ABSTRACT
Phthalates are found in different plastic materials, such as packaging, toys and medical devices. Some of these compounds are endocrine disruptors, comprising substances that are able to induce multiple hormonal disturbances and downstream developmental effects, including the disruption of androgen-dependent differentiation of the male reproductive tract and changes in pathways that regulate hormone-dependent behaviours. In a previous study, metabolites of diisopentyl phthalate (DiPeP), a potent anti-androgenic phthalate, were found in the urine of Brazilian pregnant women. Therefore, the present study aimed to evaluate the effects of DiPeP exposure during critical developmental periods on behaviours controlled by sex hormones in rats. Pregnant Wistar rats were treated with DiPeP (1, 10 or 100 mg kg day-1 ) or canola oil by oral gavage between gestational day 10 and post-natal day (PND) 21. Male offspring were tested in a behavioural battery, including the elevated plus maze task, play behaviour, partner preference and sexual behaviour. After the behavioural tests, the hypothalamus and pituitary of these animals were removed on PND 60-65 and PND 145-160 to quantify gene expression for aromatase, androgen receptor (Ar) and oestrogen receptors α (Esr1) and ß (Esr2). Male rats exposed to 1 and 10 mg kg day-1 DiPeP displayed no preference for the female stimulus rat in the partner preference test and 1 mg kg day-1 DiPeP rats also showed a significant increase in mount and penetration latencies when mated with receptive females. A decrease in pituitary Esr1 expression was observed in all DiPeP treated groups regardless of age. A reduction in hypothalamic Esr1 expression in rats exposed to 10 mg kg day-1 DiPeP was also observed. No significant changes were found with respect to Ar, Esr2 and aromatase expression in the hypothalamus. These results suggest that DiPeP exposure during critical windows of development in rats may induce changes in behaviours related to mating and the sexual motivation of males.
Subject(s)
Aromatase/biosynthesis , Behavior, Animal/drug effects , Behavior, Animal/physiology , Prenatal Exposure Delayed Effects/physiopathology , Receptors, Androgen/biosynthesis , Receptors, Estrogen/biosynthesis , Animals , Dose-Response Relationship, Drug , Female , Hypothalamus/metabolism , Lactation , Male , Pituitary Gland/metabolism , Pregnancy , Prenatal Exposure Delayed Effects/metabolism , RatsABSTRACT
Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.
Subject(s)
Cytochrome P-450 CYP1A1/biosynthesis , Estradiol/metabolism , Hyperoxia/physiopathology , Lung/embryology , 2-Methoxyestradiol , Animals , Apoptosis , Aromatase/biosynthesis , Asthma/epidemiology , Bronchopulmonary Dysplasia/epidemiology , Catechol O-Methyltransferase/biosynthesis , Cell Hypoxia/physiology , Cell Proliferation , Cells, Cultured , Cytochrome P-450 CYP1B1/biosynthesis , Estradiol/analogs & derivatives , Estradiol/biosynthesis , Estrogen Receptor alpha/biosynthesis , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/biosynthesis , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Humans , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred ICR , Muscle, Smooth/metabolism , Oxygen/metabolism , RNA, Messenger/biosynthesis , Reactive Oxygen Species/metabolism , Receptors, Estrogen/biosynthesis , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Sex Factors , Up-RegulationABSTRACT
Cultured ovarian granulosa cells are essential models to study molecular mechanisms of gene regulation during folliculogenesis. Here, we characterize primary tissue culture models for bovine granulosa cells by morphological and physiological parameters and by novel molecular luteinization markers, as transcript abundance and DNA methylation levels. The data show that: (1) collagen substrate increased the number of attached, viable cells; (2) the expression of the key transcripts of estrogen synthesis, CYP19A1, could be induced and maintained in granulosa cells from small to medium but not from large follicles, whereas (3) only granulosa cells from large but not from smaller follicles were responsive to LH; (4) serum supplementation unfavorably transformed the cellular phenotype, induced proliferation and PCNA expression, reduced or abolished the transcript abundance of steroidogenic key genes and of gonadotropin receptor genes, CYP11A1, CYP19A1, FSHR and LHCGR but, however, did not increase the abundance of the luteinization-specific marker transcripts PTGS2, PTX3, RGS2 and VNN2; but (5) by increasing the plating density, estradiol production and the abundance of CYP19A1 transcripts, in particular those derived from the main ovarian promoter P2, were decreased concurrently leaving P2-specific DNA methylation levels unchanged, whereas progesterone secretion was stimulated and the expression of both luteinization-specific marker transcripts, RGS2 and VNN2, was significantly induced. From these data, we conclude that increasing the plating density induces a different, partly complementary, physiological and gene expression profile in cultured bovine granulosa cells and drives the cells towards an early post-LH stage of luteinization, even in the absence of luteinizing agents.
