Brain Aromatase Gene Research Articles

Oxidative stress induced by hydrogen peroxide disrupts zebrafish visual development by altering apoptosis, antioxidant and estrogen related genes

Hydrogen peroxide is considered deleterious molecule that cause cellular damage integrity and function. Its key redox signaling molecule in oxidative stress and exerts toxicity on a wide range of organisms. Thus, to understand whether oxidative stress alters visual development, zebrafish embryos were exposed to H2O2 at concentration of 0.02 to 62.5 mM for 7 days. Eye to body length ratio (EBR) and apoptosis in retina at 48 hpf, and optomotor response (OMR) at 7 dpf were all measured. To investigate whether hydrogen peroxide-induced effects were mediated by oxidative stress, embryos were co-incubated with the antioxidant, glutathione (GSH) at 50 μM. Results revealed that concentrations of H2O2 at or above 0.1 mM induced developmental toxicity, leading to increased mortality and hatching delay. Furthermore, exposure to 0.1 mM H2O2 decreased EBR at 48 hpf and impaired OMR visual behavior at 7 dpf. Additionally, exposure increased the area of apoptotic cells in the retina at 48 hpf. The addition of GSH reversed the effects of H2O2, suggesting the involvement of oxidative stress. H2O2 decreased the expression of eye development-related genes, pax6α and pax6β. The expression of apoptosis-related genes, tp53, casp3 and bax, significantly increased, while bcl2α expression decreased. Antioxidant-related genes sod1, cat and gpx1a showed decreased expression. Expression levels of estrogen receptors (ERs) (esr1, esr2α, and esr2β) and ovarian and brain aromatase genes (cyp19a1a and cyp19a1b, respectively) were also significantly reduced. Interestingly, co-incubation of GSH effectivity reversed the impact of H2O2 on most parameters. Overall, these results demonstrate that H2O2 induces adverse effects on visual development via oxidative stress, which leads to alter apoptosis, diminished antioxidant defenses and reduced estrogen production.

Uma abordagem de multibiomarcadores para investigar os efeitos do paracetamol no eixo de regulação da reprodução de machos do bagre Neotropical Rhamdia quelen

Abstract: Aim Paracetamol (PCM), or acetaminophen, is one of the most used drugs for human treatment of pain and fever. Since it has often been found in the aquatic environment, the aim of this study was to investigate the effects of PCM on the reproductive axis of male Rhamdia quelen catfish. Methods Different biomarkers were evaluated in hypothalamus, liver and gonads, as well as the plasma sexual hormone quantification. The fish were exposed to three PCM concentrations: 0.25, 2.5 and 25 µg.L-1 and to a control group (solvent acetone 0.0003%). After 14 days of exposure, fish were anesthetized, for blood sampling and biometrics, and after euthanasia, the tissues were sampled. Plasma was used for 11- keto testosterone and 17β - estradiol quantification. The hypothalamus was collected for brain aromatase (cyp19a1b) gene expression; the liver for the vitellogenin (vtg) gene expression and biochemical biomarkers; and gonad for the biochemical and histological biomarkers analyses. Results No alterations were observed in the hormone’s levels, sexual maturation level or in cyp19a1b and vtg gene expression. In the liver the non-protein thiols concentration increased at 2.5 µg.L-1 of PCM, and the superoxide dismutase (SOD) activity was reduced at 0.25 µg.L-1 of PCM. In gonads, glutathione S-transferase (GST) activity decreased and SOD activity increased at 25 µg.L-1 of PCM, while the glutathione peroxidase (GPx) activity reduced after exposure to both PCM concentrations. Conclusion The results showed that environmental concentrations of PCM can cause alterations in the antioxidant system, mainly in the gonads of R. quelen males. However, without significant change in the hormones levels or in the expression of genes related to the reproduction axis. These alterations occurred at concentrations already found in aquatic environment, including in Brazil.

