Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptomic profiling of clobetasol propionate-induced immunosuppression in challenged zebrafish embryos

ABSTRACT: The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos (Danio rerio) using transcriptomic profiling. Clobetasol propionate, a highly potent synthetic corticosteroid, which is known to have immunosuppressive effects on vertebrates including zebrafish, was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection, the embryos were injected with a mixture of different pathogen-associated molecular patterns (PAMPs) consisting of Pam3CSK4 (synthetic lipopeptide), poly(I:C) (synthetic viral dsRNA) and flagellin (isolated from Bacillus subtilis). Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test (OECD 236), 20 fertilized eggs were exposed to a sub-lethal concentration (250 nM) of clobetasol propionate for 48 hours under semi-static conditions. The test comprised of three treatments with clobetasol propionate and two negative control (NC) groups and was performed in triplicates. At 46 hours post fertilization (hpf), the eggs were manually dechorinated. At 48 hpf, the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection, 8 nL of the mixed PAMPs (0.33 µg/mL Pam3CSK4, 0.33 µg/mL poly(I:C) and 0.033 µg/mL flagellin) were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs, while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. After injection, the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Danio rerio

SUBMITTER: Fabian Essfeld

PROVIDER: E-MTAB-11092 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Within the regulatory framework on the approval of new substances, the assessment of immunotoxic modes of action (MoA) is currently not covered. This is not least due to the lack of standardized methods and reliable validated biomarkers for immunotoxic effects. The experimental set up was designed to analyze the compound-specific response of zebrafish embryos to two immunomodulative reference substances, clobetasol propionate (CP, CAS 25122-46-7) and imiquimod (IMQ, CAS 99011-02-6), on the global level of gene expression. The obtained results were used to (i) identify potential biomarker candidates for the assessment of immunotoxic MoAs, (ii) identify compound-specific molecular signatures, (iii) evaluate the reliability of data acquisition using OMICs-coupled approaches and (iv) to assess the suitability of the zebrafish embryo as an alternative model for the human-representative investigation of psoriatic effects. For this, the transcriptomic profiles of embryos were bioinformatically analysed subsequent to an CP-induced immunosuppression in absence or presence of an IMQ-induced immune challenge, i.e. the simulation of a resting and an activated immune system. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to either CP (250 nM) or to IMQ (4000 nM) or to a combination of both under semi static conditions. Exposure to CP started at 2 hours post fertilization (hpf) until 72 hpf. Exposure to IMQ started at 48 hpf until 72 hpf. Untreated embryos reared in medium without CP nor IMQ dissolved was used as a negative control. All conditions comprised three biological replicates. Medium was exchanged on a daily basis by renewing half its volume. Occurrence of morphological changes and indicators of lethality as described in the OECD test guideline 236 were examined microscopically on a daily basis. At 72 hpf, all larvae were pooled for each sample for RNA extraction using a NucleoSpin RNA/Protein kit (Macherey-Nagel) following the manufacturer’s protocol. RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina NovaSeq 6000 System (Illumina) in 150 bp paired-end mode, producing a minimum of 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the Danio rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in zebrafish (Danio rerio) embryos as aquatic vertebrate non-target model exposed to sub lethal concentrations of the heavily used organophosphate insecticide Fipronil (CAS 2921-88-2). The Insecticide Resistance Action Committee (IRAC) classified Fipronil after its mode of action (MoA) in the target organism as an acetylcholinesterase (AChE) inhibitor (Group 1B). The goal is to identify toxicogenomic profiles with predictive character and potential molecular key events (KE) explaining upstream adverse effects in aquatic non-target organisms. This will provide useful information to refine and improve existing adverse outcome pathways (AOP). Furthermore, integrating the obtained profiles for this and other tested chemicals in a collective database will enable us in the future to derive predictions about the ecotoxicological hazard for chemcials with unknown apical effects, based on similarly altered transcriptomic and proteomic profiles. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of Fipronil for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 0.075 mg/L), high exposure (HE, 0.3 mg/L) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in zebrafish (Danio rerio) embryos (96 hpf) as aquatic vertebrate non-target model exposed to sub lethal concentrations of the carbamate insecticide Carbaryl (CAS 63-25-2). The Insecticide Resistance Action Committee (IRAC) classified Carbaryl after its mode of action (MoA) in the target organism as an acetylcholinesterase (AChE) inhibitor (Group 1A). The goal is to identify toxicogenomic profiles with predictive character and potential molecular key events (KE) explaining upstream adverse effects in aquatic non-target organisms. This will provide useful information to refine and improve existing adverse outcome pathways (AOP). Furthermore, integrating the obtained profiles for this and other tested chemicals in a collective database will enable us in the future to derive predictions about the ecotoxicological hazard for chemcials with unknown apical effects, based on similarly altered transcriptomic and proteomic profiles. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of Carbaryl for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 275 µg/L), high exposure (HE, 1100 µg/L) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in zebrafish (Danio rerio) embryos as aquatic vertebrate non-target model exposed to sub lethal concentrations of the heavily used organophosphate insecticide Chlorpyrifos (CAS 2921-88-2). The Insecticide Resistance Action Committee (IRAC) classified Chlorpyrifos after its mode of action (MoA) in the target organism as an acetylcholinesterase (AChE) inhibitor (Group 1B). The goal is to identify toxicogenomic profiles with predictive character and potential molecular key events (KE) explaining upstream adverse effects in aquatic non-target organisms. This will provide useful information to refine and improve existing adverse outcome pathways (AOP). Furthermore, integrating the obtained profiles for this and other tested chemicals in a collective database will enable us in the future to derive predictions about the ecotoxicological hazard for chemcials with unknown apical effects, based on similarly altered transcriptomic and proteomic profiles. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of 6PTU for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 0.75 mg/L), high exposure (HE, 3 mg/L) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for data normalization and differential gene expression analysis with DESeq2.

Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in zebrafish (Danio rerio) embryos as aquatic vertebrate non-target model exposed to sub lethal concentrations of synthetic organochloride insecticide Methoxychlor (CAS 72-43-5). It is structurally highly similar to its precursor molecule DDT (Dichlorodiphenyltrichloroethane). Like its precursor, today Methoxychlor is banned from the use as pesticide in the United States and the European Union due to its acute toxicity, high bioaccumulation potential and endocrine disruption activity. The Insecticide Resistance Action Committee (IRAC) classified Methoxychlor after its mode of action (MoA) in the target organism as a sodium channel modulator (Group 3B). Our goal is to identify toxicogenomic profiles with predictive character and potential molecular key events (KE) explaining upstream adverse effects in aquatic non-target organisms for this substance of particularly high environmental concern. This will provide useful information to refine and improve existing adverse outcome pathways (AOP). Furthermore, integrating the obtained profiles for this and other tested chemicals in a collective database will enable us in the future to derive predictions about the ecotoxicological hazard for chemcials with unknown apical effects, based on similarly altered transcriptomic and proteomic profiles. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of Methoxychlor for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 20 µg/L), mid exposure (ME, 60 µg/L), high exposure (HE, 180 µg/L) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in zebrafish (Danio rerio) embryos as aquatic vertebrate non-target model exposed to sub lethal concentrations of Abamectin (CAS 71751-41-2). Abamectin is a heavily used insecticide applied for crop protection against sucking insects (i.e. Acari). The Insecticide Resistance Action Committee (IRAC) classified Abamectin after its mode of action (MoA) in the target organism as a Glutamate-gated chloride channel (GluCl) allosteric modulator (Group 6). In vertebrates, GluCl do not exist, but they are closely related to vertebrate glycine receptors (Wolstenholme 2012). The goal is to identify toxicogenomic profiles with predictive character and potential molecular key events (KE) explaining upstream adverse effects in aquatic non-target organisms. This will provide useful information to refine and improve existing adverse outcome pathways (AOP). Furthermore, integrating the obtained profiles for this and other tested chemicals in a collective database will enable us in the future to derive predictions about the ecotoxicological hazard for chemcials with unknown apical effects, based on similarly altered transcriptomic and proteomic profiles. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of Abamectin for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE, 0.11 mg/L), mid exposure (ME, 0.22 mg/L), high exposure (HE, 0.44 mg/L) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Dataset Information

Transcriptomic profiling of clobetasol propionate-induced immunosuppression in challenged zebrafish embryos

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure