hour culture was assessed by [3H] thymidine incorporation into cancer cells and by immunocytochemical analysis of cellular cycle-related PCNA and Ki-67 antigens expression, after the incubation of the cell culture with 10, 20 and 50 nM doxorubicin (DOX), 2 nM estradiol (E2), 10 microM tamoxifen (TAM) and 1 nM, 0.01, 0.1, 1 and 10 microM of all-trans retinoid acid (ATRA). The assessment of cell viability and analysis of apoptotic and necrotic cells were performed after the 72-hour incubation of the culture with the examined substances and following apoptosis induction using acridine orange and ethidine bromide. Of the doxorubicin concentrations used in the study, 20 nM inhibited thymidine incorporation to 84.83 +/- 10.00% (control=100%). In the same culture conditions, 2 nM E2 stimulated cancer cells to 157.09 +/- 8.84%. Concentrations of 10 microM TAM and 10 microM ATRA inhibited the proliferation to 63.16 +/- 7.85% and 52.19 +/- 3.21%, respectively. A statistically significant reduction of these values was observed when 20 nM DOX was added to medium with E2 - 39.24 +/- 7.6%, TAM - 48.34 +/- 2.05% and ATRA - 21.98 +/- 1.69%, respectively; the percentage of PCNA- and Ki-67-positive cells was also reduced. Despite high antiproliferative efficacy of 20 nM DOX and 10 microM ATRA combination, the percentage of apoptotic cells was only 25 +/- 0.81%, being similar to that obtained in the culture with 20 nM DOX. The concentrations of 10, 20 and 50 nM DOX that were used to inhibit the proliferation of MCF-7 cell line were not particulary effective. The inhibitory effect was obtained when 20 nM of DOX and E2, TAM or ATRA were used simultaneously. The use of E2 caused a two-fold decrease in the percentage of proliferating cells. It was also shown that the effectiveness of DOX in combination with ATRA is significantly higher than that of DOX combined with TAM, which might suggest a valuable approach to the treatment of breast cancer.     

Secretion of estradiol-17beta by porcine mammary gland of ovariectomized steroid-treated sows

Although the mammary gland of many species secretes estradiol (E(2)), nothing is known of E(2) secretion in the porcine gland. The present study was designed to investigate whether porcine mammary gland was a source of E(2), and to test the influence of individual and combined effects of exogenous progesterone and estradiol benzoate (EB) on the secretion of E(2). Immature crossbred gilts were ovariectomized at 7 months of age followed by 4 weeks later by steroid hormone replacement therapy to produce estradiol and progesterone (P(4)) blood concentrations similar to those observed during a normal estrous cycle. Arterial and venous blood plasma (from carotid artery and anterior mammary vein, respectively) were sampled for 2h at 10 min intervals. Plasma concentrations of progesterone, androstenedione (A(4)), testosterone (T), estrone (E(1)) and estradiol were determined by RIA. In all gilts treated with progesterone alone or in combination with EB, concentrations of P(4), A(4) and E(1) in blood collected from venous outflow were lower compared to concentrations in arterial blood, whereas concentrations of E(2) were higher in blood plasma from the anterior mammary vein compared to plasma from the carotid artery. The results indicated that the porcine mammary gland secreted E(2). Increased concentrations of plasma E(2) collected only from P(4)-treated animals suggested that progesterone activated enzymes involved in steroidogenesis in porcine mammary gland, or those utilized in its metabolism.     

Effects of mandibular gland ectomy and environmental temperature of dams on eyelid opening and body weight of pups in mice

Effects of the mandibular gland ectomy and environmental temperature to pups in Jcl: ICR mice were studied. In normal environmental animal room (A room; Tem. 23 +/- 2 degrees C, R. H. 50 +/- 10%), the mammary gland weights of lactating mothers and the body weights of pups in sialoadenectomized groups were slightly smaller than that of control groups during lactating period, whereas the eyelid opening of pups was not different between both groups and all pups exhibited complete eyelid opening on 14th day after birth. On the other hand, in cold environmental animal room (B room; Tem. 15 +/- 2 degrees C, R. H. 60 +/- 20%), the mammary gland weights of lactating mothers and the body weights of pups in sialoadenectomized groups were smaller than that of sialoadenectomized groups in A room and the eyelid opening of pups was considerably delayed when compared with that of control groups. The pups exhibited complete eyelid opening at 16th day after birth. These results were caused by mandibular gland ectomy and considered to be easily influenced by environmental conditions.     

