Cytotoxicity of Mycotoxins and Their Combinations on Different Cell Lines: A Review

Mycotoxins are secondary metabolites of molds and mainly produced by species of the genera Aspergillus, Penicillium and Fusarium. They can be synthesized on the field, during harvest as well as during storage. They are fairly stable compounds and difficult to remove. Among several hundreds of mycotoxins, according to the WHO, ochratoxin A, aflatoxins, zearalenone, deoxynivalenol, patulin, fumonisins as well as T-2 and HT-2 toxins deserve special attention. Cytotoxicity is one of the most important adverse properties of mycotoxins and is generally assessed via the MTT assay, the neutral red assay, the LDH assay, the CCK-8 assay and the ATP test in different cell lines. The apoptotic cell ratio is mainly assessed via flow cytometry. Aside from the assessment of the toxicity of individual mycotoxins, it is important to determine the cytotoxicity of mycotoxin combinations. Such combinations often exhibit stronger cytotoxicity than individual mycotoxins. The cytotoxicity of different mycotoxins often depends on the cell line used in the experiment and is frequently time- and dose-dependent. A major drawback of assessing mycotoxin cytotoxicity in cell lines is the lack of interaction typical for complex organisms (for example, immune responses).


Introduction
Mycotoxins are toxic compounds mainly produced by species of the genera Aspergillus, Fusarium and Penicillium. They can be detected in numerous foodstuffs, including nuts, spices, cereals and fruits, both pre-and post-harvest. Mold growth and, consequently, mycotoxin production can be affected by numerous factors such as weather conditions. The thermal stability and ability of mycotoxins to withstand food processing is one of the main reasons for concerns. Out of several hundreds of mycotoxins most commonly occurring and threatening human and animal health, we highlight patulin, aflatoxin, ochratoxin A, fumonisin, deoxynivalenol, T-2 and HT-2 toxins, zearalenone, citrinin and enniatin [1].
Since only living cells can reduce MTT to formazan, the amount of formazan is used to assess the number of living cells [15]. The CCK-8 test is a test similar to the MTT. In this test, a highly water-soluble reagent, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophen-yl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt], is reduced by dehydrogenase in living cells to formazan (a colored product). The amount of formazan is directly correlated to the number of living cells [16]. In the ATP test, the amount of ATP is measured. In this approach, MgATP2-converts luciferin, obtained from fireflies, into a form which can be oxidized catalytically by luciferase in a chemiluminescent reaction. Light intensity (~562 nm) is directly correlated to the amount of ATP [17]. The LDH test measures lactate dehydrogenase activity in culture media. Since LDH is a cytoplasmic enzyme, it is rapidly released into culture media during necrosis or apoptosis (cell membrane damage). In this test, tetrazolium salt (yellow color) is converted by NADH to formazan dye (red color), which can be measured spectrophotometrically (~492 nm). The amount of formazan dye is directly correlated to the amount of LDH in culture media (and to the number of damaged cells) [18].
The neutral red uptake (NR, NRU) assay relies on the ability of living cells to absorb neutral red dye. The dye penetrates cells via non-ionic diffusion and is accumulated in the lysosomes. After washing, the dye is extracted from cells using acidified ethanol and measured spectrophotometrically. This assay relies on the capability of cells to retain a pH gradient which allows the dye to penetrate the cell membrane and retain in the lysosomes. Since only living cells have this ability, the amount of measured dye is directly correlated with the number of living cells [19]. The AlamarBlue™ assay is a cell viability assay using a non-toxic, weakly fluorescent blue dye, resazurin. In healthy, live cells, resazurin is reduced to a pink, highly fluorescent dye, resorufin. The intensity of fluorescence is proportional to the number of respiring, living cells. This assay is used as an alternative to other tests, for example, the MTT [20].

Cell Lines Used for Cytotoxicity Assessment
Various different cell lines are used for cytotoxicity assessment. The cell lines used in the reviewed literature are summarized in Table 1.

Results
All reviewed articles are summarized in Supplementary Table S1. 2.1. Single Mycotoxins 2.1.1. Patulin In the case of patulin, MTT was the most frequently used test, and HepG-2 was the most frequently used cell line. Results showed a decrease in cells' viability, as well as ROS formation increase, cell cycle arrest and p53 gene expression. IC50 values for patulin range from µM to mM, depending on experiment conditions, time of incubation and cell line used (full results are presented in Table 2). In case of aflatoxins, MTT was the most frequently used test, followed by LDH release and NRU, while HepG-2 and Caco-2 were the most frequently used cell lines. Results showed a decrease in cells' viability, as well as increase in the apoptotic cell ratio, increased ROS production and cell cycle arrest. IC50 values for aflatoxins range from nM to µM, depending on experiment conditions, time of incubation and cell line used (full results are presented in Table 3). Table 3. Cytotoxicity of aflatoxins tested on different cell lines.

