**3. Results**

#### *3.1. Assessment of the E*ff*ects of the Chemotherapeutic Agent 5-Fluorouracil (5-FU) on HeLa Cell Viability*

In order to study the 5-fluorouracil-induced cytotoxicity, HeLa cells were initially exposed to various concentrations of the cytostatic agen<sup>t</sup> for 24 and 48 h. Doses up to 200 μM were used causing 30% and 49% inhibition of cell growth at 24 (Figure 2a) and 48 h (Figure 2b), respectively (at the highest dose of 200 μM).

**Figure 2.** Percentage of HeLa cell viable cells after treatment with 5-fluorouracil (5-FU) at concentrations 5, 10, 25, 50, 100, 150 and 200 μM for (**a**) 24 h and (**b**) 48 h. Average results from replicate experiments ± SD (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, significantly different from the control.

#### *3.2. Increased Mitochondrial Superoxide Production in HeLa Cells is Observed after 24 h Treatment with 5-FU*

For the determination of mitochondrial superoxide production, cells were loaded with the fluorescent probe MitoSOX™ Red after exposure with a standard lethal 5-FU concentration (150 μM) for 24 h and 48 h (Figure 3). Our results indicated an increase in the mitochondrial superoxide levels in comparison with the control after 24 h cell exposure with 5-FU whereas a 48 h incubation led to a decrease in superoxide accumulation, possibly associated with cell loss due to 5-FU toxicity.

**Figure 3.** Mitochondrial superoxide levels in HeLa cells after treatment with 5-FU (150 μM) for 24 and 48 h, assessed as MitoSOX™ Red fluorescence intensity normalized to total protein content and control (no treatment with 5-FU). Average results from replicate experiments ± SD (*n* = 3). \*\* *p* < 0.01, significantly different from the control.

#### *3.3. Caspase-3 Activation after 48 h Treatment with 5-FU*

In order to determine the effect of 5-FU on the activation of caspase-3, HeLa cells were once again treated with 5-FU for 24 and 48 h and then the caspase specific activity was measured (Figure 4). HeLa cells treated for 24 h presented no significant caspase activity. On the contrary, caspase activation was remarkably increased at 48 h after treatment with 5-FU. Similar to mitochondrial superoxide levels, this observation was possibly associated with increased cell death after the 48-h treatment with 5-FU.

**Figure 4.** Caspase-3 specific activity in HeLa cells after treatment with 5-FU (150 μM) for 24 h and 48 h, normalized to control (no treatment with 5-FU). Average results from replicate experiments ± SD (*n* = 3). \*\*\* *p* < 0.001, significantly different from the control.

#### *3.4. Superoxide Accumulation in Cell Culture Determined by the Bioelectric Cell-Based Biosensor*

A significantly increased superoxide concentration determined by the bioelectric biosensor-based assay was observed following the 24 h exposure to 5-FU compared to the control (Figure 5). On the other hand, we observed a significant reduction of superoxide levels after the 48 h treatment with the anticancer agent. The results of superoxide determination in the culture medium with the biosensor assay were very highly correlated with the respective results of the MitoSOX™ Red mitochondrial superoxide assay, though positively for the 24 h treatment (r2 = 0.99) and negatively for the 48 h treatment (r2 = −0.99).

**Figure 5.** Relative superoxide accumulation in the culture medium during the 24 and 48 h incubation of HeLa cells with 5-FU, as determined with the cell-based superoxide biosensor, normalized to control (no treatment with 5-FU). Average results from replicate experiments ± SD (*n* = 3). \*\* *p* < 0.01, significantly different from the control.
