Dataset Information

Zebrafish high-throughput screening to study the impact of dissolvable metal oxide nanoparticles on the hatching enzyme, ZHE1.

ABSTRACT: The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high-throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degradation of the chorionic membrane. The data indicate that 4 of 24 metal oxide nanoparticles (CuO, ZnO, Cr2 O3 , and NiO) could interfere with embryo hatching by a chelator-sensitive mechanism that involves ligation of critical histidines in the ZHE1 center by the shed metal ions. A recombinant ZHE1 enzymatic assay is established to demonstrate that the dialysates from the same materials responsible for hatching interference also inhibit ZHE1 activity in a dose-dependent fashion. A peptide-based BLAST search identifies several additional aquatic species that express enzymes with homologous histidine-based catalytic centers, suggesting that the ZHE1 mechanistic paradigm could be used to predict the toxicity of a large number of oxide nanoparticles that pose a hazard to aquatic species.

SUBMITTER: Lin S

PROVIDER: S-EPMC4034474 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Zebrafish high-throughput screening to study the impact of dissolvable metal oxide nanoparticles on the hatching enzyme, ZHE1.

Lin Sijie S Zhao Yan Y Ji Zhaoxia Z Ear Jason J Chang Chong Hyun CH Zhang Haiyuan H Low-Kam Cecile C Yamada Kristin K Meng Huan H Wang Xiang X Liu Rong R Pokhrel Suman S Mädler Lutz L Damoiseaux Robert R Xia Tian T Godwin Hilary A HA Lin Shuo S Nel André E AE

Small (Weinheim an der Bergstrasse, Germany) 20121123 9-10

The zebrafish is emerging as a model organism for the safety assessment and hazard ranking of engineered nanomaterials. In this Communication, the implementation of a roboticized high-throughput screening (HTS) platform with automated image analysis is demonstrated to assess the impact of dissolvable oxide nanoparticles on embryo hatching. It is further demonstrated that this hatching interference is mechanistically linked to an effect on the metalloprotease, ZHE 1, which is responsible for degr ...[more]

PMID: 23180726

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Project description:The use of engineered nanomaterials (ENMs) in foods and consumer products is rising, increasing the potential for unintentional ingestion. While the cytotoxicity of many ENMs has been investigated, less attention has been given to adverse impact on the intestinal barrier integrity. Chronical disruption of gastrointestinal integrity can have far reaching health implications. Using fully differentiated Caco-2 cells, the perturbation of intestinal barrier function and cytotoxicity were investigated for 20 metal, metal oxide, and metal sulfide ENMs. Caco-2 cells were exposed to 50 μg/mL ENMs for 24 hours. ENM formulations were characterized at 0 and 24 hours, and In Vitro Sedimentation, Diffusion and Dosimetry Modeling was applied to calculate the effective dose of exposure during 24 hours. The apparent permeability coefficient (Papp) was determined for fluorescent labeled dextran (3,000 Da) and tight junction integrity was evaluated by immunofluorescence microscopy. Cytotoxicity was investigated by determining lactate dehydrogenase release (LDH) and cell metabolic activity (tetrazolium based MTS) assays. Four ENMs led to significantly increased Papp, (15.8% w/w% Ag-SiO2 nanoparticle (NP), 60 nm CdS NP, 100 nm V2O5 flakes, and 50 nm ZnO NP), while one ENM (20 nm MgO NP) decreased Papp. With the exception of CdS NP, significantly increased Papp was not connected with cell cytotoxicity. The calculated effective dose concentration was not correlated with increased Papp. Our results illustrate that while many metal, metal oxide, and metal sulfide ENMs do not adversely affect monolayer integrity or induce cytotoxicity in differentiated Caco-2 cells, a subset of ENMs may compromise the intestinal integrity. This study demonstrated the use of differentiated Caco-2 monolayer and Papp as an endpoint to identify and prioritize ENMs that should be investigated further. The interaction between ENMs and the intestinal epithelium needs to be evaluated to understand potential intestinal barrier dysfunction and resulting health implications.

Project description:BackgroundThe rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz).MethodsThe metal oxide- and silver nanoparticles were characterized in terms of agglomeration and ion release in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay, γ-H2AX and RAD51 foci formation).ResultsWe show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles.ConclusionsWe conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress.

Dataset Information

Zebrafish high-throughput screening to study the impact of dissolvable metal oxide nanoparticles on the hatching enzyme, ZHE1.

Publications

Zebrafish high-throughput screening to study the impact of dissolvable metal oxide nanoparticles on the hatching enzyme, ZHE1.

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