Unknown,Transcriptomics,Genomics,Proteomics

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Whole Adult Organism Transcriptional Profiling of Metal Exposures in Zebrafish

ABSTRACT: A convergence of technological breakthroughs in the past decade has facilitated the development of rapid screening tools for biomarkers of toxicant exposure and effect. Platforms using the whole adult organism to evaluate the genome-wide response to toxicants are especially attractive. Recent work demonstrates the feasibility of this approach in vertebrates using the experimentally robust zebrafish model. In the present study, we evaluated gene expression changes in whole adult zebrafish following an acute 24 hour exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride, and sodium dichromate concentrations corresponding to their respective 96 hr LC20, LC40 and LC60. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal. Comparative analysis identified subsets of differentially expressed transcripts both overlapping and unique to each metal. Application of gene ontology (GO) and transcription factor enrichment algorithms revealed a number of key biological processes perturbed by metal exposures and the master transcriptional regulators mediating gene expression changes. Metal exposures differentially activated biological processes associated with ribosome biogenesis, proteosomal degradation and p53 signaling cascades, while repressing oxygen-generating pathways associated with amino acid and lipid metabolism. Despite appreciable effects on gene regulation, nickel exposures did not induce any morphological alterations in zebrafish organs and tissues. Histopathological effects of cobalt remained confined to the olfactory system, while chromium targeted the gills, pharynx and intestinal mucosa. A number of enriched transcription factors mediate the observed gene response to metal exposure, including known targets such as p53, HIF1M-oM-^AM-! and the myc oncogene and novel regulatory factors such as XBP1, GATA6 and HNF3M-oM-^AM-". This work uses an experimentally innovative approach to capture global responses to metal exposures and provides mechanistic insights into metal toxicity mechanisms. Metal induced changes in gene expression in zebrafish were measured after 24 h exposures to each of three metals (nickel, chromium or cobalt). A total of 48 arrays were processed - 16 arrays per metal with 4 replicates for each exposure condition (control, low, mid, and high).

ORGANISM(S): Danio rerio

SUBMITTER: Christine Baer

PROVIDER: E-GEOD-50648 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:A convergence of technological breakthroughs in the past decade has facilitated the development of rapid screening tools for biomarkers of toxicant exposure and effect. Platforms using the whole adult organism to evaluate the genome-wide response to toxicants are especially attractive. Recent work demonstrates the feasibility of this approach in vertebrates using the experimentally robust zebrafish model. In the present study, we evaluated gene expression changes in whole adult zebrafish following an acute 24 hour exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride, and sodium dichromate concentrations corresponding to their respective 96 hr LC20, LC40 and LC60. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal. Comparative analysis identified subsets of differentially expressed transcripts both overlapping and unique to each metal. Application of gene ontology (GO) and transcription factor enrichment algorithms revealed a number of key biological processes perturbed by metal exposures and the master transcriptional regulators mediating gene expression changes. Metal exposures differentially activated biological processes associated with ribosome biogenesis, proteosomal degradation and p53 signaling cascades, while repressing oxygen-generating pathways associated with amino acid and lipid metabolism. Despite appreciable effects on gene regulation, nickel exposures did not induce any morphological alterations in zebrafish organs and tissues. Histopathological effects of cobalt remained confined to the olfactory system, while chromium targeted the gills, pharynx and intestinal mucosa. A number of enriched transcription factors mediate the observed gene response to metal exposure, including known targets such as p53, HIF1 and the myc oncogene and novel regulatory factors such as XBP1, GATA6 and HNF3. This work uses an experimentally innovative approach to capture global responses to metal exposures and provides mechanistic insights into metal toxicity mechanisms.

Project description:A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterised from mycobacteria. Mutants of M. tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal-exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FA) show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. A relatively large deltar(obs) in FA implies formation of high order apo-KmtR(n)-DNA and multiple complexes were detected by EMSA. Incubation of KmtR with cobalt inhibits DNA-complex assembly and metal-protein binding was confirmed by competition against 4-(2-pyridylazo)-resorcinol. KmtR is the second, to NmtR, characterised ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, while NmtR retains a response to nickel and cobalt under these conditions. Mixing equimolar apo-KmtR and apo-NmtR with 0.8 equivalents of nickel or cobalt gave nickel- and cobalt-dependent difference emission spectra similar to nickel(0.8)-KmtR and cobalt(0.8)-KmtR, respectively. Thus, KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB related proteins are listed in databases and a proportion can be predicted to detect metals through known allosteric sites. KmtR has none of the previously defined sites. Substitution of His(88), Glu(101), His(102), His(110) or His(111) with Gln generated KmtR-variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory-motifs revealing that only the latter should be used to predict metal-sensing. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-49

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Whole Adult Organism Transcriptional Profiling of Metal Exposures in Zebrafish

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