Brain Aromatase Activity Research Articles

Integration of metabolomics and network pharmacology to validate the mechanism of Schisandra chinensis（Turcz.）Baill - Acorus tatarinowii Schott ameliorating the Alzheimer's disease by regulating the aromatase activity to affect local estrogen in brain of AD model rats

Alzheimer's disease (AD) is a latent and progressive neurodegenerative disease. Schisandra chinensis（Turcz.）Baill - Acorus tatarinowii Schott (Sc-At) are effective in treating neurological disorders.Purpose of this study is to explore the mechanism of Sc-At in AD treatment. First, untargeted ultra-performance liquid chromatography quadrupole-time of flight/mass spectrometer (UPLC-QTOF/MS) metabolomics was employed to detect the rat brain metabolism. Then, network pharmacology was used to determine the potential anti-AD targets. Bioinformatics, and molecular docking were conducted for further analysis. A MetScape study examined the association between differential metabolites and potential targets. Finally, the targeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) metabolomics and the potential protein activity studies were carried out to elucidate the mechanisms. The results showed that Sc-At improved the neuronal cell alignment disorder in hippocampal CA1 region of AD rats. In brain metabolomics, 30 differential metabolites were screened in the study model versus blank group. The network pharmacology analyzed 54 targets of Sc-At anti-AD where, 14 were correlated with amyloid β-protein (Aβ). Aromatase was selected as an important hub target having the best binding power in molecular docking simulation predictions and also correlated with Aβ. Further tests showed that the brain aromatase activity, and the downstream product 17β-Estradiol levels were elevated in AD rats treated with Sc-At. This work may provide new perspectives for the pharmacological effects and the action mechanisms of natural compounds extracts in treating AD progression.

Treatment with androgens plus estrogens cannot reverse sex differences in song and the song control nuclei in adult canaries

Adult treatments with testosterone (T) do not activate singing behavior nor promote growth of song control nuclei to the same extent in male and female canaries (Serinus canaria). Because T acts in part via aromatization into an estrogen and brain aromatase activity is lower in females than in males in many vertebrates, we hypothesized that this enzymatic difference might explain the sex differences seen even after exposure to the same amount of T. Three groups of castrated males and 3 groups of photoregressed females (i.e., with quiescent ovaries following exposure to short days) received either 2 empty 10 mm silastic implants, one empty implant and one implant filled with T or one implant filled with T plus one with estradiol (E2). Songs were recorded for 3 h each week for 6 weeks before brains were collected and song control nuclei volumes were measured in Nissl-stained sections. Multiple measures of song were still different in males and females following treatment with T. Co-administration of E2 did not improve these measures and even tended to inhibit some measures such as song rate and song duration. The volume of forebrain song control nuclei (HVC, RA, Area X) and the rate of neurogenesis in HVC was increased by the two steroid treatments, but remained significantly smaller in females than in males irrespective of the endocrine condition. These sex differences are thus not caused by a lower aromatization of the steroid; sex differences in canaries are probably organized either by early steroid action or by sex-specific gene regulation directly in the brain.

Rapid changes in brain estrogen concentration during male sexual behavior are site and stimulus specific

Classically, estrogens regulate male sexual behavior through effects initiated in the nucleus. However, neuroestrogens, i.e., estrogens locally produced in the brain, can act within minutes via membrane-initiated events. In male quail, rapid changes in brain aromatase activity occur after exposure to sexual stimuli. We report here that local extracellular estrogen concentrations measured by in vivo microdialysis increase during sexual interactions in a brain site- and stimulus-specific manner. Indeed, estrogen concentrations rose within 10 min of the initiation of sexual interaction with a female in the medial preoptic nucleus only, while visual access to a female led to an increase in estrogen concentrations only in the bed nucleus of the stria terminalis. These are the fastest fluctuations in local estrogen concentrations ever observed in the vertebrate brain. Their site and stimulus specificity strongly confirm the neuromodulatory function of neuroestrogens on behavior.

