2.3. Assessment of Genotoxicity

Alkaline Comet assays were done according to Singh et al. [30] with some modifications [31,32,33] (for details, see Supplementary Materials and Methods). In brief, cells were carefully mixed with freshly prepared low-melting agarose, spread on slides pre-coated with agarose, and covered with a cover slip until solidification. After incubation of slides in lysis buffer and additional enzymatic treatment the human 8-oxoguanine DNA Glycosylase (hOGG1), where indicated, cellular DNA was unwound in alkaline buffer (pH > 13), and electrophoresis was performed. Nuclear DNA was stained, and DNA damage was analyzed either by Metafer CometScan (MetaSystem, Altlussheim, Germany) or by the Comet Assay III software (Perceptive Instruments, Haverhill, UK). Routinely, at least 100 nuclei were analyzed per slide. The parameter “% DNA in tail” (also called “tail intensity”) was chosen as primary readout as it was shown to be linear over a wide range and can be compared between different analysis systems. Single cell data of each slide/experimental condition were then summarized as arithmetic means, and, finally, the arithmetic means of at least three independent exposure experiments were statistically analyzed by 1- or 2-way ANOVA with post-hoc pairwise comparison tests in GraphPad Prism 8 and/or by paired and unpaired Student’s t-tests in MS Excel or SigmaStat software (Systat Software GmbH, Erkrath, Germany), considering p ≤ 0.05 as statistically significant.

For sister chromatid exchange (SCE) assays as described previously [34], the DNA of HTR-8/SVneo cells was labelled by culturing the cells in medium supplemented with 10 µM bromodeoxyuridine (BrdU) for 64 h. wEMF exposure and/or treatment with 2 µM of the PARP inhibitor AG-014699 (Selleck Chemicals, Houston, TX, USA) was done from 24–48 h, followed by 16 h of recovery. Differential staining of chromatids of metaphase spread chromosomes was done by Giemsa staining upon UV-B treatment of Hoechst 33258-labelled chromatin. Blinded for the examiner, the number of SCE, break points and chromosomes per cell were counted. Pooled SCE data and arithmetic means of the indicated number of independent experimental replica were statistically analyzed by ANOVA and Student’s t-test, respectively, using the GraphPad PRISM 8 software package.