*5.1. Reagents*

The reagent-grade chemicals and cell culture components used, Dulbecco's Modified Eagle's Medium- F12 (DMEM/F-12), fetal bovine serum (FBS), and phosphate-bu ffered saline (PBS) were supplied by Thermofisher, Gibco ™ (Paisley, UK). Methanol (MeOH, HPLC LS/MS grade), was obtained from VWR International (Fontenay-sous-Bois, France). Dimethyl sulfoxide was obtained from Fisher Scientific Co, Fisher BioReagnts ™ (Geel, Belgium). The compound (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) (MTT) for the MTT assay, penicillin, streptomycin, and Trypsin–EDTA were purchased from SigmaAldrich (St. Louis, MO, USA). Deionized water ( <18, M Ωcm resistivity) was obtained in the laboratory using a Milli-QSP ® Reagent Water System (Millipore, Beadford, MA, USA). Standard BEA (MW: 783.95 g/mol), α-ZEL, and β-ZEL (MW: 320.38 g/mol) were purchased from SigmaAldrich (St. Louis Mo. USA) (Figure 6). Stock solutions of mycotoxins were prepared in MeOH (α-ZEL and β-ZEL) and DMSO (BEA) and maintained at −20 ◦C in the dark. The final concentration of either methanol or DMSO in the medium was ≤1% (v/v) as previously established. All other reagents were of standard laboratory grade.

**Figure 6.** Chemical structures of the mycotoxins (**a**) α-ZEL, (**b**) β-ZEL, and (**c**) BEA.

### *5.2. Cell Culture*

The human neuroblastoma cell line SH-SY5Y was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's Medium/F12 (DMEM/F-12), supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were sub-cultivated after trypsinization once or twice a week and suspended in complete medium in a 1:3 split ratio. The cells were maintained as monolayers in 150 cm<sup>2</sup> cell culture flasks with filter screw caps (TPP, Trasadingen, Switzerland). Cell cultures were incubated at 37 ◦C, 5% CO2 atmosphere.

### *5.3. Mycotoxin Exposure*

Concentration of the mycotoxins and exposure time are two factors that were considered to in this study. The cells were exposed to α-ZEL, β-ZEL, and BEA mycotoxins individually for 24, 48, and 72 h at a concentration in the ranges of 0.39 to 100 μM for α-ZEL and β-ZEL and 0.009 to 25 μM for BEA, all with 1:2 dilution (Table 3). Also, the mycotoxins were assayed in combination in the following mixtures: α-ZEL + BEA, β-ZEL + BEA, α-ZEL + β-ZEL, and α-ZEL + β-ZEL + BEA at three exposure times 24, 48, and 72 h. The concentrations ranged from 1.87 to 25 μM for the binary combinations were studied and from 3.43 to 27.5 μM for the tertiary combination, including four dilutions of each mycotoxin: BEA (0.31, 0.62, 1.25, and 2.5 μM), α-ZEL and β-ZEL (1.56, 3.12, 6.25 and 12.5 μM) (Table 3). The dilution ratios of the concentrations for the binary combinations were 5:1 for α-ZEL + BEA and β-ZEL + BEA, 1:1 for α-ZEL + β-ZEL, and 5:5:1 for the tertiary combination (β-ZEL + α-ZEL + BEA) (Table 3).

**Table 3.** Concentration range (μM) of mycotoxins studied individually and in combinations. The dilution ratios were 5:1 for the combinations α-ZEL + BEA and β-ZEL + BEA, 1:1 for α-ZEL + β-ZEL, and 5:5:1 for α-ZEL + β-ZEL + BEA.


### *5.4. MTT Assay*

Cytotoxicity was examined by the MTT assay, performed as described by Ruiz et al. (2006) [37], with few modifications. The assay consists in measuring the viability of cells by determining the reduction of the yellow soluble tetrazolium salt only in cells that are metabolically active via a mitochondrial reaction to an insoluble purple formazan crystal. Cells were seeded in 96-well culture plates at 2 × 96 cells/well and allowed to adhere for 18–24 h before mycotoxin additions. Serial dilutions of α-ZEL, β-ZEL, and BEA at 1:2 dilutions were prepared with supplemented medium and added to the respective plates (Table 3). Culture medium without mycotoxins and with 1% MeOH or DMSO was used as a control. After treatment, the medium was removed, and each well received 200 μL of fresh medium containing 50 μL of MTT solution (5 mg/mL; MTT powder dissolved in phosphate-buffered saline). After an incubation time of 4 h at 37 ◦C in the darkness, the MTT-containing medium was removed, and 200 μL of DMSO and 25 μL of Sorensen's solution were added to each well before reading the optical density at 620 nm with the ELISA plate reader Multiskan EX (Thermo Scientific, MA, USA). Each mycotoxin combination plus a control were tested in three independent experiments. Mean inhibition concentration (IC50) values were calculated from full dose–response curves.

