• Exploring uptake and biodistribution of polystyrene (nano)particles in zebrafish embryos at different developmental stages.

      van Pomeren, M; Brun, N R; Peijnenburg, W J G M; Vijver, M G (2017-09)
      In ecotoxicology, it is continuously questioned whether (nano)particle exposure results in particle uptake and subsequent biodistribution or if particles adsorb to the epithelial layer only. To contribute to answering this question, we investigated different uptake routes in zebrafish embryos and how they affect particle uptake into organs and within whole organisms. This is addressed by exposing three different life stages of the zebrafish embryo in order to cover the following exposure routes: via chorion and dermal exposure; dermal exposure; oral and dermal exposure. How different nanoparticle sizes affect uptake routes was assessed by using polystyrene particles of 25, 50, 250 and 700nm. In our experimental study, we showed that particle uptake in biota is restricted to oral exposure, whereas the dermal route resulted in adsorption to the epidermis and gills only. Ingestion followed by biodistribution was observed for the tested particles of 25 and 50nm. The particles spread through the body and eventually accumulated in specific organs and tissues such as the eyes. Particles larger than 50nm were predominantly adsorbed onto the intestinal tract and outer epidermis of zebrafish embryos. Embryos exposed to particles via both epidermis and intestine showed highest uptake and eventually accumulated particles in the eye, whereas uptake of particles via the chorion and epidermis resulted in marginal uptake. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of particles.
    • Fish embryo toxicity test, threshold approach, and moribund as approaches to implement 3R principles to the acute fish toxicity test.

      Dang, ZhiChao; van der Ven, Leo T M; Kienhuis, Anne S (2017-11)
      The acute fish toxicity test (AFT) is requested by EU legal frameworks for hazard classification and risk assessment. AFT is one of the few regulatory required tests using death as an endpoint. This paper reviews efforts made to reduce, refine and replace (3Rs) AFT. We make an inventory of information requirements for AFT, summarize studies on 3Rs of AFT and give recommendations. The fish embryo toxicity test (FET) is proposed as a replacement of AFT and analyses have focused on two aspects: assessing the capacity of FET in predicting AFT and defining the applicability domain of FET. Six comparison studies have consistently shown a strong correlation of FET and AFT. In contrast, the applicability domain of FET has not yet been fully defined. FET has not yet been accepted as a replacement of AFT by any EU legal frameworks to fulfill information requirements because FET is insensitive to some chemicals. It is recommended that the outlier chemicals that do not correlate between FET and AFT should be further investigated. When necessary, additional FET data should be generated. Another effort to reduce and refine AFT is incorporation of FET into the threshold approach. Furthermore, moribund as an endpoint of fish death has been introduced in revising AFT guideline to reduce the duration of suffering for refinement. This endpoint, however, needs further work on the link of moribund and death. Global regulatory acceptance of the moribund endpoint would be critical for this development.
    • Glucocorticoid receptor-dependent induction of () inhibits zebrafish caudal fin regeneration.

      Garland, Michael A; Sengupta, Sumitra; Mathew, Lijoy K; Truong, Lisa; de Jong, Esther; Piersma, Aldert H; La Du, Jane; Tanguay, Robert L (2019-01-01)
      We previously used a chemical genetics approach with the larval zebrafish to identify small molecule inhibitors of tissue regeneration. This led to the discovery that glucocorticoids (GC) block early stages of tissue regeneration by the inappropriate activation of the glucocorticoid receptor (GR). We performed a microarray analysis to identify the changes in gene expression associated with beclomethasone dipropionate (BDP) exposure during epimorphic fin regeneration. Oncofetal cripto-1 showed > eight-fold increased expression in BDP-treated regenerates. We hypothesized that the mis-expression of cripto-1 was essential for BDP to block regeneration. Expression of cripto-1 was not elevated in GR morphants in the presence of BDP indicating that cripto-1 induction was GR-dependent. Partial translational suppression of Cripto-1 in the presence of BDP restored tissue regeneration. Retinoic acid exposure prevented increased cripto-1 expression and permitted regeneration in the presence of BDP. We demonstrated that BDP exposure increased cripto-1 expression in mouse embryonic stem cells and that regulation of cripto-1 by GCs is conserved in mammals.
    • In vitro to in vivo extrapolation of effective dosimetry in developmental toxicity testing: Application of a generic PBK modelling approach.

      Fragki, Styliani; Piersma, Aldert H; Rorije, Emiel; Zeilmaker, Marco J (2017)
      Incorporation of kinetics to quantitative in vitro to in vivo extrapolations (QIVIVE) is a key step for the realization of a non-animal testing paradigm, in the sphere of regulatory toxicology. The use of Physiologically-Based Kinetic (PBK) modelling for determining systemic doses of chemicals at the target site is accepted to be an indispensable element for such purposes. Nonetheless, PBK models are usually designed for a single or a group of compounds and are considered demanding, with respect to experimental data needed for model parameterization. Alternatively, we evaluate here the use of a more generic approach, i.e. the so-called IndusChemFate model, which is based on incorporated QSAR model parametrization. The model was used to simulate the in vivo kinetics of three diverse classes of developmental toxicants: triazoles, glycol ethers' alkoxyacetic acid metabolites and phthalate primary metabolites. The model required specific input per each class of compounds. These compounds were previously tested in three alternative assays: the whole-embryo culture (WEC), the zebrafish embryo test (ZET), and the mouse embryonic stem cell test (EST). Thereafter, the PBK-simulated blood levels at toxic in vivo doses were compared to the respective in vitro effective concentrations. Comparisons pertaining to relative potency and potency ranking with integration of kinetics were similar to previously obtained comparisons. Additionally, all three in vitro systems produced quite comparable results, and hence, a combination of alternative tests is still preferable for predicting the endpoint of developmental toxicity in vivo. This approach is put forward as biologically more plausible since plasma concentrations, rather than external administered doses, constitute the most direct in vivo dose metric.