Showing papers on "Aromatase published in 1999"

Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene

Abstract: It is well established that spermatogenesis is controlled by gonadotrophins and testosterone. However, a role for estrogens in male reproduction recently was suggested in adult mice deficient in estrogen receptor α. These mice became infertile primarily because of an interruption of fluid reabsorption by the efferent ductules of the epididymis, thus leading to a disruption of the seminiferous epithelium [Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997) Nature (London) 390, 509–512]. Despite the demonstration of the aromatase enzyme, which converts androgens to estrogens, and estrogen receptors within the rodent seminiferous epithelium, the role of aromatase and estrogen in germ cell development is unknown. We have investigated spermatogenesis in mice that lack aromatase because of the targeted disruption of the cyp19 gene (ArKO). Male mice deficient in aromatase were initially fertile but developed progressive infertility, until their ability to sire pups was severely impaired. The mice deficient in aromatase developed disruptions to spermatogenesis between 4.5 months and 1 year, despite no decreases in gonadotrophins or androgens. Spermatogenesis primarily was arrested at early spermiogenic stages, as characterized by an increase in apoptosis and the appearance of multinucleated cells, and there was a significant reduction in round and elongated spermatids, but no changes in Sertoli cells and earlier germ cells. In addition, Leydig cell hyperplasia/hypertrophy was evident, presumably as a consequence of increased circulating luteinizing hormone. Our findings indicate that local expression of aromatase is essential for spermatogenesis and provide evidence for a direct action of estrogen on male germ cell development and thus fertility.

Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain.

Abstract: The brain is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Although considerable information is now available regarding the steroidogenic capacity of the brain, little is known regarding the steroidogenic pathway and relative contributions of astrocytes, oligodendrocytes, and neurons to neurosteroidogenesis. In the present study, we investigated differential gene expression of the key steroidogenic enzymes using RT-PCR and quantitatively evaluated the production of neurosteroids by highly purified astrocytes, oligodendrocytes, and neurons from the cerebral cortex of neonatal rat brains using specific and sensitive RIAs. Astrocytes appear to be the most active steroidogenic cells in the brain. These cells express cytochrome P450 side-chain cleavage (P450scc), 17alpha-hydroxylase/C17-20-lyase (P450c17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17beta-hydroxysteroid dehydrogenase (17betaHSD), and cytochrome P450 aromatase (P450arom) and produce pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), estradiol, and estrone. Oligodendrocytes express only P450scc and 3betaHSD and produce P5, P4, and A4. These cells do not express P450c17, 17betaHSD, or P450arom or produce DHEA, T, or estrogen. Neurons express P450scc, P450c17, 3betaHSD, and P450arom and produce P5, DHEA, A4, and estrogen, but do not express 17betaHSD or produce T. By comparing the ability of each cell type in the production of neurosteroids, astrocytes are the major producer of P4, DHEA, and androgens, whereas oligodendrocytes are predominantly the producer of P5 and neurons of estrogens. These findings serve to define the neurosteroidogenic pathway, with special emphasis on the dominant role of astrocytes and their interaction with oligodendrocytes and neurons in the genesis of DHEA and active sex steroids. Thus, we propose that neurosteroidogenesis is accomplished by a tripartite contribution of the three cell types in the brain.

Estrogen: consequences and implications of human mutations in synthesis and action.