Toxicity effects of the organic UV-filter 4-Methylbenzylidene camphor in zebrafish embryos

Ultraviolet (UV) filters are widely used in personal care products and due to their lipophilicity these chemicals tend to bioaccumulate in the aquatic biota. 4-Methylbenzylidene camphor (4-MBC) is one of the most used UV-filters, and it is commonly detected in freshwater fish tissues. This substance is suspected to be an endocrine disruptor due to its interaction with Hypothalamus-Pituitary-Gonadal (HPG) and HP-Thyroid (HPT)-axis. The main objective of this study was to evaluate the effects of 4-MBC on apical endpoints, biochemical markers and on genes involved in endocrine pathways in Danio rerio. Zebrafish embryos were exposed to 4-MBC (0.083–0.77 mg/l) from 0 to 96 h post-fertilization (hpf). Hatching, heart rate and malformations were the apical endpoints assessed. Alterations on neurotransmission and oxidative stress were evaluated through acetylcholinesterase (AChE), catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. Endocrine effects were analysed by the expression of genes involved in HPG and HPT-axis of embryos exposed 96 h to the EC10 of 4-MBC (0.19 mg/l). Exposure to 4-MBC induced morphological abnormalities during embryonic development, including notochord curvature, delayed absorption of yolk sac and pericardial oedema. Concentration of 0.77 mg/l 4-MBC decreased embryo heart rate at 48h. At neurotransmission level, an induction of AChE at concentrations above 0.15 mg/l was observed. Malformations and decreased heart rate along with alterations observed at neurotransmission level might have compromised zebrafish larvae equilibrium. Glutathione S-transferase induction above 0.15 mg/l 4-MBC suggests activation of detoxification processes. Furthermore, observed brain aromatase gene down-regulation by 4-MBC suggests impairment of normal functioning of HPG axis in zebrafish.

Dynamic regulation of brain aromatase and isotocin receptor gene expression depends on parenting status

Fathering behavior is critical for offspring survival in many species across diverse taxa, but our understanding of the neuroendocrine mechanisms regulating paternal care is limited in part because of the few primarily paternal species among the common animal models. However, many teleosts display primarily paternal care, and among the teleosts, anemonefish species are particularly well suited for isolating molecular mechanisms of fathering as they perform parental care in isolation of many other typically competing behaviors such as territorial defense and nest building. The goal of this study was to determine the extent to which whole brain gene expression levels of isotocin receptors, arginine vasotocin receptors, and aromatase as well as circulating levels of the bioactive sex steroid hormones estradiol (E2) and 11-ketotestosterone (11KT) vary in association with parenting behavior in Amphiprion ocellaris. Brain aromatase and IT receptor gene expression were higher in both males and females that were parenting versus not. IT receptor expression was overall higher in males than females, which we interpret is a reflection of the greater parental effort that males display. Aromatase was overall higher in females than males, which we conclude is related to the higher circulating E2, which crosses into the brain and increases aromatase transcription. Results suggest both aromatase and IT receptors are dynamically upregulated in the brains of A. ocellaris males and females to support high levels of parental effort.

Triclosan Lacks Anti-Estrogenic Effects in Zebrafish Cells but Modulates Estrogen Response in Zebrafish Embryos.

Triclosan (TCS), an antimicrobial agent widely found in the aquatic environment, is suspected to act as an endocrine disrupting compound, however mechanistic information is lacking in regards to aquatic species. This study assessed the ability of TCS to interfere with estrogen receptor (ER) transcriptional activity, in zebrafish-specific in vitro and in vivo reporter gene assays. We report that TCS exhibits a lack of either agonistic or antagonistic effects on a panel of ER-expressing zebrafish (ZELH-zfERα and -zfERβ) and human (MELN) cell lines. At the organism level, TCS at concentrations of up to 0.3 µM had no effect on ER-regulated brain aromatase gene expression in transgenic cyp19a1b-GFP zebrafish embryos. At a concentration of 1 µM, TCS interfered with the E2 response in an ambivalent manner by potentializing a low E2 response (0.625 nM), but decreasing a high E2 response (10 nM). Altogether, our study suggests that while modulation of ER-regulated genes by TCS may occur in zebrafish, it does so irrespective of a direct binding and activation of zfERs.