#### *5.5. Experimental Design and Combination Index*

The isobologram analysis (Chou–Talalay model) was used to determine the type of interaction (synergism, additive effect, and antagonism) that occurred when the mycotoxins studied were in combination. This model allows characterizing the interactions induced by combinations of mycotoxins in different cell lines and with different mycotoxins but it does not allow the elucidation of the mechanisms by which these types of interaction are produced. The median effect/combination index (CI) isobologram equation by Chou (2006) [31] and Chou and Talalay (1984) [30] permitted analyzing drug combination effects. The isobologram analysis involves plotting the dose–effect curves for each compound and its combinations in multiple diluted concentrations. Parameters such as *Dm* (median effect dose), *fa* (fraction affected by concentration), and *m* (coefficient signifying the shape of the dose–effect relationship) are relevant in the equation [30]. Therefore, the method considers both potency (*Dm*) and shape (*m*) parameters.

Chou and Talalay (1984) [30] introduced the term combination index (CI). CI values <1, =1, and >1 indicate synergism, additive effects, and antagonism of the combination, respectively. CalcuSyn software version 2.1. (Biosoft, Cambridge, UK, 1996–2007) was used to study the types of interactions assessed by the isobologram analysis. The IC25, IC50, IC75, and IC90 are the doses required to produce toxicity at 25%, 50%, 75%, and 90%, respectively.

#### *5.6. Extraction of* α*-ZEL,* β*-ZEL, and BEA from the Culture Media*

To determine the intracellular accumulation of the mycotoxins studied, an extraction procedure of the culture media was carried out following the method described by Juan-García et al. (2015 and 2016) [27,28], with several modifications. Briefly, 0.8 mL of culture medium was collected and transferred into a polypropylene tube, 1.5 mL of ethyl acetate was added, and the mixture was shaken for 2 min with an Ultra-Turrax Ika T18 basic (Staufen, Germany). Afterwards, the mixture as sonicated in an ultrasound cleaning bath (VWR, USC1700TH) for 10 min. Finally, the mixture was centrifuged at ~5600× *g* for 5 min at 22 ◦C (Centrifuge 5810R, Eppendorf, Germany). The supernatant phase was collected. The liquid–liquid extraction process was repeated three times. Finally, the total volume obtained (approx. 4.5 mL) was evaporated to dryness at 45 ◦C in an N2 stream with a TurboVap-LV (Zymark, Allschwil, Switzerland) and then re-dissolved in 0.25 mL of a mixture of methanol and water (70:30, v/v) by vortexing vigorously (15 s), before being transferred into a vial for LC–ESI–qTOF-MS injection.

#### *5.7. Determination of BEA,* β*-ZEL, and* α*-ZEL by LC–ESI–qTOF-MS*

The analysis was performed using an LC–ESI–qTOF-MS system, consisting of an LC Agilent 1200-LC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a vacuum degasser, an autosampler, and a binary pump. The columns were a Gemini NX-C18 column (150 × 2 mm, i.d. 3 μm, Phenomenex, Torrance, California) and a guard column C18 (4 × 2 mm, i.d. 3 μM).

Mobile phases consisted of milli-Q water with 0.1% of formic acid as solvent system A and acetonitrile and 0.1% of formic acid as solvent system B, with the following gradient elution: 3 min, 70% B; in 2 min 70–80% B; in 1 min ge<sup>t</sup> 90% of B, maintained 4 min; 90–100% B 4 min and maintained 2 min; in 2 min decrease to 50% B; in 2 min 90% B, maintained 2 min. The flow rate used was 0.250 mL min−1, and the total run time was 22 min. The sample volume injected was 20 μL.

MS analysis was carried out using a 6540 Agilent Ultra- High-Definition Accurate-Mass q-TOF-MS, equipped with an Agilent Dual Jet Stream electrospray ionization (Dual AJS ESI) interface in negative and positive ionization modes. Operation conditions were as follows: sheath gas temperature 350 ◦C at a flow rate of 8 L/min, capillary voltage 3500 V, nebulizer pressure 45 psig, drying gas 10 L/min, gas temperature 300 ◦C, skimmer voltage 65 V, octopole RF peak 750 V, and fragmentor voltage 130 V. Analyses were performed using AutoMS/MS mode with fixed collision energy (10, 20 and 30) and in mass range of 50–1700 *<sup>m</sup>*/*<sup>z</sup>*. Acquisition rate was 3 spectra/second. Acquisition data were processed with Agilent MassHunter Workstation software.