Abstract: Recent developments have advanced our knowledge of the role of estrogen in the male. Studies of the mutations in CYP19, the gene encoding aromatase, in six females and two males and a mutant estrogen receptor alpha in a man are described. These observations provide illuminating new insights into the critical role of estrogen in the male (as well as female) in the pubertal growth spurt and skeletal maturation, and in the importance of estrogen sufficiency in the accrual and maintenance of bone mass. The weight of evidence supports an effect of androgens on the latter processes, but this effect has not been quantitated. There is a discordance in the estrogen-deficient male between skeletal growth and skeletal maturation and the accrual of bone mass and density. Estrogen synthesis by the testis is limited before puberty, and estrogen deficiency does not affect the age of pubertal onset. Estrogen deficiency in men leads to hypergonadotropism, macroorchidism, and increased testosterone levels. Estrogen lack has a significant effect on carbohydrate and lipid metabolism, and estrogen resistance was associated with evidence of premature coronary atherosclerosis in a man. These observations have highlighted the role of extraglandular estrogen synthesis and intracrine and paracrine actions. In the human, in contrast to nonprimate vertebrates, aromatase deficiency and estrogen resistance (alpha) does not seem to affect gender identity or psychosexual development. The clinical repercussions of mutations in CYP19 on the fetal-placental unit have highlighted the major role of placental aromatase in the protection of the female fetus from androgen excess, thus preventing androgen-induced pseudohermaphrodism and virilization of the mother. These features are compared with the virilization that occurs in utero in the female spotted hyena. The novel features of the aromatase deficiency syndrome in the affected female--in the fetus, during childhood, and at puberty--are discussed, including virilization at puberty and development of polycystic ovaries. The severity of the syndrome correlates with the severity of impairment of aromatase formation in expression systems. Finally, the structural consequences of missense mutations in CYP19 are described in accordance with a model of the structure of human aromatase.

Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus).

Abstract: In order to study the physiological implication of sex steroid hormones in gonadal sex differentiation in fish, we first investigated the potential role of estrogens using two fish models: the rainbow trout (Oncorhynchus mykiss) and a tilapia species (Oreochromis niloticus). All experiments were carried out on genetically all-male (XY) and all-female (XX) populations. In vivo treatments with an aromatase inhibitor (ATD, 1,4,6- androstatriene-3–17-dione) result in 100% masculinization of an all-female population in rainbow trout (dosage 50 mg/kg of food) and 75.3% in tilapia (dosage 150 mg/kg of food). In tilapia, the effectiveness of the aromatase inhibition by ATD is demonstrated by the marked decrease of the gonadal aromatase activity in treated animals versus control. No masculinization is obtained following treatment with an estrogen receptor antagonist (tamoxifen) in both species. Aromatase and estrogen receptor gene expression was studied in rainbow trout by semi-quantitative RT-PCR in gonads sampled before, during and after sex-differentiation. Aromatase mRNA is specifically detected in female gonads, 3 weeks before the first sign of histological sex-differentiation, i.e., first female meiosis. Aromatase expression in male gonads is at least a few hundred times less than in female gonads. Estrogen receptor gene is expressed in both male and female gonads at all stages with no dimorphic expression between sexes. Specific aromatase gene expression before ovarian differentiation was also demonstrated using virtual Northern blot, with no expression detected in male differentiating gonads. From these results it can be concluded that estrogen synthesis is crucial for ovarian differentiation, and transcription of the aromatase gene can be proposed as a key step in that process in fish. Mol. Reprod. Dev. 54:154–162, 1999. © 1999 Wiley-Liss, Inc.

Estrogen and the developing mammalian brain.

Abstract: In recent years, the knowledge of how estrogen interferes with mammalian brain functions and development has broadened substantially. In the adult brain, estrogen is not only involved in the neuroendocrine feedback regulation at the hypothalamic and pituitary level but also in the control of motor and cognitive functions. More recently, estrogen was found to act as a protective factor for neurodegenerative disorders such as Parkinson's and Alzheimer's disease. In contrast to these regulatory and protective functions, estrogen plays a different role during neuronal development. After the demonstration that the estrogen-synthesizing enzyme aromatase and both nuclear estrogen receptors are expressed in many brain areas during ontogeny, it was soon realized that estrogen modulates neuronal differentiation, notably by influencing cell migration, survival and death, and synaptic plasticity of neurons. These effects were initially seen in the classical target area for estrogen, the hypothalamus, but successive studies revealed the neurotrophic potential of estrogen also in other brain regions. The focus of this review will be to summarize estrogen formation and the role of estrogen during mammalian brain development. Moreover, cellular mechanisms involved in these neurotrophic effects will be discussed, giving special emphasis to "nongenomic" estrogen signaling and cross-coupling of estrogen signaling with those of growth factors.