Type of Test Cell Line Results Reference
CCK-8 BME Aflatoxins AFB1 and AFM1 exhibited cytotoxic properties in a dose-and time-dependent manner at various concentrations after 24 and 48 h of incubation. They also induced apoptosis and increased the ratio of cells in the G1 and G2 phases. [32] MTT, LDH release Caco-2, Hep-G2, SK-N-SH Both aflatoxins AFB1 and AFM1 decreased the viability of cells by damaging the cell membrane. [33] MTT Caco-2 Aflatoxin AFM1 inhibited cell viability in a dose-and time-dependent manner after 24, 48 and 72 h of incubation. [34] MTT, LDH release BRL 3A AFB1 reduced cell viability in a dose-and time-dependent manner. AFB1 also increased LDH activity, apoptotic cell ratio and ROS production. [35] MTT, NRU Caco-2, Raw264.7, MDBK AFB1 exhibited cytotoxic properties against MDBK, reducing cell viability by 21% after 48 h of incubation with AFB1 at a concentration of 3.8 µg/mL. No significant decrease in cell viability was observed in Raw264.7 and Caco-2 cell lines. [36] Cell Proliferation Reagent WST-1 BME-UV1 Aflatoxin B1 is cytotoxic against the BME-UV1 cell line in a doseand time-dependent manner, with LC50 values of 687 and 180 nM after 24 and 48 h, respectively. [37] MTT, NRU BME-UV1 Aflatoxin caused a decrease in cell viability in a dose-and timedependent manner. NRU tests showed that after 72 h of incubation, cell viability was decreased by more than 70% in all concentrations tested. The MTT test also showed a significant decrease in cell viability in all concentrations tested after 24 h of incubation. [38] MTT Caco-2 Aflatoxin B1 and M1 exhibited cytotoxic properties against the Caco-2 cell line. The MTT assay showed a significant dose-and time-dependent decrease in cell viability, both differentiated and undifferentiated cells, when treated with mycotoxins. It was shown that aflatoxin B1 is more cytotoxic than aflatoxin M1. [39] MTT, LDH release PK-15 Aflatoxin B1 exhibited dose-and time-dependent cytotoxic properties. The MTT test showed that after 48 h of incubation, the IC50 for aflatoxin B1 was 38.8 µM. Regarding the LDH release, an AFB1 in concentration of 24.9 µM caused an increase in LDH release by 30% after 24 h of incubation. [40] Cell Proliferation ELISA BrdU Kit, Flow cytometry MAC-T Incubation with AFB1 significantly decreased cell proliferation in a dose-dependent manner. Since the ratio of cells in sub-G1, S and G2/M phases was elevated, it was assumed that AFB1 inhibited cell proliferation by inhibiting the cell cycle. Flow cytometry also showed that incubation with AFB1 induced apoptosis in MAC-T cells. [41] high content screening BF-2 AFB1 reduced cell viability in a dose-dependent manner, with IC50 estimated at 11.11 µM. Moreover, AFB1 generated strong oxidative stress. [42] Cell Proliferation Reagent WST-1 HepG-2, BEAS-2B AFB1 decreased HepG-2 cell viability, with a IC50 estimated at 1 µM; however, after exposure of BEAS-2B cells to AFB1, cell viability was at 90% compared to the control group in all tested concentrations. [43] MTT SK, MDCK, HeLa Cytotoxicity was defined as a decrease in cell viability by 20%. This effect was achieved by incubating AFB1 for 24 h at a concentration of 25 µg/mL with HeLa cells, but it could not be achieved with other cells.
[31] AlamarBlue™ assay HepG-2, RAW 264.7 After 48 h of incubation with AFB1, the AlamarBlue™ assay showed a dose-dependent reduction in cell viability, with a IC50 of 3.12 ppm. [45] high content assay HepG-2 Results showed that AFB1 deceased cell viability in a dose-dependent manner, with a significant increase in cell viability reduction at low concentrations and a similar reduction at the three highest concentrations tested. [46] MTT, LDH release primary hepatocytes of Cyprinus carpio The results of the MTT assay showed a time-and dose-dependent decrease in cell viability. Moreover, the activity of LDH in cultured medium was elevated after incubation with AFB1 compared to the control group. [47]

Ochratoxin A
In case of ochratoxin A, MTT was the most frequently used test, followed by CCK-8, while HepG-2 and Caco-2 were the most frequently used cell lines. Results showed a decrease in cells' viability, as well as an increase in the apoptotic cell ratio, increased ROS production and cell cycle arrest. IC50 values for ochratoxin A range from 1.86 µM to >200 µM, depending on experiment conditions, time of incubation and cell line used (full results are presented in Table 4). Table 4. Cytotoxicity of ochratoxin A tested on different cell lines.