Subject(s)
Granulosa Cells/cytology , Granulosa Cells/physiology , Luteinization/physiology , Androstenedione/pharmacology , Animals , Aromatase/biosynthesis , Aromatase/genetics , Cattle , Cell Count , Cells, Cultured , Collagen/chemistry , Cytological Techniques/methods , DNA Methylation , Estradiol/metabolism , Female , Follicle Stimulating Hormone/pharmacology , Gene Expression Regulation , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Insulin-Like Growth Factor I/pharmacology , Luteinizing Hormone/pharmacology , Progesterone/metabolismABSTRACT
In breast cancer, the interaction between estrogen-producing breast adipose fibroblasts (BAFs) and estrogen-dependent epithelial tumor cells is pivotal. Local estrogen production is catalyzed by aromatase, which is differentially regulated in disease-free and tumorigenic breast tissue. The use of aromatase inhibitors to block local estrogen production has proven effective in treatment of estrogen-dependent breast cancer. However, a major problem during breast cancer treatment is the sudden onset of menopause and many women seek for alternative medicines, such as the soy isoflavone genistein. In this study, we show that genistein can induce estrogen-dependent MCF-7 tumor cell growth and increase breast cancer-associated aromatase expression and activity in vitro. We have previously developed an in vitro breast cancer model where the positive feedback loop between primary BAFs and estrogen-dependent MCF-7 tumor cells is operational, thereby representing a more natural in vitro model for breast cancer. In this model, genistein could negate the growth inhibitory action of the aromatase inhibitor fadrozole at physiologically relevant concentrations. These data suggest that soy-based supplements might affect the efficacy of breast cancer treatment with aromatase inhibitors. Considering the high number of breast cancer patients using soy supplements to treat menopausal symptoms, the increasing risk for adverse interactions with breast cancer treatment is of major concern and should be considered with care.
Subject(s)
Aromatase/biosynthesis , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Estrogens/physiology , Genistein/pharmacology , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Coculture Techniques , Dogs , Enzyme Induction/drug effects , Enzyme Induction/physiology , Female , HumansABSTRACT
Aromatase, the key enzyme in estrogen biosynthesis, converts androstenedione to estrone and testosterone to estradiol. The enzyme is expressed in various tissues such as ovary, placenta, bone, brain, skin, and adipose tissue. Aromatase enzyme is encoded by a single gene CYP 19A1 and its expression is controlled by tissue-specific promoters. Aromatase mRNA is primarily transcribed from promoter I.4 in normal breast tissue and physiological levels of aromatase are found in breast adipose stromal fibroblasts. Under the conditions of breast cancer, as a result of the activation of a distinct set of aromatase promoters (I.3, II, and I.7) aromatase expression is enhanced leading to local overproduction of estrogen that promotes breast cancer. Aromatase is considered as a potential target for endocrine treatment of breast cancer but due to nonspecific reduction of aromatase activity in other tissues, aromatase inhibitors (AIs) are associated with undesirable side effects such as bone loss, and abnormal lipid metabolism. Inhibition of aromatase expression by inactivating breast tumor-specific aromatase promoters can selectively block estrogen production at the tumor site. Although several synthetic chemical compounds and nuclear receptor ligands are known to inhibit the activity of the tumor-specific aromatase promoters, further development of more specific and efficacious drugs without adverse effects is still warranted. Plants are rich in chemopreventive agents that have a great potential to be used in chemotherapy for hormone dependent breast cancer which could serve as a source for natural AIs. In this brief review, we summarize the studies on phytochemicals such as biochanin A, genistein, quercetin, isoliquiritigenin, resveratrol, and grape seed extracts related to their effect on the activation of breast cancer-associated aromatase promoters and discuss their aromatase inhibitory potential to be used as safer chemotherapeutic agents for specific hormone-dependent breast cancer.