Mixture Concentration-Response Modeling Reveals Antagonistic Effects of Estradiol and Genistein in Combination on Brain Aromatase Gene (cyp19a1b) in Zebrafish.

Comprehension of compound interactions in mixtures is of increasing interest to scientists, especially from a perspective of mixture risk assessment. However, most of conducted studies have been dedicated to the effects on gonads, while only few of them were. interested in the effects on the central nervous system which is a known target for estrogenic compounds. In the present study, the effects of estradiol (E2), a natural estrogen, and genistein (GEN), a phyto-estrogen, on the brain ER-regulated cyp19a1b gene in radial glial cells were investigated alone and in mixtures. For that, zebrafish-specific in vitro and in vivo bioassays were used. In U251-MG transactivation assays, E2 and GEN produced antagonistic effects at low mixture concentrations. In the cyp19a1b-GFP transgenic zebrafish, this antagonism was observed at all ratios and all concentrations of mixtures, confirming the in vitro effects. In the present study, we confirm (i) that our in vitro and in vivo biological models are valuable complementary tools to assess the estrogenic potency of chemicals both alone and in mixtures; (ii) the usefulness of the ray design approach combined with the concentration-addition modeling to highlight interactions between mixture components.

Social factors and aromatase gene expression during adult male-to-female sex change in captive leopard grouper Mycteroperca rosacea

Social factors and aromatase gene expression in the leopard grouper Mycteroperca rosacea was studied when captive fish were separated by sex during the reproductive (April–June) and post-reproductive (July–September) seasons. Monosex females, monosex males, and mixed-sex, held in social sextet units were analyzed for sex steroids throughout confinement. At the end of the experiment, the gonad-sex was defined by histology, and gonad and brain aromatase gene expressions were quantified. Only males held in the monosex social units changed sex. Histology showed one male remained unchanged, six were found in a transitional sexual stage, in which two had intersex-predominantly-testes, and four had a more defined intersex ovo-testes pattern, and 11 were immature de novo females (neofemales). Neofemales and most intersex fish did not survive. In spring, 11-ketosterone showed a specific male profile, which suggests that male-to-female sex change was not triggered during the reproductive season. The low steroid levels in summer made it impossible to associate the sex change to a gonad hormonal shift; in September, gonad aromatase gene expression was not significantly different among groups. However, brain aromatase expression in intersex fish was significantly higher than monosex females, mixed-sex females, and neofemale groups. These results suggest that in the absence of female hormonal compounds, and at a time when male gonad steroidogenesis was diminished, the brain mediated male-to-male social-behavioral interactions, including stress, by increasing aromatization, resulting in derived intersex-male, which triggered more aromatization, followed by a sex change.

Cloning of gonadal aromatase gene Cyp19a in an endemic fish (Schizothorax kozlovi) of the Upper Yangtze River, and temperature effects on its expression

In order to elucidate the role of Cyp19a in sex differentiation of Schizothorax kozlovi, the full length cDNA of Cyp19a was cloned from the mature ovary of S. kozlovi by using rapid amplification of cDNA ends method, and then its relative mRNA expression levels among tissues and temperature groups were determined by using quantitative real-time PCR. The complete Cyp19a cDNA of 1795 bp of S. kozlovi was obtained, which encoded 517 amino acids and belonged to gonadal aromatase. Its deduced amino acid sequence had the above 70 % identity compared with gonadal aromatase genes of teleost fishes, but only 62–67 % when compared with brain aromatase genes of fishes. It was expressed only in heart and gonad, but no expression in other tissues, presenting relatively high tissue specificity. It also exhibited sex-specific expression pattern in gonads, but no sex differences in heart. Comparing with the Cyp19a expression levels at 12 days post hatching (dph), significant temperature effects were revealed in low temperature group (10 °C) at 18 dph, and in high temperature group (26 °C) at 40 dph. It suggested that gonadal aromatase Cyp19a gene may play important roles on the feminization or masculinization of S. kozlovi affected by temperature during the early developmental stage.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata)

