## 4 Discussion
Thus, it becomes clear that direct plasma treatment provides the highest reactivity since all listed devices previously reported in the literature are based on indirect plasma treatment. Despite only the more frequent excited states of molecular nitrogen having been detected in plasma OES, other species such as NO, oxygen, and dissociation products of air such as atomic nitrogen and hydrogen are expected to be present [59]. There is also the production of ozone usually observed in corona discharges in air [60]. There are certainly other active species but they are not observable in the OES mainly due to quenching processes, emission outside the selected wavelength range [61], or simply because they are not being excited [62]. Thus, it is suggested that active chemical species produced inside plasma are directly delivered into the liquid phase, by solubilization and diffusion, where they reach cells, or react to produce secondary RS [56]. These can be scavenged by cancer cells and trigger biochemical reactions inside them. Since an oxidant environment can trigger cell death, it is possible that the intracellular RS, directly or indirectly induced by plasma, can be seen as responsible for the cytotoxicity of plasma. This seems to be the most common explanation for the cytotoxicity of CAP-based anticancer therapy [13; 22; 54; 63; 64; 65; 66; 67]. According to our data, there is an increase in a time-dependent manner of the concentration of peroxides while the concentration of nitrites remains similarly high during the tested exposition times. However, nitrites are difficult to quantify since the typical half-time of 1 to 5 min renders it less reliable in time-consuming colorimetric assays. Even when these limitations are considered, when we confront the peroxides and nitrites concentrations (Figure 4a,b) with each cell line biological response (Appendix A Figures A1 and A2), we may hypothesize hydrogen peroxide having a greater importance than nitrite. Since the cytotoxicity of plasma increases with longer exposures, it seems plausible that mediators responsible for plasma effects should also increase with longer treatment durations. Therefore, hydrogen peroxide may be the major player in plasma cytotoxic effects, since its concentration increases with longer plasma exposures while nitrite concentrations do not correlate with the plasma's effect. In Figure 4, we can see that there is a tendency for nitrites to decrease and peroxides to increase with FBS supplementation and that this effect is higher in RPMI than in DMEM. The half time of hydrogen peroxide is dependent upon the presence of trace transition metals on the media. Ferrous non-chelated iron is known to react and consume peroxides through the Fenton and Haber-Weiss reaction [68] and its presence in DMEM but not in RPMI can explain why peroxide concentration is lower in the former. Different results are seen regarding the effect of FBS on peroxides and nitrite concentration in the literature since FBS is highly variable in composition according to the producer and batch [69; 70]. In our work, the concentration of peroxides increased with FBS supplementation from 5 to 15%, and the opposite was seen with nitrites. Some studies show that FBS can scavenge nitrites [51] or peroxides [43; 71], depending on the experiment. Our results do not support this hypothesis. However, while FBS can scavenge RS, it does not protect cells from death, since exposure to plasma/plasma reactive species induces changes in the composition of the serum (such as lipid peroxidation, oxidation of proteins, amino acids) which are themselves cytotoxic [71]. When we compare the effect of plasma in different cell lines, there are two considerations to make: (1) the cytotoxicity of plasma-activated medium and (2) the intrinsic susceptibility of cell lines. As previously shown, RPMI and supplementation with FBS increase peroxide concentration.