Subject(s)
Aromatase Inhibitors/pharmacology , Aromatase/biosynthesis , Biological Products/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Estrogens/biosynthesis , Neoplasms, Hormone-Dependent/drug therapy , Phytoestrogens/pharmacology , Promoter Regions, Genetic/drug effects , Female , Humans , PregnancyABSTRACT
In an earlier study in rodents, we showed that the aromatase that converts androgens to estrogens in the preoptic area and bed nucleus of stria terminalis was significantly increased in concentration after exposure to anabolic-androgenic steroids. To confirm whether this occurs in primates, we conducted a positron emission tomographic study using macaque monkeys. Male rhesus monkeys were treated with nandrolone decanoate for 3 weeks. To measure aromatase concentrations, we performed positron emission tomographic imaging using a 11C-labeled specific aromatase inhibitor, [11C]vorozole. After treatment with nandrolone, significant increase in [11C]vorozole binding was observed in the hypothalamus but not other areas including the amygdala, which is also aromatase enriched. These findings in monkeys are consistent with those we obtained earlier in rats. These findings strongly suggest that aromatase in the hypothalamus may play a crucial role in the emotional instability of anabolic-androgenic steroids abusers.
Subject(s)
Anabolic Agents/pharmacology , Androgens/pharmacology , Aromatase/biosynthesis , Hypothalamus/drug effects , Hypothalamus/enzymology , Triazoles , Animals , Aromatase/metabolism , Carbon Radioisotopes , Hypothalamus/diagnostic imaging , Macaca mulatta , Male , Positron-Emission Tomography/methodsABSTRACT
Syntheses and structure-activity relationships (SAR) of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain azaindole moieties as A-ring mimetics and display various degrees of in vitro dissociation between gene transrepression and transactivation. Collagen induced arthritis studies in mouse have demonstrated that in vitro dissociated compounds (R)-16 and (R)-37 have steroid-like anti-inflammatory properties with improved metabolic side effect profiles, such as a reduced increase in body fat and serum insulin levels, compared to steroids.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Pyridines/chemical synthesis , Pyrroles/chemical synthesis , Receptors, Glucocorticoid/agonists , Steroids/chemistry , Adipose Tissue/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aromatase/biosynthesis , Aromatase/genetics , Aromatase Inhibitors/pharmacology , Arthritis, Experimental/drug therapy , Biological Availability , Cells, Cultured , Enzyme Induction , Female , Humans , Hydrogen Bonding , Insulin/blood , Interleukin-1/pharmacology , Interleukin-6/antagonists & inhibitors , Interleukin-6/biosynthesis , Interleukin-6/genetics , Mice , Mice, Inbred BALB C , Models, Molecular , Pyridines/adverse effects , Pyridines/pharmacology , Pyrroles/adverse effects , Pyrroles/pharmacology , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity Relationship , Transcriptional ActivationABSTRACT
OBJECTIVE: To study the effect of peritoneal fluid from women with (PF-E) and without (PF-C) endometriosis on P(450)Arom expression in endometrial cells. DESIGN: Experimental study. SETTING: University research unit. PATIENT(S): Forty women of reproductive age with (n = 22) or without (control; n = 18) endometriosis. INTERVENTION(S): Peritoneal fluid and eutopic endometrial samples were obtained during surgery from women with (n = 13 and 9, respectively) and without (n = 4 and 14, respectively) endometriosis. MAIN OUTCOME MEASURE(S): Expression study for P(450)Arom, steroid factor 1 (SF-1), chicken ovalbumin upstream transcription factor I (COUP-TFI), and COUP-TFII messenger RNA (reverse transcriptase-polymerase chain reacion) and/or protein (immunoblot) in isolated endometrial epithelial cells transfected or not with expression vector containing SF-1, COUP-TFI, or COUP-TFII complementary DNAs. RESULT(S): Basal messenger RNA and/or protein expression of P(450)Arom and SF-1 were augmented in endometriosis, and that of COUP-TF was diminished. In control cells, (Bu)(2)cAMP and PF-E increased P(450)Arom and SF-1 expression (but not COUP-TF expression) in a dose-dependent way, an effect not observed with PF-C, adsorbed PF-E, or 10(-5) M indomethacin. Transfected cells confirmed these results. Any treatments modified the studied molecules in endometriosis cells. CONCLUSION(S): These data indicate that molecules contained in PF-E favor an estrogenic microenvironment, suggesting a role in the etiopathogenesis of endometriosis enabling the survival, maintenance, and growth of endometrial implants in the ectopic locations.
