**2. Results**

#### *2.1. Cytotoxicity Assay of Individual and Combined Mycotoxins*

The cytotoxicity e ffects of α-ZEL, β-ZEL, and BEA mycotoxins on SH-SY5Y cells were evaluated by the MTT assays over 24, 48, and 72 h. Figure 1 shows the time- and concentration-dependent decrease in cell viability after exposure to each mycotoxin individually, while IC50 values are shown in Table 1. After 24 h, the IC50 value could be calculated only for β-ZEL and was 94.3 ± 2.0 μM; after 48 h of exposure, the IC50 values were 20.8 ± 0.5 μM for α-ZEL and 9.1 ± 1.8 μM for β-ZEL. After 72 h of exposure, the IC50 values were 14.0 ± 1.8 μM, 7.5 ± 1.2 μM. and 2.5 ± 0.2 μM for α-ZEL, β-ZEL, and BEA, respectively. According to the IC50 values obtained at 72 h, BEA showed the highest cytotoxic effect on SH-S5Y5 cells (Table 1).

**Figure 1.** Cytotoxicity of the mycotoxins α-ZEL (**a**), β-ZEL (**b**), and BEA (**c**) individually at 24 h, 48 h, and 72 h. All values are the results of three independent experiments with eight replicates and are expressed as mean ± SD; *p* ≤ 0.05 (\*), *p* ≤ 0.01 (\*\*), *p* ≤ 0.001 (\*\*\*).

**Table 1.** Medium inhibitory concentration (IC50 ± SD) of α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), and beauvericin (BEA) for SH-SY5Y cells after 24, 48, and 72 h of exposure, determined by the MTT assay. Three independent experiments were performed with eight replicates each.


The cytotoxic effect of binary and tertiary combinations of α-ZEL, β-ZEL, and BEA on SH-SY5Y cells was evaluated by the MTT assays over 24, 48, and 72 h. The dose–response curves of the two- and three-mycotoxin combinations are shown in Figures 2 and 3, which demonstrate higher cytotoxicity of the combinations compared with individual mycotoxin. Figure 2 shows the concentration-dependent decrease in SH-SY5Y cell viability upon combined treatment with α-ZEL + BEA (5:1) (Figure 2a), β-ZEL + BEA (5:1) (Figure 2b), α-ZEL + β-ZEL (1:1) (Figure 2c); Figure 3 shows the results for α-ZEL + β-ZEL + BEA (5:5:1).

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**Figure 3.** Cytotoxicity of the mycotoxin combination of α-ZEL + β-ZEL + BEA (5:5:1) at 24 h (**a**), 48 h, (**b**) and 72 h (**c**). All values are the results of three independent experiments with eight replicates and are expressed as mean ± SD; *p* ≤ 0.05 (\*), *p* ≤ 0.01 (\*\*), *p* ≤ 0.001 (\*\*\*). BEA: line and square; β-ZEL: line and diamond; α-ZEL: line and triangle; Mixture: line and ×.

The α-ZEL + BEA combination at the highest concentration induced a decrease in cell proliferation at 24 h of exposure (Figure 2a) of 35% with respect to the e ffect α-ZEL tested individually and of 37% with respect to the e ffect BEA. After 48 h of exposure, the decrease in cell proliferation was 67% with respect to that measured for α-ZEL and 36% with respect to that measured for BEA. After 72 h of exposure, the viability decreased 53% with respect to α-ZEL and 43% with respect to BEA. After 24 h of exposure, the β-ZEL + BEA combination (Figure 2b) decreased cell proliferation by about 55% and 29% at the highest concentration with respect to β-ZEL and BEA tested individually, respectively. After 48 h of exposure, the highest concentration of the combination reduced cell proliferation by 11% with respect to BEA tested individually. Also, at 72 h of exposure, the combination decreased cell proliferation by approximately 36% with respect to BEA individually tested. Such e ffect was not noticed after 48 and 72 h with respect to b-ZEL. In Figure 2c, the α-ZEL + β-ZEL combination after 24 h of exposure showed 17% of decrease in cell proliferation compared to β-ZEL individually assayed. After 48 and 72 h of exposure, the highest concentration of the combination reduced cell proliferation by 60% and 50%, respectively, compared to α-ZEL tested alone, whereas, this did not happen with respect to β-ZEL after 48 and 72 h of exposure. Figure 3 shows the dose–response curves for the tertiary combination of α-ZEL, β-ZEL, and BEA at 24, 48, and 72 h of exposure in SH-SY5Y cells. At 24 h of exposure, cell proliferation decreased by 16%, 44%, and 18% compared to cells exposed to α-ZEL, β-ZEL, and BEA alone. After 48 and 72 h of exposure, a significant reduction in cell proliferation, corresponding to 57% and 51%, was observed with respect to α-ZEL alone, and a reduction of 26% and 41% was observed with respect to BEA alone, while such e ffect was not observed with respect to β-ZEL alone.