Type of Test Cell Line Results Reference
MTT Caco-2 Results after 24, 48 and 72 h of incubation showed that OTA inhibited cell viability in a dose-and time-dependent manner, which was comparable to the cytotoxicity of AFM1. [34] MTT HK-2, HepG-2 OTA caused a significant viability decrease in HK-2 cells and HepG-2 cells in a dose-dependent manner in all tested concentrations. [48] CCK-8, flow cytometry, western blot Het-1A OTA at concentrations of 5 µM and 10 µM caused reductions in Het-1A cells after 24 h of incubation. The results also showed an increase in apoptosis cell ratio via flow cytometry. Western blot indicated increased expression levels of Bax, cleaved caspase-3, cleaved caspase-9 and cytochrome c, as well as decreased expression levels of Bcl-2, Bcl-xl, caspase-3 and caspase-9, which is typical for apoptosis. [49] MTT, NRU Caco-2, Raw264.7, MDBK After 48 h of incubation with OTA at 10 µg/mL concentration, cell viability was decreased by 7, 33 and 55% in Caco-2, Raw264 and MDBK cells, respectively, which was confirmed by both assays. [38] CCK-8, flow cytometry BRL After incubation with OTA at concentrations between 17 and 30 µM, cells' viability decreased in a dose-dependent manner, with IC50 = 37.8 µM. Additionally, flow cytometry showed an increased ratio of apoptotic cells after incubation with OTA.
[50] [28] MTT PBM OTA at concentration of 12.5 mg/L significantly decreased both cell metabolic activity and proliferation (37.6 and 42.8%, respectively, compared to the control). [53] AlamarBlue™, CFDA-AM, NRU RTH-149, PLHC-1, H4IIE OTA had no strong cytotoxic properties against the tested cell lines, with IC50 values for NRU of 5.47 and 21.65 µg/mL for PLHC-1 and RTH-149, respectively (the remaining assays showed IC50 > 40.4 µg/mL for all cell lines). [54] MTT K562, PBL, PBG Significant decrease in K562 cell viability between 16 and 48 h of exposure at a concentration of 25 µg/mL. This decrease in cell viability was dose-dependent, which could also be observed in PBL and PBG cells incubated with OTA (although the cytotoxicity of OTA against these cell lines was lower). [55] Triple Assay (AlamarBlue™, CFDA-AM, NRU)

RTGiLL-W1
After 24 h of incubation, OTA had a similar cytotoxicity towards mitochondria (EC50 106.12 µM) and lysosomes (EC50 108.84 µM) and a significantly lower cytotoxicity towards plasma membrane (EC50 > 247.65 µM). [44] MTT HepG-2 Significant, dose-dependent decrease in cell viability, GSH and MDA levels and GSH-Px activity. [56] MTT, NRU RPTC, HKC, PKC, LLC-PK1 NRU assay showed that after 24 and 48 h of incubation, the viability of RPTC and HKC cells decreased, with a significant difference between males and females (from which cells were isolated); however, after 72 and 96 h, this difference was no longer visible. In PKC cells, OTA exhibited a similar cytotoxicity across all incubation periods in males and a slightly higher cytotoxicity in females after 48 h of incubation. Moreover, in LLC-PK1 cells, OTA caused a similar cytotoxicity across all incubation times. The MTT assay supported these results; however, it was less sensitive than the NRU assay. [57] MTT, flow cytometry HEK 293 Dose-dependent decrease in cell viability, with a IC50 of 16 µM.
In addition, flow cytometry showed that OTA caused a significant accumulation of cells in the S phase, thus disrupting the cell cycle. [58] MTT PK15 OTA significantly increased the cell death ratio after 48 h of incubation. This study also showed a decrease in the concentration of thiol groups (SH) as well as a down-regulation of Hsp70 and Hsp27 expression. [59] MTT, AO/EB staining HepG-2 After 24 h of incubation, OTA in a concentration range of 0-300 µM significantly decreased cell viability in a dose-dependent manner, with a IC50 of 210 µM. Additionally AO/EB staining revealed an increased ratio of apoptotic and necrotic cells (with a significant majority of apoptotic cells).
[60] In the case of fumonisins, MTT was the most frequently used test, followed by LDH release, while HepG-2 and Caco-2 were the most frequently used cell lines. Results showed that, even at high concentration, fumonisins are significantly less cytotoxic than other tested mycotoxins (full results are presented in Table 5). Table 5. Cytotoxicity of fumonisins tested on different cell lines.