Subject(s)
Aromatase/biosynthesis , Ascitic Fluid/pathology , Ascitic Fluid/physiology , Endometriosis/pathology , Endometrium/metabolism , Peritoneal Diseases/pathology , Adult , Aromatase/genetics , COUP Transcription Factors/genetics , COUP Transcription Factors/metabolism , Case-Control Studies , Cell Separation , Cells, Cultured , Endometriosis/metabolism , Endometrium/cytology , Endometrium/drug effects , Endometrium/enzymology , Enzyme Induction , Female , Humans , Middle Aged , Peritoneal Diseases/metabolism , Steroidogenic Factor 1/genetics , Steroidogenic Factor 1/metabolismABSTRACT
The gender-specific expression pattern of aromatase and 5alpha-reductases (5alpha-R) during brain development provides neurons the right amount of estradiol and DHT to induce a dimorphic organization of the structure. Polychlorinated biphenyls (PCBs) are endocrine disruptive pollutants; exposure to PCBs through placental transfer and breast-feeding may adversely affect the organizational action of sex steroid, resulting in long-term alteration of reproductive neuroendocrinology. The study was aimed at: a) evaluating the hypothalamic expression of aromatase, 5alpha-R1 and 5alpha-R2 in fetuses (GD20), infant (PN12), weaning (PN21) and young adult (PN60) male and female rats exposed to PCBs during development; b) correlating these parameters with the time of testicular descent, puberty onset, estrous cyclicity and copulatory behavior; c) evaluating possible alterations of some non reproductive behaviors (locomotion, learning and memory, depression/anxiety behavior). A reconstituted mixture of four indicator congeners (PCB 126, 138, 153 and 180) was injected subcutaneously to dams at the dose of 10 mg/kg daily from GD15 to GD19 and then twice a week till weanling. The results indicated that developmental PCB exposure produced important changes in the dimorphic hypothalamic expression of both aromatase and the 5alpha-Rs, which were still evident in adult animals. We observed that female puberty onset occurs earlier than in control animals without cycle irregularity, while testicular descent in males was delayed. A slight but significant impairment of sexual behavior and an important alteration in memory retention were also noted specifically in males. We conclude that PCBs might affect the dimorphic neuroendocrine control of reproductive system and of other neurobiological processes.
Subject(s)
Aromatase/biosynthesis , Cholestenone 5 alpha-Reductase/biosynthesis , Endocrine Disruptors/toxicity , Hypothalamus/enzymology , Memory/drug effects , Polychlorinated Biphenyls/toxicity , Prenatal Exposure Delayed Effects/chemically induced , Sexual Behavior, Animal/drug effects , Aging/drug effects , Aging/metabolism , Animals , Dose-Response Relationship, Drug , Endocrine Disruptors/pharmacokinetics , Female , Hypothalamus/drug effects , Hypothalamus/growth & development , Lactation , Male , Maternal Exposure/adverse effects , Maze Learning/drug effects , Motor Activity/drug effects , Polychlorinated Biphenyls/pharmacokinetics , Pregnancy , Prenatal Exposure Delayed Effects/enzymology , Prenatal Exposure Delayed Effects/physiopathology , Rats , Rats, Sprague-Dawley , Reproduction/drug effects , Sex CharacteristicsABSTRACT
Although male sex is a well-recognized risk factor for stroke, the role of androgens in cerebral ischemia remains unclear. Therefore, we evaluated effects of testosterone on infarct size in both young adult and middle-aged rats (Wistar, 3-month versus 14-month old) and mice (C57/BL6, 3-month versus 12-month old) subjected to middle cerebral artery occlusion. In young adult groups, castrates displayed less ischemic damage as compared with intact males and castrates with testosterone replacement (Cortex: 24% in castrates versus 42% in intact versus 40% with testosterone; Striatum: 45% versus 73% versus 70%) at 22 h reperfusion. Surprisingly, supplementing testosterone in middle-aged rats to the physiologic levels ordinarily seen in young males reduced infarction (Cortex: 2% with testosterone versus 31%; Striatum: 38% with testosterone versus 68%). Testosterone effects on infarct size were blocked by the androgen receptor (AR) antagonist flutamide and further confirmed in young versus middle-aged mice. Baseline cerebral aromatase mRNA levels and activity were not different between young and middle-aged rats. Aromatase activity increased in ischemic tissue, but only in young males. Lastly, stroke damage was not different in aging aromatase knockout mice versus wild-type controls. Our findings indicate that testosterone's effects in experimental stroke are age dependent, mediated via AR, but not cerebral aromatase.
