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Imprint
High-throughput methods for the estimation of toxicological effects of nanomaterials
Project
Project code: BfR-ZEBET-08-1329-475
Contract period: 01.11.2011
- 30.07.2015
Purpose of research: Applied research
For the assessment of the toxicological potential of new nanomaterials which are produced in multiple variations in vitro methods are indispensable. Microscopy-based high-throughput methods are used in this project to evaluate nanomaterials faster and more reliable. Using fluorescent dyes multiple endpoints (proliferation, cell death, activation of stress kinases, transcription factors and others) can be analysed simultaneously. Contributions to the mode of action how nanomaterials induce toxicity are expected. The microscopy-based high-throughput apoptosis assay has been established for various cell lines (A549, RAW264.7, HCT116) and various nanoparticles (NP) such as SiO2, ZnO, Ag, TiO2, Fe-oxides, CeO2. Cell count, number of early-apoptotic, late apoptotic and necrotic cells are determined reproducibly. The toxicity of the SiO2 NPs was concentration- and time-dependent and dependent on the biomolecular corona. The results were verified by conventional cytotoxicity assays (LDH release, Alamar Blue reduction). The use of other fluorescent dyes for other endpoints was established: Ca2+ influx with Fluo-4 AM, the formation of superoxide anion radical with dihydroethidium (DHE) and immunofluorescence to detect the phosphorylated H2AX protein as a marker of DNA damage. Microscopy-based high-throughput screening provides more information in a shorter time, thus improving the efficiency of in vitro tests for nanomaterials. In addition, the validity is increased since individual cells can be examined, as well as the safety, since the microscopic analysis is less susceptible to misinterpretation. The method is therefore far superior to the toxicological in vitro standard methods. Mechanisms of NPs can be analyzed more quickly, whereby the safety of nanomaterials can be improved. In future, the establishment of additional immuno-histochemical methods in high throughput should improve the validity of this approach for the toxicological testing enormously, especially with regard to genotoxicity and mechanisms of nanomaterials.
Section overview
Subjects
- Toxicology
Framework programme
Funding programme
Excutive institution
BfR - Centre for Documentation and Evaluation of Alternatives to Animal Experiments (BfR - ZEBET)
