Zebrafish transgenerational methylation profiling in response to TCDD, 5-AZA and MeHg
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ABSTRACT:
ORGANISM(S): Danio rerio
SUBMITTER: Tim Williams
PROVIDER: E-MTAB-2057 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Methylmercury (MeHg) is a potent neurotoxin and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. We investigated the gene expression profile in adult female zebrafish whole brain induced by acute (96 hr) MeHg exposure. Fish were exposed by injection to 0 or 0.5 M-BM-5g MeHg/g. Gene expression changes in the brain were examined using a two-color 22,000 feature zebrafish microarray. At a significance level of p<0.01, 79 genes were up-regulated and 76 genes were down-regulated in response to MeHg exposure. Individual genes exhibiting altered expression in response to MeHg exposure implicate effects on glutathione metabolism and GABA-A receptors in the mechanism of MeHg neurotoxicity. Gene ontology (GO) terms significantly enriched among altered genes included protein folding, cell redox homeostasis, and steroid biosynthetic process. The most affected biological functions were related to the nervous system development and function, as well as lipid metabolism and molecular transport. These results support the involvement of oxidative stress and effects on protein structure in the mechanism of action of MeHg in the female brain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxins and investigate responsive genes as potential biomarkers of MeHg exposure. Wild-type strain AB-1 zebrafish (Zebrafish International Resource Center, University of Oregon, Eugene, OR) were cultured at the Columbia Environmental Research Center (CERC), USGS, for MeHg exposures. Adult female zebrafish were injected with 0 M-NM-<g/g or 0.5 M-NM-<g/g MeHg in 2 M-BM-5L Na2CO3 (pH 6.98)/g body weight. After 96 hr, fish were anesthetized using ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma, St. Louis, MO). Whole brains were removed, flash frozen with liquid nitrogen and stored at 80M-BM-0C. For the microarray experiment, two zebrafish brains were pooled per sample. Four pooled samples were taken from fish treated with 0.5 M-NM-<g/g of MeHg, and the other five were taken from control fish treated with sodium carbonate. Array hybridizations were performed using a reference design, where each sample was compared to a reference sample. The reference sample consisted of equal amounts of RNA from control and treated female brains. Five replicates for the control and four replicates for the treated were analyzed. cDNA synthesis, cRNA labeling, amplification and hybridization were performed following the manufacturerM-bM-^@M-^Ys kits and protocols (Agilent Low RNA Input Fluorescent Linear Amplification Kit and Agilent 60-mer oligo microarray processing protocol; Agilent, Palo Alto, CA).
2010-09-30 | E-GEOD-22662 | biostudies-arrayexpress
Project description:To fully understand molecular toxicity of TCDD in an in vivo animal model, adult zebrafish were exposed to TCDD at 10 nM for 96 h and the livers were sampled for RNA-sequencing based transcriptomic profiling. A total of 1,058 differently expressed genes were identified based on fold-change>2 and TPM (transcripts per million) >10. Among the top 20 up-regulated genes, 10 novel responsive genes were identified and verified by qRT-PCR analysis on independent samples. Transcriptomic analysis indicated several deregulated pathways associated with cell cycle, endocrine disruptors, signal transduction and immune systems. Comparative analyses of TCDD-induced transcriptomic changes between fish and mammalian models revealed that proteomic pathway is consistently up-regulated while calcium signaling pathway and several immune-related pathways are generally down-regulated. Transcriptome profiling of treated sample (TCDD) and control sample (DMSO) were generated by deep sequencing using 3' RNA-SAGE with SOLiD system
2013-11-12 | E-GEOD-49915 | biostudies-arrayexpress
Project description:Adult zebrafish can completely regenerate their caudal fin following amputation. This complex process is initiated by the formation of an epithelial would cap over the amputation site by 12 hours post amputation (hpa). Once the cap is formed, mesenchymal cells proliferate and migrate from sites distal to the wound plane and accumulate under the epithelial cap forming the blastemal structure within 48 hpa. Blastemal cells proliferate and differentiate, replacing the amputated tissues, which are populated with angiogenic vessels and innervating nerves during the regenerative outgrowth phase which is completed around 14 days post amputation (dpa). Regenerative outgrowth does not occur in TCDD-exposed zebrafish. To identify the molecular pathways that are perturbed by TCDD exposure, male zebrafish were i.p. injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3 and 5 dpa for mRNA abundance analysis. Microarray analysis and quantitative real time PCR revealed that wound healing and regeneration alone altered the expression of nearly 900 genes by at least two fold between 1 and 5 dpa. TCDD altered the abundance of 370 genes at least two fold. Among these, several known aryl hydrocarbon responsive genes were identified in addition to several genes involved in extracellular matrix composition and metabolism. The profile of misexpressed genes is suggestive of impaired cellular differentiation and extracellular matrix composition potentially regulated by Sox9b. Experiment Overall Design: Regenerating fins were isolated at 1, 3 and 5 days post amputation. Three replicates were collected at each time point. 10 fins were pooled to comprise one replicate. Fish were dosed at 0 days post amputation with vehicle control alone or 50 ng/g TCDD. Experiment Overall Design: 1 Day Post Amputation Vehicle Exposed: GSM85187, GSM85188, and GSM85189 Experiment Overall Design: 1 Day Post Amputation TCDD Exposed: GSM85190, GSM85191, and GSM85192 Experiment Overall Design: 3 Days Post Amputation Vehicle Exposed: GSM85193, GSM85194, and GSM85195 Experiment Overall Design: 3 Day Post Amputation TCDD Exposed: GSM85196, GSM85197, and GSM85198 Experiment Overall Design: 5 Days Post Amputation Vehicle Exposed: GSM85199, GSM85200, and GSM85201 Experiment Overall Design: 5 Days Post Amputation TCDD Exposed: GSM85202, GSM85203, and GSM85204
