## 3 Results
### The Effect of L-DOPA on Viability of Differentiated Caco-2  
L-DOPA is one of the compounds that are of wide interest to scientists in the context of its activity in the digestive system. In the intestinal epithelial cells, L-DOPA is converted by DOPA-decarboxylase to dopamine, which can act via dopamine receptors located in the gastrointestinal tract (GIT). The Caco-2 cell line is a well-established human intestinal cell model that has been used for years for investigating the gastrointestinal-related activity of a range of different biomolecules [12]. Although the toxicity of L-DOPA to Caco-2 cells was already tested, the applied protocol included the use of undifferentiated Caco-2 cells subjected to overnight culture prior to the toxicity experiment [22]. The authors used the standard MTT procedure in order to calculate the IC\({}_{50}\) value for L-DOPA. The Caco-2 cells were incubated for the 72 h at the presence of different L-DOPA concentrations ranged from 0 to 120 \(\mu\)g/mL. As a result, the IC\({}_{50}\) value calculated after 72 h exposure of Caco-2 cells to various doses of L-DOPA was 81.2 \(\pm\) 4.81 \(\mu\)g/mL. Moreover, the level of released LDH was also tested and was around 180% of control (not treated cells) after incubation with 120 \(\mu\)g/mL of L-DOPA for 72 h [22]. In our in-house toxicity study, the L-DOPA was added first to undifferentiated cells (24 h post-seeding) in the following range of concentrations: 0.1, 1, 5, 10, 100, and 200 ug/mL. The viability of cells was assessed next using the MTS procedure after 72 h of incubation (Figure 7). The calculated from our study IC\({}_{50}\) value was 59.47 ug/mL and was comparable to the literature [22]. For real-time evaluation of the L-DOPA activity against differentiated Caco-2 cells, we followed the previously optimized procedure with cells seeded in the concentration of 2000 cells per well and incubated in 20% FBS during adhesion and early proliferation phases (Figure 5A). The media changing from 20% to 1% FBS was performed at 75 h or 96 h of incubation (Figure 8). In both experiments the cells reached the highest confluence with a maximum CI between 3.5 and 5.5. The Caco-2 cells were treated next with L-DOPA (100 ug/mL) at either 406 h (first experiment, Figure 8A) or 404 h of incubation (second experiment, Figure 8B). In the first experiment, the quick decline was noticed within 72 h  
Figure 6: Concentration and time-dependent inhibitory effect of TNF-\(\alpha\) on epithelial resistance of the monolayer of Caco-2 cells. Data from one experiment in triplicate (**A**). The CI curves of Caco-2 with cell density 2000/well treated by 100 ng/mL of TNF-\(\alpha\) at 450 h (cyan). Untreated, control cells were marked by red. The cells were incubated in full media (20% of FBS) which were replaced next by 1% of FBS at 260 h. The baseline (violet) includes a cell-free medium well. Data from one experiment in triplicate (**B**). of treatment with L-DOPA, confirming its toxicity against Caco-2 cells in the used dose of 100 ug/mL (Figure 8A). The toxic effect of L-DOPA on cells started around 60 h from the addition. The similar loss of viability was observed in the repeated independent experiment (Figure 8B). However, the real-time toxic effect of L-DOPA on cells was observed earlier than in the previous experiment, at around 40 h from the addition. The comparison of CI curves and the statistical analysis after the addition of L-DOPA (100 ug/mL) are shown in Figure 8C,D. The temporary increase in CI immediately after the addition of L-DOPA was observed only in the first experiment (Figure 8A). Therefore, we suspect that this effect may be not directly related to cells but rather to high concentration of the tested compound introduced to E-plate 16 in fresh media and the response of highly sensitive gold electrodes on the rapid changes in environment, such as it was observed during media changing in the previous experiments (Figures 4-6).