Subject(s)
Aging/metabolism , Stroke/metabolism , Testosterone/metabolism , Androgen Antagonists/pharmacology , Animals , Aromatase/biosynthesis , Aromatase/genetics , Castration , Flutamide/pharmacology , Infarction, Middle Cerebral Artery/complications , Infarction, Middle Cerebral Artery/enzymology , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Male , Mice , Mice, Knockout , Polymerase Chain Reaction , Rats , Rats, Wistar , Sex Factors , Stroke/enzymology , Stroke/etiology , Stroke/pathology , Testosterone/pharmacologyABSTRACT
To date, 10 promoters were reported to regulate the expression of the human aromatase (CYP19) gene, giving rise to transcripts with an identical coding region but tissue-specific first exons comprising unique 5'-untranslated regions. We describe the identification and characterization of a new CYP19 exon I, designated exon I.8, in a 5'-rapid amplification of complementary DNA ends-generated library of human THP-1 monocytic cells. A construct containing exon I.8 and its 5'-flanking sequence was sufficient to drive transcription in THP-1 cells. This novel promoter was located approximately 2-kb upstream of promoter I.4 and approximately 75-kb upstream of the common splice junction. We detected several I.8-containing splice variants, 2 of which also contained a sequence from exon I.4. Analysis of human tissues revealed a unique pattern of promoter I.8 usage. The placenta contained the highest level of I.8-specific transcripts. This work underscores the complexity of the mechanisms that regulate normal and pathologic aromatase expression.
Subject(s)
Aromatase/genetics , Promoter Regions, Genetic/genetics , Aromatase/biosynthesis , Aromatase/metabolism , Base Sequence , Cell Line, Tumor , Exons/genetics , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Neoplastic/genetics , Humans , Leukemia, Myeloid/enzymology , Leukemia, Myeloid/genetics , Molecular Sequence DataABSTRACT
AIM: To investigate the effects of 17beta-estradiol (E2), Peganum harmala extract (PHE) and caloric restriction (CR) on various testis parameters during aging. METHODS: Twelve month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR (40%). RESULTS: Our results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes: gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats. CONCLUSION: Besides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species (ROS) produced in large quantities in the aged testis.
Subject(s)
Aging/physiology , Caloric Restriction , Estrogens/pharmacology , Testis/drug effects , Testis/growth & development , Animals , Antioxidants/metabolism , Aromatase/biosynthesis , Aromatase/genetics , Estradiol/metabolism , Estradiol/pharmacology , Lipid Peroxidation/drug effects , Male , Oxidative Stress/drug effects , Peganum/chemistry , Plant Extracts/pharmacology , RNA/biosynthesis , RNA/genetics , Rats , Rats, Wistar , Receptors, Estrogen/biosynthesis , Receptors, Estrogen/genetics , Testis/enzymology , Testosterone/metabolism , Thiobarbituric Acid Reactive Substances/metabolismABSTRACT
Aromatase P450 (P450(arom)) is overexpressed in endometriosis, endometrial cancers and uterine fibroids. With weak estrogen agonists/antagonists and some other enzymatic activities, isoflavones are increasingly advocated as a natural alternative to estrogen replacement therapy (ERT) and are available as dietary supplements. Puerarin is major isoflavonoid compound isolated from Pueraria lobata, a Chinese medicine known as Gegen. Our clinical study shows that puerarin can be used in the treatment of endometriosis, which improves pain and infertility. Assuming that the effect of puerarin on endometriosis may result from the regulation of aromatase expression or activity, we carried out this study to test the effects of puerarin on aromatase in Ishikawa and RL95-2 cell lines. Our data have demonstrated a significant decrease of P450(arom) expression at both mRNA and protein levels by low dose puerarin treatment in both cell lines. Besides, we found that the -410/-401bp and -565/-559bp regions of aromatase promoter II contained the critical cis-acting elements, binding AP-1 and c-jun. We also found that puerarin exerted a time-course effect on the inhibition of c-jun mRNA, which parallelled that of P450(arom). To further confirm if c-jun is responsible for the P450(arom) regulation by puerarin, we knocked down c-jun expression using siRNA and it indicates that c-jun acts as a considerable transcription factor in regulating P450(arom) expression and activity. Accordingly, the suppression of P450(arom) expression and activity by puerarin treatment may associate with the downregulation of transcription factor AP-1 or c-jun.
