Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozole (original) (raw)
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Environmental Toxicology and Chemistry, 2009
As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 g/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 g/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.
General and Comparative Endocrinology, 2017
Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish. Adult female fathead minnows (Pimephales promelas) were exposed via water to 0, 5, or 50 μg fadrozole/L for a time-course of 0.5, 1, 2, 4, and 6 h, or 0 or 50 μg fadrozole/L for a timecourse of 6, 12, and 24 h. We examined ex vivo ovarian 17β-estradiol (E2) and testosterone (T) production, and plasma E2 concentrations from each study. Expression profiles of genes known or hypothesized to be impacted by fadrozole including aromatase (cytochrome P450 [cyp] 19a1a), steriodogenic acute regulatory protein (star), cytochrome P450 side-chain cleavage (cyp11a), cytochrome P450 17 alpha hydroxylase/17,20 lyase (cyp17), and follicle stimulating hormone receptor (fshr) were measured in the ovaries by quantitative real-time polymerase chain reaction (QPCR). In addition, broader ovarian gene expression was examined using a 15k fathead minnow microarray. The 5 μg/L exposure significantly reduced ex vivo E2 production by 6 h. In the 50 μg/L treatment, ex vivo E2 production was significantly reduced after just 2 h of exposure and remained depressed at all time-points examined through 24 h. Plasma E2 concentrations were significantly reduced as early as 4 h after initiation of exposure to either 5 or 50 μg fadrozole/L
Physiological …, 2009
Teleost fish represent unique models to study the role of neuroestrogens because of the extremely high activity of brain aromatase (AroB; the product of cyp19a1b). Aromatase respectively converts androstenedione and testosterone to estrone and 17-estradiol (E2). Specific inhibition of aromatase activity by fadrozole has been shown to impair estrogen production and influence neuroendocrine and reproductive functions in fish, amphibians, and rodents. However, very few studies have identified the global transcriptomic response to fadrozole-induced decline of estrogens in a physiological context. In our study, sexually mature prespawning female goldfish were exposed to fadrozole (50 g/l) in March and April when goldfish have the highest AroB activity and maximal gonadal size. Fadrozole treatment significantly decreased serum E2 levels (4.7 times lower; P ϭ 0.027) and depressed AroB mRNA expression threefold in both the telencephalon (P ϭ 0.021) and the hypothalamus (P ϭ 0.006). Microarray expression profiling of the telencephalon identified 98 differentially expressed genes after fadrozole treatment (q value Ͻ0.05). Some of these genes have shown previously to be estrogen responsive in either fish or other species, including rat, mouse, and human. Gene ontology analysis together with functional annotations revealed several regulatory themes for physiological estrogen action in fish brain that include the regulation of calcium signaling pathway and autoregulation of estrogen receptor action. Real-time PCR verified microarray data for decreased (activin-A) or increased (calmodulin, ornithine decarboxylase 1) mRNA expression. These data have implications for our understanding of estrogen actions in the adult vertebrate brain. aromatase; microarray; fish; brain MAIN ESTROGENS estradiol-17 (E2) and estrone (E1) play fundamental regulatory roles in neuroendocrine and reproductive systems. Through binding to its nuclear estrogen receptors (nER)-␣ and nER-, E2 regulates transcriptional process of target genes whose promoters contain estrogen-responsive elements (ERE) (57). In addition to this classical nuclear action, E2 also has membrane actions in which a unique membrane receptor is utilized to rapidly activate a series of signaling pathways (52, 70). Since the signaling pathway activation will ultimately influence the transcriptional activities of downstream transcription factors including nERs, membrane actions of E2 provide another mode to regulate genomic gene expression (70). Fish are unique models in which to study E2 action
Aquatic Toxicology, 2016
The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10 g/L, 32 g/L, 100 g/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48 h, 96 h, 28 days and 63 days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10 g/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxygenase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.
Sensitive and robust gene expression changes in fish exposed to estrogen – a microarray approach
BMC Genomics, 2007
Background Vitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure. Results Hepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10 ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and...
Aquatic Toxicology, 2014
The steroidogenic enzyme aromatase catalyzes the conversion of androgens to estrogens and therefore plays a central role in reproduction. In contrast to most vertebrates, teleost fish have two distinct forms of aromatase. Because brain aromatase activity in fish is up to 1000 times that in mammals, fish may be especially susceptible to negative effects from environmental endocrine-disrupting chemicals (EDCs) that impact aromatase activity. In this study, the effects of estradiol (E 2 ), ethynylestradiol (EE 2 ), octylphenol (OP), and androstatrienedione (ATD) on reproduction and aromatase activity in brains and gonads from the marine fish cunner (Tautogolabrus adspersus) was investigated. The purpose of the study was to explore the relationship between changes in aromatase activity and reproductive output in a marine fish, as well as compare aromatase activity to two commonly used indicators of EDC exposure, plasma vitellogenin (VTG) and gonadosomatic index (GSI). Results with E 2 , EE 2 , and ATD indicate that aromatase activity in cunner brain and ovary are affected differently by exposure to these EDCs. In the case of E 2 and EE 2 , male brain aromatase activity was signficantly increased by these treatments, female brain aromatase activity was unaffected, and ovarian aromatase activity was significantly decreased. Treatment with the aromatase inhibitor ATD resulted in significantly decreased aromatase activity in male and female brain, but had no significant impact on ovarian aromatase activity. Regardless of test chemical, a decrease or an increase in male brain aromatase activity relative to controls was associated with decreased egg production in cunner and was also correlated with significant changes in GSI in both sexes. E 2 and EE 2 significantly elevated plasma VTG in males and females, while ATD had no significant effect. Treatment of cunner with OP had no significant effect on any measured endpoint. Overall, results with these exposures indicate EDCs that impact aromatase activity also affect reproductive output in spawning cunner.