Role of astroglia in estrogen regulation of synaptic plasticity and brain repair.

Abstract: Astroglia are targets for estrogen and testosterone and are apparently involved in the action of sex steroids on the brain. Sex hormones induce changes in the expression of glial fibrillary acidic protein, the growth of astrocytic processes, and the degree of apposition of astroglial processes to neuronal membranes in the rat hypothalamus. These changes are linked to modifications in the number of synaptic inputs to hypothalamic neurons. These findings suggest that astrocytes may participate in the genesis of androgen-induced sex differences in synaptic connectivity and in estrogen-induced synaptic plasticity in the adult brain. Astrocytes and tanycytes may also participate in the cellular effects of sex steroids by releasing neuroactive substances and by regulating the local accumulation of specific growth factors, such as insulin-like growth factor-I, that are involved in estrogen-induced synaptic plasticity and estrogen-mediated neuroendocrine control. Astroglia may also be involved in regenerative and neuroprotective effects of sex steroids, since astroglia formation after brain injury or after peripheral nerve axotomy is regulated by sex hormones. Furthermore, the expression of aromatase, the enzyme that produces estrogen, is induced de novo in astrocytes in lesioned brain areas of adult male and female rodents. Since astroglia do not express aromatase under normal circumstances, the induction of this enzyme may be part of the program of glial activation to cope with the new conditions of the neural tissue after injury. Given the neuroprotective and growth-promoting effects of estrogen after injury, the local production of this steroid may be a relevant component of the reparative process.

Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex differentiation in the Japanese flounder (Paralichthys olivaceus)

Abstract: The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determination mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex differentiation in the Japanese flounder, we generated two populations of larvae, both having XX (genetic females) but each growing up to display all phenotypic females or males, by rearing the larvae at normal (18 degrees C) or high (27 degrees C) water temperatures from days 30 to 100 after hatching respectively. The larvae (XX) were produced artificially by mating normal females (XX) with gynogenetic diploid males (XX) which had been sex-reversed to phenotypic males by 17alpha-methyltestosterone. To study the role of P450arom in sex determination in the flounder, we first isolated a P450arom cDNA containing the complete open reading frame from the ovary. RT-PCR showed that P450arom mRNA was highly expressed in the ovary and spleen but weakly in the testis and brain. Semi-quantitative analyses of P450arom mRNA in gonads during sex differentiation showed that there was no difference in the levels of P450arom mRNA between the female and male groups when the gonad was sexually indifferent (day 50 after hatching). However, after the initiation of sex differentiation (day 60), the mRNA levels increased rapidly in the female group, whereas they decreased slightly in the male group. Similarly, estradiol-17beta levels rose remarkably in the female group, yet remained constant in the male group. These results suggest that induction of sex reversal of genetically female larvae to phenotypic males by rearing them at a high water temperature caused a suppression of P450arom gene expression. Furthermore, we suggest that the maintenance of P450arom mRNA at very low levels is a prerequisite for testicular differentiation, while the increased levels are indispensable for ovarian differentiation.

Temperature-dependent sex determination and gonadal differentiation in reptiles

Abstract: In many reptile species, sexual differentiation of gonads is sensitive to temperature during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models among which turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Present studies deal with cloning (complementary DNAs) and expression (messenger RNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results indicate that homologues of AMH, DAX1, SF1, SOX9 and WT1 genes with the same function(s) as in mammals exist in reptiles. How these genes could interact with aromatase is being examined.

Sources of oestrogen in the testis and reproductive tract of the male.

Abstract: The cytochrome P450 aromatase (P450arom) is the terminal enzyme responsible for the irreversible transformation of androgens into oestrogens and is present in the endoplasmic reticulum of various tissues throughout at least the phylum of vertebrates. The CYP 19 gene is unique and its expression is regulated in a tissue and more precisely in a cell-specific fashion via the alternative use of several promoters located in the first exons. The P450arom has been immunolocalized in germ cells of the mouse, brown bear and rooster. According to age, aromatase activity has been measured in immature and mature rat Leydig cells as well as in Sertoli cells, whereas in the pig, ram and human aromatase is mainly present in Leydig cells. In the adult rat testis, four complementary approaches (RTPCR, in situ hybridization, immunocytochemistry and the tritiated water assay) demonstrate that not only somatic cells but also mature germ cells represent a source of oestrogen synthesis. Taking into account the widespread distribution of oestrogen receptors (ER alpha & ER beta) in testicular cells and the genital tract of the male on the one hand, and the cross-talk between sex steroids and growth factors, and between membrane receptors and nuclear receptors for steroids on the other hand, it is anticipated that understanding of the pathophysiological roles of these 'female' hormones in the male will advance understanding of the hormonal regulation of male reproductive function. One of the future goals is to define oestrogen-targeted genes in the male gonad and indeed, a lot of work is now focused on this specific area in order to clarify the role of oestrogens in the reproductive tract of the male as well as to elucidate the regulation of aromatase gene expression.

Stimulation of Aromatase P450 Promoter (II) Activity in Endometriosis and Its Inhibition in Endometrium Are Regulated by Competitive Binding of Steroidogenic Factor-1 and Chicken Ovalbumin Upstream Promoter Transcription Factor to the Same cis-Acting Element

Abstract: In stromal cells of endometriosis, marked levels of aromatase P450 (P450arom) mRNA and activity are present and can be vigorously stimulated by (Bu)2cAMP or PGE2 to give rise to physiologically significant estrogen biosynthesis. Since eutopic endometrial tissue or stromal cells lack P450arom expression, we studied the molecular basis for differential P450arom expression in endometriosis and eutopic endometrium. First, we demonstrated by rapid amplification of cDNA 5'-ends that P450arom expression in pelvic endometriotic lesions is regulated almost exclusively via the alternative promoter II. Then, luciferase reporter plasmids containing deletion mutations of the 5'-flanking region of promoter II were transfected into endometriotic stromal cells. We identified two critical regulatory regions for cAMP induction of promoter II activity: 1) a-214/-100 bp proximal region responsible for a 3.7-fold induction, and 2) a -517/ -214 distal region responsible for potentiation of cAMP response up to 13-fold. In the -214/-100 region, we studied eutopic endometrial and endometriotic nuclear protein binding to a nuclear receptor half-site (NRHS, AGGTCA) and an imperfect cAMP response element (TGCACGTCA). Using electrophoretic mobility shift assay, cAMP response element-binding activity in nuclear proteins from both endometriotic and eutopic endometrial cells gave rise to formation of identical DNA-protein complexes. The NRHS probe, on the other hand, formed a distinct complex with nuclear proteins from endometriotic cells, which migrated at a much faster rate compared with the complex formed with nuclear proteins from eutopic endometrial cells. Employing recombinant proteins and antibodies against steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF), we demonstrated that COUP-TF but not SF-1 bound to NRHS in eutopic endometrial cells, whereas SF-1 was the primary NRHS-binding protein in endometriotic cells. In fact, COUP-TF transcripts were present in both eutopic endometrial (n = 12) and endometriotic tissues (n = 8), whereas SF-1 transcripts were detected in all endometriotic tissues (n = 12), but in only 3 of 15 eutopic endometrial tissues. Moreover, we demonstrated a dose-dependent direct competition between SF-1 and COUP-TF for occupancy of the NRHS, to which SF-1 bound with a higher affinity. Finally, overexpression of SF-1 in eutopic endometrial and endometriotic cells strikingly potentiated baseline and cAMP-induced activities of -517 promoter II construct, whereas overexpression of COUP-TF almost completely abolished these activities. In conclusion, COUP-TF might be one of the factors responsible for the inhibition of P450arom expression in eutopic endometrial stromal cells, which lack SF-1 expression in the majority (80%) of the samples; in contrast, aberrant SF-1 expression in endometriotic stromal cells can override this inhibition by competing for the same DNA-binding site, which is likely to account for high levels of baseline and cAMP-induced aromatase activity.