2007-10-05 | E-GEOD-3667 | biostudies-arrayexpress
Project description:2,3,7,8-TCDD (TCDD) is a reproductive toxicant and endocrine disruptor in nearly all vertebrates, yet the mechanisms by which TCDD induces these reproductive alterations have not been fully characterized. Fish are among the most sensitive vertebrates to the toxic effects of TCDD, and even subtle physiologic changes induced by TCDD can impair reproduction. Previously, we have shown that chronic, sub-lethal exposure to TCDD decreased reproductive capacity, reduced serum estradiol and vitellogenin concentrations, and altered follicular development. Here we investigate the transcriptional changes in zebrafish ovary as they relate to observed attenuated estradiol concentrations and ovarian development. We used quantitative RT-PCR to assess TCDDâs effects on the expression of several candidate genes important in the regulation of follicular development and steroidogenesis. Additionally, global changes in gene expression in the ovary caused by TCDD exposure were identified using Affymetrix Gene Chip Analysis. Our data suggest that TCDD may inhibit follicle maturation via attenuated gonadotropin responsiveness and/or depressed estradiol biosynthesis. Additionally, genes involved in glucose and lipid metabolism, regulation of transcription, and immune function were dysregulated by at least 2-fold suggesting that TCDD alters various integrated networks of signaling pathways. Approximately 89% of dysregulated transcripts contain putative AHR response elements (AHRE) within 5kb upstream of the predicted transcriptional start site suggesting ovarian toxicity is AHRE driven. Furthermore, approximately 49% of dysregulated transcripts contain putative estrogen response elements (ERE) suggesting that dysregulation of estrogen-responsive genes may also contribute to TCDD-induced attenuated follicular development. Patterns in gene expression were correlated with putative EREs and AHREs, and suggest that impacts on the regulation of transcription may play a large role in TCDDâs ovarian toxicity. Taken together, these data illustrate the complexity of TCDDâs ovarian toxicity. Experiment Overall Design: A total of eight samples were analyzed including three TCDD doses (10, 40, 100 ppb, dietary exposure) and vehicle control samples. cRNA targets were synthesized from pooled total RNA isolated from five ovaries from different individuals in each treatment group. Each individual contributed equally to a given pool. Two technical replicates were run for each treatment group. Several genes of interest arising from this microarray study were validated by Quantitative RT-PCR with individual ovary samples to assess biological variability.
2009-04-30 | E-GEOD-4859 | biostudies-arrayexpress
Project description:Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species and its effects are especially prominent in early life stages of fish. TCDD activates the aryl hydrocarbon receptor (AHR), a transcription factor that mediates most if not all TCDD responses. We investigated the transcriptional response in the developing zebrafish jaw following TCDD exposure using DNA microarrays. Zebrafish larvae were exposed to TCDD at 96 h postfertilization (hpf) and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4 and 12 h postexposure (hpe). Numerous chondrogenic transcripts were misregulated by TCDD in the jaw. Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different. Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw. We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting formation of the embryonic jaw. Morpholino knock down of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype. Heterozygous sox9b deletion mutant embryos were sensitized to TCDD. Lastly, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos. These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation. Experiment Overall Design: Three independent replicate microarray time course experiments were performed comparing transcript levels between TCDD-exposed and control zebrafish. For each experiment, zebrafish were exposed to TCDD for 1 h starting at 96 hpf as described above. For each time point (97, 98, 100 and 108 hpf) and treatment jaw samples were pooled from 10 dissections for RNA isolation and hybridization with Affymetrix zebrafish arrays (Affymetrix, Santa Clara, CA). Each microarray contains roughly 14,900 probes corresponding to approximately 30% of the zebrafish genome. For each array, total RNA (1 µg) was isolated from 10 jaw microdissections with the QIAGEN RNeasy Mini kit following the manufacturerâs protocol (QIAGEN, Valencia, CA). The One-Cycle Target Labeling and Control Reagents kit was used to synthesize cDNA and biotinylated cRNA following the manufacturerâs protocol (Affymetrix, Santa Clara, CA). Biotin-labeled cRNA (15 µg) was fragmented and hybridized onto Affymetrix Zebrafish Genechip Arrays following the protocol in the Affymetrix Genechip Expression Analysis Technical Manual. Following hybdrization, the arrays were washed and stained with streptavidin-phycoerythrin on an Affymetrix Fluidics Station 400 using the protocol EukGE WS2v4. Arrays were scanned with an Agilent Gene Array Scanner.
