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Proteome Integral Solubility Alteration assay in zebrafish embryo for exploring exposure to bioactive compounds including pollutants and chemicals mixtures

ABSTRACT: The One Health initiative connects human and animal health with their shared environment. The development of novel methodology to unbiased identification of mechanisms of action of chemicals and chemicals mixtures would address one of today´s challenges of environmental and human toxicology. Zebrafish embryos offer a unique opportunity to explore the proteome integral solubility alteration (PISA) assay to study the proteins that increase or decrease their solubility as protein targets of the bioactive compounds in an organism at an developmental stage. We presented here 4 different applications of the PISA method in zebrafish embryo with different types of compounds of environmental and health concern: i) endocrine disrupting chemical, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to outline the pathways compromised under exposure; ii) complex chemical mixtures composed by TCDD + alpha-endosulfan + bisphenol A (BPA) previously studied in vitro cells lines to evaluate single chemical versus mixture; iii) novel compounds from blue-green circular economy to map the organismal response, and a iv) novel drug directed to anti-cancer activity in lymphoma models to discharge side-effects at non-target organs. Our results demonstrated the unique opportunities of PISA assay in zebrafish for toxicology. We remark some differences to its application for drug target deconvolution. First, we evaluate both targets arising from an increase in solubility and in those decreasing, as both would be involved in modulation the cellular functions. Second, the method could be applicable to bioactive compound before chemical characterization or a chemical mixture of unknown stoichiometry. Third, the results from zebrafish embryo could provide a first glimpse of unspecific interactions with proteins in non-target tissues.The introduction of PISA assay in toxi- and ecotoxicoproteomics offers high-resolution and throughput in support with the 3R, and 3M principles.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

SUBMITTER: Ana Maria Carrasco del Amor

LAB HEAD: Susana Cristobal

PROVIDER: PXD021670 | Pride | 2022-02-28

REPOSITORIES: Pride

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Project description:Acetylcholinesterase (AChE) inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of muscular activity. Whether AChE inhibitors and related neurotoxic compound also provoke specific changes in gene transcription patterns that allow to establish a mechanistic link useful for diagnostic purposes has, however, yet not been investigated. Therefore, as a proof of principle, we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM), in zebrafish embryos and compared the response with two non-AChE inhibiting control compounds, 1,4-dimethoxybenzene (DMB) and 2,4-dinitrophenol (DNP). A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive gene in response to AChE inhibitors. Comparison of expression in wild-type, ache and fixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR) activity. Furthermore, modulators of intracellular calcium levels led to an hspb11 transcriptional up-regulation which suggest that elevated intracellular calcium levels and the resulting increase in muscular activity triggering hspb11 up-regulation. During early zebrafish development, hspb11 was specifically expressed in muscle pioneers and morpholino-knockdown resulted in defects in slow muscle myosin organization. These findings demonstrate that a combination of transcriptome and functional analyzes in the zebrafish embryo is able to correlate gene expression changes to the mechanism of action of neurotoxic compounds. Zebrafish embryos were exposed for 24 h and 48 h (26-50 and 2-50 hpf) to 5,83 µM of azinphos-methyl (APM), 509 µM of 1,4-dimethoxybenzene (DMB) and 14,2 µM 2,4-dinitrophenol (DNP), the respective LC10 of each compound, in order to determine mode of action specific responding genes, or not exposed and parallel treated as controls. 50 zebrafish embryos were used per treatment, and 4 biological independent replicates were analysed, respectively.

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Proteome Integral Solubility Alteration assay in zebrafish embryo for exploring exposure to bioactive compounds including pollutants and chemicals mixtures

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