Exposure of zebrafish embryos to organic solvents, dimethylformamide and dimethyl sulfoxide
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ABSTRACT: Current aquatic chemical testing guidelines recognise that solvents can potentially interfere with the organism or environmental conditions of aquatic ecotoxicity tests and therefore recommend concentration limits for their use. These recommendations are based on evidence of adverse solvent effects in apical level tests. The growing importance of sub-apical and chronic endpoints in future test strategies, however, suggests the limits may need re-assessment. To address this concern, microarrays were used to determine the effects of organic solvents, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), upon the transcriptome of zebrafish (Danio rerio) embryos. Embryos were exposed for 48 h to 0.025 and 0.1 ml/L DMF or DMSO. Effects on survival and development after 24 and 48 h were assessed microscopically with no effects on mortality or morphology. However, analysis of 48-h embryonic RNA revealed large numbers of differentially expressed genes for both solvent at both concentrations. The enrichment of differentially expressed genes was found for metabolic, development and other key biological processes, some of which could be linked to observed morphological effects at higher solvent concentrations. These findings emphasise the need to remove or lower as far as possible, the concentrations of solvent carriers in ecotoxicology tests.
Project description:Current aquatic chemical testing guidelines recognise that solvents can potentially interfere with the organism or environmental conditions of aquatic ecotoxicity tests and therefore recommend concentration limits for their use. These recommendations are based on evidence of adverse solvent effects in apical level tests. The growing importance of sub-apical and chronic endpoints in future test strategies, however, suggests the limits may need re-assessment. To address this concern, microarrays were used to determine the effects of organic solvents, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), upon the transcriptome of zebrafish (Danio rerio) embryos. Embryos were exposed for 48 h to 0.025 and 0.1 ml/L DMF or DMSO. Effects on survival and development after 24 and 48 h were assessed microscopically with no effects on mortality or morphology. However, analysis of 48-h embryonic RNA revealed large numbers of differentially expressed genes for both solvent at both concentrations. The enrichment of differentially expressed genes was found for metabolic, development and other key biological processes, some of which could be linked to observed morphological effects at higher solvent concentrations. These findings emphasise the need to remove or lower as far as possible, the concentrations of solvent carriers in ecotoxicology tests. Balanced loop design consisting of five separate conditions - two exposure concentrations for each of the two solvents and a control treatment - using four replicates for each condition all of which were dye swapped, resulting in a total of twenty independant samples on twenty arrays.
Project description:The per-and polyfluoroalkyl substances (PFAS) are of significant global concern due to their highly ubiquitous and persistent nature, bioaccumulation in organisms, and potential toxicity. The aquatic environment is known as an important sink for PFAS resulting in high concentrations in aquatic organisms. However, little is known about the developmental windows of sensitivity in which the PFAS chemicals are biologically active, in addition to the toxicity endpoints that best reflect chemical hazard. In this study, zebrafish (Danio rerio) were exposed to a 0.33% DMSO vehicle control, 1uM chlorpyrifos (CAS 2921-88-2) positive control, and eight concentrations (0-100 uM, half-log dilutions) of three environmentally relevant PFAS compounds in concentration-response: PFOS (CAS 1763-21-1), PFOA (CAS 45285-51-6), and PFHxS (CAS 355-46-4). There was also a group of unexposed zebrafish aliquoted from a single pool into all exposure plates to serve as a quality assurance measure. The goal of this study was to generate transcriptomic point of departure (tPOD; a benchmark dose/concentration -based treatment level below which a concerted gene expression response is not observed) estimates for zebrafish exposed to PFAS compounds as a health protective exposure level for risk assessment. Through the use of short-term embryo/larval plate-based high-throughput toxicity tests, tPODs were determined across seven distinct developmental windows (6-24 hours post fertilization (hpf), 6-48 hpf, 24-48 hpf, 6-120 hpf, 24-120 hpf, 48-120 hpf, 96-120 hpf) to assess how common experimental design variables (e.g., different exposure durations, exposure at different developmental stages) affect point of departure estimates.