Subject(s)
Aromatase Inhibitors , Aromatase/biosynthesis , Isoflavones/pharmacology , Proto-Oncogene Proteins c-jun/biosynthesis , Transcription Factor AP-1/biosynthesis , 5' Flanking Region , Aromatase/genetics , Blotting, Western , Cell Line , Down-Regulation/drug effects , Endometrium/cytology , Endometrium/drug effects , Epithelial Cells/drug effects , Estradiol/pharmacology , Female , Humans , Luciferases/biosynthesis , Luciferases/genetics , RNA/biosynthesis , RNA/isolation & purification , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Neuronal aromatase, the enzyme that catalyzes the conversion of androgens to estrogens, is involved in brain sexual differentiation, the regulation of reproductive behavior, and gonadotropin secretion. We have previously reported that aromatase P450 (AromP450) protein expression is enhanced by both androgens and estrogens in the principal nucleus of the bed nucleus of the stria terminalis (prBST) and posterodorsal part of the medial amygdaloid nucleus (pdMAm) of the adult rat but is not altered in the central amygdaloid nucleus (CeAm) even after sex-steroid withdrawal or supplementation. Here, we have evaluated, via in situ hybridization with digoxigenin-labeled cRNA probes, the sex-steroidal regulation of brain AromP450 mRNA in the prBST, pdMAm, and CeAm of orchidectomized and adrenalectomized adult male rats treated with sesame oil, testosterone (1 mg/rat/day), dihydrotestosterone (1 mg/rat/day), or 17beta-estradiol (2 microg/rat/day) for 6 days. AromP450-mRNA expression in the prBST and pdMAm was markedly reduced in orchidectomized/adrenalectomized rats treated with sesame oil but strongly enhanced by testosterone or dihydrotestosterone and significantly reinstated by 17beta-estradiol. These results are essentially consistent with those of AromP450 protein expression and thus indicate that enhanced AromP450-protein expression in the prBST and pdMAm reflects transcriptional upregulation and/or post-transcriptional stabilization of its mRNA by sex steroids. In the CeAm, despite moderate AromP450-protein expression, the mRNA has never been detected with or without sex-steroidal manipulations, indicating that the putative sex-steroid-insensitive AromP450 mRNA in the CeAm may be distinct from that in the prBST and pdMAm or, if it occurs at all, expressed at much lower levels.
Subject(s)
Aromatase/genetics , Brain/metabolism , Gene Expression Regulation, Enzymologic , Gonadal Steroid Hormones/pharmacology , Animals , Aromatase/biosynthesis , Brain Chemistry , Dihydrotestosterone/pharmacology , Estradiol/pharmacology , Gene Expression , Hormones/blood , In Situ Hybridization/methods , Male , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Wistar , Steroids/blood , Testosterone/pharmacologyABSTRACT
Aromatase is a member of the cytochrome P450 superfamily of enzymes which catalyses the rate-limiting step in the biosynthesis of estrogens. A number of clinical studies have highlighted the importance of local estrogen production in adipose tissue. In particular, in the postmenopausal woman, the degree of her estrogenization is mainly determined by the extent of her adiposity and it is this extragonadal source of estrogen that likely contributes to breast cancer development and progression. The mechanisms regulating aromatase expression in adipose tissue however, have not been fully elucidated. In this study, we have characterised the expression of aromatase and its activity in a human preadipocyte cell strain, SGBS. Aromatase is expressed in SGBS cells and its expression and activity are strongly stimulated by forskolin (FSK) and phorbol 12-myristate-13-acetate (PMA) treatment. Consistent with this, FSK and PMA treatment also increased activation of the proximal aromatase promoter, promoter II. These findings mimic those that have previously been shown in isolated primary human preadipocytes. These data suggest that SGBS cells are a valuable model with which to further elucidate the mechanisms regulating aromatase expression, and therefore local estrogen synthesis in human adipose tissue.
Subject(s)
Adipocytes/enzymology , Aromatase/biosynthesis , Adipocytes/metabolism , Adipose Tissue/metabolism , Aromatase/metabolism , Cell Line , Colforsin/pharmacology , Cytochrome P-450 Enzyme System/metabolism , DNA, Complementary/metabolism , Disease Progression , Estrogens/metabolism , Humans , Promoter Regions, Genetic , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tetradecanoylphorbol Acetate/pharmacology , Time FactorsABSTRACT
Estrogen is crucial in preparing of pregnancy, and its role in the maintenance of pregnancy has yet to be elucidated. During the course of pregnancy, the placenta is responsible for the provision of estrogen. The hormone biosynthesis is catalyzed by cytochrome P450 (CYP) 19 or aromatase. In the present study, we screened several common dietary components and identified the grape polyphenol resveratrol to be a potential inhibitor in the hormone synthesis. In a recombinant protein system resveratrol inhibited the aromatase activity with an IC(50) value of approximately 40 microM. Subsequent analysis was performed in the human placental JEG-3 cells, and 25 microM resveratrol significantly reduced the mRNA abundance in these cells. Since the transcriptional control of CYP19 gene is tissue-specific and the proximal promoter region of exon Ia has previously been shown to be crucial in CYP19 expression in placental cells, we also evaluated the promoter activity of this gene. Reporter gene assays revealed that resveratrol repressed the transcriptional control of promoter Ia. The present study illustrated the possibility that dietary supplementation of resveratrol interfered with the normal functioning of placental cells.