The isobologram analysis was used to determine the type of interaction between α-ZEL, β-ZEL, and BEA. The values of the parameters *Dm*, *m*, and *r* of the double and triple combinations, as well as of the mean combination index (CI) are shown in Table 2. The IC50, IC75, and IC90 are the doses required to inhibit proliferation at 25%, 50%, 75%, and 90%, respectively. These CI values were calculated automatically by the computer software CalcuSyn. The CI fractional e ffect (*fa*) curves for α-ZEL, β-ZEL, and BEA combinations in SH-SY5Y cells are shown in Figure 4. Synergism for all concentration of the α-ZEL + BEA (5:1) mixture after 24 and 48 h of exposure was demonstrated; however, after 72 h of exposure, an additive e ffect for the α-ZEL + BEA combination was observed (Figure 4a, Table 2). The β-ZEL + BEA (5:1) mixture showed synergism after 24 h of exposure; however, after 48 and 72 h it showed antagonism at high concentrations and moderate synergism at low concentrations (Figure 4b, Table 2). The mixture of α-ZEL + β-ZEL showed antagonism after 24 h of exposure at all concentrations assayed but at 48 and 72 h, it showed antagonism at high concentration and a moderate synergism at low concentration (Figure 4c, Table 2). The tertiary mixture, after 24 h of exposure, showed antagonism at high concentration and synergism at low concentration, while after 48 h, it showed synergism and after 72 h, antagonism at all concentrations assayed (Figure 4d, Table 2).

Cytotoxicity after 24 h of incubation decreased in this order: α-ZEL + BEA > β-ZEL + BEA > α-ZEL + β-ZEL + BEA > α-ZEL + β-ZEL. After 48 and 72 h of incubation, the ranking was α-ZEL + BEA > β-ZEL + BEA >α-ZEL + β-ZEL > α-ZEL + β-ZEL + BEA.

#### *2.2.* α*-ZEL,* β*-ZEL, and BEA Present in Cell Medium after Treatment in Binary and Tertiary Combination*

The medium of SH-SY5Y cells containing α-ZEL, β-ZEL, and BEA after treatments (individual and combined after 24, 48, and 72h) was collected from each well. The amount of each mycotoxin remaining in the medium was calculated as a percentage with respect to the respective amount used in the exposure assays. In this sense, we determined whether the amounts were above or below 50% of those used for treatment (Figure 5). In individual exposures, the amounts of BEA and β-ZEL in the medium were below 50% at 48 and 72 h (Figure 5b,c), while, at 24 h, their concentrations tended to be higher and >50% for both mycotoxins. For α-ZEL, the concentration in the medium was maintained above 50% at all times studied (Figure 5a). This evidenced that a lower amount of α-ZEL exerted the examined effect compared to the amount necessary for BEA and β-ZEL, as higher amounts of α-ZEL were detectable in the medium at all times and concentrations.

In the binary combination α-ZEL + BEA (5:1), the amounts of each mycotoxin after 24 and 48 h were below 50% (Figure 5d.1,d.2), although the amount of BEA was higher than that of α-ZEL once the concentration assayed overpassed 0.62 μM for BEA and 3.12 μM for α-ZEL, revealing that the effects exerted by this mixture in neuroblastoma cells depended on both mycotoxins and were due more to α-ZEL than to BEA. This tendency at 72 h was more accentuated, as the amount of BEA in the medium was above 50% for all concentrations, while that of α-ZEL was below 50% (Figure 5d.3).

Also, for the combination β-ZEL + BEA (5:1), the mycotoxin's percentage remaining in the media was the same as that found for α-ZEL + BEA; however, β-ZEL was detected in higher amount than BEA in all scenarios, revealing that the effect of this mixture and was due more to BEA than to β-ZEL (Supplementary Figure S1A). On the other hand, for the binary combination of ZEN metabolites, α-ZEL + β-ZEL (1:1), the amounts of mycotoxins recovered were below 50%, and slightly superior for α-ZEL than for β-ZEL. This revealed that both mycotoxins contributed to the effect of this mixture in SH-SY5Y cell line (Supplementary Figure S1B). For the tertiary combination (α-ZEL + β-ZEL + BEA, (5:5:1)), the mycotoxins' percentages detected were also below 50% of the administered concentration, and this percentage was higher for higher concentrations administered and lower time of exposure (Figure 5e). This revealed that high amounts of α-ZEL and β-ZEL accessed the neuroblastoma cells, and the effect was due more to β-ZEL at 48 and 72 h, according to the results in Figures 3 and 5.

**Table 2.** The parameters *Dm*, *m*, and *r* are the antilog of x-intercept, the slope, and the linear correlation of the median-effect plot, which means the shape of the dose–effect curve, the potency (IC50), and the conformity of the data to the mass action law, respectively [30,31]. *Dm* and *m* values are used for calculating the combination index (CI) value (CI < 1, =1, and >1 indicate synergism (Syn), additive (Add) effect, and antagonism (Ant), respectively. IC50, IC75, and IC90 are the doses required to inhibit proliferation at 50%, 75%, and 90%, respectively. CalcuSyn software automatically provided theses values.


**Figure 5.** Percentage of α-ZEL, β-ZEL, and BEA remaining in the medium of SH-SY5Y cells after treatment for 24, 48, and 72 h at different concentrations individually or in combination by LC–ESI–qTOF-MS. (**a**) α-ZEL; (**b**) β-ZEL; (**c**) BEA; (**d**) α-ZEL + BEA and (**e**) α-ZEL + β-ZEL + BEA.
