**Individual and Combined E** ff**ect of Zearalenone Derivates and Beauvericin Mycotoxins on SH-SY5Y Cells**

#### **Fojan Agahi, Guillermina Font, Cristina Juan \* and Ana Juan-García**

Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, València, Spain; agahifozhan@gmail.com (F.A.); crisjua3@uv.es (G.F.); ana.juan@uv.es (A.J.-G.)

**\*** Correspondence: Cristina.juan@uv.es

Received: 9 February 2020; Accepted: 25 March 2020; Published: 27 March 2020

**Abstract:** Beauvericin (BEA) and zearalenone derivatives, α-zearalenol ( α-ZEL), and β-zearalenol (β-ZEL), are produced by several *Fusarium* species. Considering the impact of various mycotoxins on human's health, this study determined and evaluated the cytotoxic e ffect of individual, binary, and tertiary mycotoxin treatments consisting of α-ZEL, β-ZEL, and BEA at different concentrations over 24, 48, and 72 h on SH-SY5Y neuronal cells, by using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide). Subsequently, the isobologram method was applied to elucidate if the mixtures produced synergism, antagonism, or additive effects. Ultimately, we determined the amount of mycotoxin recovered from the media after treatment using liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry (LC–ESI–qTOF-MS). The IC50 values detected at all assayed times ranged from 95 to 0.2 μM for the individual treatments. The result indicated that β-ZEL was the most cytotoxic mycotoxin when tested individually. The major e ffect detected for all combinations assayed was synergism. Among the combinations assayed, α-ZEL + β-ZEL + BEA and α-ZEL + BEA presented the highest cytotoxic potential with respect to the IC value. At all assayed times, BEA was the mycotoxin recovered at the highest concentration in individual form, and β-ZEL + BEA was the combination recovered at the highest concentration.

**Keywords:** SH-SY5Y cells; zearalenone derivates; beauvericin; MTT; qTOF–MS/MS

**Key Contribution:** β-ZEL alone presented the highest cytotoxicological potency than α-ZEL and BEA on SH-SY5Y neuronal cells; α-ZEL + β-ZEL + BEA and α-ZEL + BEA presented the highest cytotoxic potential with respect to the IC value.
