Transcriptome and Proteome Analyses Reveal Stage-Specific DNA Damage Response in Embryos of Endangered Sturgeon

DNA damage during early life stages may have a negative effect on embryo development, inducing malformations that have long-lasting effects during adult life. Therefore, in the current study, we analyzed the effect of DNA damage induced by genotoxicants (camptothecin (CPT) and olaparib) at different stages of embryo development. We analyzed the survival, DNA fragmentation, transcriptome, and proteome of the endangered sturgeon Acipenser ruthenus. Sturgeons are non-model fish species that can provide new insights into the DNA damage response and embryo development. The transcriptomic and proteomic patterns changed significantly after exposure to genotoxicants in a stage-dependent manner. The results of this study indicate a correlation between phenotype formation and changes in transcriptomic and proteomic profiles. CPT and olaparib downregulated oxidative phosphorylation and metabolic pathways, and upregulated pathways involved in nucleotide excision repair, base excision repair, and homologous recombination. We observed the upregulated expression of zona pellucida sperm-binding proteins in all treatment groups, as well as the upregulation of several glycolytic enzymes. The analysis of gene expression revealed several markers of DNA damage response and adaptive stress-response, which could be applied in toxicological studies on fish embryo. This study is the first complex analysis of the DNA damage response in endangered sturgeons.

Specificity of time- and dose-dependent morphological endpoints in the fish embryo acute toxicity (FET) test for substances with diverse modes of action: the search for a “fingerprint”

The fish embryo acute toxicity (FET) test with the zebrafish (Danio rerio) embryo according to OECD TG 236 was originally developed as an alternative test method for acute fish toxicity testing according to, e.g., OECD TG 203. Given the versatility of the protocol, however, the FET test has found application beyond acute toxicity testing as a common tool in environmental hazard and risk assessment. Whereas the standard OECD guideline is restricted to four core endpoints (coagulation as well as lack of somite formation, heartbeat, and tail detachment) for simple, rapid assessment of acute toxicity, further endpoints can easily be integrated into the FET test protocol. This has led to the hypothesis that an extended FET test might allow for the identification of different classes of toxicants via a “fingerprint” of morphological observations. To test this hypothesis, the present study investigated a set of 18 compounds with highly diverse modes of action with respect to acute and sublethal endpoints. Especially at higher concentrations, most observations proved toxicant-unspecific. With decreasing concentrations, however, observations declined in number, but gained in specificity. Specific observations may at best be made at test concentrations ≤ EC10. The existence of a “fingerprint” based on morphological observations in the FET is, therefore, highly unlikely in the range of acute toxicity, but cannot be excluded for experiments at sublethal concentrations.

Chorion Alterations in Eyed-Stage Salmonid Eggs Farmed in La Araucanía, Chile: A Retrospective Study

The chorion is the primary envelop that protects the fish embryo against mechanical actions, pathogens, and abrupt changes in physical and chemicals conditions of the incubation medium. During embryo development, chorion alterations are not rare, but the occurrence of these is scarcely reported. Increased frequency of chorion alterations can result in increased embryo mortality and thus decreased reproductive performance and losses for fish farms. In this study, we characterize different chorion alterations observed in samples collected over 14 years from 12 salmon and trout farms located in the region of La Araucanía in southern Chile, which sent live eyed-stage embryos (‘eyed-eggs’) for quality analysis to our laboratory. We found soft chorion as the most common alteration observed, being present in the whole 14-year series analyzed in Atlantic Salmon (Salmo salar) and affecting up to 35.0% of the samples examined in a year. This alteration also affected up to 20.0 and 5.7% of Coho Salmon (Oncorhynchus kisutch) and Rainbow Trout (Oncorhynchus mykiss) samples analyzed in a year, respectively. We also found an increase of other chorion alterations, including perforated and white-spotted chorion in Atlantic and Coho Salmon, in the last 8 years. Among the three species, Rainbow Trout exhibited fewer chorion alterations. As the embryonated eggs analyzed here were obtained from broodstocks maintained under standard industrial conditions, these alterations might be linked to changes in environmental conditions affecting the incubation water that need to be further investigated.

Do Single-Component and Mixtures Selected Organic UV Filters Induce Embryotoxic Effects in Zebrafish (Danio rerio)?

UVs are important ingredients in common cosmetic products (e.g., sunscreens, hairsprays, soap). After their use, they can enter the aquatic ecosystem and negatively affect non-target aquatic organisms. The aim of our study was to evaluate acute embryotoxicity of selected organic UVs 2-phenylbenzimidazole-5-sulfonic acid (PBSA), ethylhexyl methoxycinnamate (EHMC), octocrylene (OC), 4-methylbenzylidene camphor (4-MBC) and benzophenone-3 (BP-3). The chemicals were tested both as a single substance and their mixtures. The types of mixtures were chosen as follows: the combination of OC and 4-MBC; the combination of PBSA, EHMC and BP-3 and the combination of all five UV filters. The embryotoxicity was evaluated using a modified method of the Fish Embryo Acute Toxicity Test-OECD guideline 236 and zebrafish (Danio rerio) was selected as a suitable fish model organism. The toxic effects were studied by assessing mortality, hatching and the occurrence of malformations at 24, 48, 72 and 96 h post fertilization. The obtained results indicate that especially the mixture of OC and 4-MBC presents a potential risk of embryotoxicity for zebrafish due to a significant increase in mortality, which was 41.7% in the experimental group exposed to 10 μg/L at 96 h post fertilization. Based on our results, the most effected sub-lethal endpoints were hatching and malformation (e.g., edema of pericard, bent spine, yolk edema), but with no statistically significant effect. These results differ within groups with single UVs and with their mixtures, suggesting the interaction of these substances when they are exposed together.