Testicular compensatory hypertrophy in the hemicastrated calf: effects of exogenous estradiol

Unilaterally orchidectomized (hemicastrated) bull calves were studied to monitor possible changes in serum concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the phase of testicular compensatory growth, to examine the characteristics of LH and FSH binding to the testis of the post-pubertal animal, and to determine whether any of these responses were altered by exogenous estradiol. Twenty-four calves were assigned randomly at one week of age to a 2 X 2 factorial experiment involving intact control (I) and hemicastrated animals (H), as well as estradiol-implanted intact (I+E2) and hemicastrated animals (H+E2). Relative to I, testis growth was accelerated in H and suppressed in I+E2 and H+E2. Mean testis weights at 27 weeks of age were 42 +/- 4, 72 +/- 6, 12 +/- 1 and 14 +/- 1 g for the four respective treatment groups. Serum FSH, but not serum LH, was positively associated with the accelerated testis growth of H. LH and FSH binding per testis were both enhanced approximately twofold in the testis from hemicastrated animals relative to those from intact calves. In contrast, estradiol markedly suppressed the number of LH-binding sites per testis in both I and H calves, but only suppressed the number of FSH-binding sites per testis in H calves. LH-affinity constants were not affected by treatment, whereas those for FSH were significantly decreased by estradiol. In conclusion, neonatal hemicastration results in elevated serum FSH, testicular compensatory hypertrophy, and an increased number of gonadotropin receptors in the bovine testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological responses of tamoxifen in the fetal and newborn vagina and uterus of the guinea-pig and in the R-27 mammary cancer cell line

The biological and morphological responses of tamoxifen were studied in two models: the uterus and vagina of fetal and newborn guinea-pigs: R-27 cells--a mammary cancer cell line (tamoxifen resistant) derived from the MCF-7 cancer cell line. Tamoxifen (TAM) alone or in combination with estradiol (E2) was administered to pregnant (50-52 days of gestation) or to newborn (2-day-old) guinea-pigs for a long period (12 days). TAM alone produced a great trophic effect on the uterus and vagina which was markedly enhanced when TAM was administered together with E2. Histological studies showed that TAM provokes morphological changes in both the endometria and the myometria and this effect was also greater when TAM was administered together with E2. In the fetal uterus and vagina, the ultrastructural studies showed that TAM induces morphological alterations in different cytoplasmic organelles. This effect was much more intense in newborns where TAM provoked a significant vacuolization of the epithelial cells. Concerning progesterone receptor (PR) in the fetal or newborn tissues (uterus or vagina) TAM provoked a less intense effect than those provoked by E2, but TAM did not block the effect provoked by E2. It was observed that [3H]TAM binds specifically to the estrogen receptor (ER) of fetal guinea pig uterus and this complex is partially recognized by a monoclonal antibody which recognizes the activated form of this receptor, supporting the suggestion that the biological action of TAM is mediated by the ER. The biological and ultrastructural effects provoked by TAM (1 X 10(-6) M), estriol (E3)(5 X 10(-8) M) and the combination of TAM + E3 were studied in the R-27 mammary cancer cell line in culture. E3 stimulated the PR content by 7-10 times. However, TAM did not provoke a significant decrease in the concentration of PR, and in the mixture of TAM + E3 the concentration of PR was of the same order as that in E3 treatment. Ultrastructural observations indicate an intense concentration of ribosomes in the pericytoplasmic area after exposure to E3 and with exposure to TAM an increase in vacuoles and a significant enlargement of the size of the mitochondria were observed. It is concluded that TAM in the target tissues of fetal and newborn guinea pigs acts as a real estrogen and in the R-27 mammary cancer cell line TAM does not block the effect provoked by E3, however it does provoke intense ultrastructural modifications.     

Leptin concentration in plasma and in milk during the interpartum period in the mare

The aim of this work is to investigate on plasma profiles of leptin and estradiol 17beta during the interpartum period and leptin concentrations in the milk and in the colostrum during the period from parturition to the successive delivery in mare. Leptin plasma concentration varied from 5.1+/-2.3 ng/ml after the first parturition (week 0) to 3.0+/-0.7 at week 21 (p<0.05), then it increased to maximal level at week 49 (6.9+/-1.0 ng/ml, p<0.05). Leptin concentration in the colostrum and in the milk has been significantly (p<0.05) higher than that in plasma samples at week 1 (milk 8.8+/-2.3 versus plasma 5.2+/-0.6 ng/ml) and between week 12 and 17. This difference may be explained with a local leptin production at mammary level and supports a role of leptin in the mammary gland and/or in foal intestine. Estradiol 17beta increased from week 15 (17.9+/-2.3 pg/ml) up to 487.9+/-67.7 pg/ml at week 43. Plasma estradiol 17beta rise anticipated by 4 weeks plasma leptin increase and it does not seem to be positively correlated to leptin secretion.     

