Microarray analysis of di-n-butyl phthalate and 17α ethinyl-oestradiol responses in three-spined stickleback testes
Ontology highlight
ABSTRACT:
ORGANISM(S): Gasterosteus aculeatus
SUBMITTER: Jenni Prokkola
PROVIDER: E-MTAB-3951 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Sex steroids play a key role in triggering sec differentiation in fish and the use of exogenous hormone treatment leads to partial or complete sex reversal. This phenomenon has attracted attention since the discovery taht even low environmental doses of exogenous steroids can adversely affect gonad morphology (ovotestis development) and induce reproductive failure. Modern genomic-based technologies have enhanced opportunities to find mechanisms of action (MOA) and dentify biomarkers for the toxic action of a compound. The goal of this study are to improve the understanding of feminization in fish by analyzing gene expression patterns in the gonads of rainbow trout fry after a chronic exposure to several doses (0.01, 0.1, 1 and 10 ?g/L) of ethynylestradiol (EE2) and to offer target genes as potential biomarkers of ovotestis development. An all-male population of Rainbow trout fry was exposed during 76 days (from 60 to 136 days post-fertilization (dpf)) to five nominal concentrations of 17?-ethynylestradiol (0-solvent control-, 0.01, 0.1, 1 and 10 ?g EE2/L of water), using 3 tanks per condition. In total, 30 samples were analyzed independantly: 6 samples per concentration tested (two samples per tank, three tanks per concentration), each sample being a pool of 10 pairs of gonads.
2015-06-25 | E-GEOD-58519 | biostudies-arrayexpress
Project description:Microarrays are used to assess the impacts of aquatic contaminants in both laboratory and field studies, which necessitates the validation of the comparability of microarray data generated by different laboratories before this tool is adopted for regulatory toxicology or environmental monitoring. Fundamental issues which need answering before microarray technology can be used in environmental monitoring programs include defining whether these molecular changes translate into adverse effect at the individual or population level, as well as how large a magnitude (threshold) and how to quantify (fold change versus p value) a modification or modifications in gene expression which constitute an adverse effect. Here, male FHM were exposed to 25 ng/L EE2 for 96 hr, and six laboratories received flash frozen liver to conduct a microarray analysis on using a 60K Agilent microarray. This study aimed to assess the congruency of microarray data across six independent laboratories given both control and EE2 exposed samples, and to identify gold standard estrogen-responsive genes triggered by treatment with an environmentally relevant EE2 dose. 8 biological replicates per group. 2 groups, control and EE2 treated.
2016-05-31 | E-GEOD-81348 | biostudies-arrayexpress
Project description:In Sparus aurata, seasonal temperature variations outside the normal thermal range, may trigger physiological responses leading to pathologies and death. In the present study two groups of wild sea bream were exposed for 21 days to two temperature regimes: 16 ± 0.3 °C (control group) and 6.8 ± 0.3 °C (cold-exposed group). Samples were collected during the acute phase (0, 6 and 24 hours after temperature drop) and upon chronic exposure (21 days). A comparative analysis of gene expression was conducted between S. aurata exposed to 6.8°C. Two groups of wild sea breams (120±16 g body weight; 21±1 cm total length) caught in Valle Bonello were kept in duplicate groups in four 5 m3 circular recycling tanks and acclimated over one month at 16 °C (Di Marco et al. 2010). Fish were then exposed for 21 days to two different temperature treatments: 16 ± 0.3 °C (control groups) and 6.8 ± 0.3 °C (cold groups). Both groups were fasted during the trial. Warm and cold water temperature conditions were maintained by supplying tanks with two separate re-circulating systems equipped with mechanical and biological filters. Cold water conditions were achieved by inducing two consecutive temperature drops: water was firstly gradually cooled from 16 °C to 12 °C at a rate of 1°C day -1 and then reduced from 12 °C to 6.8 °C within 24 h. This temperature was then kept for 21 days. Ten fish per treatment were sampled at different intervals during acute: 0 (T1), 6 (T2) and 24 hours (T3), and chronic exposure (21 days; T4) to low temperature. Fish were firstly anaesthetized in a solution of tricaine methanesulphonate (300 mg/l; MS 222, Finquel) in a 30-litre bucket and then euthanized by severing their spinal cord. Liver samples (about 30 mg) were collected from each specimen, incubated in RNA later at 4 °C for 24 h and then stored at -20 °C. Total RNA was extracted from tissue samples using the RNAeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentration was determined using a UV-Vis spectrophotometer NanoDrop® ND-1000 (NanoDrop Technologies, Wilmington, USA). RNA integrity and quality was evaluated by means of Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and Expert software according to Ferraresso et al. (2008). RNA integrity number (RIN) index was calculated for each sample that provides a numerical assessment of RNA integrity. This value facilitates the standardization of the quality interpretation; only RNAs with RIN >8 were used for further microarray experiments. Once single RNA samples were extracted and measured, 3 pools (Pool_A consisting of four individuals, Pool_B and _C of three) per each sampling-time point were prepared. This was aimed at getting a sufficient statistical power which is reached by a minimum of three biological replicates per analysed point and at reducing microarray analysis cost. To perform DNA microarray analyses has been used the microarray platform GPL6467 (Ferraresso et al. 2008). Sample labelling and hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol. For each of 24 liver pools 500 ng of total RNA were linearly amplified and labelled with Cy3-dCTP, using the Low RNA Input Linear Amplification Kit PLUS one-color (Agilent Technologies). A total of 24 Agilent 4 x 44K sea bream microarrays (12 samples from both control and cold groups) were then hybridized. An Agilent G2565BA DNA microarray scanner was used to scan arrays at 5 µm resolution, Feature Extraction Software 9.5.1 was then used to process and analyse array images. The software returns a series of spot quality measures in order to evaluate the goodness and the reliability of spot intensity estimates. Filtering and cyclic lowess normalization were performed using R statistical software (http://www.r-project.org/).