Project description:The aim of this experiment was to identify the genes involved in the detoxification of the toxic pollutant and explosive compound 2,4,6-trinitrotolune (TNT). Fourteen-day-old, liquid culture grown, Arabidopsis seedlings, ecotype Col0 (NASC stock code N1093), were dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF) solvent, or 60 ul DMF only. After six hours, RNA was extracted and used for the microarray analysis. Further details and characterisation of glucosyltransferases identified using this method are presented in citation below.
Project description:The aim of this experiment was to identify the genes involved in the detoxification of the toxic pollutant and explosive compound 2,4,6-trinitrotoluene (TNT). Fourteen-day-old, liquid culture grown, Arabidopsis seedlings, ecotype Col0 (NASC stock code N1093), were dosed with 60 uM TNT dissolved in 60 ul dimethyl formamide (DMF) solvent, or 60 ul DMF only. After six hours, RNA was extracted and used for the microarray analysis. Further details and characterisation of glucosyltransferases identified using this method are presented in citation below. 6 samples were used in this experiment
Project description:Daphnia magna has been used extensively to evaluate organism- and population-level responses to pollutants in acute toxicity and reproductive toxicity tests. We have previously reported that exposure to juvenile hormone (JH) agonists results in a reduction of reproductive function and production of male offspring in a cyclic parthenogenesis, D. magna. Recent advances in molecular techniques have provided tools to better understand the responses to pollutants in aquatic organisms including D. magna. DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to JH agonists; methoprene (125, 250 and 500 ppb), fenoxycarb (0.5, 1 and 2 ppb), and epofenonane (50, 100 and 200 ppb). Exposure to these JH analogs resulted in chemical specific patterns of gene expression. The heat map analyses based on hierarchical clustering revealed a similar pattern between treatments with a high dose of methoprene and with epofenonane. In contrast, treatment with low to middle doses of methoprene resulted in similar profiles to fenoxycarb treatments. Hemoglobin and JH epoxide hydrolase genes clustered to be JH-responsive genes. These data suggest that fenoxycarb has high activity as a JH agonist, methoprene shows high toxicity and epofenonane works through a different mechanism compared with other JH analogs, agreeing with data of previously reported toxicity tests. In conclusion, D. magna DNA microarray is useful for classification of JH analogs and identification of JH-responsive genes. Juvenile hormone agonists induced gene expression in daphnids neonates was measured at 48 hours after exposure to methoprene (125, 250, 500 ppb), fenoxycarb (0.5, 1, 2 ppb), epofenonane (50, 100, 200) and DMF as a control. Three independent experiments were performed at each chemicals.
Project description:The lack of accurate in vitro assays for predicting in vivo toxicity of chemicals together with new legislations demanding replacement and reduction of animal testing has triggered the development of alternative methods. This study aimed at developing a transcriptomics-based in vitro prediction assay for in vivo genotoxicity. The transcriptomics changes induced in the human liver cell line HepG2 by 34 compounds after treatment for 12h, 24h and 48h were used for the selection of gene-sets that can discriminate between in vivo genotoxins (GTX) and in vivo non-genotoxins (NGTX). By combining publicly available results for these chemicals from standard in vitro genotoxicity studies with transcriptomics, we developed several prediction models. These models were validated by means of an additional set of 28 chemicals. The study investigated differential gene expression in HepG2 cell line mRNA following 12 hours of exposure to 34 different compounds and their solvents; 24 and 48 hours of exposure to 62 different compounds and their solvents. Three biological replicates per compound/solvent. In total 560 arrays .