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Social Interaction Influences Blood Cortisol Values and Brain Aromatase Genes in the Protandrous False Clown Anemonefish,Amphiprion ocellaris

Anemonefish, Amphiprion spp., are socially controlled, protandrous sex changers with a monogamous mating system. Under certain conditions, sexually immature anemonefish with ambisexual gonads differentiate directly into males or females. Formation and maintenance of social rank in a group are considered key requirements for the induction of sex change or differentiation. Generally, each animal living in a social group experiences a different level of social stress in accordance with its social rank, and we hypothesize that the stress situation of individual anemonefish influences its sex determination. Groups of three sexually immature anemonefish were placed into each of five experimental tanks and kept for 10 days to allow for social rank formation and behavioral observation. The fish were then euthanized, and blood and brain samples were collected from each fish. The social rank of each individual was distinguishable from day 1 of the experiment. Aggressive behaviors were most frequent and blood Cortisol values were higher in dominant individuals. The transcription of mRNA for stress-related genes, i.e., those encoding for glucocorticoid and arginine vasotocin receptors, was higher in the brains of dominant individuals than in other social ranks. Furthermore, we detected higher transcription levels of gonad and brain aromatase genes, which encode the enzyme that converts androgens into estrogens, in the brains of dominant individuals. These results suggest that social rank reflects the blood Cortisol value, which in turn leads to sex differentiation by manipulating transcription of genes, including aromatase genes, in the brain.

Increase in estrogen signaling in the early brain of orange-spotted grouper Epinephelus coioides: a mini-review

Despite neurosteroidogenic enzymes are playing important roles in the regulation of brain development and function, the potential link between brain and gonad by the action of steroid hormones during gonadal sex differentiation is still not clear in teleosts. In this mini-review, we summarized our understanding on the early brain development related to the synthesis of neurosteroids and receptor signaling during gonadal sex differentiation in protogynous orange-spotted grouper, Epinephelus coioides (functional females for the first 6years of life and start to sex change around the age of 7years) and protandrous black porgy (functional males for the first 2years of life but begin to change sex during the third year). We found a similar profile in the increased expression of brain aromatase gene (aromatatse B or cyp19a1b), aromatase activity, estradiol (E(2)), and estrogen signaling in the brain of both grouper and black porgy fish during gonadal sex differentiation. In contrast to mammals, teleost fish Cyp19a1b expressed in a unique cell type, a radial glial cell, which is acted as progenitors in the brain of developing and adult fish. In agreement with these pioneer studies, we demonstrated that the grouper cyp19a1b/Cyp19a1b was expressed in radial glial cells. Further, in vivo data in the grouper brain showed that exogenous E(2) upregulated Cyp19a1b immunoreactivity (ir) in radial glial cells. These data suggest the possible roles of Cyp19a1b and E(2) in early brain development which is presumably related to gonadal sex differentiation.

FTZ-F1 and FOXL2 up-regulate catfish brain aromatase gene transcription by specific binding to the promoter motifs

Cytochrome P450 aromatase (cyp19) catalyzes the conversion of androgens into estrogens. Teleosts have distinct, ovarian specific ( cyp19a1a) and brain specific ( cyp19a1b) cyp19 genes. Previous studies in teleosts demonstrated regulation of cyp19a1a expression by the NR5A nuclear receptor subfamily as well as a fork head transcription factor, FOXL2. In the present study, we investigated the involvement of fushi tarazu factor 1, FTZ-F1, a NR5A subfamily member, and FOXL2 in the regulation of cyp19a1b expression in brain of the air-breathing catfish, Clarias gariepinus. Based on the synchronous expression pattern of cyp19a1b, FTZ-F1 and FOXL2 in the brain, we isolated the 5′ upstream region of cyp19a1b to analyse regulatory motifs. Promoter motif analysis revealed FTZ-F1/NR5A1 and FOXL2 binding nucleotide sequences. Transient transfection studies showed that FTZ-F1 and FOXL2 together enhanced the transcriptional activity of cyp19a1b gene in mammalian cell lines. Mutation in either of their putative binding sites within the cyp19a1b promoter abolished this effect. Electrophoretic gel mobility shift experiments indicated that FTZ-F1 and FOXL2 proteins bind to the synthesized radio-labelled oligomers used as probes and mobility shifted upon addition of their respective antibodies. Chromatin immunoprecipitation assay confirmed the binding of both these transcription factors to their corresponding cis-acting elements in the upstream region of cyp19a1b. To our knowledge, this study is the first report on the transcriptional regulation of cyp19a1b by FTZ-F1 and FOXL2 in a teleost fish.