Developing and applying a classification system for ranking the biological effects of endocrine disrupting chemicals on male rockfish Sebastiscus marmoratus in the Maowei Sea, China

Endocrine disrupting compounds (EDCs) in marine environments has become a major environmental concern. Nonetheless, the biological effects of EDCs on organisms in coastal environments remain poorly characterized. In this study, biomonitoring of EDCs in male fish Sebastiscus marmoratus was carried out in the Maowei Sea, China. The results showed that the concentration of 4-nonylphenol (4-NP) was below the detection limit, the concentrations of 4-tert-octylphenol (4-t-OP) and bisphenol A (BPA) in seawater were moderate compared with those in other global regions, and the possible sources are the municipal wastewater discharge. Nested ANOVA analyses suggest significant differences of the brain aromatase activities and plasma vitellogenin (VTG) expression between the port area and the oyster farming area. A new fish expert system (FES) was developed for evaluating the biological effects of EDCs on fish. Our findings show that the FES is a potential tool to evaluate the biological effects of marine pollutants.

Developmental reproductive toxicity and endocrine activity of propiconazole in the Xenopus tropicalis model

Environmental pollutants and especially endocrine disrupting chemicals (EDCs) are implicated as one of the drivers of the amphibian declines. To advance the understanding of the risks of EDCs to amphibians, methods to determine endocrine-linked adverse effects are needed. The aims were to 1) develop a partial life-cycle assay with the model frog Xenopus tropicalis to determine endocrine perturbation and adverse developmental effects, and 2) determine effects of propiconazole in this assay. Propiconazole is a pesticide with multiple endocrine modes of action in vitro. Its potential endocrine activity and adverse effects in amphibians remain to be elucidated. Tadpoles were exposed to 0, 33 and 384 μg propiconazole/L during critical developmental windows until completed metamorphosis. At metamorphosis, a sub-sample of animals was analysed for endpoints for disruption of estrogen/androgen (sex ratio, brain aromatase activity) and thyroid pathways (time to metamorphosis). The remaining individuals were kept unexposed for 2 months post-metamorphosis to analyze effects on sexual development including gonadal and Müllerian duct maturity and gametogenesis. At metamorphosis, brain aromatase activity was significantly increased in the high-dose group compared to control. In both propiconazole groups, an increased proportion of individuals reached metamorphosis faster than the mean time for controls, suggesting a stimulatory effect on the thyroid system. At 2 months post-metamorphosis, testis size, sperm and Müllerian duct maturity were reduced in the low-dose males, and the liver somatic index in males was increased in both propiconazole groups, compared with controls. In conclusion, our results show that propiconazole exposure caused endocrine perturbations and subsequent hepatic and reproductive effects evident at puberty, indicating persistent disruption of metabolism and male reproductive function. Our findings advance the development of methodology to determine endocrine and adverse effects of EDCs. Moreover, they increase the understanding of endocrine perturbations and consequent risk of adverse effects of azoles in amphibians.

Phosphorylation of human placental aromatase CYP19A1.

Aromatase CYP19A1 catalyzes the synthesis of estrogens in endocrine, reproductive and central nervous systems. Higher levels of 17β-estradiol (E2) are associated with malignancies and diseases of the breast, ovary and endometrium, while low E2 levels increase the risk for osteoporosis, cardiovascular diseases and cognitive disorders. E2, the transcriptional activator of the estrogen receptors, is also known to be involved in non-genomic signaling as a neurotransmitter/neuromodulator, with recent evidence for rapid estrogen synthesis (RES) within the synaptic terminal. Although regulation of brain aromatase activity by phosphorylation/dephosphorylation has been suggested, it remains obscure in the endocrine and reproductive systems. RES and overabundance of estrogens could stimulate the genomic and non-genomic signaling pathways, and genotoxic effects of estrogen metabolites. Here, by utilizing biochemical, cellular, mass spectrometric, and structural data we unequivocally demonstrate phosphorylation of human placental aromatase and regulation of its activity. We report that human aromatase has multiple phosphorylation sites, some of which are consistently detectable. Phosphorylation of the residue Y361 at the reductase-coupling interface significantly elevates aromatase activity. Other sites include the active site residue S478 and several at the membrane interface. We present the evidence that two histidine residues are phosphorylated. Furthermore, oxidation of two proline residues near the active site may have implications in regulation. Taken together, the results demonstrate that aromatase activity is regulated by phosphorylation and possibly other post-translational modifications. Protein level regulation of aromatase activity not only represents a paradigm shift in estrogen-mediated biology, it could also explain unresolved clinical questions such as aromatase inhibitor resistance.

