Large-Scale Image Analysis for Investigating Spatio-Temporal Changes in Nuclear DNA Damage Caused by Nitrogen...

The effective clinical application of atmospheric pressure plasma jet (APPJ) treatments requires a well-founded methodology that can describe the interactions between the plasma jet and a treated sample and the temporal and spatial changes that result from the treatment. In this study, we developed a large-scale image analysis method to identify the cell-cycle stage and quantify damage to nuclear DNA in single cells. The method was then tested and used to examine spatio-temporal distributions of nuclear DNA damage in two cell lines from the same anatomic location, namely the oral cavity, after treatment with a nitrogen APPJ. One cell line was malignant, and the other, nonmalignant. The results showed that DNA damage in cancer cells was maximized at the plasma jet treatment region, where the APPJ directly contacted the sample, and declined radially outward. As incubation continued, DNA damage in cancer cells decreased slightly over the first 4 h before rapidly decreasing by approximately 60% at 8 h post-treatment. In nonmalignant cells, no damage was observed within 1 h after treatment, but damage was detected 2 h after treatment. Notably, the damage was 5-fold less than that detected in irradiated cancer cells. Moreover, examining damage with respect to the cell cycle showed that S phase cells were more susceptible to DNA damage than either G1 or G2 phase cells. The proposed methodology for large-scale image analysis is not limited to APPJ post-treatment applications and can be utilized to evaluate biological samples affected by any type of radiation, and, more so, the cell-cycle classification can be used on any cell type with any nuclear DNA staining.