Type of Test Cell Line Results Reference
MTT, LDH release BRL 3A Exposure to fumonisin FB1 resulted in slightly increased cell viability, even at high concentrations. The LDH activity was also unaffected by fumonisin FB1. [35] MTT HK-2, HepG-2 No significant decrease in cell viability in HK-2 and HepG-2 cells in the presence of fumonisin B1. [48] MTT CHO-K1, V79, C5-O, Caco-2, HepG-2 FB1 exhibited cytotoxic properties against all studied cell lines (sensitivity to FB1 after 72 h of exposure to 100 µg/mL: MTT PBM FB1 at a concentration of 125 mg/L significantly decreased both cell metabolic activity and proliferation (55.6 and 56.1%, respectively, compared to the control).
[53] This suggests that the cytotoxicity of FB1 against porcine lymphocytes is generally weak.
[ 66] 2.1.5. Deoxynivalenol In the case of deoxynivalenol, MTT was the most frequently used test, followed by NRU, while HepG-2 was the most frequently used cell line, followed by Caco-2. Results showed a decrease in cells' viability, as well as an increase in the apoptotic cell ratio and increased ROS production. IC50 values for deoxynivalenol range from nM to µM, depending on experiment conditions, time of incubation and cell line used (full results are presented in Table 6). MTT, LDH release BRL 3A Cell viability decreased in a dose-and time-dependent manner.
Results also showed that DON increased LDH activity, apoptotic cell ratio and ROS production. [35] MTT, NRU Caco-2 DON exhibited cytotoxic properties cells at a concentration of 1 µM. This effect was dose-dependent. Results also showed DON inhibited protein and DNA synthesis in a concentration-dependent manner, with IC50 values of 5 and 1.7 µM for protein and DNA synthesis, respectively. The production of MDA (lipid peroxidation marker) was also increased in the presence of DON, indicating increased oxidative stress in cells. [67] NRU IPEC-12 Significant drop in proliferative cell viability at a concentration of 0.1 mg/mL after 24 h; however, no significant viability decrease was observed in differentiated cells, even after 24 h of incubation with DON at a concentration of 10 mg/mL. An increase in the ratio of apoptotic cells was also confirmed. [68] MTT  [44] NRU, SRB, WST-1 RTgill-W1, IPEC-1, IPEC-2, HepG-2 NR and SRB assays showed that DON reduced RTgill-W1 cell viability. At a concentration 40 µmol/L, viability was reduced by 63 and 52% (SRB and NR assay, respectively). Regarding IPEC-1 and IPEC-2 cells, DON decreased cell viability in a dose-dependent manner in both lines. The WST-1 assay showed that at a concentration 6.9 µmol/L, viability was reduced by 65.6 and 60.9% (for IPEC-1 and IPEC-2 cells, respectively). In HepG2, DON caused a dose-dependent decrease in cell viability. The WST-1 assay showed that at a concentration 3.5 µmol/l, viability was decreased by 39.7%. [72] Table 6. Cont.

Type of Test Cell Line Results Reference
AlamarBlue™ HepG-2, RAW 264.7 After 48 h of incubation, AlamarBlue™ assay showed a dose-dependent reduction in cell viability, with a IC50 of 0.23 ppm. [45] high content assay HepG-2 After 24 h of incubation, DON decreased cell viability in a dose-dependent manner, with a significant increase in cell viability reduction at low concentrations and a similar reduction at the three highest concentrations tested. [46] MTT, NRU Caco-2 2.1.6. T-2 and HT-2 Toxins In the case of T-2 and HT-2 toxins, MTT was the most frequently used test, followed by LDH release and NRU, while HepG-2 was the most frequently used cell lines followed by Vero and Leyding cells. Results showed a decrease in cells' viability, as well as an increase in the apoptotic cell ratio, increased ROS production. It was also shown that a strong cytotoxic effect occurs even at low concentrations (full results are presented in Table 7). Table 7. Cytotoxicity of T-2 and HT-2 toxins tested on different cell lines.