Reproductive traits in the spur-thighed tortoise (Testudo graeca terrestris): new tools for the enhancement of reproductive success and survivorship

The Mediterranean tortoise (Testudo graeca) is listed as "Vulnerable" on the IUCN Red List. Reproductive characteristics and means to increase offspring production were studied in T. graeca terrestris in a semi-natural environment. Courtship and mating occurred during early spring for about 4 weeks, followed by a laying season of approximately 2 months, with a second, shorter mating period in the fall. During the first mating, calcified eggs were already present in the uterus; we inferred that sperm from both mating seasons were stored in the oviduct for fertilization of eggs of the second laying cycle and of the following year. Average egg production was 3.8+/-0.3 eggs/year. Most females laid all of their eggs in a single clutch, but 18% laid in a second clutch, 11-21 days later. X-ray radiography revealed calcified eggs in the uterus about 4 weeks before oviposition. All eggs in the uterus were calcified simultaneously and were laid in a single clutch; if a second clutch developed, those eggs were also calcified simultaneously. Based on endoscopic examinations, ovaries were active throughout the entire year. Plasma progesterone concentrations in females were very low and were detected only soon after oviposition ( 440 +/-141 pg/ml). Plasma estradiol concentrations in females varied from 4.1 +/-1.5 pg/ml to 70.2 +/-29.4 pg/ml, with no clear seasonal pattern. Maintaining tortoises at a low environmental temperature (9 +/-1 degrees C versus 28 +/- 1 degrees C) reduced plasma estradiol concentrations. Giving 2mg/kg tamoxifen (TAM) increased plasma estradiol to 220 +/-33 pg/ml when treatment was given in September but not in late October, winter or spring. Treatment with TAM increased the number of eggs laid during the following laying season to 7.3 +/- 1.0 eggs/year, laid in one to three clutches. In males, plasma testosterone concentrations had a seasonal pattern with the onset of a rise in July from 2 to >4ng/ml, a continued increase to a peak of 12.8+/-5.3 ng/ml during November and a decline thereafter. Artificial incubation in sand at 29 +/-1 degrees C shortened the natural incubation time of 103+/-3.1 days to 83.5 +/- 1.3 days, increased hatching rate from 28 to 53%, and increased survival rate from 51 to 71% at 40 weeks of age. In summary, this study provides options for increasing reproductive performance, hatchability and offspring survival in captive Mediterranean tortoises, and may offer new tools for conservation of animals that are on the verge of extinction.     

Potentiation of the antitumor effect of tamoxifen by combination with the antiprogestin onapristone

The present study was undertaken to investigate the antitumor effect of a combination of the antiestrogen tamoxifen (TAM) and either the antiprogestin onapristone (ON) or the progestogen megestrol acetate (MEG) in experimental mammary tumor models. Rats bearing DMBA- or NMU-induced mammary tumors were treated with ON or MEG either alone or in combination with TAM for four weeks. In the DMBA-tumor model, treatment with ON or TAM alone caused tumor remissions, whereas the combination of ON and TAM was almost as effective as ovariectomy (100% remission) and led to a remission of 86–100%. The combination of TAM and ON was distinctly more effective than that of TAM and MEG. A similar potentiation of the antitumor effect of TAM and ON was observed in the NMU-tumor model. In DMBA-tumors, the concentration of progesterone receptors was found to increase after treatment for three days with TAM and ON. In hosts bearing DMBA-tumors, treatment with the combination of TAM and ON caused a reduction in ovarian and uterine weights. In the same animals, the basal level of progesterone was decreased in spite of a slight increase in the LH level. These findings suggest that the high antitumor effect of the combination of TAM and ON compared to the corresponding monotherapies can be related not only to the interaction of antihormones and receptors, but also to the up-regulation of PR and to a decrease in progesterone production. These data clearly suggest the sense of a combination of TAM with an antiprogestin in breast cancer treatment.     