Toxicological Sciences, 2006
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 (ERa) 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 ERa 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 ERa 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 nonreceptor-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.
Environmental Health Perspectives, 2009
Background: Several chemicals in the environment have the potential to inhibit aromatase, an enzyme critical to estrogen synthesis. oBjectives: The objective of this study was to provide a detailed characterization of molecular and biochemical responses of female fathead minnows to a model aromatase inhibitor, fadrozole (FAD). Methods: Fish were exposed via water to 0, 3, or 30 µg FAD/L for 8 days and then held in clean water for 8 days, with samples collected at four time points during each 8-day period. We quantified ex vivo steroid production, plasma steroids, and plasma vitellogenin (Vtg) concentrations and analyzed relative transcript abundance of 10 key regulatory genes in ovaries and 3 in pituitary tissue by real-time polymerase chain reaction. results: Ex vivo 17β-estradiol (E 2 ) production and plasma E 2 and Vtg concentrations were significantly reduced after a single day of exposure to 3 µg or 30 µg FAD/L. However, plasma E 2 concentrations recovered by the eighth day of exposure in the 3-µg/L group and within 1 day of cessation of exposure in the 30-µg/L group, indicating concentration-and time-dependent physiologic compensation and recovery. Concentration-dependent increases in transcripts coding for aromatase (A isoform), cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and follicle-stimulating hormone receptor all coincided with increased E 2 production and recovery of plasma E 2 concentrations. conclusions: Results of this research highlight the need to consider compensation/adaptation and recovery when developing and interpreting short-term bioassays or biomarkers or when trying to predict the effects of chemical exposures based on mode of action.
Fish Physiology and Biochemistry, 2003
Cytochrome P450-aromatase (CYP19) is the critical steroidogenic enzyme controlling estrogen biosynthesis. In teleosts, two genes for CYP19 are found with CYP19A1 expressed mainly in the ovary and CYP19A2 expressed in both brain and pituitary. The promoters for these two homologous genes are considerably different. Exposure to estrogenic endocrine disrupting chemicals (EDC) such as ethinylestradiol and nonylphenol resulted in strong up-regulation of the CYP19A2 gene whereas the expression of CYP19A1 was largely unaffected. Moreover, CYP19A2 transcript abundance was shown to vary during the spawning cycle of both the catfish and the zebrafish which implies an association of the CYP19A2 gene in brain-pituitary endocrinology. Together these findings suggest that EDCs could affect the reproductive physiology of fish through disrupting the gene expression of CYP19A2 in the brain.
General and Comparative Endocrinology, 2014
The endocrine disrupting effects of estrogenic compounds in surface waters on fish, such as feminization of males and altered sex ratios, may also occur in aquatic invertebrates. However, the underlying mechanisms of action and toxicity, especially in native freshwater mussels (Order Unionoida), remain undefined. This study evaluated the effects of a 12-day exposure of 17␣-ethinylestradiol (EE2), a synthetic estrogen in oral contraceptives commonly found in surface waters, on the behavior, condition, metabolism, and reproductive status of Lampsilis fasciola. Adult mussels of both sexes were exposed to a control and two concentrations of EE2 (0 ng/L, 5 ng/L considered to be environmentally relevant, and 1000 ng/L designed to provide a positive metabolic response), and samples of gill tissue were taken on days 4 and 12; gills were used because of the variety of critical processes they mediate, such as feeding, ion exchange, and siphoning. Observations of mussel behavior (mantle display, siphoning, and foot movement) were made daily, and condition of conglutinates (packets of eggs and/or glochidia) released by females was examined. No significant effects of EE2 on glochidia mortality, conglutinate condition, female marsupial gill condition, or mussel foot extension were observed. However, exposure to both concentrations of EE2 significantly reduced male siphoning and mantle display behavior of females. Metabolomics analyses identified 207 known biochemicals in mussel gill tissue and showed that environmentally relevant EE2 concentrations led to decreases in glycogen metabolism end products, glucose, and several essential fatty acids in females after 12 days, indicating reductions in energy reserves that could otherwise be used for growth or reproduction. Moreover, males and females showed significant alterations in metabolites involved in signal transduction, immune response, and neuromodulation. Most of these changes were apparent at 1000 ng/L EE2, but similar metabolites and pathways were also affected at 5 ng/L EE2. Components of the extracellular matrix of gill tissue were also altered. These results demonstrate the utility of metabolomics when used in conjunction with traditional physiological and behavioral toxicity test endpoints and establish the usefulness of this approach in determining possible underlying toxicological mechanisms of EE2 in exposed freshwater mussels.