Dataset Information

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

ABSTRACT: Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC(10)) exposure for 48?h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate "pattern-only" PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress.

SUBMITTER: Hanisch K

PROVIDER: S-EPMC3200224 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

Hanisch Karen K Küster Eberhard E Altenburger Rolf R Gündel Ulrike U

International journal of proteomics 20101014

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed ...[more]

PMID: 22084678

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Project description:Glutathione (GSH), the most abundant vertebrate endogenous redox buffer, plays key roles in organogenesis and embryonic development, however, organ-specific GSH utilization during development remains understudied. Monochlorobimane (MCB), a dye conjugated with GSH by glutathione-s-transferase (GST) to form a fluorescent adduct, was used to visualize organ-specific GSH utilization in live developing zebrafish (Danio rerio) embryos. Embryos were incubated in 20 μM MCB for 1 h and imaged on an epifluorescence microscope. GSH conjugation with MCB was high during early organogenesis, decreasing as embryos aged. The heart had fluorescence 21-fold above autofluorescence at 24 hpf, dropping to 8.5-fold by 48 hpf; this increased again by 72 hpf to 23.5-fold, and stayed high till 96 hpf (18-fold). The brain had lower fluorescence (10-fold) at 24 and 48 hpf, steadily increasing to 30-fold by 96 hpf. The sensitivity and specificity of MCB staining was then tested with known GSH modulators. A 10-min treatment at 48 hpf with 750 μM tert-butylhydroperoxide, caused organ-specific reductions in staining, with the heart losing 30% fluorescence, and, the brain ventricle losing 47% fluorescence. A 24 h treatment from 24-48 hpf with 100 μM of N-Acetylcysteine (NAC) resulted in significantly increased fluorescence, with the brain ventricle and heart showing 312% and 240% increases respectively, these were abolished upon co-treatment with 5 μM BSO, an inhibitor of the enzyme that utilizes NAC to synthesize GSH. A 60 min 100 μM treatment with ethacrynic acid, a specific GST inhibitor, caused 30% reduction in fluorescence across all measured structures. MCB staining was then applied to test for GSH disruptions caused by the toxicants perfluorooctanesulfonic acid and mono-(2-ethyl-hexyl)phthalate; MCB fluorescence responded in a dose, structure and age-dependent manner. MCB staining is a robust, sensitive method to detect spatiotemporal changes in GSH utilization, and, can be applied to identify sensitive target tissues of toxicants.

Project description:BackgroundDevelopment and application of transcriptomics-based gene classifiers for ecotoxicological applications lag far behind those of biomedical sciences. Many such classifiers discovered thus far lack vigorous statistical and experimental validations. A combination of genetic algorithm/support vector machines and genetic algorithm/K nearest neighbors was used in this study to search for classifiers of endocrine-disrupting chemicals (EDCs) in zebrafish. Searches were conducted on both tissue-specific and tissue-combined datasets, either across the entire transcriptome or within individual transcription factor (TF) networks previously linked to EDC effects. Candidate classifiers were evaluated by gene set enrichment analysis (GSEA) on both the original training data and a dedicated validation dataset.ResultsMulti-tissue dataset yielded no classifiers. Among the 19 chemical-tissue conditions evaluated, the transcriptome-wide searches yielded classifiers for six of them, each having approximately 20 to 30 gene features unique to a condition. Searches within individual TF networks produced classifiers for 15 chemical-tissue conditions, each containing 100 or fewer top-ranked gene features pooled from those of multiple TF networks and also unique to each condition. For the training dataset, 10 out of 11 classifiers successfully identified the gene expression profiles (GEPs) of their targeted chemical-tissue conditions by GSEA. For the validation dataset, classifiers for prochloraz-ovary and flutamide-ovary also correctly identified the GEPs of corresponding conditions while no classifier could predict the GEP from prochloraz-brain.ConclusionsThe discrepancies in the performance of these classifiers were attributed in part to varying data complexity among the conditions, as measured to some degree by Fisher's discriminant ratio statistic. This variation in data complexity could likely be compensated by adjusting sample size for individual chemical-tissue conditions, thus suggesting a need for a preliminary survey of transcriptomic responses before launching a full scale classifier discovery effort. Classifier discovery based on individual TF networks could yield more mechanistically-oriented biomarkers. GSEA proved to be a flexible and effective tool for application of gene classifiers but a similar and more refined algorithm, connectivity mapping, should also be explored. The distribution characteristics of classifiers across tissues, chemicals, and TF networks suggested a differential biological impact among the EDCs on zebrafish transcriptome involving some basic cellular functions.

Dataset Information

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

Publications

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

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