Use of aromatase inhibitors in breast carcinoma.

Abstract: Aromatase, a cytochrome P-450 enzyme that catalyzes the conversion of androgens to estrogens, is the major mechanism of estrogen synthesis in the post-menopausal woman. We review some of the recent scientific advances which shed light on the biologic significance, physiology, expression and regulation of aromatase in breast tissue. Inhibition of aromatase, the terminal step in estrogen biosynthesis, provides a way of treating hormone-dependent breast cancer in older patients. Aminoglutethimide was the first widely used aromatase inhibitor but had several clinical drawbacks. Newer agents are considerably more selective, more potent, less toxic and easier to use in the clinical setting. This article reviews the clinical data supporting the use of the potent, oral competitive aromatase inhibitors anastrozole, letrozole and vorozole and the irreversible inhibitors 4-OH androstenedione and exemestane. The more potent compounds inhibit both peripheral and intra-tumoral aromatase. We discuss the evidence supporting the notion that aromatase inhibitors lack cross-resistance with antiestrogens and suggest that the newer, more potent compounds may have a particular application in breast cancer treatment in a setting of adaptive hypersensitivity to estrogens. Currently available aromatase inhibitors are safe and effective in the management of hormone-dependent breast cancer in post-menopausal women failing antiestrogen therapy and should now be used before progestational agents. There is abundant evidence to support testing these compounds as first-line hormonal therapy for metastatic breast cancer as well as part of adjuvant regimens in older patients and quite possibly in chemoprevention trials of breast cancer.

Localized Expression of Aromatase in Human Vascular Tissues

Abstract: The atheroprotective effects of estrogen are well established and the presence of an estrogen receptor in vascular tissues has recently been reported. Therefore, we investigated the localization of the estrogen-producing enzyme aromatase in vascular tissues to assess the possible contribution of endocrine, paracrine, and autocrine modes of action. Aromatase was found in human vascular smooth muscle cells (SMCs) but not in endothelial cells on in situ hybridization. These observations were further supported by quantitative analysis of aromatase mRNA and the activity in 15 human vascular specimens. Only trace levels of expression were detected in the 3 infants examined, whereas 0.0088 to 0.0806 amol/ microg RNA of aromatase mRNA and 12.9 to 122.3 fmol. h-1. mg-1 protein of the activity were detected in 12 of the adult individuals. The switching of tissue-specific exon 1 of the human aromatase gene was also observed in some cases. Aromatase was found to be expressed only in cultured SMCs and not in cultured endothelial cells of human aorta and pulmonary artery and to be regulated through dexamethasone and the signaling pathways of protein kinase A and C. Study results revealed the localized expression of aromatase in vascular SMCs, which indicated a possible direct action of locally produced estrogen in an autocrine or paracrine manner, with possible cross talk between smooth muscle and endothelial cells.

Aromatase and breast cancer susceptibility.