Effect of estradiol and relaxin on growth of fibroblastic cells isolated from guinea pig mammary glands

Fibroblasts were isolated from the mammary glands of guinea pigs and grown in 96-well culture plates. They were treated with a factorial arrangement of porcine relaxin (0.0, 0.5, 1.0 or 1.5 micrograms/ml) and estradiol-17 beta (0, 200, 400 or 600 pg/ml). Tritiated thymidine or uridine was added to a final activity of 25 nCi per well and the cells incubated at 37 degrees C for 48 h. Cells were then harvested onto filter paper and counted for tritium. Controls (0.0 micrograms/ml relaxin and 0 pg/ml estradiol) incorporated 3.7 nCi of tritiated thymidine and 4.8 nCi tritiated uridine. Both relaxin and estradiol altered the incorporation of thymidine and uridine. There was also an interaction between the two hormones. Thymidine incorporation with no estradiol and 1.5 micrograms/ml relaxin was 129% of controls. The optimum incorporation of thymidine occurred with 0.5 micrograms/ml relaxin and 400 pg/ml estradiol. This combination of hormones gave a response of 145% of controls. Uridine incorporation followed a different pattern. Relaxin alone at a concentration of 1.5 micrograms/ml gave a near-optimum response of 141% of control. The optimum combination of relaxin and estradiol for uridine incorporation was 1.5 micrograms/ml relaxin and 400 pg/ml estradiol, which gave a response of 156% of controls. These data indicated that relaxin and estradiol alter DNA and RNA synthesis in mammary fibroblasts and thus may be important in controlling the growth of the mammary gland stroma.     

Antibodies interacting with the proteins of the mammary cancer virus (MMTV) in the sera of healthy women

Using enzyme-linked immunoassay (ELISA), sera of 405 women without breast cancer were screened for the presence of antibodies to mouse mammary tumour virus proteins. 16 donors with positive sera were identified (3.96 +/- 0.97%). An attempt was made to establish the correlation between the age, ovarian Activity, benign breast disease and other factors and the appearance of antibodies to mouse mammary tumour virus. No statistically significant differences have been revealed among women aged 20-29 (3.92%), 30-39 (4.69%), 40-49 (4.08%) and 50-59 (2.38%), as well as in premenopausal (4.31%) and postmenopausal (4.5%) women and women with simple fibromatous disease (4.76%) and without any mammary gland pathology.     

Comparison of background factors relevant to mammary tumorigenesis between DDD/1-Mtv-2/Mtv-2 congenic and DDD/1 mice

To elucidate the possible participation of the Mtv-2 gene in the background factors relevant to mammary tumorigenesis, DDD/1-Miv-2/Mtv-2 (DDD-Mtv-2) congenic and DDD/1 background mice were compared for endocrine and immune organs, mammary gland development, expression of mouse mammary tumor virus (MMTV)-gp 52 antigen, hyperplastic alveolar nodules (HAN) and mammary tumor incidence. The congenic strain had been established by introducing Mtv-2 from GRS/AJms (GR) into DDD/1 mice by repeated 12 backcrosses. Body, thymus, spleen, uterus, ovary, adrenal and pituitary weights, histology of the ovary and mammary gland development showed no differences ascribable to Mtv-2 between both strains of mice at 4,6 and 12 months of age. In contrast, MMTV-gp 52 antigen expression, HAN and mammary tumor occurred in DDD-Mtv-2 but not in DDD/1 mice. These results showed that the Mtv-2 gene stimulates mammary tumorigenesis by constitutional production of endogenous MMTV which transforms mammary epithelial cells but not by influencing the background factors relevant to mammary tumorigenesis. Furthermore, the expression of Mtv-2 appeared to be lower on the DDD/1 than on the GR background.     

Two-dimensional gel analysis of polypeptides from normal,preneoplastic and neoplastic mouse mammary tissues

Summary High-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) was employed to reveal tumor-associated polypeptide changes, using the BALB/c C4 line mouse mammary model system, for which phenotypic and immunogenic alterations accompanying tumor progression are well defined. In the first set of experiments, polypeptide patterns from 20 µg whole tissue lysates of normal mammary gland, C4 preneoplastic hyperplatic alveolar nodule outgrowth (HAN) and spontaneous tumor from C4 HAN were compared. In order to normalize for differential cellularity and extracellular protein content in the whole tissues, our analysis included polypeptide patterns from serum, increased concentration of protein from whole normal mammary gland, and primary cultures of epithelial cells from normal gland, HAN and tumor. Using a computer-based image-analysis system, 90 polypeptides were identified in C4 tumor that were absent in C4 HAN, normal mammary gland and serum. None of the 90 polypeptides could be shown to represent a definite qualitative change in the protein composition of tumor epithelium as they were found to be either present in a higher concentration of protein from whole normal gland, or present in the primary epithelial culture from HAN, or absent in the primary epithelial culture from tumor.Conversely in the second set of experiments, when epithelial cultures were used as the starting point for comparisons to locate tumor-associated polypeptides, none of the 15 polypeptides that were present in cultures from three different tumors, and absent in the culture from normal mammary gland was specific to C4 tumor, as they were present in whole tissues of normal gland.Thus our experimental approach detected significant quantitative but no qualitative polypeptide changes in whole tumor tissue, or in tumor-derived epithelial cell cultures. This finding may reflect the limitations of the two-dimensional PAGE method, and warrants caution in the use of such gel analysis alone to identify tumor-associated proteins.Supported by NIH grant CA42522     

