Project description:Tritium is an ubiquist radionuclide which can be found in the environment due to natural and anthropogenic activities, particularly in aquatic ecosystems. In this context, tritium effects on aquatic species such as fish have to be characterized. HTO (tritiated water) effects were therefore investigated in zebrafish, Danio rerio, a common model in toxicology and ecotoxicology with a fully sequenced genome. Experiments were conducted on early life stages. Larvae were exposed to 0.4 and 4 mGy/h of HTO until 10 days post fertilization. Tritium internalization was quantified and effects were investigated using a proteomic analysis. The global analysis of the proteome was performed after protein extraction at 7 and 10 dpf on zebrafish eggs exposed from 3 hpf to 10 dpf.
Project description:Fathead minnow and zebrafish are among the most intensively studied fish species in environmental toxicogenomics. To aid the assessment and interpretation of subtle transcriptomic effects from treatment conditions of interest, there needs to be a better characterization and understanding of the natural variation in gene expression among fish individuals within populations. Little effort, however, has been made in this area. Leveraging the transcriptomics data from a number of our toxicogenomics studies conducted over the years, we conducted a meta-analysis of nearly 600 microarrays generated from the ovary tissue of untreated, reproductively mature fathead minnow and zebrafish samples. As expected, there was considerable batch-to-batch transcriptomic variation; this “batch-effect” appeared to impact the fish transcriptomes randomly. The overall level of variation within-batch was quite low in fish ovary tissue, making it a suitable system for studying chemical stressors with subtle biological effects. The within-batch variation, however, differed considerably among individual genes and molecular pathways. This difference in variability is probably both technical and biological, thus suggesting a need to take into account both the expression levels and variance in evaluating and interpreting the transcriptional impact on genes and pathways by experimental conditions. There was significant conservation of both the genomes and transcriptomes between fathead minnow and zebrafish. The conservation to such a degree would enable not only a comparative biology approach in studying the mechanisms of action underlying environmental stressors, but also effective sharing of a large amount of existing public transcriptomics data for future development of toxicogenomics applications.
Project description:Natural Variation in Fish Transcriptomes: Comparative Analysis of the Fathead Minnow (Pimephales promelas) and Zebrafish (Danio rerio)
Project description:All vertebrates have multiple genes encoding for different CASQ isoforms. Increasing interest has been focused on mammalian and human CASQ genes since mutations of both cardiac (CASQ2) and skeletal (CASQ1) isoforms cause different, and sometime severe, human pathologies Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work expression, biochemical properties and cellular and sub-cellular localization of Danio rerio native CASQ isoforms are investigated. By quantitative PCR three mRNAs were detected in skeletal muscle and one mRNA in heart. Three zebrafish CASQs were identified by mass spectrometry and they share properties with mammalian skeletal and cardiac CASQs. Skeletal calsequestrins were found primarily, but not exclusively, at the sarcomere Z-line level where Terminal Cisternae of Sarcoplasmic reticulum are located.
Project description:In triplicate for each condition, 12 WT and acbd6 F0 crispant Danio rerio (zebrafish) embryos were incubated with 20 μM YnMyr for 24 h, either between 48-72 hpf or 96-120 hpf. After labelling, zebrafish were washed twice with fresh egg water, deyolked, flash frozen in liquid nitrogen and stored at -80°C until further analysis.
Project description:Pharmaceutical chemicals used in human medicine are released into surface waters via municipal effluents and pose a risk for aquatic organisms. Among these substances are selective serotonin reuptake inhibitors (SSRIs) which can affect aquatic organisms at sub ppb concentrations. To better understand biochemical pathways influenced by SSRIs, evaluate changes in the transcriptome, and identify gene transcripts with potential for biomarkers of exposure to SSRIs; larval zebrafish Danio rerio were exposed (96 h) to two concentrations (25 and 250 µg/L) of the SSRIs, fluoxetine and sertraline, and changes in global gene expression were evaluated (Affymetrix GeneChip® Zebrafish Array). Significant changes in gene expression (>=1.7 fold change, p<0.05) were determined with Partek® Genomics Suite Gene Expression Data Analysis System and ontology analysis was conducted using Molecular Annotation System 3. The number of genes differentially expressed after fluoxetine exposure was 288 at 25 µg/L and 131 at 250 µg/L; and after sertraline exposure was 33 at 25 µg/L and 52 at 250 µg/L. Five genes were differentially regulated in all treatments relative to control, suggesting that both SSRIs share some similar molecular pathways. Among them, expression of the gene coding for FK506 binding protein 5 (FKBP5), which is annotated to stress response regulation, was highly down-regulated in all treatments (results confirmed by qRT-PCR). Gene ontology analysis indicated that regulation of stress response and cholinesterase activity were critical functions influenced by these SSRIs, and suggested that changes in the transcription of FKBP5 or acetylcholinesterase could be useful biomarkers of SSRIs exposure in wild fish. Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained at the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the UT Insititutional Animal Care and Use Committee (Protocol #1690-1007). Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl. Embroyos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (<15 minutes), such that larvae used in experiments were of similar age at the time of exposure. All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27 +/- 1 C and 14:10h light:dark photoperiod. Larval zebrafish (72 hpf) were exposed for 96 h in 200ml fish water containing appropirate amount of SSRI stock (i.e. fluoxetine or sertraline). There were four SSRIs treatments (25 and 250 ug/L fluoxetine and 25 and 250 ug/L sertraline) and one control (no SSRIs) with triplicate beakers and each beaker contained about 100 larval fish. During exposure for 96 hours, beakers were kept covered to prevent water evaporation and fish were not fed (i.e., fish consumed their yolk sac).
Project description:In triplicate for each condition, 12 WT and acbd6 F0 crispant Danio rerio (zebrafish) embryos were incubated with 20 μM YnMyr for 24 h, either between 48-72 hpf or 96-120 hpf. After labelling, zebrafish were washed twice with fresh egg water, deyolked, flash frozen in liquid nitrogen and stored at -80°C until further analysis.
Project description:We used a Diet-Induced-Obesity approach using the zebrafish (Danio rerio) based on overfeeding to analyze the liver transcriptomic modulation in the disease and to determine how the obesity affects the immune response against an acute inflammatory stimulus as the lipopolysacharide (LPS). Overfed zebrafish were obese and showed signs of steatosis in their liver. Furthermore, the gene modulation profile resembled to that observed in humans. After the inflammatory stimulus, there was a clear differential immune response in normal and overfed fish. In normal fish, the response to LPS reported a typical host defensive reaction comparable to the one occurring in stimulated mammals whereas there was not any significantly modulated gene when comparing the expression of liver from LPS-stimulated with non-stimulated obese zebrafish.