Type of Test
Cell Line Results Reference

CCK-8 porcine Leydig cells
After 24 h of incubation; the IC50 values were 0.0209 and 0.0401 µM for T-2 and HT-2 toxin, respectively. Cytotoxic effect was dose-dependent. [76] MTT HepG-2 The IC50 values obtained by the MTT assay were 68.6 ± 4.8 nM and 61.9 ± 2.4 nM at 24 and 48 h, respectively; T-2 toxin decreased cell viability in a dose-and time-dependent manner. [77] MTT, LDH release SerW3 That T-2 toxin exhibits cytotoxic properties in a dose-and timedependent manner. Cytotoxicity was assessed after 24 and 48 h of incubation at concentrations of 12, 120 and 1200 ng/mL. The LDH cytotoxicity assay also confirmed cell membrane damage in cells exposed to T-2 toxin, suggesting that T-2 toxin has cytotoxic properties.

Type of Test Cell Line Results Reference
MTT, LDH release TM3 Leydig cells T-2 decreased cell viability in a dose-dependent manner. Cell viability was decreased to 82.95, 63.47 and 32.79% for 1, 10 and 100 nM, respectively, compared to the control. Moreover, T-2 toxin increased the LDH release at concentrations of 10 and 100 nM. Flow cytometry also showed an increased ratio in apoptotic cells after incubation with T-2 toxin. [79]

RPTCE, NHLF
Both T-2 and HT-2 toxin exhibited a similar cytotoxicity, with IC50 values of 0.2 and 0.8 µM for T-2 and HT-2 toxin, respectively, in the RPTEC cell line and 0.5 and 0.7 µM for T-2 and HT-2 toxin, respectively, in the NHLF cell line. Based on increased caspase-3 activity and the results from staining with Hoechst 33,258 dye, T-2 and HT-2 toxins promoted apoptotic cell death in both cell lines. [81]

CCK-8 TM4
Results showed dose-dependent decrease in cell viability, with a IC50 of 8.1 nM after 24 h of incubation. Further analyses of apoptosis rate, caspase-3, caspase-8 and caspase-9 activity showed that T-2 toxin promoted apoptotic cell death. Moreover, ROS levels were significantly increased, and antioxidant enzymes levels were decreased in cells incubated with T-2 toxin. [82]

MTT, NRU Vero
After 24, 48 and 72 h of incubation, a significant dose-and time-dependent decrease in cell viability was observed. The IC50 calculated based on the MTT assay ranged from 7 to 12 nM, whereas that calculated based on the NRU assay ranged from 4 to 5 nM. [70] MTT SK, MDCK, HeLa Cytotoxicity was defined as a decrease in cell viability by 20%. This effect was achieved by incubating T-2 toxin for 24 h at a concentration 100 µg/mL with HeLa cells and at a concentration 0.4 µg/mL with SK cells, but it could not be obtained with MDCK cells. Additionally, the cytotoxicity of HT-2 toxin was tested against SK cells; 20% decrease in cell viability after 24 h of incubation with HT-2 toxin was observed at a concentration of 3.1 µg/ml.

Zearalenone
In the case of zearalenone, MTT was the most frequently used test, followed by NRU, while HepG-2 was the most frequently used cell line, followed by Caco-2 and CHO-K1. Results showed a decrease in cells' viability, as well as an increase in the apoptotic cell ratio, inhibition in protein and DNA synthesis and increased ROS production. IC50 values for zearalenone range from~10 µM to >100 µM, depending on experiment conditions, time of incubation and cell line used (full results are presented in Table 8). [55] MTT SK, MDCK, HeLa Cytotoxicity was defined as a decrease in cell viability by 20%. This effect could not be achieved in any of the three cell lines after 24 h of incubation at concentrations within the tested range (0.01-100 µg/mL cell culture medium). [31]

Type of Test Cell Line Results Reference
Triple Assay (AlamarBlue™, CFDA-AM, NRU)

RTGill-W1
ZEN was primarily cytotoxic against lysosomes (EC50 25.19 µM), followed by plasma membrane (EC50 71.48 µM) and mitochondria (EC50 135.14 µM). [44] MTT SK, Vero, MDCK, BEL At low concentrations, ZEN had little or no effect on cell viability after 2 and 24 h of incubation. At the higher concentration (100 µg/mL), a significant drop in cell viability was observed after 16 h (up to 40% of the control). In MDCK cells, 100 µg/mL of ZEN resulted in a significant decrease in cell viability after 2 and 16 h of incubation, but after 24 h, viability reached a level of 110% compared to the control. At lower concentrations, ZEN slightly stimulated cell viability after 2 and 24 h of incubation. In SK cells, a high ZEN concentration resulted in a significant drop in cell viability (up to 20% of the control), whereas at lower concentrations, ZEN stimulated cell viability (up to 160% of the control after 16 h). [72]