Subject(s)
Aromatase Inhibitors/toxicity , Aromatase/metabolism , Estrogens/metabolism , Placenta/drug effects , Stilbenes/toxicity , Aromatase/biosynthesis , Aromatase/genetics , Cell Line , Dose-Response Relationship, Drug , Enzyme Repression , Genes, Reporter , Humans , Luciferases/genetics , Placenta/enzymology , Placenta/metabolism , Promoter Regions, Genetic/drug effects , RNA, Messenger/biosynthesis , Recombinant Proteins/metabolism , Resveratrol , Transcription, Genetic/drug effects , TransfectionABSTRACT
CONTEXT: Uterine leiomyomata are common tumors that cause irregular uterine bleeding and pregnancy loss and depend on estrogen for growth. Aromatase catalyzes the conversion of androgens to estrogens. Aromatase expression is regulated via alternatively used promoters in the placenta (I.1 and I.2a), fat (I.4, I.3, and II), bone (I.6), and gonads (II). A prostaglandin E(2)/cAMP-dependent pathway regulates coordinately the proximal promoters I.3/II, whereas glucocorticoids and cytokines regulate the distal promoter I.4. Use of each promoter gives rise to a population of aromatase mRNA species with unique 5'-untranslated regions (5'-UTRs). Uterine leiomyoma tissue, but not normal myometrium, overexpresses aromatase leading to estrogen-stimulated cell proliferation. Aromatase inhibitor treatment shrank uterine leiomyomata in a few women. OBJECTIVE AND DESIGN: Promoter I.4 was reported to regulate aromatase expression in uterine leiomyomata from a group of Japanese women. Here, we used two independent techniques to identify the promoters that regulate aromatase expression in uterine leiomyomata (n = 30) from 23 African-American, Hispanic, and white women. RESULTS: Rapid amplification of 5'-cDNA ends of aromatase mRNA species revealed the following distribution of promoter usage in leiomyomata: promoters I.3/II, 61.5%; I.2a, 15.4%; I.6, 15.4%; and I.4, 7.7%. Real-time PCR, which quantifies mRNA species with promoter-specific 5'-UTRs, revealed the following distribution for each 5'-UTR as a fraction of total aromatase mRNA: I.3/II, 69.6%; I.4, 7.3%; and other promoters, 23.1%. CONCLUSIONS: The primary in vivo aromatase promoter in leiomyoma tissues in non-Asian U.S. women is the prostaglandin E(2)/cAMP-responsive I.3/II region. Alternative signals may stimulate aromatase expression that is a common biological phenotype in uterine leiomyomata.
Subject(s)
Aromatase/biosynthesis , Aromatase/genetics , Leiomyoma/enzymology , Leiomyoma/genetics , Promoter Regions, Genetic/genetics , Uterine Neoplasms/enzymology , Uterine Neoplasms/genetics , 5' Untranslated Regions/genetics , Adult , Female , Gene Amplification , Gene Expression Regulation, Enzymologic/physiology , Humans , Middle Aged , RNA, Complementary/biosynthesis , RNA, Complementary/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In birds and mammals, aromatase activity in the preoptic-hypothalamic region (HPOA) is usually higher in males than in females. It is, however, not known whether the enzymatic sex difference reflects the differential activation of aromatase transcription or some other control mechanism. Although sex differences in aromatase activity are clearly documented in the HPOA of Japanese quail (Coturnix japonica), only minimal or even no differences at all were observed in the number of aromatase-immunoreactive (ARO-ir) cells in the medial preoptic nucleus (POM) and in the medial part of the bed nucleus striae terminalis (BSTM). We investigated by in situ hybridization the distribution and possible sex differences in aromatase mRNA expression in the brain of sexually active adult quail. The distribution of aromatase mRNA matched very closely the results of previous immunocytochemical studies with the densest signal being observed in the POM, BSTM and in the mediobasal hypothalamus (MBH). Additional weaker signals were detected in the rostral forebrain, arcopallium and mesencephalic regions. No sex difference in the optical density of the hybridization signal could be found in the POM and MBH but the area covered by mRNA was larger in males than in females, indicating a higher overall expression in males. In contrast, in the BSTM, similar areas were covered by the aromatase expression in both sexes but the density of the signal was higher in females than in males. The physiological control of aromatase is thus neuroanatomically specific and with regard to sex differences, these controls are at least partially different if one compares the level of transcription, translation and activity of the enzyme. These results also indirectly suggest that the sex difference in aromatase enzyme activity that is present in the quail HPOA largely results from differentiated controls of enzymatic activity rather than differences in enzyme concentration.