Project description:The identification of endocrine disruptive properties of chemicals certain to enter the aquatic environment relies on toxicity tests with fish, assessing adverse effects on reproduction and sexual development. The demand for quick, reliable endocrine disruption (ED) assays favored the use of fish embryos as alternative test organisms. We investigated the application of a transcriptomics-based assay for estrogenic and anti-androgenic chemicals with zebrafish embryos. Two reference compounds, 17a-ethinylestradiol and flutamide, were tested to evaluate the effects on development and the transcriptome after 48h-exposures. Comparison of the transcriptome response with other estrogenic and anti-androgenic compounds (genistein, bisphenol A, methylparaben, linuron, prochloraz, propanil) showed commonalities and differences in regulated pathways, enabling us to classify the estrogenic and anti-androgenic potencies. This demonstrates that different mechanism of ED can be assessed already in fish embryos. Newly fertilized embryos (<2hpf) were exposed for 48 hours to 0.65mg/l (EC10) and 0.8mg/l (EC20) 17-alpha ethinylestradiol (EE2), 1.2mg/l (EC10) and 1.4mg/l (EC20) flutamide, 8 mg/l (EC10)and 8.5mg/l (EC20) bisphenol A(BPA), 0.8 mg/l (EC10) and 1.1mg/l (EC20) propanil, 19.8mg/l (EC10) and 24.4mg/l (EC20) methylparaben, 1.2 mg/l (EC10) and 1.3mg/l (EC20) linuron as well as to 1.7mg/l (EC10) and 2 mg/l (EC20) prochloraz. Water controls were included in all studies and acetone solvent controls were applied when indicated (AC). For each study, four replicates (a,b,c,d) were performed per condition, each consisting of 24 pooled embryos.
Project description:Recent advances in mass spectrometry (MS) have enabled quantitative proteomics to become a powerful tool in the field of drug discovery, especially when applied toward proteome-wide target engagement studies. Similar to temperature gradients, increasing concentrations of organic solvents stimulate unfolding and precipitation of the cellular proteome. This property can be influenced by physical association with ligands and other molecules, making individual proteins more or less susceptible to solvent-induced denaturation. Herein, we report the development of proteome-wide solvent shift assays by combining the principles of solvent-induced precipitation (Zhang et al., 2020) with modern quantitative proteomics. Using this approach, we developed solvent proteome profiling (SPP), which is capable of establishing target engagement through analysis of SPP denaturation curves. We readily identified the specific targets of compounds with known mechanisms of action. As a further efficiency boost, we applied the concept of area-under-the-curve analysis to develop solvent proteome integral solubility alteration (solvent-PISA) and demonstrate that this approach can serve as a reliable surrogate for SPP. We propose that by combining SPP with alternative methods, like thermal proteome profiling, it will be possible to increase the absolute number of high-quality melting curves that are attainable by either approach individually thereby increasing the fraction of the proteome that can be screened for evidence of ligand binding.
Project description:The aim of this mRNA expression profiling experiment was to screen for ecotoxicogenomic fingerprints in juvenile daphnids (daphnia magna) as aquatic vertebrate non-target model exposed to sub lethal concentrations of Fipronil. Fipronil is a widely used insecticide applied for crop protection against field pests and Ectoparasites. The Insecticide Resistance Action Committee (IRAC) classified Fipronil after its mode of action (MoA) in the target organism as a GABA-gated chloride channel blocker (Group 2). The goal is to identify toxicogenomic profiles with predictive character and identify potential molecular key events (KE) explaining upstream adverse effects. 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 acute immobilisation test (OECD 202), 50 juvenile Daphnids were exposed to two sub lethal concentrations of Fipronil for 48 hours under static conditions. Each test comprised of a low exposure (LE), high exposure (HE) and a negative control (NC) group and was performed in triplicates. At 48 hours after introducing the daphnids into the test solutions, RNA and protein was extracted from living daphnids with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before messenger 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. magna reference genome (daphmag2.4) using 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 count 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 juvenile daphnids (daphnia magna) as aquatic vertebrate non-target model exposed to sub lethal concentrations of Imidacloprid. Imidacloprid is is a popular insecticide widely applied in agriculture and by veterinarians against ectoparasites.The Insecticide Resistance Action Committee (IRAC) classified Imidacloprid after its mode of Action (MoA) in the target organism as a nicotinic acetylcholine receptor (nAChR) competitive modulator (Group 4). The goal is to identify toxicogenomic profiles with predictive character and identify potential molecular key events (KE) explaining upstream adverse effects. 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 acute immobilisation test (OECD 202), 50 juvenile Daphnids were exposed to two sub lethal concentrations of Imidacloprid for 48 hours under static conditions. Each test comprised of a low exposure (LE), high exposure (HE) and a negative control (NC) group and was performed in triplicates. At 48 hours after introducing the daphnids into the test solutions, RNA and protein was extracted from living daphnids with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before messenger 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. magna reference genome (daphmag2.4) using 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 count normalization and differential gene expression analysis with DESeq2.