MTT
HepG-2 Significant, dose-dependent decrease in cell viability, GSH content and GSH-Px activity was observed. [56] AlamarBlue™ HepG-2, RAW 264.7 After 48 h of incubation, the AlamarBlue™ assay showed a dose-dependent reduction in cell viability, with a IC50 calculated at 6.45 ppm. [45] high content assay HepG-2 ZEN decreased cell viability in a dose-dependent manner, with a significant increase in cell viability reduction between concentrations of 5.77 × 10 −2 µg/mL and 6.41 × 10 −3 µg/mL and a similar reduction at the two highest concentrations tested. [ 86] 2.1.8. Citrinin In the case of citrinin, MTT was the most frequently used test. Results showed a decrease in cells' viability, as well as an increase in the apoptotic cell ratio, increased ROS production and cell cycle arrest. IC50 values for citrinin varies depending on experiment conditions, time of incubation and cell line used (full results are presented in Table 9). Table 9. Cytotoxicity of citrinin tested on different cell lines.

MTT SK
Cytotoxicity was defined as a decrease in cell viability by 20%. However, this effect could not be achieved within the tested concentration range after 24 h of incubation. [31] MTT PBM CIT at a concentration of 125 mg/L significantly decreased both cell metabolic activity and proliferation (41.8 and 44.2%, respectively, compared to the control).

Type of Test Cell Line Results Reference
MTT, flow cytometry HEK 293 Dose-dependent decrease in cell viability, with a IC50 estimated at 189 µM observed. In addition, flow cytometry showed that CIT caused a significant accumulation of cells in the G2/M phase, thus disrupting the cell cycle. [58] MTT PK-15 CIT significantly increased the cell death ratio after 48 h of incubation. This study also showed a decrease in the concentration of thiol groups (SH) and an up-regulation of Hsp70 and Hsp27 expression. [59] Nuclei counted via hemocotometer MDBK, PFBK The viability of MDBK cells was significantly reduced after 24 h of incubation with CIT at concentrations of 150-300 µM (IC50 calculated at 140 µM). In PFBK cells, CIT caused a significant decrease in cell viability after 24 h when used at concentrations of 0.5-1 mM (IC50 calculated at 380 µM). [87] MTT, AO/EB staining HepG-2 CIT in a concentration of 0-300 µM significantly decreased cell viability in a dose-dependent manner, with a IC50 calculated at 155 µM. Additionally, AO/EB staining revealed an increased ratio of apoptotic and necrotic cells. [60] MTT Vero Significant decrease in cell viability, albeit at relatively high concentrations (no effects were visible at concentrations ≤ 60 µM), was observed. The IC50 was estimated at 220 µM. [61] MTT and trypan blue exclusion assay HL-60 After 24 h of incubation, a significant decrease in cell viability was observed at concentrations ≥ 50 µM (no significant effect was observed at a concentration of 25 µM). Cell viability was reduced to 11.9% compared to the control at 100 µM (MTT assay). [88] MTT human osteoblasts Decrease in cell viability by 40-70% compared to the control was observed. Moreover, the JNK pathway (which is essential for apoptosis in some cell types) was activated in a dose-dependent manner after incubation with CIT for 1 h. [89] CCK-8, CASY cell counter assay IHKE CIT exhibited cytotoxic effects, with EC50 values calculated at 56.3 and 27.7 µmol/L (for CCK-8 and CASY assays, respectively). Moreover, a significant increase in caspase-3 activity (indicator of apoptosis) was observed after incubation with CIT at concentrations ≥ 5 µmol/L.

