IN VITRO TOXICOLOGICAL EFFECTS OF FUMONISIN B1 ALONE AND COMBINED WITH OTHER MYCOTOXINS

Mycotoxins, secondary metabolites produced by moulds, mainly Aspergillus spp., Fusarium spp. and Penicillium spp., are common contaminants of food and feed. The aim of this project was to evaluate: (i) the potential endocrine disruptor effects of fumonisin B1 (FB1), beauvericin (BEA), deoxynivalenol (DON) and zearalenone (ZEA) metabolites �-zearalenol (�-ZEA) and �zearalenol (�ZEA), alone and combined, using a bovine granulosa cell (GC) in vitro model and (ii) the individual and combined effects of FB1 and BEA on the intestinal barrier using Caco-2 cells cultured in vitro on semipermeable inserts. The results obtained indicated that FB1 alone at all tested doses (0; 0.5; 1; 1.5; 3; 6 �M) had no effects on GC proliferation and progesterone (P4) production. In the presence of �ZEA at30 ng/mL (0.094 �M), FB1 at 30 ng/mL (0.042 �M) showed a stimulatory effect on GC numbers. Cell proliferation decreased after exposure to �ZEA alone at 5.0 mg/mL (15.6 �M) and FB1 with �-ZEA and �ZEA at the same concentration. Regarding steroid production, FB1 at 30 ng/mL (0.042 �M ) and 100 ng/mL (0.13 �M amplified the inhibitory effect of �ZEA at 30 ng/mL (0.094 �M) on estradiol (E2) production, while FB1 alone increased (P<0.05) IGF1-induced E2 production. FB1 in combination with �ZEA decreased (P < 0.05) E2 production. FB1 at 1, 1.5 and 3 �M slightly inhibited (P < 0.05) E2 production. BEA at concentrations � 3 �M was found to strongly decrease (P < 0.05) both steroid production and FB1 did not influence the effects of BEA. At 10 �M both mycotoxins decreased (P < 0.001) serum-induced GC proliferation. At 30 �M, BEA showed inhibitory effects on FSH plus IGF-1-induced CYP11A1 and CYP19A1 mRNA abundance (P < 0.05), whereas FB1 at 30 �M had no effect on CYP11A1 and CYP19A1 gene expression. As regards the effects of FB1 and BEA, alone and combined, on the Caco-2 intestinal barrier model data showed a TEER decrease after 1 h and 2 h of Bl exposure to BEA at 0.5 and 1.5 �M and after 24 h of Bl exposure to BEA at 0.5 �M, whereas after 24 h of Bl exposure, BEA at 3 and 6 �M was found to significantly (P < 0.05) increase TEER. FB1 had no effect on the intestinal barrier integrity and when combined with BEA the TEER increase induced by BEA was no longer observed. Cytokine release was observed only after exposure to BEA alone, and not in combination with FB1, with an increase of IL-6 and IL8 release after apical exposure to 3 and 6 �M and after basolateral exposure to 1.5, 3, 6 �M for IL-6 and only to 6 �M for IL-8. TNF- � release was induced by Ap (0.5 -1.5 �M) and Bl (1.5 �M) exposure to BEA. Overall, these results provide information on in vitro toxicological effects of Fusarium mycotoxins.