Profiling site-specific cell states of Ewing Sarcoma xenografts in zebrafish

A key challenge in cancer research is to identify functional cell states as they relate to specific tissue microenvironments. In this work, we present a quantitative high-resolution imaging assay of cancer cell morphology in zebrafish xenografts to probe functional adaptation to variable cell extrinsic cues and molecular interventions. We focus on Ewing Sarcoma, a pediatric cancer driven by a single oncogenic fusion protein EWSR1-FLI1, and with little to no additional somatic mutations, making it a prototypical form of cancer whose adaptation is likely driven by acute, non-genomic mechanisms. By applying machine learning approaches to 3D cell shapes, we find systematic shifts in the distribution of cell morphological states between seeding sites in the fish embryo, as well as between cells with differential expression of EWSR1-FLI1 in an environmentally sensitive fashion. We propose a model where Ewing Sarcoma cancer cell plasticity is sensitive both to expression fluctuation of EWSR1-FLI1 and signals from the surrounding tissue microenvironment, with either or both factors possibly contributing to metastatic potential.

Toxic or Not Toxic, That Is the Carbon Quantum Dot’s Question: A Comprehensive Evaluation with Zebrafish Embryo, Eleutheroembryo, and Adult Models

Carbon quantum dots (CQDs) are emerging novel nanomaterials with a wide range of applications and high biocompatibility. However, there is a lack of in-depth research on whether CQDs can cause acute or long-term adverse reactions in aquatic organisms. In this study, two different types of CQDs prepared by ammonia citrate and spermidine, namely CQDAC and CQDSpd, were used to evaluate their biocompatibilities. In the fish embryo acute toxicity test (FET), the LD50 of CQDAC and CQDSpd was about 500 and 100 ppm. During the stage of eleutheroembryo, the LD50 decreased to 340 and 55 ppm, respectively. However, both CQDs were quickly eliminated from embryo and eleutheroembryo, indicating a lack of bioaccumulation. Long-term accumulation of CQDs was also performed in this study, and adult zebrafish showed no adverse effects in 12 weeks. In addition, there was no difference in the hatchability and deformity rates of offspring produced by adult zebrafish, regardless of whether they were fed CQDs or not. The results showed that both CQDAC and CQDSpd have low toxicity and bioaccumulation to zebrafish. Moreover, the toxicity assay developed in this study provides a comprehensive platform to assess the impacts of CQDs on aquatic organisms in the future.

The Role of Polymeric Coatings for a Safe-by-Design Development of Biomedical Gold Nanoparticles Assessed in Zebrafish Embryo

In the biomedical field, gold nanoparticles (GNPs) have attracted the attention of the scientific community thanks to their high potential in both diagnostic and therapeutic applications. The extensive use of GNPs led researchers to investigate their toxicity, identifying stability, size, shape, and surface charge as key properties determining their impact on biological systems, with possible strategies defined to reduce it according to a Safe-by-Design (SbD) approach. The purpose of the present work was to analyze the toxicity of GNPs of various sizes and with different coating polymers on the developing vertebrate model, zebrafish. In particular, increasing concentrations (from 0.001 to 1 nM) of 6 or 15 nm poly-(isobutylene-alt-maleic anhydride)-graft-dodecyl polymer (PMA)- or polyethylene glycol (PEG)-coated GNPs were tested on zebrafish embryos using the fish embryo test (FET). While GNP@PMA did not exert significant toxicity on zebrafish embryos, GNP@PEG induced a significant inhibition of embryo viability, a delay of hatching (with the smaller size NPs), and a higher incidence of malformations, in terms of tail morphology and eye development. Transmission electron microscope analysis evidenced that the more negatively charged GNP@PMA was sequestered by the positive charges of chorion proteins, with a consequent reduction in the amount of NPs able to reach the developing embryo and exert toxicological activity. The mild toxic response observed on embryos directly exposed to GNP@PMA suggest that these NPs are promising in terms of SbD development of gold-based biomedical nanodevices. On the other hand, the almost neutral GNP@PEG, which did not interact with the chorion surface and was free to cross chorion pores, significantly impacted the developing zebrafish. The present study raises concerns about the safety of PEGylated gold nanoparticles and contributes to the debated issue of the free use of this nanotool in medicine and nano-biotechnologies.

Brazilian silverside, Atherinella brasiliensis (Quoy & Gaimard,1825) embryos as a test-species for marine fish ecotoxicological tests

The fish embryo test (FET) is an alternative to the classic freshwater toxicity test used to assess environmental hazards and risks to fish. This test has been standardized and adopted by the Organization for Economic and Cooperation and Development (OECD). As salinity may affect the substances’ toxicity, we describe the development of an alternative euryhaline test species for embryonic ecotoxicological tests: the Brazilian silverside Atherinella brasiliensis (Quoy & Gaimard, 1825). This species is broadly distributed along the coast of South America and is able to inhabit a broad range of environmental and saline conditions. Ours is the first study on the maintenance of a native South American species for natural reproduction and the generation of embryos for tests. The embryos used are transparent and possess fluorescent cells which have only been seen in a few species and which may be used as markers, making it an alternative assessment tool for the lethal and sublethal substances in marine and estuarine environments. We provide a detailed description and analysis of embryonic development under different salinities and temperatures. The embryos and larvae developed in similar ways at different salinities, however as temperatures increased, mortality also increased. We considered the effects of the reference toxicants Zn2+ and SDS using a protocol similar to the FET that was standardized for zebrafish. Brazilian silverside embryos are as sensitive as freshwater, or euryhaline fish, to the surfactant but are more resistant to metals prior to hatching. We were able to show the advantages of the Brazilian silverside as a model for a marine fish embryo test (FETm) with high levels of reproducibility and little contaminated waste.