2014-08-27 | E-GEOD-51442 | biostudies-arrayexpress
Project description:Total RNA was extracted from liver tissues of lab-reared threespine stickleback. First generation fish originating from the Baltic Sea near Helsinki (Finland) were bred using a paternal half-sib design. At 6 months individuals were randomly assigned to either a treatment or control group: the temperature of treated fish (T) was raised 1oC per hour over the course of 6h to a final temperature of 23oC, then maintained for 1h at final temperature; control (C) fish were maintained at 17oC under similar holding conditions. Immediately following experimental (or sham) treatment, fish were euthanized by anaesthetic overdose. Liver tissues were immediately dissected and flash frozen in liquid nitrogen for subsequent RNA extraction. Annotated R scripts defining the normalization procedures are available as additional files (see http://www.ebi.ac.uk/arrayexpress/files/E-MTAB-3098). Additional files also include a targets file to assist with reading raw data into R (Targs.txt), and a metadata file (matrix.df.csv) to facilitate construction of design matrices used by the M-bsnmM-b package.
2014-12-03 | E-MTAB-3098 | biostudies-arrayexpress
Project description:The present study explores the potential of compound-specific gene-upregulation profiles in the ubiquitous purple nonsulfur bacterium Rhodospirillum rubrum S1H as biomarkers for exposure to surface water contaminants, i.e. high production-volume pharmaceuticals. Even though the pharmaceuticals [i.e., acetylsalicylic acid (ASA), diclofenac (DCF), and 17M-NM-1-ethinylestradiol (EE2)] did not affect the bacterial growth kinetics at environmentally-relevant concentrations (86nM), whole-genome microarray analyses revealed the upregulation of 128, 49, and 47 genes upon exposure to DCF, ASA, and EE2, respectively. A strong overlap (27-48%) was observed between transcriptional responses, but a total of 93 genes were found to be upregulated in a compound-specific manner. Hence, we were able to identify 74 and 15 potential biomarker genes for DCF and ASA, respectively. DCF specifically induced genes involved mainly in stress response, signal transduction, response regulation, the electron transport chain, and transcription, while ASA specifically induced genes predominantly involved in signal transduction, response regulation, and trans-membrane translocation. Moreover, our findings validated triclosan-specific biomarker genes that were identified previously. As only 4 genes were specifically-upregulated for EE2, no representative biomarker profile was identified. This study illustrates that a pollutant-specific molecular response can be generated in R. rubrum S1H, which could become a relevant model-microorganism to screen for the ecological impact of surface water contaminants in situ. KEYWORDS: environmental impact studies, risk assessment, biosensor, wastewater, micropollutant, aspirin Two-condition experiments. Comparing samples after induction of three pharmaceuticals each with a non-induced samples. Biological triplicate. Each array contains 3 technical replicates.
2013-08-23 | E-GEOD-23794 | biostudies-arrayexpress
Project description:Fish respond to stressors by means of an array of changes that involve many biological compartments including molecular, cellular, metabolic, physiological and behavioral acclimation. Among these changes, the immune system also experiences significant alterations that are characterized by an induction of the innate non-specific responses and changes in the acquired specific response. The present investigation was undertaken to check whether the innate response of the complement system is affected after a combination of an immune challenge and a husbandry stressor. In European seabass, two different transcripts of the complement C3 protein, which correspond to two tissue-specific isoforms, have been detected in liver. They may come from post-transcriptional modifications at the pre-mRNA level or from alternative splicing in order to generate a diversified repertoire of protein isoforms in some fish species according to the tissue were they are located. After cloning the hepatic C3, the quantification of seabass isoforms showed specific responses, thus confirming the possible specialized roles of the C3 different isoforms in terms of defence immunity. On one hand, a stronger esbC3_2 expression was observed in front to the viral challenge, suggesting a specific participation of this molecule in front to viruses. This observation is in agreement with previous studies in trout macrophages in our lab, demonstrating that the immune response to viral infections was stronger than to bacterial ones. On the other hand, the esbC3_1 expression appeared stronger after bacterial challenge, especially under high density conditions. Our results confirm the hypothesis of an immune-activation of specific isoforms of the C3 protein of the complement system as a non-specific response in front to either immune or other type of stressors. In order to confirm our hypothesis, we created an oligonucleotide microarray (Agilent Technologies) containing 6275 annotated transcripts, each with x3 specific probes, and 4516 ESTs with 1 probe/target sequence. Gene expression profiles obtained from the livers of fish held under high density culture conditions highlighted a significant contrast in the liver response. In total, 54 transcripts were differentially expressed (GDE; fold change FC >2) in treatment over the control (p<0.01)/201 (p<0.05). Oligonucleotide probes were used to construct a high-density seabass microarray based on the Agilent 4 × 44 K design format (http://www.agilent.com/), which covers 13,199 unique transcripts of Dicentrarchus labrax. Oligonucleotides, 60-mer plus a “linker”, are synthesized directly on glass slides using the Agilent’s SurePrint Tecnology; oligo design was carried out by Agilent and three or more non-overlapping probes were designed for each EST cluster. The number of transcripts represented by three is 6,275, while 6,924 targets EST have only one probe.