Augmented efficacy of tamoxifen in rat breast tumorigenesis when gavaged along with riboflavin, niacin, and CoQ10: effects on lipid peroxidation and antioxidants in mitochondria

Reactive oxygen species (ROS) play a major role in causing mitochondrial changes linked to cancer and metastasis. Uptake of antioxidants by tissue to reduce the ROS production could be instrumental in controlling cancer. Tamoxifen (TAM), a nonsteroidal anti-estrogen drug most used in the chemotherapy and chemoprevention of breast cancer. Riboflavin, niacin and coenzyme Q10 (CoQ10) are proved to be potent antioxidants and protective agents against many diseases including cancer. The objective of this research is to determine the therapeutic efficacy of combinatorial therapy on mammary carcinoma bearing rats in terms of the mitochondrial lipid peroxidation and antioxidant status especially MnSOD. Female albino rats of Sprague-Dawley strain were selected for the investigation. Mammary carcinoma was induced with 7,12-dimethyl benz(a)anthracene (DMBA: 25 mg), and the treatment was started by the oral administration of TAM (10 mg/kg body weight/day) along with riboflavin (45 mg/kg body weight/day), niacin (100 mg/kg body weight/day) and CoQ10 (40 mg/kg body weight/day) for 28 days. The levels of lipid peroxides, activities of enzymic and non-enzymic antioxidants were measured in the mitochondria isolated from the mammary gland and liver of control and experimental rats. Rats treated with DMBA showed an increase in mitochondrial lipid peroxidation (mammary gland 52.3%; liver 25.1%) accompanied by high malondialdehyde levels along with lowered activities of mitochondrial enzymic antioxidants [superoxide dismutase (mammary gland 19.9%; liver 24.8%), catalase (mammary gland 50%; liver 19.7%), glutathione peroxidase (mammary gland 47.8%; liver 31.1%)] and non-enzymic antioxidants [reduced glutathione (mammary gland 14.3%; liver 13.3%), Vitamin C (mammary gland 6.49%; liver 21.4%) and E (mammary gland 20.3%; liver 22.2%)]. Administration of combinatorial therapy restored lipid peroxide level and the activities of enzymic and non-enzymic antioxidants to near normalcy. In addition, antitumour activity was also found to be enhanced which is evident from the increased expression of tumour suppressor gene MnSOD thereby preventing cancer cell proliferation. These results suggested that TAM treatment is the most effective during co-administration of riboflavin, niacin and CoQ10 in terms of mitochondrial antioxidant and antitumour activity.     

Effects of estrogens and selective estrogen receptor modulators on vascular reactivity in the perfused mesenteric vascular bed

Estrogens and selective estrogen receptor modulators (SERMs), such as raloxifene (RAL) and tamoxifen (TAM), acutely relax arteries, but the long-term effects of estrogens and SERMs on vascular reactivity in the mesenteric vasculature have not been well defined. In this study, we used an isolated, perfused mesenteric vascular bed technique to investigate the effect of chronic treatment of estrogens and SERMs on vascular reactivity of the mesenteric bed. Ovariectomized female Sprague-Dawley rats were treated by gavage with vehicle (control, 2-hydroxypropyl-beta-cyclodextrin), ethinyl estradiol, estradiol benzoate, equilin (EQ), TAM, or RAL for 3 wk. EQ and TAM increased vasoconstriction in response to all three vasoconstrictors tested (KCl, norepinephrine, and 5-HT). Ethinyl estradiol increased vasoconstriction in response to KCl and 5-HT, whereas responses to estradiol benzoate and RAL were less consistent. Only EQ (134 +/- 4 mmHg) and TAM (104 +/- 4 mmHg) changed mean arterial blood pressure compared with control (117 +/- 4 mmHg). These data demonstrate that 3-wk gavage treatment with estrogens and SERMs affects vascular reactivity in the mesenteric vascular bed. However, the three formulations of estrogen did not produce equivalent effects, and the effects of the SERMs were different from those of the estrogens.