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up-regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 x 10(-6), 40 x 10(-6) and 80 x 10(-6) M of benomyl-treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase-based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 x 10(-6) M concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds.

Characterization of a cis‐acting element involved in cell‐specific expression of the zebrafish brain aromatase gene

The cytochrome P450 Aromatase is the key enzyme catalyzing the conversion of androgens into estrogens. In zebrafish, the brain aromatase is encoded by cyp19b. Expression of cyp19b is restricted to radial glial cells bordering forebrain ventricles and is strongly stimulated by estrogens during development. At the promoter level, we have previously shown that an estrogen responsive element (ERE) is required for induction by estrogens. Here, we investigated the role of ERE flanking regions in the control of cell-specific expression. First, we show that a 20 bp length motif, named G x RE (glial x responsive element), acts in synergy with the ERE to mediate the estrogenic induction specifically in glial cells. Second, we demonstrate that, in vitro, this sequence binds factors exclusively present in glial or neuro-glial cells and is able to confer a glial specificity to an artificial estrogen-dependent gene. Taken together, these results contribute to the understanding of the molecular mechanisms allowing cyp19b regulation by estrogens and allowed to identify a promoter sequence involved in the strong estrogen inducibility of cyp19b which is specific for glial cells. The exceptional aromatase activity measured in the brain of teleost fish could rely on such mechanisms.

Cloning and differential expression of estrogen receptor and aromatase genes in the self-fertilizing hermaphrodite and male mangrove rivulus, Kryptolebias marmoratus

The mechanisms underlying sex determination and differentiation in fishes are labile in response to environmental parameters. Sex-specific phenotypes are largely regulated by sex steroids, and the inhibition or the stimulation of aromatase can reverse sex as well as alter secondary sexual characteristics in fishes. Among vertebrates, the mangrove rivulus is the only known self-fertilizing hermaphrodite. Throughout most of its range, rivulus appear to exist as clonally reproducing hermaphrodites. However, outcrossing has been documented in Belize, where up to 25% of rivulus collected are males. The direct development of (primary) males occurs when embryos are incubated at 18 degrees C and hermaphrodites develop into secondary males when held at 28 degrees C. Given the importance of sex steroids, their receptors, and aromatase in sex determination and differentiation of fishes, we cloned, sequenced, and quantified the expression of estrogen receptors (ERalpha, ERbeta) and ovarian (AroA) and brain (AroB) aromatase genes. Hermaphrodites had increased ERalpha, ERbeta, AroA, and AroB gene expression in the liver, gonad, gonad, and brain respectively, compared to males. These data are consistent with the gene expression data reported for other species and are reflective of the presence of ovarian tissue in the hermaphrodites. Interestingly, we show the elevated expression of brain aromatase in the hermaphrodite brain. The role of the dimorphic expression of brain aromatase in the regulation of sex-specific characteristics is intriguing and requires further research. Because of the uniqueness of its reproductive biology, rivulus is an excellent model for elucidating the mechanisms regulating vertebrate sex determination and sexual differentiation.

