*2.3. Double-Stranded DNA Breaks*

High levels of oxygen and nitrogen free radicals can lead to DNA damage, including DBSs. The number of double-stranded DNA breaks (DSBs) was assessed in the FHA by measuring the size of the nuclear halo (chromatin dispersion). The results obtained after 1 h incubation are presented on sample photos with the nuclear halo and analysis of relative NDF in Figure 4.

In neuron-like cells treated with glucose for 1 h, the increase in DBSs was generally lower than after incubation with insulin in all used concentrations. The dependence of disruption on concentration has been demonstrated for all concentrations and substances tested—the higher the concentration, the stronger the chromatin dispersion halo. The highest DBSs were observed after administration of 250 M insulin (x-fold—3.59), while the lowest DBSs levels were measured after treatment with 50 mM glucose (x-fold—2.0). The observed effect was statistically significant (*p* < 0.001) in all experimental settings.

Results obtained after 24 h incubation are presented as sample photos with the chromatin dispersion and analysis of relative NDF in Figure 5.

**Figure 4.** Analysis of double-stranded DNA breaks in neuron-like cells after 1 h incubation with glucose or insulin. (**1**) Sample microphotographs of a nuclear diffusion halo: A—control (untreated cells), B—glucose 50 mM, C—glucose 100 mM and D—glucose 150 mM. (**2**) Sample microphotographs of nuclear diffusion halo: A—control (untreated cells), B—insulin 50 μM, C—insulin 100 μM and D—insulin 150 μM. (**3**) A comparison of relative NDF for neuronal-like cells incubated for 1 h with glucose or insulin. Statistically significant differences compared to the untreated neuron-like cells: \*\*\* *p* < 0.001.

Similar to the results obtained in the DCF-DA, the incubation of neuron-like cells with glucose or insulin for 24 h resulted in less DNA strand damage than incubation for 1 h. After 24 h incubation with all three concentrations of insulin, the damaged DNA strand was observed to regenerate. There was a strong concentration dependence in both glucose and insulin. Glucose caused weak DNA damage with maximum levels after 24 h incubation with 150 mM glucose (x-fold—1.43). Insulin treatment had the opposite effect, leading to the regeneration of DNA double strands, with minimal values after 24 h incubation with 50 μM (x-fold—1.43).

#### *2.4. S100B Protein Concentration*

The study assessed the effects of glucose and insulin on the intracellular and extracellular S100B protein levels in neuron-like cells. The obtained concentrations of S100B protein after 24 h incubation with a broad range of glucose concentrations are shown in Figure 6. The extracellular concentration is presented per the amount of cells seeded in one well (1 × <sup>10</sup>4), while the intracellular concentration was adjusted per 5 <sup>μ</sup>g of total protein concentration in 100 μL of cell lysates measured by BCA assay.

**Figure 5.** Analysis of double-stranded DNA breaks in neuron-like cells after 24 h incubation with glucose or insulin. (**1**) Sample microphotographs of a nuclear diffusion halo: A—control (untreated cells), B—glucose 50 mM, C—glucose 100 mM and D—glucose 150 mM. (**2**) Sample microphotographs of nuclear diffusion halo: A—control (untreated cells), B—insulin 50 μM, C—insulin 100 μM and D—insulin 150 μM. (**3**) A comparison of relative NDF for neuronal-like cells incubated for 24 h with glucose or insulin. Statistically significant differences compared to the untreated neuron-like cells: \*\* *p* < 0.01, \*\*\* *p* < 0.001.

**Figure 6.** S100B protein concentration in neuron-like cells after incubation with different glucose concentrations (50, 100 and 200 mM). Control—untreated cells. (**A**) extracellular S100B levels per 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells; (**B**) intracellular S100B levels per 5 μg of total cellular protein concentration. Statistically significant differences compared to the untreated neuron-like cells: \*\* *p* < 0.01, \*\*\* *p* < 0.001.

At 50–200 mM glucose concentrations, significantly lower S100B protein levels (for around 10%) in the supernatant were observed compared to the control. In the case of extracellular S100B protein levels, there was no a dose-response relation observed. All obtained outcomes for extracellular S100B protein concentrations were statistically significant with *p* < 0.01 or *p* < 0.001. Cellular levels of S100B protein after incubation with glucose show an opposite trend compared to data obtained for supernatants, which stays in line with the results observed. As shown in Figure 6B, all treatments caused a rise in S100B intracellular concentrations, which reached their maximum after the administration of 100 mM glucose (432 pg/5 μg total protein). The increase after 50 mM glucose treatment is 23%, the next 100 mM of glucose caused a rise of 49% and, after 200 mM glucose, the upsurge of 45%. Figure 6B depicts the normal distribution of values of S100B concentrations after glucose administration confirmed by a bell-shaped dose-response dependence. Significant differences in intracellular S100B values were obtained in the case of 200 mM glucose treatment (*p* < 0.05).

Figure 7 presents the changes in S100B protein levels identified after 24 h incubation with a range of insulin concentrations. The extracellular concentration is presented per the amount of cells seeded in one well (1 × 104), while the intracellular concentration was adjusted per 5 μg of total protein concentration in 100 μL of cell lysates measured by BCA assay.

**Figure 7.** S100B protein concentration in neuron-like cells after incubation with different insulin concentrations (50, 100 and 250 <sup>μ</sup>M). Control—untreated cells. (**A**) Extracellular S100B levels per 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells; (**B**) intracellular S100B levels per 5 μg of total cellular protein concentration. Statistically significant differences compared to the untreated neuron-like cells: \*\*\* *p* < 0.001.

The incubation with each selected insulin concentration (50–250 μM) significantly diminished the S100B protein level in the supernatants, and the strongest effect was observed after the administration of 50 <sup>μ</sup>M (1769 pg/1 × 104 cells). There is a weak direct proportional correlation between increasing insulin concentration and S100B protein extracellular levels with maximum concentration after 250 <sup>μ</sup>M insulin treatment (1810 pg/1 × <sup>10</sup><sup>4</sup> cells). All obtained data for extracellular S100B protein concentrations were statistically significant with *p* < 0.001. As illustrated in Figure 7B, the highest value of S100B protein was detected after administration of 100 μM insulin (341 pg/5 μg total protein), while after the administration of 250 μM insulin, the increase was the lowest compared to the control (326 pg/5 μg total protein and 219 pg/5 μg total protein, respectively). Significant differences (*p* < 0.05) in intracellular S100B values were obtained in cases of all insulin treatments. A bell-shaped graph can also be observed in the case of incubation with insulin in Figure 7B, and these values correlate favorably with data obtained with glucose. It is interesting to note that treatment incubation with insulin caused similar effects to glucose—a decrease in extracellular and increase in intracellular S100 levels.

#### *2.5. Statistical Correlation between ROS, NO and DBSs Results*

Correlation coefficients between DNA damage and ROS or NO levels were determined and shown in Table 1.

**Table 1.** Correlations between measured parameters ROS, NO and DBSs in neuron-like cells after glucose or insulin treatment for 1 and 24 h.


Based on the calculated Pearson correlation coefficients in all tested concentrations, correlations between DCF-A, FHA and Griess results for glucose after 1 h incubation were strongly positive and statistically significant, whereas correlations between the results obtained after 24 h incubation with all tested glucose concentrations were negative. After insulin treatment, a strong correlation was observed only between ROS and DNA damage after 1 h incubation as well as after 24 h treatment. In the case of insulin, there was a positive correlation only between ROS level and DNA damage after 1 h and 24 h incubation.
