Genotoxicity: Comet Assay

The alkaline version of the single-cell microgel electrophoresis (comet) assay was used to show DNA strand breaks and alkali labile as well as incomplete excision repair sites in single cells.42

Based on the previously performed cytotoxicological analysis by the MTT assay and Annexin V assay, ZnO NPs were tested in HUVEC at sub-cytotoxic doses of 1, 5 and 10 µg/mL. Additionally, a negative control was done with pure culture medium, whereas the 200 µM directly alkylating methyl methanesulfonate (MMS, Sigma-Aldrich) served as a positive control. Test concentrations were applied for 24 hrs. Three subsequent treatment times for 1 hr with intermittent washing steps and regeneration periods of another hour were done in the repetitive exposure setting (Figure 2).

Experimental design of repetitive cell exposure to ZnO NPs. HUVEC were exposed for 1 hr three subsequent times with washing steps and regeneration periods of 1 hr in between, followed by a 24-hr regeneration period. MTT assay was performed after 1 hr of treatment, after 3 hrs of treatment and after the 24-hr regeneration period. Comet assay was performed after 3 hrs of treatment.

The comet assay was performed and analyzed as previously described.43,44 For every test concentration, 50 randomly chosen single cells on each of 2 slides (resulting in a total of 100 cells per test concentration) were semi-automatically analyzed with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) using a green excitation filter, a dichromatic beam splitter (580 nm long pass) and an emission filter (590 nm long pass) at a magnification of 400x. The software COMET 5.5 image system (Kinetic Imaging, Liverpool, UK) was used. Percent of DNA in tail (TD), tail length (TL) and Olive tail moment (OTM) as a multiplication product of the median migration distance and the percentage of DNA in the tail served as outcome parameters.45 In our study, the OTM was used for statistical analysis.