# Low-Dose Oxidant Toxicity and Oxidative Stress in Human Papillary Thyroid Carcinoma Cells K1
## 3 Results  
Low-Dose Oxidants Reduce Metabolic Activity, Proliferation, and Viability in a Human Papillary Thyroid Carcinoma Cell Line  
Plasma-oxidized liquids are considered promising anti-cancer tools based on their ability to target the cellular redox state via reactive species. The present study aimed to investigate responses of a human papillary thyroid carcinoma cell line K1 under different redox conditions. Besides exposure to mixed, plasma-derived ROS/RNS in plasma-oxidized DMEM (oxDMEM), metabolic activity, proliferation, viability, and redox responses were evaluated after application of H\({}_{2}\)O\({}_{2}\) and SNP, an RNS donor, alone (Figure 1a). ROS/RNS composition and quantity in plasma-oxidized liquids is influenced by several factors, including feed gas and admixtures, energy supply, treatment time and also the jet-to-target distance. In the present study, the distance between effluent tip and liquid surface was 23.3 mm (corresponding to a nozzle-to-surface distance of 35 mm). The jet was operated with argon at 3 slm (Figure 1b). Sufficient amounts of plasma-oxidized DMEM were generated by exposing 1.4 mL DMEM to plasma for 30, 60, or 120 s. oxDMEM was prepared freshly before the start of experiment (Figure 1c) and added to the cells for 24 h. Exposure to oxDMEM reduced the metabolic activity of K1 cells significantly in a dose-dependent manner (Figure 2a). Similar effects were observed for H\({}_{2}\)O\({}_{2}\) (Figure 2b) and SNP (Figure 2c), although high concentrations of H\({}_{2}\)O\({}_{2}\) (>1 mM) in particular were needed to reduce the metabolic activity significantly. A hallmark of cancers is their sustained proliferative signaling, supported by metabolic reprogramming, ensuring supply of substrates for macromolecule synthesis. The proliferative activity of K1 cells was assessed 24 h after treatment using the trypan blue exclusion assay (Figure 3a). Again, oxDMEM reduced cellular proliferation to a significant extent in a dose-dependent manner. Similar tendencies were observed for H\({}_{2}\)O\({}_{2}\) and SNP (Figure 3b). Live-dead discrimination using trypan blue staining was done to differentiate tumorigenic and tumortoxic action of the applied oxidants. At 24 h after exposure to oxDMEM, H\({}_{2}\)O\({}_{2}\), or SNP, enhanced toxicity could be observed, correlating with the applied dose. Toxicity was lowest after exposure to H\({}_{2}\)O\({}_{2}\) (Figure 3c). Low-Dose Oxidants Induce Membrane Lipid Peroxidation without Alterations in the Antioxidant Capacity  
Plasma induces cell death by irreversible damage of cellular macromolecules. A substantial impact of ROS on fatty acid chains yields cleavages and decreased membrane stiffnesses, amplifying their entry into the cells. Cellular membrane oxidation was evaluated 24 h after exposure to 60 s oxDMEM, 1800 uM H\({}_{2}\)O\({}_{2}\), or 400 uM SNP in 25 cm\({}^{2}\) cell culture flasks. Relative detection of lipid peroxides in K1 cell lysates was done based on tert-butyl  
Figure 3: **Proliferation and viability of K1 cells after exposure to different oxidants. ** (**a**) Representative _brightfield_ images of K1 cells 24 h after exposure to untreated (control) or plasma-oxidized DMEM (oxDMEM); (**b**) number of cells 24 h after exposure to different dosages of oxDMEM, H\({}_{2}\)O\({}_{2}\), and SNP; (**c**) percentage of dead cells 24 h after exposure to different dosages of oxDMEM, H\({}_{2}\)O\({}_{2}\), and SNP. Graphs show mean \(\pm\) standard error of the mean (SEM). Violin plots show median and individual values. Statistical analysis was performed using one-way analysis of variances (ANOVA) with [PERSON]’s post hoc testing (* \(p\) \(<\) 0.05, **_p_ \(<\) 0.01, and *** \(p\) \(<\) 0.001). C = control. Figure 2: **Metabolic activity of K1 cells after exposure to different oxidants. ** (**a–c**) Assessment of metabolic activity 24 h after exposure to different dosages of oxDMEM (**a**), H\({}_{2}\)O\({}_{2}\) (**b**), and SNP (**c**). Bar graphs show mean \(\pm\) standard error of the mean (SEM).