[62]
AlamarBlue™ A549 CIT reduced A549 cell viability in a dose-and time-dependent manner. After 6 h of incubation with 50 µg/mL, CIT viability dropped to 80% of that of the control, whereas after 24 h and 72 h, it dropped to 40 and 20% of that of the control, respectively. [90]

Enniatins
In the case of enniatins, MTT was the most frequently used test, followed by Alamar-Blue™, while Caco-2 was the most frequently used cell line, followed by HepG-2. Results showed a decrease in cells' viability, as well as an increase in the apoptotic cell ratio and cell cycle arrest. IC50 values for enniatins vary, depending on experiment conditions, time of incubation, cell line used and specific enniatin tested (full results are presented in Table 10). Flow cytometry IPEC-J2 Flow cytometry showed that ENA1 was the most cytotoxic enniatin, whereas ENAB was the least cytotoxic one. The cytotoxicity of enniatins followed the order ENA > ENA1 > ENB1 > ENB (30, 86, 93, 95% of viable cells compared to the control, respectively). [92] AlamarBlue, BrdU HepG-2, MRC-5 AlamarBlue™ assay showed that MRC-5 cells were more susceptible to enniatins, with IC50 for MRC-5 estimated at 3.65, 6.4, 5.85 and 4.6 µM (for ENA, ENA1, ENB and ENB1, respectively) and for HepG-2 at 8.35, 14.9, 321.3 and 22.6 µM (for ENA, ENA1, ENB and ENB1, respectively). This is supported by the results from the BrdU test, albeit with the exception of enniatin B. The IC50 values for MRC-5 were estimated at 0.7, 1.25, 2.25 and 1.3 µM (for ENA, ENA1, ENB and ENB1, respectively) and for HepG-2 at 2.05, 3.4, 1 and 3.15 µM (for ENA, ENA1, ENB and ENB1, respectively). Additionally, the cytotoxicity of ENB2 and ENB3 against HepG-2 cells was tested via the BrdU assay, resulting in IC50 values of 8.45 and 8.9 µM for ENB2 and ENB3, respectively. [75] MTT Caco-2 ENA and ENA had little to no effect on Caco-2 cell viability, whereas ENA1 and ENB1 decreased cell viability in a dose-dependent manner, with IC50 values of 12.3 and 19.5 µM, respectively. [93] MTT Caco-2, HepG-2, HT29 Almost all examined enniatins decreased cell viability in a dose-dependent manner. After 48 h of incubation, the IC50 values for Caco-2 cells were 9.3, 2.7, 2.6, 5.3 and 2.9 µM (for ENA, ENA1, ENA2, ENB1 and ENB4, respectively), whereas those for HT29 were 8.2, 1.4, 2.8, 3.7 and 15 µM (for ENA, ENA1, ENB, ENB1 and ENB4, respectively). The IC50 values for HepG-2 cells were 4.6, 2.6, 8.5 and 12.7 µM (for ENA, ENA1, ENB1 and ENB4, respectively), and those for ENJ3 could not be calculated in any of the cell lines; also, the IC50 values for ENA2 in HT29 cells, ENB in Caco-2 cells and ENA2, ENB in HepG-2 cells could not be determined.  Additionally, ENB at a concentration of 1.25 µM or higher caused an increase in the ratio of cells in G1 phase, thus suggesting cell cycle inhibition. Moreover, ENB also increased the ratio of apoptotic and necrotic cells. [97]

Multiple Mycotoxins
In the case of mycotoxins' mixtures, their combined effect can be calculated using the combination index (CI), a method derived from the median effect principle [98,99]. The CI value is calculated from the general equation: where: n (CI) x is combination index for n components at x% of cell proliferation inhibition (D) j is the dose of n mycotoxins that causes x% cell proliferation inhibition in the combination (D x ) j is the dose of each n mycotoxin individually that causes x% cell proliferation inhibition CI < 0.9, CI = 0.9-1.1, CI > 1.1 generally indicate synergistic, additive and antagonistic effects, respectively. Additionally, when the CI indicates a synergistic effect, dose reduction indices (DRI) can be calculated. DRI indicate a fold of reduction of dose of each component at a given effect level compared to a dose of each component individually and can be calculated from equation [100]: where: DRI is dose reduction indices D m is median effect dose

Combination of Two Mycotoxins
In the case of combinations of two mycotoxins, MTT was the most frequently used test, while HepG-2 was the most frequently used cell line. Results showed that cytotoxicity of binary combinations strongly depend on components (while some mixtures exhibit synergistic effects, others exhibit additive ore antagonistic effects). Moreover, a concentration of individual components seems to have an effect on mixture cytotoxicity (full results are presented in Table 11). MTT Caco-2 PAT + OTA OTA was predominantly responsible for the cytotoxicity of the mixture; however, at higher concentrations of patulin, the cytotoxicity of the mixture was lower than that of patulin and OTA alone, suggesting an antagonistic effect. [28] MTT IPEC-J2 In cytotoxic concentrations (measured previously for individual mycotoxins), there was no increased cytotoxicity in mixtures containing DON. A mixture of FB1 and ZEN was more cytotoxic than those two mycotoxins applied individually. At non-cytotoxic concentrations (measured for individual mycotoxins), all mixtures were cytotoxic against IPEC-J2 cells.

