Yolk Sac of Zebrafish Embryos as Backpack for Chemicals?

The zebrafish embryo (Danio rerio) has developed into one of the most important nonsentient animal models for the hazard assessments of chemicals, but the processes governing its toxicokinetics (TK) are poorly understood. This study compares the uptake of seven test compounds into the embryonic body and the yolk sac of the zebrafish embryo using TK experiments, a dialysis approach, thermodynamic calculations, and kinetic modeling. Experimental data show that between 95% (4-iodophenol) and 67% (carbamazepine) of the total internal amount in 26 h post fertilization (hpf) embryos and between 80 and 49% in 74 hpf embryos were found in the yolk. Thus, internal concentrations determined for the whole embryo overestimate the internal concentration in the embryonic body: for the compounds of this study, up to a factor of 5. Partition coefficients for the embryonic body and a one-compartment model with diffusive exchange were calculated for the neutral test compounds and agreed reasonably with the experimental data. For prevalently ionic test compounds at exposure pH (bromoxynil, paroxetine), however, the extent and the speed of uptake were low and could not be modeled adequately. A better understanding of the TK of ionizable test compounds is essential to allow assessment of the validity of this organismic test system for ionic test compounds.

Canine and human sarcomas exhibit predominant FGFR1 expression and impaired viability after inhibition of signaling.

Fibroblast growth factor receptors (FGFRs) are important in malignant progression of several human epithelial tumors. However, little is known about FGFRs in canine or human soft tissue sarcomas. Thus, our aim was to investigate expression of FGFRs and their involvement in cell survival in sarcomas of both species. FGFR1-4 and FGFRL1 transcripts as well as IIIb/IIIc splice variants of FGFR1-3 were evaluated in 3 canine- and 6 human sarcoma cell lines and 19 spontaneous canine sarcomas by SYBRqPCR. FGFR1 protein expression was assessed by immunohistochemistry. Growth inhibitory effects of FGFR1 inhibitor PD166866 and dominant negative recombinant FGFR adenoviral expression constructs (dnFGFR) on tumor cell lines were analyzed. Profiling of multiple FGFR transcripts detected comparable co-expression in most of human and canine sarcoma cell lines and canine tumor specimens. This indicates existence of closely related regulation mechanisms for FGFR expression in sarcomas of both species. FGFR1 with splice variant IIIc was consistently expressed with highest transcript levels. In 88% of the spontaneous tumor samples a heterogeneous FGFR1 protein expression was observed. Significant growth inhibition and cell death was seen after infection with dnFGFR1 in canine and human sarcoma cells, but not with dnFGFR3 and 4. PD166866 showed selective cytotoxicity with IC50 values between 12.1 and 26.4 µM. FGFR1 inhibition blocked ligand-induced tyrosine phosphorylation of ERK1/2 mitogen-activated protein kinase isoforms. This study emphasizes the important role FGFR1, especially splice variant IIIc, likely plays in sarcomas. Inhibitory small molecules could be of potential use for targeted therapy in aggressive sarcomas of both species.

Investigation of Peroxisome Proliferator-Activated Receptor Genes as Requirements for Visual Startle Response Hyperactivity in Larval Zebrafish Exposed to Structurally Similar Per- and Polyfluoroalkyl Substances (PFAS).

BACKGROUND: Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. OBJECTIVES: The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. METHODS: Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to 0.43-7.86µM PFOS, 7.87-120µM PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration-response (BMCT) modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to 7.86µM PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed. RESULTS: Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, BMCT estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. DISCUSSION: This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. https://doi.org/10.1289/EHP13667.

Yolk-Water Partitioning of Neutral Organic Compounds in the Model Organism Danio rerio.

Yolk is the most important temporary biological compartment of the early life stages of fish embryos. The sorption strength of a chemical to yolk components may significantly influence the distribution of that chemical in the fish embryo. We determined yolk-water partition coefficients (Kyolk/water , in liters of water per kilogram of yolk, normalized to dry wt) for 70 neutral organic chemicals. The log Kyolk/water values range from 0.76 to 6.56. On the basis of these values, we developed polyparameter linear free energy relationship models to predict yolk-water partitioning for a broad range of neutral organic chemicals with a root mean squared error of 0.37 and r2 of 0.919. These models can be applied for the prediction of internal concentrations at equilibrium (neglecting biotransformation and active transport) in the zebrafish embryo test system. Environ Toxicol Chem 2020;39:1506-1516. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cancer-Cell-Intrinsic cGAS Expression Mediates Tumor Immunogenicity.

Sensing of cytoplasmic DNA by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) results in production of the dinucleotide cGAMP and consecutive activation of stimulator of interferon genes (STING) followed by production of type I interferon (IFN). Although cancer cells contain supra-normal concentrations of cytoplasmic DNA, they rarely produce type I IFN spontaneously. This suggests that defects in the DNA-sensing pathway may serve as an immune escape mechanism. We find that cancer cells produce cGAMP that is transferred via gap junctions to tumor-associated dendritic cells (DCs) and macrophages, which respond by producing type I IFN in situ. Cancer-cell-intrinsic expression of cGAS, but not STING, promotes infiltration by effector CD8+ T cells and consequently results in prolonged survival. Furthermore, cGAS-expressing cancers respond better to genotoxic treatments and immunotherapy. Thus, cancer-cell-derived cGAMP is crucial to protective anti-tumor CD8+ T cell immunity. Consequently, cancer-cell-intrinsic expression of cGAS determines tumor immunogenicity and makes tumors hot. These findings are relevant for genotoxic and immune therapies for cancer.