Abstract: Based on experimental and epidemiological evidence it is hypothesized that estrogen increases breast cancer risk by increasing mitotic activity in breast epithelial cells. Aromatase is crucial to the biosynthesis of estrogens and may therefore play a role in breast cancer development. Supporting data for an etiological role of aromatase in breast tumor biology are several-fold. First, the association between weight and postmenopausal breast cancer risk may be mediated by aromatase. Secondly, a pilot study found a higher aromatase expression in normal breast adipose tissue from breast cancer cases as opposed to healthy women. Thirdly, experimental data in animals suggest that aromatase activity predisposes mammary tissue to preneoplastic and neoplastic changes. In a multiethnic cohort study conducted in Los Angeles and on Hawaii we investigated (i) whether the plasma estrone to androstenedione (E1/A) ratio in different ethnic groups was associated with ethnic differences in breast cancer incidence, and (ii) whether genetic variation in the CYP19 gene encoding the P450 aromatase protein was associated with breast cancer risk. The age- and weight-adjusted ethnic specific E1/A ratios x 100 among women without oophorectomy were 7.92 in African-Americans, 8.22 in Japanese, 10.73 in Latinas and 9.29 in non-Latina Whites (P=0.09). The high E1/A ratio in Latina women was not associated with a high breast cancer incidence; in fact Latina women had the lowest breast cancer incidence in the cohort observed so far. We found no consistent association of an intronic (TTTA)n repeat polymorphism with breast cancer risk in different ethnic groups. This polymorphism was not associated with differences in the plasma E1/A ratio in a way that would predict its functional relevance. We describe a newly identified TTC deletion in intron 5 of the CYP19 gene that is associated with the (TTTA)n repeat polymorphism. Neither this polymorphism, nor a polymorphism at codon 264 in exon VII of the CYP19 gene, was associated with breast cancer. We did not identify any genetic variation in exon VIII in 54 African-American subjects. We identified rare genetic variants of unknown functional relevance in the promoter 1.4 of the CYP19 gene in 3 out of 24 Latina women. Further investigation into the role of aromatase in breast cancer etiology is important, given that the potential use of aromatase inhibitors as breast cancer chemopreventives depends on these results.

Adrenal androgens stimulate the proliferation of breast cancer cells as direct activators of estrogen receptor alpha.

Abstract: Estrogens stimulate the proliferation of many breast tumors and cell lines derived from them. Antiestrogens have therefore become a powerful therapeutic agent to treat breast tumors that express estrogen receptor (ER) alpha. In addition, aromatase inhibitors are now used in postmenopausal women to block the in situ conversion of adrenal androgens to estrogens. This approach can only be successful if ER-alpha in a particular tumor is not directly stimulated by adrenal androgens. We have examined this possibility using several different cell lines as model systems: (a) wild-type MCF7 cells, an ER-alpha-dependent human breast cancer cell line; (b) MCF7SH cells, an estrogen-independent MCF7 variant; (c) Ishikawa cells, an ER-alpha-containing human uterine cell line; (d) ER-negative HeLa cells; and (e) budding yeast. Transactivation assays with transfected ER-alpha reporter genes reveal a direct activation of ER-alpha by dehydroepiandrosterone (DHEA), 5alpha-androstene-3beta,17beta-diol, testosterone, and the two nonaromatizable androgens, dihydrotestosterone and 5alpha-androstane-3beta,17beta-diol. The involvement of other steroid receptors could be ruled out with specific antihormones. Moreover, the same set of ligands stimulates the proliferation of the two breast cancer cell lines. At subsaturating and physiologically relevant concentrations of DHEA, DHEA stimulates the proliferation of MCF7SH cells, which correlates with a substantial, albeit submaximal, transcriptional response. Thus, adrenal androgens must also be considered as risk factors in postmenopausal women.

Leptin directly stimulates aromatase activity in human luteinized granulosa cells.

