*4.4. IC<sup>50</sup> Value*

Represents concentration of AA that leads to 50% cell growth reduction. To calculate the IC<sup>50</sup> value, cells were treated with serially diluted AA using log 2 dilution starting at 400 mM. Cells without (w/o) AA treatment were used as a control. Cells were incubated in 96 well plates for 9 h at 30 ◦C w/o shaking, and *OD*<sup>600</sup> was determined every hour. For the calculation, the freely available online calculator was used, Quest Graph™ IC50 Calculator (https://www.aatbio.com/tools/ic50-calculator/, last accessed on 22 November 2021; AAT Bioquest, Sunnyvale, CA, USA).

#### *4.5. Characterization of the Yeast Morphology*

Yeast morphology analysis was performed as previously described [50]. Briefly, cells incubated with AA for 9 h with and w/o ASA pre-treatment were visualized under brightfield microscopy at 40× magnification (Leica DMI 6000, Leica microsystems, Wetzlar, Germany). Microscopy images were captured, and cell morphometric analyses were determined by the ImageJ software v. 1.52r (National Institutes of Health, CA, USA).

Cell volume (*V*; µm<sup>3</sup> ) was calculated according to Equation (3):

$$V = \frac{4}{3}\pi L W^2 \tag{3}$$

where *L* represents the cell length, and *W* is the cell width.

Cell surface (*S*; µm<sup>2</sup> ) was calculated according to Equation (4):

$$S = 2\pi \left( W^2 + LW \frac{\arcsin \varepsilon}{\varepsilon} \right) \tag{4}$$

where, factor *ε* is calculated as *ε* = √ *L* <sup>2</sup>−*W*<sup>2</sup> *L* .

#### *4.6. Spot Test*

To solid YES media 0, 0.1, 1, 10, and 20 mM of AA was added. Cells from the overnight culture were divided into two groups, one was left untreated, and the second was pretreated with 10 mM AsA. Serially diluted cells resulting approximately in 10,000, 1000, 100, and 10 cells/spot, were placed on plates. After 2–3 days of incubation at 30 ◦C, the size and density of spots were compared.

#### *4.7. Preparation of the Cell Extract for Biochemical Analyses*

Control and AA-treated cells with and w/o AsA preincubation were collected by centrifugation at 8500 rpm for 90 s, washed 3 times with sterile H2O, and resuspended in PBS (pH 7.0). Cells were either directly used for further analyses or stored at −80 ◦C. Cell homogenization was achieved by sonication (Digital Sonifier 450, Branson Ultrasonics Corp, Danbury, CT, USA) at 3 × 30 s intervals (repeated 1 s pulses followed by a 1 s pause giving 15 pulses within 30 s, and power 80 W representing 20% of the full power capacity) on ice. Cell debris was removed after 15 min centrifugation at 14,000× *g* and 4 ◦C. In the collected supernatant the protein level, metabolic activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were determined.

#### *4.8. Metabolic Activity*

Metabolic activity of yeast was calculated according to [53] with a few modifications. Briefly, yeast suspensions were centrifugated for 90 s at 10,000× *g* and washed with PBS (pH 7.0). Pellets were resuspended in 1 mL 0.5% 2,3,5-triphenyltertrazolium chloride (TTC) diluted in PBS and incubated for 20 h in the dark at 30 ◦C. After incubation, the pellet was washed twice with PBS, and generated red formazan was extracted by addition of 1 mL ethanol:acetone (2:1) mixture prior to cell lysis by sonication. Absorbance was measured at 485 nm and metabolic activity was calculated as relative units (r.u.) of absorbance per mg protein.

#### *4.9. Cell Viability*

Cells un- and pre-treated with AsA exposed to AA for 6 h were washed in PBS and 0.05% of methylene blue (Sigma–Aldrich) with 0.1% natrium citrate (Sigma–Aldrich) was added for 5 min. Methylene blue is able to penetrate only dead cells, hence blue-stained cells are considered as dead. Microscopic slides were prepared and a percentual portion of the dead to living cells was determined.

#### *4.10. Biochemical Analysis*

Agilent Cary 60 UV/VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) was used to determine CAT activity represented as the stepwise decrease in the absorbance at 240 nm for 90 s which determines the H2O<sup>2</sup> decomposition. Addition of 50 µL of 30 mM H2O<sup>2</sup> to 100 µL of the solution containing sample initialized reaction; the final volume of the reaction was 600 µL. The molar absorption coefficient of 36 mM−<sup>1</sup> cm−<sup>1</sup> was used to calculate specific catalase activity.