Acute exposure to 4-OH-A, not PCB1254, alters brain aromatase activity but does not adversely affect growth in zebrafish

Acute developmental exposure to pharmaceuticals or environmental contaminants can have deleterious, long lasting effects. Many of these compounds are endocrine disruptors (EDCs) that target estrogen signaling, with effects on reproductive and non-reproductive tissues. We recently reported that zebrafish larvae transiently exposed to the pharmaceutical EDC 4-OH-A display visual deficits as adults. Here, we examine whether these long-term effects are due to compound-induced morphological and/or cellular changes. Zebrafish aged 24 h, 48 h, 72 h, or 7 days post-fertilization (larvae) or 3-4mos (adults) were exposed to either 4-OH-A or PCB1254 for 24 h. After that time, notochord length, eye diameter, inter-eye distance, and heart rate were measured from larvae; and aromatase (estrogen synthase) activity was measured in homogenates of adult brain tissue. In general, indices of larval growth and development were not altered by 24 h exposure to either compound. 4-OH-A potently inhibited aromatase activity, while PCB1254 did not, with inhibition continuing even after removal from treatment. These results support differential function of EDCs and indicate that developmental exposure to 4-OH-A causes sustained inhibition of aromatase, which could be associated with altered adult behaviors.

Aromatase immunolocalization and activity in the lizard's brain: Dynamic changes during the reproductive cycle

The purpose of the present study is to highlight the role of aromatase in the neuroendocrine control of the reproductive cycle of the male lizard Podarcis sicula during the three significant phases, i.e. the pre-reproductive, reproductive, and post-reproductive stages. Using immunohistochemical, biochemical, and hormonal tools, we have determined the localization and the activity of P450 aromatase (P450 aro) in the lizard's brain together with the determination of hormonal profile of sex steroids, i.e. testosterone and 17β-estradiol. The present data demonstrated that the localization of P450 is shown in brain regions involved in the regulation of the reproductive behavior (medial septum, preoptic area, and hypothalamus). Its activity, as well as the intensity of the signal, is modified according to the period of reproduction, resulting in functional dynamic changes. P450 aro activity and signal intensity decrease in the pre-reproductive period and progressively increase during the reproductive stage until it reaches the maximum peak level at the post-reproductive phase. P450 aro determines a local estrogen synthesis, balancing the testosterone and estradiol levels, and therefore its role is crucial, since it plays an important role in the neuroendocrine/behavioral regulation of the reproductive processes in the male lizard P. sicula.

Effects of a novel partner and sexual satiety on the expression of male sexual behavior and brain aromatase activity in quail

This study was designed to determine whether changes in sexual motivation acutely regulate brain estrogen synthesis by aromatase. Five experiments (Exp.1–5) were first conducted to determine the effect of recent mating and of the presentation of a new female (Coolidge effect) on sexual motivation. Exp.1–2 showed that 10 min or overnight access to copulation decreases measures of male sexual motivation when male subjects were visually exposed to the female they had copulated with and this effect is not counteracted by the view of a new female. Exp.3 showed that sexual motivation is revived by the view of a new female in previously unmated males only allowed to see another female for 10 min. After mating for 10 min (Exp.4) or overnight (Exp.5) with a female, males showed a decrease in copulatory behavior that was not reversed by access to a new female. Exp.6 and 7 confirmed that overnight copulation (Exp.6) and view of a novel female (Exp.7) respectively decreases and increases sexual behavior and motivation. Yet, these manipulations did not affect brain aromatase activity except in the tuberal hypothalamus. Together these data confirm that copulation or prolonged view of a female decrease sexual motivation but a reactivation of sexual motivation by a new female can only be obtained if males had only seen another female but not copulated with her, which is different in some degree from the Coolidge effect described in rodents. Moreover changes in brain aromatase do not simply reflect changes in motivation and more complex mechanisms must be considered.