Abstract: Leptin, the obese (ob) gene product, is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors. Leptin may also have a stimulatory effect on reproductive function. Furthermore, leptin receptor mRNA is expressed in the ovary, suggesting a direct effect on its function. The present study examines the direct role of leptin on the oestrogen-producing activity in human luteinized granulosa cells. The cells were obtained from in-vitro fertilization pre-ovulatory follicles, precultured for 24 h in the presence of 5% charcoal-treated serum, and incubated for 48-96 h in a serum-free medium containing recombinant human leptin, follicle stimulating hormone (FSH), and/or insulin-like growth factor-I (IGF-I). A single addition of leptin (0. 5-10 ng/ml) stimulated aromatase activity with the incubation time of up to 96 h. The addition of leptin (1 ng/ml) further augmented the stimulation by a single addition of FSH (100 ng/ml) or IGF-I (100 ng/ml), or a combination of both. A single addition of leptin (1 ng/ml) or a combination of leptin (1 ng/ml), FSH (100 ng/ml), and IGF-I (100 ng/ml) gave rise to an increase in each parameter of oestrogen-producing activity measured, i.e. P450arom mRNA level, P450arom protein level, aromatase specific activity, and the oestradiol concentration in the culture supernatant. However, the production of progesterone did not change. These results indicate that leptin stimulates oestrogen production by increasing P450arom mRNA and P450arom protein expression and, consequently, aromatase activity by its direct action on the human luteinized granulosa cells.

Aromatase and gynecomastia.

Abstract: An imbalance between estrogen action relative to androgen action at the breast tissue level results in gynecomastia. Enhancement of aromatization of androgens to estrogens is important in the pathogenesis of gynecomastia associated with obesity, aging, puberty, liver disease, thyrotoxicosis, 17-oxosteroid reductase deficiency. Klinefelter's syndrome, and neoplasms of the testes, adrenals and liver. A primary aromatase excess syndrome with exuberant gynecomastia had been found both sporadically and in a familial setting. Although aromatase inhibition would appear to be an important class of drugs to treat gynecomastia, relatively little published data with these drugs exist and most concern the use of delta1-testolactone, which reduces the size of the breast glandular tissue, but does not completely ameliorate the problem. Studies with the newer generation of more potent aromatase inhibitors need to be carried out.

Inhibition of aromatase activity by flavonoids.

Abstract: In searching for potent cancer chemopreventive agents from synthetic or natural products, 28 randomly selected flavonoids were screened for inhibitory effects against partially purified aromatase prepared from human placenta. Over 50% of the flavonoids significantly inhibited aromatase activity, with greatest activity being demonstrated with apigenin (IC50: 0.9 microg/mL), chrysin (IC50: 1.1 microg/mL), and hesperetin (IC50: 1.0 microg/mL).

Estrogen production in endometriosis and use of aromatase inhibitors to treat endometriosis.

Abstract: Estrogen is the most important known factor that stimulates the growth of endometriosis. Estrogen delivery to endometriotic implants was classically viewed to be only via the circulating blood in an endocrine fashion. We recently uncovered an autocrine positive feedback mechanism, which favored the continuous production of estrogen and prostaglandin (PG)E2 in the endometriotic stromal cells. The enzyme, aromatase, is aberrantly expressed in endometriotic stromal cells and catalyzes the conversion of C19 steroids to estrogens, which then stimulate cyclooxygenase-2 to increase the levels of PGE2. PGE2, in turn, is a potent inducer of aromatase activity in endometriotic stromal cells. Aromatase is not expressed in the eutopic endometrium. Aromatase expression in endometriosis and its inhibition in eutopic endometrium are controlled by the competitive binding of a stimulatory transcription factor, steroidogenic factor-1, and an inhibitory factor, chicken ovalbumin upstream promoter-transcription factor to a regulatory element in the aromatase P450 gene promoter. In addition, we find that endometriotic tissue is deficient in 17beta-hydroxysteroid dehydrogenase type 2, which is normally expressed in eutopic endometrial glandular cells and inactivates estradiol-17beta to estrone. This deficiency is another aberration that favors higher levels of estradiol-17beta in endometriotic tissues in comparison with the eutopic endometrium. The clinical relevance of local aromatase expression in endometriosis was exemplified by the successful treatment of an unusually aggressive form of recurrent endometriosis in a postmenopausal woman using an aromatase inhibitor.