Brain Cytochrome P450 Aromatase Gene Isoforms and Activity Levels in Atlantic Salmon After Waterborne Exposure to Nominal Environmental Concentrations of the Pharmaceutical Ethynylestradiol and Antifoulant Tributyltin

In this study, the effects of two environmental endocrine disruptors, the synthetic pharmaceutical estrogen (ethynylestradiol, EE2) and antifoulant (tributyltin, TBT) representing two different modes of action on the endocrine system, were studied on brain steroidogenic pathway of juvenile Atlantic salmon (Salmo salar). Neurosteroidogenesis was studied using brain aromatase gene isoforms and enzyme activity, in parallel with typical xenoestrogen responses, such as brain estrogen receptor (ERalpha) and plasma vitellogenin (Vtg) levels. Fish were exposed to nominal waterborne EE2 (5 and 50 ng/l) and TBT (50 and 250 ng/l) concentrations dissolved in dimethyl sulfoxide (DMSO), singly and in combination. Gene expressions were quantified using real-time PCR with gene-specific primers, aromatase activity was analyzed using the tritiated water-release assay, and plasma Vtg was analyzed using competitive ELISA. Our data show that EE2 induced a concentration-specific modulation of P450aromA, P450aromB, and aromatase activity in addition to ERalpha and plasma Vtg levels in juvenile salmon at day 3 postexposure. TBT exposure caused both the elevation and inhibition of P450aromA, P450aromB, and aromatase activity levels, depending on concentration, at day 7 postexposure. TBT elevated and inhibited ERalpha and plasma Vtg and also antagonized EE2-induced expression of the studied variables at day 7 postexposure. Interestingly, the carrier vehicle DMSO modulated the receptor-mediated and non-receptor-mediated estrogenic responses at day 7 postexposure, compared to day 3. In general, these findings suggest that the exposed animals are experiencing impaired steroidogenesis and modulations of receptor-mediated endocrine responses. Given the integral role of neurosteroids in homeostatic process, growth, metabolism, reproduction, and development of central nervous system and function, these effects may have serious impact on this endocrine pathway and potentially affect organismal reproductive performance and health. In conclusion, the regulation of steroidogenesis is a fundamental mechanism involved in the biosynthesis of important biological compounds, irrespective of organ; therefore, the search for the molecular targets of xenoestrogens, given singly and also in combination, in these pathways will increase our understanding of organismal endocrine disruption and potential consequences.

Short-term exposure to low concentrations of the synthetic androgen methyltestosterone affects vitellogenin and steroid levels in adult male zebrafish ( Danio rerio)

Short-term effects of methyltestosterone (MT) on the endocrine system of adult male zebrafish ( Danio rerio) were examined. Males were exposed to 0, 4.5, 6.6, 8.5, 19.8, 35.9, 62.3 ng MT/l and ethinylestradiol (EE2) (26.4 ng/l) for 7 days. Several physiological endpoints that may be affected by endocrine disrupters were analysed, specifically vitellogenin (VTG) concentration, estradiol (E2), testosterone (T), and 11-ketotestosterone (KT) content, brain aromatase activity and gene expression of CYP19A1 and CYP19A2 in the testis. Exposure to the lowest MT concentration (4.5 ng MT/l), and the EE2 increased the concentration of VTG significantly compared to solvent control group. Exposure to higher concentrations of MT did not increase VTG levels. Endogenous KT and T levels decreased significantly in a concentration-dependent manner in response to the MT exposure and the lowest effective concentrations were 6.4 and 8.5 ng MT/l, respectively. The levels of KT and T were also significantly suppressed by EE2 when compared to the solvent control group. Significant decreases in endogenous E2 levels were found in some MT groups but it was not possible to distinguish a simple concentration–response relationship. No effects of MT or EE2 on the brain aromatase activity or on testicular gene expression of CYP19A1 and CYP19A2 were detected. The results show that androgens such as MT can act as endocrine disrupters even at very low concentrations.