Cognitive Effects of Aromatase and Possible Role in Memory Disorders.

Diverse cognitive functions in many vertebrate species are influenced by local conversion of androgens to 17β-estradiol (E2) by aromatase. This enzyme is highly expressed in various brain regions across species, with some inter-species variation in terms of regional brain expression. Since women with breast cancer and men and women with other disorders are often treated with aromatase inhibitors (AI), these populations might be especially vulnerable to cognitive deficits due to low neuroE2 synthesis, i.e., synthesis of E2 directly within the brain. Animal models have been useful in deciphering aromatase effects on cognitive functions. Consequences of AI administration at various life cycle stages have been assessed on auditory, song processing, and spatial memory in birds and various aspects of cognition in rodent models. Additionally, cognitive deficits have been described in aromatase knockout (ArKO) mice that systemically lack this gene throughout their lifespan. This review will consider evidence to date that AI treatment in male and female rodent models, birds, and humans results in cognitive impairments. How brain aromatase regulates cognitive function throughout the lifespan, and gaps in current knowledge will be considered, along with future directions to better define how aromatase might guide learning and memory from early development through the geriatric period. Better understanding the importance of E2 synthesis on neurobehavioral responses at various ages will likely aid in the discovery of therapeutic strategies to prevent potential cognitive deficits, including Alzheimer's Disease, in individuals treated with AI or those possessing CYP19 gene polymorphisms, as well as cognitive effects of normal aging that may be related to changes in brain aromatase activity.

Joint effects of chronic exposure to environmentally relevant levels of nonylphenol and cadmium on the reproductive functions in male rockfish Sebastiscus marmoratus

Nonylphenol (NP) and Cadmium (Cd) are two common contaminants that can be detected in aquatic environments. Nevertheless, the combined toxicity of NP and Cd at environmentally relevant concentrations in aquatic organisms has not been thoroughly characterized to date. In the present study, the interactions between NP and Cd on male Sebastiscus marmoratus were studied. After 21 days of exposure, the brain aromatase activity was observed to be significantly induced by 100 ng/L NP and 40 μg/L Cd, whereas all of the concentrations of co-treatment resulted in an increase in brain aromatase activity. Additionally, NP could also reduce plasma testosterone concentration, while NP, Cd and their mixture could induce plasma 17β-estradiol (E2) concentration and VTG concentration. The interactions between NP and Cd on the reproductive physiology were antagonism. Our results also support the notion of using these indicators as biomarkers for exposure to EDCs and further extend the boundary of biomonitoring to environmental levels.

Effects of bisphenol A (BPA) on brain-specific expression of cyp19a1b gene in swim-up fry of Labeo rohita

Estrogen regulates numerous developmental and physiological processes and effects are mediated mainly by estrogenic receptors (ERs), which function as ligand-regulated transcription factor. ERs can be activated by many different types endocrine disrupting chemicals (EDCs) and interfere with behaviour and reproductive potential of living organism. Estrogenic regulation of membrane associated G protein-coupled estrogen receptor, GPER activity has also been reported. Bisphenol A (BPA), a ubiquitous endocrine disruptor is present in many household products, has been linked to many adverse effect on sexual development and reproductive potential of wild life species. The present work is aimed to elucidate how an environmentally pervasive chemical BPA affects in vivo expression of a known estrogen target gene, cyp19a1b in the brain, and a known estrogenic biomarker, vitellogenin (Vg) in the whole body homogenate of 30 days post fertilization (dpf) swim-up fry of Labeo rohita. We confirm that, like estrogen, the xenoestrogen BPA exposure for 5–15 days induces strong overexpression of cyp19a1b, but not cyp19a1a mRNA in the brain and increase concentration of vitellogenin in swim-up fry. BPA also induces strong overexpression of aromatase B protein and aromatase activity in brain. Experiments using selective modulators of classical ERs and GPER argue that this induction is largely through nuclear ERs, not through GPER. Thus, BPA has the potential to elevate the levels of aromatase and thereby, levels of endogenous estrogen in developing brain. These results indicate that L. rohita swim-up fry can be used to detect environmental endocrine disruptors either using cyp19a1b gene expression or vitellogenin induction.