Subject(s)
Aromatase/biosynthesis , Coturnix/physiology , Preoptic Area/anatomy & histology , Preoptic Area/metabolism , RNA, Messenger/biosynthesis , Septal Nuclei/anatomy & histology , Septal Nuclei/metabolism , Animals , Cloning, Molecular , DNA, Complementary/biosynthesis , Data Interpretation, Statistical , Female , Gene Expression Regulation, Enzymologic/physiology , In Situ Hybridization , Male , Phosphorylation , Preoptic Area/enzymology , Septal Nuclei/enzymology , Sex CharacteristicsABSTRACT
Estrogens are biosynthesised from androgens by the CYP450 enzyme complex called aromatase. Aromatase is expressed in the ovary, placenta, brain, bone, adipose tissue and breast tissue. In breast cancer, intratumoural aromatase is the source for local estrogen production in the tissue. Inhibition of aromatase is an important approach for reducing growth stimulatory effects of estrogens in estrogen-dependent breast cancer. The potent and selective third-generation aromatase inhibitors anastrozole, letrozole and exemestane were introduced to the market as endocrine therapy in postmenopausal patients failing anti-estrogen therapy alone, or multiple hormonal therapies. Anastrozole and letrozole are both non-steroidal aromatase inhibitors that compete with the substrate for binding to the enzyme active site. Exemestane is a mechanism-based steroidal inhibitor that mimics the substrate, is converted by the enzyme to a reactive intermediate, and results in inactivation of aromatase. These third-generation aromatase inhibitors are currently approved as first-line therapy for the treatment of postmenopausal women with metastatic estrogen-dependent breast cancer. The use of an aromatase inhibitor as initial therapy, or after treatment with tamoxifen, is now recommended as adjuvant hormonal therapy for postmenopausal women with hormone-dependent breast cancer. Several clinical studies of aromatase inhibitors focus on the use of these agents in the adjuvant setting, for the treatment of early breast cancer. Recently published results show improved responses with these agents compared with tamoxifen.
Subject(s)
Aromatase Inhibitors/therapeutic use , Aromatase/physiology , Breast Neoplasms/drug therapy , Anastrozole , Androstadienes/therapeutic use , Animals , Aromatase/biosynthesis , Aromatase/genetics , Breast Neoplasms/enzymology , Female , Humans , Letrozole , Nitriles/therapeutic use , Postmenopause , Randomized Controlled Trials as Topic , Triazoles/therapeutic useABSTRACT
Aromatase is the enzyme that converts androgen to estrogen. It is expressed at higher levels in breast cancer tissues than normal breast tissues. Grape seed extract (GSE) contains high levels of procyanidin dimers that have been shown in our laboratory to be potent inhibitors of aromatase. In this study, GSE was found to inhibit aromatase activity in a dose-dependent manner and reduce androgen-dependent tumor growth in an aromatase-transfected MCF-7 (MCF-7aro) breast cancer xenograft model, agreeing with our previous findings. We have also examined the effect of GSE on aromatase expression. Reverse transcription-PCR experiments showed that treatment with 60 mug/mL of GSE suppressed the levels of exon I.3-, exon PII-, and exon I.6-containing aromatase mRNAs in MCF-7 and SK-BR-3 cells. The levels of exon I.1-containing mRNA, however, did not change with GSE treatment. Transient transfection experiments with luciferase-aromatase promoter I.3/II or I.4 reporter vectors showed the suppression of the promoter activity in a dose-dependent manner. The GSE treatment also led to the down-regulation of two transcription factors, cyclic AMP-responsive element binding protein-1 (CREB-1) and glucocorticoid receptor (GR). CREB-1 and GR are known to up-regulate aromatase gene expression through promoters I.3/II and I.4, respectively. We believe that these results are exciting in that they show GSE to be potentially useful in the prevention/treatment of hormone-dependent breast cancer through the inhibition of aromatase activity as well as its expression.