Total superoxide dismutase (SOD) activity was assayed according to [54] with slight modifications. Briefly, 100 µL of homogenized sample solution was added into 880 µL of a reactive mixture of 50 mM phosphate buffer (pH 7.8) containing 1 mM EDTA, 13 mM L-methionine, and 75 µM NBT (nitroblue tetrazolium). Finally, 20 µL of 2 mM riboflavin was added and the reaction was started by light irradiation (5000 lux) for 10 min at 20 ◦C. Absorbance of the samples was measured spectrophotometrically at 560 nm.

Malondialdehyde (MDA) content that represents lipid peroxidation was evaluated as previously described by [50]. Briefly, the TBA solution (15% trichloroacetate (TCA) containing 0.375% (*w*/*v*) thiobarbituric acid (TBA) was added to the supernatant of each sample and incubated at 95 ◦C for 30 min. The sample was rapidly cooled on ice and centrifuged at 8500 rpm for 60 s, the absorbance of the supernatant was measured at 532 and 600 nm at the Agilent Cary 60 UV/VIS spectrophotometer. The molar absorption coefficient 153 mM−<sup>1</sup> cm−<sup>1</sup> was used to calculate MDA content in nmol µg <sup>−</sup><sup>1</sup> protein.

The Bradford assay [55] was used to determine protein concentration at 600 nm using bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA) as a standard.

#### *4.11. Determination of ROS Generation*

Generation of the total ROS was performed as previously described by [56] with slight modifications. Control and AA-treated cell cultures with and w/o AsA (10 mM) preincubation were adjusted to *OD*<sup>600</sup> = 1, washed with PBS, and incubated with 10 µM H2DCFDA (Sigma–Aldrich) at 30 ◦C in the dark for 1 h without shaking. H2DCFDA is a compound that is oxidized by ROS to a highly fluorescent DCF, which is fluorescently detectable at 498 nm wavelength, thus providing an estimate of ROS levels in the cell. The excessive fluorescent dye was washed off and the cells were resuspended in PBS. Fluorescence filter with 490 nm excitation wavelength at the Glomax Multi Detection System (Promega Corporation, Madison, WI, USA) was used for the DCF fluorescence detection. The measured values were normalized to the untreated cells.

#### *4.12. Detection of Apoptosis and Necrosis*

Evaluation of the yeast cells' apoptosis was performed according to [57] with some modifications. Annexin V- FITC (fluorescein isothiocyanate) -conjugated that specifically binds to phosphatidylserine (PS) residues, was used to detect the externalization of PS which is an apoptosis marker. Propidium iodide (PI) (Sigma Aldrich) penetrates dead cells and serves as a marker to differentiate between apoptosis and necrosis. After 1 h AA exposure with and without AsA treatment, yeast cells were collected by centrifugation and washed twice with phosphate-buffered saline (PBS), pH 6.8. Washed cells were resuspended in sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl2, and 35 mM K2HPO4), pH 6.8 to a final concentration of 1 <sup>×</sup> <sup>10</sup><sup>7</sup> cells mL−<sup>1</sup> . For cell wall digestion, cells were incubated with 10 µg mL−<sup>1</sup> zymolyase (Roche) in sorbitol buffer for 1 h at 37 ◦C. Afterward, 1 mL of spheroplasts were centrifuged at 500 rpm for 5 min and resuspended in 60 µL of incubation buffer (containing the Annexin-V-FITC and PI) and incubated for 10 min at room temperature (RT). Cells were visualized by the fluorescence microscope (Leica DMI 6000, Leica microsystems, Wetzlar, Germany). Two independent experiments were evaluated, each containing at least 150 cells. Cells were observed in the stepwise-selected microscope fields to avoid counting the same cells.

#### *4.13. Determination of Ion Composition*

Ion composition was evaluated by the use of ICP-OES (ICP-OES 720, Agilent Technologies Australia (M) Pty Ltd., Santa Clara, CA, USA) as previously described [48]. Briefly, yeast cells exposed to 0-, 1-, and 10-mM AA for 3 and 9 h with and without AsA preincubation were washed three times with deionized water and incubated at 55 ◦C for 12 h. Weighted yeast pellets were placed into PTFE digestion tubes and 3 mL of extra pure HNO<sup>3</sup> was added for pressure microwave digestion by the ETHOS-One (Milestone, Srl., Sorisole (BG), Italy) microwave digestion system. Mineralized samples were filtered through a quantitative Munktell filter paper No. 390 (Munktell & Filtrak, Bärenstein, Germany) into 25 mL volumetric flasks and filled with deionized H2O in 4 biological replicates. Afterwards, the ion content detected by ICP-OES was calculated to µg g−<sup>1</sup> of the yeast dry matter and expressed as the content ratio to the untreated control.