Estrogen-regulated expression of P450arom genes in the brain and ovaries of adult female Indian climbing perch, Anabas testudineus.

Cytochrome P450arom (CYP19), a product of cyp19a1 gene, catalyzes the conversion of androgens to estrogens and is essential for regulation of reproductive function in vertebrates. In the present study, we isolated partial cDNA encoding the ovarian (cyp19a1a) and brain (cyp19a1b) P450arom genes from adult female perch, Anabas testudineus and investigated their regulation by estrogen in vivo. Results demonstrated that cyp19a1a and cyp19a1b predominate in ovary and brain respectively, with quantity of both attuned to reproductive cycle. To elucidate estrogen-regulated expression of cyp19a1b in brain and cyp19a1a in ovary, dose- and time-dependent studies were conducted with estrogen in vitellogenic-stage fish in the presence or absence of specific aromatase inhibitor fadrozole. Results demonstrated that treatment of fish with 17β-estradiol (E2; 1.0 μM)) for 6 days caused significant upregulation of cyp19a1b transcripts, aromatase B protein, and aromatase activity in brain in a dose- and time-dependent manner. Ovarian cyp19a1a mRNA, aromatase protein, and aromatase activity, however, was less responsive to E2 than brain. Treatment of fish with an aromatase inhibitor fadrozole for 6 days attenuated both brain and ovarian cyp19a1 mRNAs expression and stimulatory effects of E2 was also significantly reduced. These results indicate that expression of cyp19a1b in brain and cyp19a1a in ovary of adult female A. testudineus was closely associated to plasma E2 levels and seasonal reproductive cycle. Results further show apparent differential regulation of cyp19a1a and cyp19a1b expression by E2/fadrozole manipulation.

Rapid changes in brain aromatase activity in the female quail brain following expression of sexual behaviour.

In male quail, oestrogens produced in the brain (neuro-oestrogens) exert a dual action on male sexual behaviour: they increase sexual motivation within minutes via mechanisms activated at the membrane but facilitate sexual performance by slower, presumably nuclear-initiated, mechanisms. Recent work indicates that neuro-oestrogens are also implicated in the control of female sexual motivation despite the presence of high circulating concentrations of oestrogens of ovarian origin. Interestingly, aromatase activity (AA) in the male brain is regulated in time domains corresponding to the slow "genomic" and faster "nongenomic" modes of action of oestrogens. Furthermore, rapid changes in brain AA are observed in males after sexual interactions with a female. In the present study, we investigated whether similar rapid changes in brain AA are observed in females allowed to interact sexually with males. A significant decrease in AA was observed in the medial preoptic nucleus after interactions that lasted 2, 5 or 10minutes, although this decrease was no longer significant after 15minutes of interaction. In the bed nucleus of the stria terminalis, a progressive decline of average AA was observed between 2 and 15minutes, although it never reached statistical significance. AA in this nucleus was, however, negatively correlated with the sexual receptivity of the female. These data indicate that sexual interactions affect brain AA in females as in males in an anatomically specific manner and suggest that rapid changes in brain oestrogens production could also modulate female sexual behaviour.

Estrogen-regulated expression of cyp19a1a and cyp19a1b genes in swim-up fry of Labeo rohita

P450 aromatase is the terminal enzyme in the steroidogenic pathway and catalyzes the conversion of androgens to estrogens. The expression of cyp19a1 genes in brain and gonad of Indian major carp, Labeo rohita swim-up fry was measured by quantitative real-time polymerase chain-reaction. Results demonstrated that cyp19a1b and cyp19a1a predominate in brain and gonad respectively. Treatment of fry with an aromatase inhibitor fadrozole for 6days attenuated brain cyp19a1b expression, but not cyp19a1a of gonad. Fadrozole also attenuated brain aromatase activity. Treatment with 17β-estradiol (E2) for 6days resulted in up-regulation of brain cyp19a1b transcripts in a dose- and time-dependent manner, but not cyp19a1a. Whole-body concentration of vitellogenin also increased in response to E2. Altogether, these results indicate L. rohita swim-up fry can be used to detect environmental estrogens either using vitellogenin induction or cyp19a1b gene expression.