2008-11-13 | E-GEOD-11893 | biostudies-arrayexpress
Project description:Zebrafish have the remarkable ability to regenerate body parts including the heart, spinal cord and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage-larvae also possess the ability to regenerate the caudal fin. A comparative genomic analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of Retinoic acid (RA), as one of the highly induced genes across the three regeneration platforms. Experiment Overall Design: The caudal fin of zebrafish larvae at 2days post fertilization were amputated. Caudal fin tissue at 2dpf and regenerating fins were isolated at 1, 2and 3 days post amputation. Three replicates were collected at each time point. 150 fins were pooled to comprise one replicate.
2008-04-07 | E-GEOD-10188 | biostudies-arrayexpress
Project description:The purpose of this experiment is to understand which transcripts are differentially expressed following exposure to TCDD. We used microarrays to understand global changes in gene signal intensity Adult zebrafish (danio rerio) were injected (i.p) to vehicle control or TCDD (70 ng/g) one day prior to heart ventricle amputation. A subset of fish were exposed but the hearts were not amputated. Amputated hearts were collected at 6 hours post amputation and 7 days post amputation and processed for microarray analysis
2011-11-29 | E-GEOD-33981 | biostudies-arrayexpress
Project description:Dietary supplementation with ω-3 polyunsaturated fatty acids (ω-3 PUFAs), specifically the fatty acids docosahexaenoic acid (DHA; 22:6 ω-3) and eicosapentaenoic acid (EPA; 20:5 ω-3), is known to have beneficial health effects including improvements in glucose and lipid homeostasis and modulation of inflammation. To evaluate the efficacy of two different sources of ω-3 PUFAs, we performed gene expression profiling in the liver of mice fed diets supplemented with either fish oil or krill oil. We found that ω-3 PUFA supplements derived from a phospholipid krill fraction (krill oil) downregulated the activity of pathways involved in hepatic glucose production as well as lipid and cholesterol synthesis. The data also suggested that krill oil-supplementation increases the activity of the mitochondrial respiratory chain. Surprisingly, an equimolar dose of EPA and DHA derived from fish oil modulated fewer pathways than a krill oil-supplemented diet and did not modulate key metabolic pathways regulated by krill oil, including glucose metabolism, lipid metabolism and the mitochondrial respiratory chain. Moreover, fish oil upregulated the cholesterol synthesis pathway, which was the opposite effect of krill supplementation. Neither diet elicited changes in plasma levels of lipids, glucose or insulin, probably because the mice used in this study were young and were fed a low fat diet. Further studies of krill oil supplementation using animal models of metabolic disorders and/or diets with a higher level of fat may be required to observe these effects. Twenty-one microarrays: three diets (CO, FO, KO) x seven mice per diet x one microarray per mouse
2011-07-21 | E-GEOD-29572 | biostudies-arrayexpress
Project description:Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis as well as to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. To assess the response to chemicals at different stages of development, zebrafish (Danio rerio) embryos at 1, 2, 3, 4, 5, and 6 days post-fertilization (dpf) were exposed to 0.1% dimethyl sulfoxide (DMSO), 2 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or 10 uM tert-butylhydroquinone (tBHQ) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray (Agilent 22k). By microarray, 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated embryos at 4 dpf. Of these, 220 were 2-fold or greater up-regulated and 108 were 2-fold or greater down-regulated by tBHQ. For TCDD, 17 probes were 2-fold or greater up-regulated and 6 were 2-fold or greater down-regulated. Genes induced by tBHQ, included genes involved in glutathione synthesis and utilization, signal transduction, and DNA damage / stress response. Keywords: Danio rerio, TCDD, tBHQ, oxidative stress, gene expression Separate groups of 30 embryos generated from TL adults were placed in 15 ml egg water in a 10-cm glass petri and at 1, 2, 3, 4, 5, and 6-dpf were exposed for 6 hr to 0.1% dimethyl sulfoxide (DMSO), 2 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or 10 uM tert-butylhydroquinone (tBHQ) (4 replicates per compound per time point). Embyros were frozen immediately after the 6 hr exposure and RNA isolated for microarray analysis. A Universal RNA Reference Sample was created by mixing equal amounts of total RNA from 2 replicates each from all toxicants (TCDD, tBHQ, DMSO) and timepoints (1, 2, 3, 4, 5, 6-dpf). Based on qRT-PCR results, the 4 dpf timepoint was selected for microarray analysis, in which the RNA samples (4 replicates per compound) were Cy3 labeled and co-hybridized with Cy5 labeled Universal RNA Reference Sample to generate data for analysis of variance.
2010-04-03 | E-GEOD-10157 | biostudies-arrayexpress
Project description:Gene respons in the central nervous system of zebrafish embryos exposed to the neurotoxicant methyl mercury Two-condition experiment, control vs MeHg exposure embryos (60 µg/l), Biological replicates: 4, dye swaps
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