Unknown,Transcriptomics,Genomics,Proteomics

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D. magna exposed to fluoranthene and pyrene - both as single compounds and binary mixtures

ABSTRACT: Daphnia are an important and widely studied model species in ecological and toxicological studies throughout the world and an official (OECD) recommended test organism. Their small size, wide distribution and easy growth conditions make this an organism ideal for functional genomics based studies, including metabolic profiling and transcriptomics. In this study we used an integrated systems approach in which transcriptomic, metabolomic and energetic responses of juvenile (4 days old) daphnids were evaluated in response to exposure to two poly aromatic hydrocarbons (pyrene and fluoranthene) and binary mixtures thereof. In addition, these responses were linked to responses measured during chronic experiments (21 days) assessing survival, growth and reproductive traits. Custom Daphnia magna microarrays were used to assess transcriptomic changes. Hierarchical cluster analysis did not result in a clear distinction between the single compounds suggesting similar molecular modes of action. Cluster analysis with both the single compounds and the binary mixture treatments resulted in a separation of treatments based on differences in toxic ratios rather than component differences. Changes in the metabolic profiles of the organisms were investigated using Nuclear Magnetic Resonance spectroscopy and Gas and Liquid Chromatography Mass Spectrometry. These multivariate metabolomic datasets were analysed with Principal Components Analysis and Partial Least Squares Discriminant Analysis. The major metabolite changes responsible for the differences observed indicated a disturbance in aminosugar metabolism in all cases. The study demonstrates the potential of âomicsâ to provide screening tools for monitoring of the freshwater environment â in invertebrate species - which is reasonably rapid, cost-effective and has the potential to greatly increase the amount of information obtained from aquatic toxicology testing. Two independent experiments were performed, each with two biological replicates. As a result, every treatment was measured four times with the exception of the two highest mixtures doses where we only had two available replicates. In general, a universal reference design was used in which the reference sample was a pool composed of aliquots from all samples. Dye-bias effects were accounted for by labeling two samples of every treatment with Cy3 and two with Cy5. Exp1Rep1 (reference Cy3, sample Cy5); Exp1Rep2 (reference Cy5, sample Cy3); Exp2Rep1 (reference Cy5, sample Cy3) and Exp2Rep2 (reference Cy3, sample Cy5)

ORGANISM(S): Daphnia magna

SUBMITTER: Tine Vandenbrouck

PROVIDER: E-GEOD-16869 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the Aryl Hydrocarbon Receptor (AHR) in a structurally-dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at a concentration that induces developmental malformations by 120 hours post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burden was analyzed at these time points using GC-MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the smallest number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure and may provide a path for unraveling the toxicity of complex PAH mixtures. Gene expression was measured in zebrafish embryos after exposure to PAHs. Embryos were batch-exposed in groups of 40 to 25 μM BAA, 25 μM DBT, 25 μM PYR or 1% DMSO vehicle control starting at 6 hpf and collected at 24 or 48 hpf. Four independent biological replicates were prepared for each treatment. The reference was a pool of zebrafish embryos exposed to DMSO control until 24 and 48 hpf.

Project description:Structural analogues are assumed to elicit toxicity via similar predominant modes of action (MOAs). Currently, MOA categorization of chemicals in environmental risk assessment is mainly based on the physico-chemical properties of potential toxicants. It is often not known whether such classification schemes are also supported by mechanistic biological data. In this study, the toxic effects of two groups of structural analogues (alcohols and anilines) with predefined MOA (narcotics and polar narcotics) were investigated at different levels of biological organization (gene transcription, energy reserves and growth). Chemical similarity was not indicative of a comparable degree of toxicity and a similar biological response. Categorization of the test chemicals based on the different biological responses (growth, energy use and gene transcription) did not result in a classification of the predefined narcotics versus the predefined polar narcotics. Moreover, gene transcription based clustering profiles were indicative of the observed effects at higher level of biological organization. Furthermore, a small set of classifier genes could be identified that was discriminative for the clustering pattern. These classifier genes co-varied with the organismal and physiological responses. Compared to the physico-chemistry based MOA classification, integrated biological multi-level effect assessment can provide the necessary MOA information that is crucial in high-quality environmental risk assessment. Our findings support the view that transcriptomics tools hold considerable promise to be used in biological response based mechanistic profiling of potential (eco)toxicants. The hybridization design was a universal reference design (a mixture of aliquots from control and exposed samples), which is recommended when class discovery is the main purpose of the experiment. One of the three biological replicates of each exposure condition was labeled with one dye, the remaining two samples were labeled with the second dye.

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D. magna exposed to fluoranthene and pyrene - both as single compounds and binary mixtures

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