Project description:Impacts of engineered nanomaterial (ENM) exposure is known to be dependent upon physico-chemical characteristics of the material but is also significantly dependent upon the organism exposed, with unclear reasons for these differences. Although studies have identified some common mechanistic impacts across species (e.g., oxidative stress), these do not necessarily correlate to adverse outcomes, which may differ by orders of magnitude across species. Differences in toxicological response could potentially be due to species-specific biochemical mechanisms, magnitude of response of common pathways across species, or differences in the exposure and uptake of nanomaterials. In-depth analysis across studies is made difficult as multiple species are not often included in the same study, nanomaterials from multiple sources differ in their characteristics, and variation in exposure duration and media confounds the ability to draw synthetic conclusions from existing cross-species data. The current study examines mechanisms of response across model species Danio rerio, Daphnia magna, and Chironomus riparius to a single-source complex metal oxide, lithium cobalt oxide nanosheets. Each species has previously been shown to have differing sensitivities to NM exposure. RNA-sequencing was used to identify impacted pathways and physiological functions, revealing nanomaterial-specific biochemical responses not replicated by ion exposures, as has also been shown by standard toxicology tests, and importantly that nanomaterial uptake does not fully explain cross-species differences. Significant variation in biochemical response may explain differences, both in the magnitude of response of unifying mechanisms across species and novel pathways impacted in a sensitive or tolerant species. Analysis indicates both commonly identified responses to ENM, including stress, changes in energy metabolism, apoptosis, and immune functions and lesser-explored or novel responses involved in cardiovascular system, hormone, and central nervous system impacts. This comparison demonstrates new universal mechanisms of toxicity for this model ENM and provides insight into how biomolecular responses across species can play a role in varying sensitivity to nanomaterials in general.

Project description:Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment. Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment. Four 14-day Chironomus riparius sediment toxicity tests were conducted, one with each toxicant. The surviving larvae were individually flash frozen in liquid nitrogen. For each toxicant we analyzed the gene expression of larvae exposed to low, intermediate and high concentrations. We also included a control and a solvent control. For each treatment we analyzed 10 replicates (individual larvae).

Project description:Whole-transcriptome gene-expression analyses are commonly performed in species that have a sequenced genome and for which microarrays are commercially available. To do such analyses in species with no or limited genome data, i.e. non-model organisms, necessary transcriptomics resources, i.e. an annotated transcriptome and a validated gene-expression microarray, must first be developed. The aim of the present study was to establish an advanced approach for developing transcriptomics resources for non-model organisms by combining next-generation sequencing (NGS) and microarray technology. We applied our approach to the non-biting midge Chironomus riparius, an ecologically relevant species that is widely used in sediment ecotoxicity testing. We sampled extensively covering all C. riparius developmental stages as well as toxicant exposed larvae and obtained from a normalized cDNA library 1.5 M NGS reads totalling 501 Mbp. Using the NGS data we developed transcriptomics resources in several steps. First, we designed 844 k probes directly on the NGS reads, as well as 76 k probes targeting expressed sequence tags of related species. These probes were tested for their affinity to C. riparius DNA and mRNA, by performing two biological experiments with a 1 M probe-selection microarray that contained the entire probe-library. Subsequently, the 1.5 M NGS reads were assembled into 23,709 isotigs and 135,082 singletons, which were associated to ~55 k, respectively, ~61 k gene ontology terms and which corresponded together to 22,593 unique protein accessions. An algorithm was developed that took the assembly and the probe affinities to DNA and mRNA into account, what resulted in 59 k highly-reliable probes that targeted uniquely 95% of the isotigs and 18% of the singletons. Concluding, our approach allowed the development of high-quality transcriptomics resources for C. riparius, and is applicable to any non-model organism. It is expected, that these resources will advance ecotoxicity testing with C. riparius as whole-transcriptome gene-expression analysis are now possible with this species. 1x 1M CGH array with Cy3 labeled C. riparius gDNA and Cy5 labeled A. gambiae gDNA. The microarray was designed against C. riparius mRNA sequencing reads, and has been used to identify trustworthy sequencing reads to design an expression array. This 1M array is therefore not functionally annotated.

Project description:The goal of this study was to investigate the effects of magnetic iron cobalt oxide nanoparticles (cobalt doped Fe3O4 nanoparticles with increasing amounts of cobalt) after pulmonary exposure while in parallel presenting a proteomics platform that is easily transferable to large-scale nanotoxicology screening as part of an integrated assessment and testing approach for regulation of nanoparticles. Bronchoalveolar lavage fluid (BALF) is a proximal biofluid that can be used to monitor airway inflammation and toxic responses in the lung. It is routinely sampled for differential lung diagnostics and has been discussed as a source for early detection of lung cancer. In order to assess effects of metal oxide nanoparticles upon inhalation, bronchoalveolar lavage fluid from mice dosed by single intratracheal instillation was collected and subjected to classical biocompatibility assays as well as proteome analysis. Magnetic oxide nanoparticles with iron and cobalt oxide (Fe3-xCoxO4) at different ratios (1:0, 3:1, 1:1, 1:3, 0:1) were tested at two concentrations (54 µg, 162 µg per animal) and two time points after instillation (day 1, day 3). As a positive control carbon black nanomaterial known to induce lung inflammation was included for both time points, but only at the high dosage (162 µg per animal). Proteomics experiments were divided into three parts – test of reproducibility, discovery and screening phase. The reproducibility of the newly introduced Evosep One LC system was evaluated by re-measuring of technical replicates (n=16). During the discovery phase, selected representative samples with 3 biological replicates per group (total n=9) including pure iron oxide nanoparticles, pure cobalt oxide nanoparticles and vehicle controls were subjected to in-depth proteome profiling by extensive pre-fractionation and including isobaric tandem mass tag (TMT) labeling following a classical LC-MS/MS setup. This step allowed us to identify affected pathways and generate hypotheses regarding mechanisms of the effects of nanoparticles. During screening, all samples of the study were measured label-free as single-shot injections separated on short gradients of 21 min using the robust, high-throughput Evosep One LC system. This step allowed a fast screening of the 5 different types of magnetic metal oxide nanoparticles on BALF, at two concentrations and two time points together with their representative controls (total n=166). All screening measurements were completed in only 2.7 days.

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Protein Fe-S centers as a molecular target of toxicity of a complex transition metal oxide nanomaterial with downstream impacts on metabolism and growth

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