Glutamate released in the preoptic area during sexual behavior controls local estrogen synthesis in male quail

Estrogens are known to act rapidly, probably via membrane estrogen receptors, to induce fast effects on physiological and behavioral processes. Engaging in some of these behaviors, such as sexual behavior, results in an acute modulation of the production of estrogens in the brain by regulating the efficiency of the estrogen synthase enzyme, aromatase. We recently demonstrated that aromatase activity (AA) in the male quail brain is rapidly inhibited in discrete brain regions including the medial preoptic nucleus (POM) following exposure to a female. Evidence from in vitro studies point to glutamate release as one of the mechanisms controlling these rapid regulations of the aromatase enzyme. Here, we show that (a) the acute injection of the glutamatergic agonist kainate into the POM of anesthetized male quail inhibits AA and (b) glutamate is released in the POM during copulation. These results provide the first set of in vivo data demonstrating a role for glutamate release in the rapid control of AA in the context of sexual behavior.

Region-specific rapid regulation of aromatase activity in zebra finch brain.

Recent studies demonstrate that rapid modulation of the estrogen synthetic enzyme aromatase, regulates hypothalamic (HYP) estrogen production, and subsequent neurophysiology and reproductive behavior. In songbirds, in addition to expression in the HYP, aromatase is expressed at high levels in several brain regions notably in the hippocampus (HP) and caudomedial nidopallium (NCM), where estrogens affect learning and memory and auditory processing, respectively. Previous studies, largely in quail HYP, show that aromatase activity is acutely down-regulated by Ca2+ -dependent phosphorylation. Here, using zebra finches (Taeniopygia guttata), we ask if similar mechanisms are at work in the songbird HYP and if there are sex as well as regional differences in aromatase modulation. Using invitro assays to measure activity in homogenates or in partially purified supernatants containing microsomes and synaptosomes of the HP, HYP, and NCM, we examined effects of Ca2+ , Mg2+ , ATP, NADPH, and an inhibitor of kinase activity. We report a rapid down-regulation of aromatase activity in the presence of phosphorylating conditions across all three brain regions and both sexes. However, regional differences were seen in response to some phosphorylating factors, some of which were improved by partial purification of the homogenates. Furthermore, while low concentrations of ATP inhibited aromatase activity, unexpectedly, inhibition was no longer seen with high ATP concentrations. These results provide evidence for a regional and temporal specificity in the rapid modulation of aromatase activity that may bear on local neuroendocrine function. Aromatase activity in male and female zebra finch hippocampus, hypothalamus, and caudomedial nidopallium is rapidly regulated by Ca2+ -dependent phosphorylation. Low ATP and Mg2+ decrease activity, whereas nicotinamide adenine dinucleotide phosphate (NADPH), high ATP, and inhibition of protein kinase C increase activity. Evidence suggests this may occur at the synapse. These results provide a mechanism for rapid regulation of behavior via brain estrogen synthesis.

Effects of short-term exposure to the model anti-androgen, flutamide on reproductive function based endpoints in female Murray rainbowfish (Melanotaenia fluviatilis)