Aromatase deficiency caused by a novel P450arom gene mutation: impact of absent estrogen production on serum gonadotropin concentration in a boy.

Abstract: We identified a new point mutation in the CYP19 gene responsible for aromatase (P450arom) deficiency in a 46,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a 1-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of androstenedione at birth, a striking decrease occurred by 4 weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.

Two Organochlorine Pesticides, Toxaphene and Chlordane, Are Antagonists for Estrogen-related Receptor α-1 Orphan Receptor

Abstract: Estrogen-related receptor (ERR) alpha-1 shares a high amino acid sequence homology with estrogen receptor alpha Although estrogens are not ligands of ERR alpha-1, our recent results suggest that toxaphene and chlordane, two organochlorine pesticides with estrogen-like activity, behave as antagonists for this orphan nuclear receptor The two compounds increased ERR alpha-1-mediated expression of the reporter enzyme beta-galactosidase in a yeast-based assay The screen was developed by expressing the hERR alpha-1-yeast Gal 4 activation domain fusion protein in yeast cells carrying the beta-galactosidase reporter plasmid, which contains an ERR alpha-1-binding element In transfection experiments using mammalian cell lines, such as the SK-BR-3 breast cancer cell line, the compounds were found to have an antagonist activity against ERR alpha-1-mediated expression of the reporter chloramphenicol acetyltransferase In contrast to the findings with ERR alpha-1, the two compounds were found to slightly induce the estrogen receptor a-mediated expression of chloramphenicol acetyltransferase in SK-BR-3 cells In a ligand-independent manner, the ERR alpha-1 activity in SK-BR-3 cells was induced 3-fold by cotransfection with the GRIP1 coactivator expression plasmid Toxaphene was found to be capable of suppressing the GRIP1 coactivator-induced ERR alpha-1 activity in SK-BR-3 cells In addition, a stable ERR alpha-1 expressing HepG2 hepatoma cell line was generated, and the aromatase activity in the transfected cell line was found to be twice that in the untransfected cell line The enzyme aromatase converts androgens to estrogens, and aromatase expression in HepG2 cells is regulated in part by an ERR alpha-1-modulating promoter A 24-h incubation of an ERR alpha-1-transfected HepG2 cell line with 10 microM toxaphene reduced its aromatase activity to the level in the untransfected cell line Because toxaphene is not an inhibitor of aromatase, it is thought that the decrease of the aromatase activity in ERR alpha-1 transfected HepG2 cells following toxaphene treatment resulted from a suppression of the aromatase expression by toxaphene acting as the antagonist of ERR alpha-1 Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention Their antagonistic effects on ERR alpha-1 should not be overlooked

Constitutional genetic variation at the human aromatase gene (Cyp19) and breast cancer risk

Abstract: The activity of the aromatase enzyme, which converts androgens into oestrogens and has a major role in regulating oestrogen levels in the breast, is thought to be a contributing factor in the development of breast cancer. We undertook this study to assess the role of constitutional genetic variation in the human aromatase gene (Cyp19) in the development of this disease. Our genotyping of 348 cases with breast cancer and 145 controls (all Caucasian women) for a published tetranucleotide repeat polymorphism at intron 4 of the Cyp19 gene revealed the presence of six common and two rare alleles. Contingency table analysis revealed a significant difference in allelic distribution between cases and controls (χ2 5df = 13.52, P = 0.019). The allele measuring 171 bp was over-represented in cases; of 14 individuals homozygous for this allele, 13 were cases. These individuals had a higher incidence of cancer in family members and an earlier age at diagnosis than other cases. In sequencing Cyp19's coding exons and regulatory regions, we discovered a perfect association between a silent polymorphism (G→A at Val80) and the high-risk genotype. Our conclusion is that constitutional genetic variation at the Cyp19 locus is associated with the risk of developing breast cancer, with the 171-bp allele serving as the high-risk allele. © 1999 Cancer Research Campaign