#### *4.14. Immunostaining and Fluorescence Microscopy*

Chromosome segregation was analyzed by the use of fluorescence microscopy as previously described [58]. Shortly, yeast strain *JG15457* with the chromosome II marked with GFP was grown in YES medium at 30 ◦C and 150 rpm to achieve exponential growth. Cells were collected after 6 h of incubation, fixed by 2% PFA, and stained with primary TAT1 mouse monoclonal anti-tubulin and rabbit polyclonal anti-GFP antibodies, DNA was visualized using DAPI. Analyses were performed at the fluorescence microscope (Leica DMI 6000, Leica microsystems, Wetzlar, Germany) equipped with a digital camera. At least 200 cells in the anaphase stage of the mitotic cell cycle were evaluated for correct or incorrect segregation of chromosome II.

#### *4.15. Statistical Analysis*

Data are expressed as the mean ± standard deviation (SD). Statistical significance of obtained differences was analyzed by the ANOVA Duncan's and Fisher LSD post-hoc test using the Statistica 10 software (StatSoft Inc., Tulsa, OK, USA). Lavene's and Cochran's tests were used to evaluate data homogeneity and normality distribution of the results. Limits of the statistical significance were set up to *p* < 0.05 \*, 0.01 \*\*, 0.001 \*\*\*. Time-dependent ionome changes were calculated for each ion as a ratio to the untreated control. Absolute ion concentrations were then *Z-score*-transformed to adjust for the differences in magnitude between different ions according to Formula (5):

$$Z-score^{Individual} = \frac{\left(\text{Concentration}^{individual} - \text{Mean concentration}\right)}{SD} \tag{5}$$

*Z-scores* were used for cluster analysis by the average linkage clustering method with Pearson distance measurement. Pearson correlations between individual ions were calculated. *Z-score* and ion correlations were visualized by Heatmapper [59].

#### **5. Conclusions**

In this study, we investigated the underlining toxic effect of AA on cell vital functions by multiapproach analyses covering large-scale biological processes and stress responses. AA-induced enhancement of ROS production led to oxidative stress in *S. pombe* resulting in cell cycle arrest and alterations in chromosome segregation. Additionally, the cell disturbing activity of AA was confirmed by its negative effect on ion balance maintenance. Supplementation of ascorbic acid significantly protected cells against AA-mediated cytotoxicity due to its direct ROS scavenging activity and intracellular Ca uptake support. Our complex study extended the knowledge of AA-induced toxicity from the known metabolic disorders to disrupted ionome balance and altered chromosome segregation resulting in cell cycle arrest without marked signs of apoptosis. Additionally, we show that AsA treatment not only reduced ROS production but also improved ionome homeostasis and prevented errors in chromosome segregation during mitosis. Though, our results suggest that the use of AsA, a natural antioxidant supplementation, could significantly attenuate AA-induced cytotoxicity which might have health-protective implications in situations of dietary AA exposure. However, as AA is known to accumulate in the cell, further investigations of low-dose AA exposure for a longer period are required.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/molecules27134307/s1, Figure S1: Cell growth intensity upon AA addition with and w/o AsA pre-treatment, Figure S2: Relative growth rate of cells ex-posed to AA with and w/o AsA pre-treatment, Figure S3: Generation time (gt), Figure S4: Determination of IC50 upon AA exposure with and w/o AsA pre-treatment, Figure S5: Spot test, Figure S6: Cell viability upon AA addition with and w/o AsA pre-treatment, Figure S7: Protein content, Figure S8: CAT/MDA ratio.

**Author Contributions:** Conceptualization, M.K., M.P. and A.N.; methodology, M.K., A.N.; validation, M.P., M.K. and A.T.; formal analysis, M.K., A.N., R.K.; investigation, M.P.; resources, A.T.; data

curation, M.K.; writing—original draft preparation, M.P.; writing—review and editing, M.K., A.N., A.T.; visualization, M.P., M.K.; project administration, A.T.; funding acquisition, A.N., M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the SUA grant agency projects under grants 12-GASPU-2021, and GA FAPZ 2/2021; and the project "Drive4SIFood" of the European Union Operational Program Integrated Infrastructure Managing Authority Ministry of Transport and Construction of the Slovak Republic under grant number ITMS2014+ 313011V336.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary Materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are available from the authors or from commercial sources.

#### **References**