The aim of the present study was to evaluate the responses of female Murray rainbowfish (Melanotaenia fluviatilis) to the model anti-androgen, flutamide in a short-term exposure. Adult female Murray rainbowfish were exposed to nominal (measured) concentrations of 125 (104), 250 (163), 500 (378) and 1000 (769)µg/L of flutamide for seven days in a semi-static set-up. Plasma vitellogenin (VTG), 11-keto testosterone (11-KT) and 17β-estradiol (E2) concentrations, brain aromatase activity and ovarian histology were assessed following the exposure. No treatment-related mortality was found in rainbowfish and there was no effect of flutamide on the developmental stage of the ovaries. Histological investigation revealed absence of mature oocytes in flutamide-treated fish. In addition, a significant reduction in the sizes of the vitellogenic oocytes was found after treatment with 500 and 1000µg/L flutamide. The circulating levels of VTG and the activity of aromatase in the brain were also significantly reduced in fish treated with 500 and 1000µg/L flutamide. Treatment with higher concentrations of flutamide reduced the levels of 11-KT and E2 in plasma. The results from this study demonstrate that a short-term exposure to the model anti-androgen, flutamide can adversely affect the reproductive function based on end-points such as plasma VTG, 11-KT and E2; brain aromatase activity and sizes of the oocytes in female Murray rainbowfish. Further, a positive correlation between these experimental variables suggests hormonal imbalance.

Effects of nonylphenol and ethinylestradiol on copper redhorse (Moxostoma hubbsi), an endangered species

The copper redhorse, Moxostoma hubbsi, is an endangered species endemic to Quebec. The presence of contaminants, in particular endocrine disrupting chemicals (EDCs), in its habitat has been advanced as partly responsible for the reproductive difficulties encountered by the species. In the present study, immature copper redhorse were exposed to the estrogenic surfactant nonylphenol (NP; 1, 10 and 50µg/l) and the synthetic estrogen 17α-ethinylestradiol (EE2; 10ng/l) for 21 days in a flow-through system. The endpoints investigated included general health indicators (hepatosomatic index and hematocrit), thyroid hormones, sex steroids, brain aromatase activity, plasma and mucus vitellogenin (VTG), cytochrome P4501A protein expression and ethoxyresorufin-O-deethylase activity, heat shock protein 70 (HSP70) and muscle acetylcholinesterase. Exposure to 10ng EE2/l significantly increased brain aromatase activity. Exposure to 50µg NP/l resulted in a significant reduction of plasma testosterone concentrations and a significant induction of hepatic HSP70 protein expression. NP at 50µg/l also induced plasma and mucus VTG. The presence of elevated VTG levels in the surface mucus of immature copper redhorse exposed to NP, and its correlation to plasma VTG, supports the use of mucus VTG as a non-invasive biomarker to evaluate copper redhorse exposure to EDCs in the environment and contribute to restoration efforts of the species. The results of the present study indicate that exposure to high environmentally relevant concentrations of NP and EE2 can affect molecular endpoints related to reproduction in the copper redhorse.

Effects of the commercial antiandrogen flutamide on the biomarkers of reproduction in male Murray rainbowfish (Melanotaenia fluviatilis)

The endocrine responses in male Murray rainbowfish (Melanotaenia fluviatilis) were evaluated after exposures to biologically active concentrations of the nonsteroidal pharmaceutical, flutamide. Fish were exposed to nominal concentrations of 125 µg/L, 250 µg/L, 500 µg/L, and 1000 µg/L of flutamide for 7 d, after which plasma vitellogenin concentration; brain aromatase activity; and hepatic expression of the genes for vitellogenin, choriogenin, and androgen and estrogen receptors were assessed. Qualitative assessment of the testes of the fish exposed to flutamide exhibited hindrance in the transformation of spermatogonia to spermatozoa and increased testicular anomalies, such as multinucleated and pyknotic cells and interstitial fibrosis. An increase in the hepatosomatic index with respect to the controls was noted after treating the fish with flutamide at all concentrations. Vitellogenin was induced in plasma in the 1000 µg/L flutamide group. The activity of aromatase in the brain declined significantly after exposures to flutamide at all concentrations. Males exposed to 1000 µg/L of flutamide showed a downregulation in the genes encoding androgen receptors α and β. The expression of the gene for the estrogen receptor α was induced and of vitellogenin was downregulated after treatment with 250 µg/L to 1000 µg/L of flutamide. The results suggest that 7-d exposures to 125 µg/L to 1000 µg/L flutamide can impair the reproductive endocrine system in male Murray rainbowfish at multiple levels by an antiandrogenic mode of action.