Combination of Three Mycotoxins
In the case of combinations of three mycotoxins, MTT was the most frequently used test, while HepG-2 and Caco-2 were the most frequently used cell line. Results showed that cytotoxicity of tertiary combinations strongly depend on components (while some mixtures exhibit synergistic effects, others exhibit additive ore antagonistic effects). Moreover, a concentration of individual components seems to have effect on mixture cytotoxicity (full results are presented in Table 12). The combination of PAT + DON + T-2 exhibited stronger cytotoxic properties than each toxin individually, irrespective of the concentration. This combination reduced cell viability by 30 to 57% compared to PAT, 12 to 46% compared to T-2 and 30 to 38% compared to DON applied individually. [21] MTT Caco-2 AFM1 + OTA + ZEN Combination of AFM1 + OTA + ZEN had stronger cytotoxic properties against Caco-2 cells than each mycotoxin applied individually. [34] MTT, NRU Caco-2, RAW264.7, MDBK OTA + AFB1 + FB1 After 48 h of incubation, concentrationdependent decrease in cells' viability was observed. MDBK cells' viability decreased by 26% and 14% (for MTT and NRU, respectively). There was no cytotoxic effect in raw264.7 cells, and viability of caco-2 cells decreased by 9% at highest tested concentration. [36] MTT IPEC-J2 DON + FB1 + ZEN At cytotoxic concentrations (measured for individual mycotoxins), the mixture was less cytotoxic than DON applied individually. However, at non-cytotoxic concentrations (measured for individual mycotoxins), the mixture was significantly more cytotoxic than the individual mycotoxins. [65] MTT Caco-2 All tertiary mixtures reduced cell viability in a dose-dependent manner by approximately 40% [91] high content screening BF-2 ZEN + DON + AFB1 This combination had a higher cytotoxicity than its components when tested individually. [42] MTT PBM OTA + FB1 + CIT This combination exhibited stronger cytotoxic properties than any of these mycotoxins tested individually. It significantly decreased both cell metabolic activity and proliferation (31.2 and 33.1%, respectively, compared to the control). [53] AlamarBlue™ HepG-2, RAW 264.7 AFB1 + ZEN + DON After 48 h of incubation, results showed that this combination exhibited stronger cytotoxic properties than its individual components against both cell lines. [45] high content assay HepG-2 ZEN + DON + AFB1 After 24 h of incubation, this combination was more cytotoxic than its components tested individually at higher tested concentrations, whereas no differences in cytotoxicity were observed at lower concentrations (compared to individual mycotoxins). [46]

Combination of Four Mycotoxins
Prosperini et al. [91] tested the cytotoxicity of mixtures of four enniatins against Caco-2 cells. The MTT assay showed that this mixture reduced cell viability in a dose-dependent manner by approximately 40%.

Conclusions
In conclusion, among various negative properties, cytotoxicity is one of the most important one of mycotoxins. To assess this cytotoxicity, the most frequently chosen assay is MTT, followed by the neutral red assay, the CKK-8 assay, the AlamarBlue™ assay and the LDH cytotoxicity assay. For apoptotic cell ratio assessment, flow cytometry is most frequently being used. As shown in this review, most mycotoxins exhibit cytotoxic properties in a dose-and time-dependent manner; however, the concentration of those mycotoxins range from nM to µM. The cell line chosen in the mycotoxin cytotoxicity study is also important because different cell lines react differently to certain mycotoxins. Under natural conditions, several mycotoxins often co-occur, making it important to assess the cytotoxic effects of different combinations of mycotoxins. Based on the findings of this review, such combinations often exhibit different levels of cytotoxicity compared to the individually applied mycotoxins, with a stronger cytotoxicity. A major drawback of the cytotoxicity assessment in cell lines is the lack of interaction with different cell types and mechanisms naturally occurring in complex organisms (for example, immunology responses). This problem could be partially solved by using primary cell lines instead of continuous cell lines (since they retain many characteristics of cells in vivo; this however generates a problem with reproducibility of the results due to, e.g., viral or bacterial contamination) or by using 3D printed models of tissues to emulate the natural environment better.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxins14040244/s1, Table S1: Summary of reviewed literature, cell lines and cytotoxicity assays used and mycotoxins tested.