2011-04-23 | E-GEOD-28812 | biostudies-arrayexpress
Project description:17alpha-ethinylestradiol (EE2) is one of the most potent estrogens that have the ability to interfere with the endocrine system of fish. The objective was to investigate the effects and mechanisms of action caused by 60 days of dietary exposure to 0.2 mg EE2/kg and 0.07 mg EE2/kg feed in female largemouth bass (LMB) during the reproductive season. Microarrays and pathway analyses were performed on hepatic tissues to identify genes and biological processes altered in female LMB by EE2 exposure. The hypothesis was that the two concentrations of EE2 would produce dose-response changes in sensitive genes. Body and ovary weights were measured and blood was collected for measurement of plasma steroid hormones (17beta-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. The 0.2 mg EE2/kg feed exposure reduced the gonadosomatic index (GSI) by 75%, and plasma levels of E2 and T were reduced by over 90%. Plasma VTG was increased by approximately 100% (from 4 to 8mg/ml) by the 0.2 mg/kg treatment. T levels, from the 0.07 mg EE2/kg feed, reduced GSI by approximately 30% and circulating E2 and T by ~80% but did not affect VTG concentrations. We found 1,594 and 1,165 genes were significantly affected (p<0.05) by the 0.07 mg EE2/kg feed and 0.2 mg EE2/kg feed, respectively. Gene ontology (GO) analysis revealed that there were different biological processes regulated by the two concentrations of EE2. Pathway analysis showed that the 0.07 mg EE2/kg feed exposure caused differential regulation of genes associated with fatty acid biosynthesis and glycolysis, indicating some metabolic effects. In contrast, the 0.2 mg EE2/kg feed exposure altered transcription of genes involved in immune response and apoptosis, suggesting a toxic response at this concentration. These results suggest that the two concentrations demonstrated distinct physiological responses, with the higher concentration inducing complete endocrine disruption in LMB. These findings demonstrate the usefulness of microarrays to identify possible biomarkers and modes of toxic action to dietary exposure in LMB. Two concentrations of EE2 would produce dose-response changes in sensitive genes. Female LMB were fed 5 days per week for 60 days with floating pellets that contained 0.07 or 0.2 mg/kg of EE2.
2011-03-24 | E-GEOD-28143 | biostudies-arrayexpress
Project description:The objective of this study was to investigate pathway signatures altered in the livers of female largemouth bass (LMB) and their potential links with biological responses by dietary exposure to 0.2 mg EE2/Kg (1% of their body weight) over two months using a transcriptomics approach. A high concentration of dietary 17alpha-ethinylestradiol (EE2) can activate key signaling pathways in response to oxidative damage may occur regardless of tumorigenesis and cancer. Female LMB received about 1.2g EE2/day/fish (from EE2-laced feed containing 0.2 mg EE2/Kg) for 60 days.
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Project description:The present work was designed to assess the effects of high plasma cortisol levels induced by slow-release cortisol implants in the mRNA transcription of the GR in the different organs of the Sparus aurata, including liver. For that purpose fish were intraperitoneally injected with the implants containing two different concentrations of cortisol (50 or 200 µg/g body weight) and blood and organs were sampled after 7 and 14 days of implantation. Only fish with 200 µg/g implants exhibited a significant rise in the plasma cortisol. For microarray analysis we used livers of gilthead sea bream (N=36 fish). Fish were injected with 200 µg/g body weight of cortisol and sampled after 7 and 14 days (n=6 for each condition). RNA samples were grouped into pools of 2 animals for each time point. The analysis were carried out considering the transcripts differentially expressed in the group of fish implanted during 7 days with cortisol comparing to control group. Additionally, the transcripts differentially expressed at day 14 were compared with day 7.
2011-07-19 | E-GEOD-30782 | biostudies-arrayexpress