Evaluation of apoptosis induced by nanoparticles and fine particles in RAW 264.7 macrophages: Facts and artefacts

• Published in: Toxicology in vitro Vol. 26; no. 2; pp. 323 - 334
• Main Authors: Wilhelmi, Verena, Fischer, Ute, van Berlo, Damiën, Schulze-Osthoff, Klaus, Schins, Roel P.F., Albrecht, Catrin
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.03.2012
• Subjects: Animals; Apoptosis; Apoptosis - drug effects; Assay validation; Biological Assay; Caspase 3 - metabolism; Caspase 7 - metabolism; Cell Line; Cell Survival - drug effects; DNA Fragmentation; In vitro testing; Macrophages - drug effects; Macrophages - metabolism; Magnesium Oxide - toxicity; Mice; Nanoparticles; Nanoparticles - toxicity; Necrosis; Silicon Dioxide - toxicity; Titanium - toxicity; Zinc Oxide - toxicity; Nanoparticles; In vitro testing; Assay validation; Necrosis; Apoptosis
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2011.12.006

► Specific nanoparticles cause artefacts in in vitro apoptosis/necrosis assays. ► Crystalline silica is an appropriate positive control in nanotoxicology testing. ► MgO is a candidate negative nanosize control for in vitro screening assays. ► Flow cytometry analysis in nanotoxicology requires exclusion of particle events. Current hazard characterisation of nanoparticles (NP) is predominantly based on in vitro test systems, being established for small molecules of drugs and chemicals. However, specific physicochemical properties of NP may result in interference with assay components, biomarkers, or detection systems. In the present study, six types of (nano)particles were screened in RAW 264.7 macrophages by common cytotoxicity methods (WST-1, LDH). Our specific focus was on the investigation of apoptosis (analysis of hypodiploid DNA, phosphatidylserine exposure, caspase 3/7 activation, and Cell Death Detection ELISA). Assays were validated by the well-known apoptosis inducer staurosporine. Our results show that ZnO, DQ12 quartz and amorphous silica are cytotoxic with strong indications for apoptotic effects in RAW 264.7 macrophages, whereas toxicity was absent for MgO. For fine as well as ultrafine TiO2, no apoptotic effects could be detected except for induction of DNA fragmentation. The results of our study demonstrate the necessity to control on a case-by-case basis for assay interference to avoid misinterpretation of specific in vitro test findings. To obtain valid statements on the potential induction of apoptosis by specific NP the measurement of multiple endpoints is a prerequisite.