Assessment of Xenoestrogens Using Three Distinct Estrogen Receptors and the Zebrafish Brain Aromatase Gene in a Highly Responsive Glial Cell System

The brain cytochrome P450 aromatase (Aro-B) in zebrafish is expressed in radial glial cells and is strongly stimulated by estrogens (E2); thus, it can be used in vivo as a biomarker of xenoestrogen effects on the central nervous system. By quantitative real-time polymerase chain reaction, we first confirmed that the expression of Aro-B gene is robustly stimulated in juvenile zebrafish exposed to several xenoestrogens. To investigate the impact of environmental estrogenic chemicals on distinct estrogen receptor (ER) activity, we developed a glial cell-based assay using Aro-B as the target gene. To this end, the ER-negative glial cell line U251-MG was transfected with the three zebrafish ER subtypes and the Aro-B promoter linked to a luciferase reporter gene. E2 treatment of U251-MG glial cells cotransfected with zebrafish ER-α and the Aro-B promoter–luciferase reporter resulted in a 60- to 80-fold stimulation of luciferase activity. The detection limit was < 0.05 nM, and the EC50 (median effective concentration) was 1.4 nM. Interestingly, in this glial cell context, maximal induction achieved with the Aro-B reporter was three times greater than that observed with a classical estrogen-response-element reporter gene (ERE-tk-Luc). Dose–response analyses with ethynylestradiol (EE2), estrone (E1), α-zeralenol, and genistein showed that estrogenic potency of these agents markedly differed depending on the ER subtype in the assay. Moreover, the combination of these agents showed an additive effect according to the concept of concentration addition. This confirmed that the combined additive effect of the xenoestrogens leads to an enhancement of the estrogenic potency, even when each single agent might be present at low effect concentrations. In conclusion, we demonstrate that our bioassay provides a fast, reliable, sensitive, and efficient test for evaluating estrogenic potency of endocrine disruptors on ER subtypes in a glial context.

Brain aromatase in Japanese medaka ( Oryzias latipes): Molecular characterization and role in xenoestrogen-induced sex reversal

In female fish estrogen is required for the development of primary and secondary sex characteristics and is derived from the aromatization of androgens by aromatase. There are two isoforms of aromatase in several teleost species, brain and ovarian. The objective of this study was two-fold: clone and sequence the coding and promoter region of brain aromatase in medaka, and determine the effects of exposure to an environmental estrogen ( o, p-DDT) on sex determination and brain aromatase transcription and activity. The brain aromatase coding sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) and PCR-based genomic DNA walking was used to clone the promoter of the brain aromatase gene. The promoter sequence revealed potential binding sites for the estrogen receptor and for transcription factors involved in primary neurogenesis and sex determination. Medaka fry were exposed to increasing o, p-DDT concentrations (0–5.5 μg/L) from days 1 to 15 after hatch and brain aromatase expression and activity were measured on days 5, 9, and 14. A complete male-to-female sex reversal occurred at 5.5 μg/L o, p-DDT and aromatase activity and expression data showed a significant five-fold increase at this concentration at day 14. This information suggests that brain aromatase is involved in the abnormal sexual differentiation of fish treated with xenoestrogens.

Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene

Compared with adult mammals, the brain of teleost fish is characterized by an extremely high capacity to aromatize androgens into estrogens, and this metabolic activity results from the expression of a specific brain aromatase (AroB) generated by the cyp19b gene. In this study, we first generated antibodies to zebrafish AroB and used them to map AroB-positive structures in the brain of adult zebrafish. We show that AroB is exclusively expressed in radial glial cells, mainly in the olfactory bulbs, telencephalon, preoptic area, and hypothalamus. Second, we investigated in vivo and in vitro the mechanisms involved in the estradiol (E2) regulation of the cyp19b gene. By means of whole-mount hybridization and immunohistochemistry on zebrafish embryos and larvae, we confirmed the E2-dependent upregulation of the cyp19b gene, and we show that E2 triggers AroB expression in radial glial cells mainly in the preoptic area and mediobasal hypothalamus of 48 hpf (hours post fertilization) and 108 hpf larvae. In addition, an in vitro analysis of 0.5 kb of the promoter region of the cyp19b gene demonstrated that this E2-dependent regulation involves a direct transcriptional action of estrogen receptors requiring estrogen-responsive elements. However, the data obtained on different cell lines demonstrate that a glial cell context is necessary for full E2 induction. The correlation between our in vivo and in vitro data suggests that the E2-dependent upregulation of AroB is favored by a glial cell context.