The mycotoxin enniatin B1 (ENN B1) is widely present in grain-based feed and food products. In the present study, we have investigated how this lipophilic and ionophoric molecule can affect the lysosomal stability and chaperone-mediated autophagy (CMA) in wild-type (WT) and in lysosome-associated membrane proteins (LAMP)-1/2 double-deficient (DD) mouse embryonic fibroblasts (MEF). The cell viability and lysosomal pH were assessed using the Neutral Red (NR) cytotoxicity assay and the LysoSensor® Yellow/Blue DND-160, respectively. Changes in the expression of the CMA-related components LAMP-2 and the chaperones heat shock cognate (hsc) 70 and heat shock protein (hsp) 90 were determined in cytosolic extracts by immunoblotting. In the NR assay, LAMP-1/2 DD MEF cells were significantly less sensitive to ENN B1 than WT MEF cells after 24 h exposure to ENN B1 at levels of 2.5-10 μmol/L. Exposure to ENN B1 at concentrations below the half maximal effective concentration (EC50) (1.5-1.7 μmol/L) increased the lysosomal pH in WT MEF, but not in LAMP-1/2 DD cells, suggesting that lysosomal LAMP-2 is an early target of ENN B1-induced lysosomal alkalization and cytotoxicity in MEF cells. Additionally, cytosolic hsp90 and LAMP-2 levels slightly increased after exposure for 4 h, indicating lysosomal membrane permeabilization (LMP). In summary, it appeared that ENN B1 can destabilize the LAMP-2 complex in the lysosomal membrane at concentrations close to the EC50, resulting in the alkalinization of lysosomes, partial LMP, and thereby leakage of CMA-associated components into the cytosol.
Keywords: Chaperone-mediated autophagy (CMA); Cytotoxic activity; Enniatin B1 (ENN B1); Lysosomal pH; Mouse embryonic fibroblasts (MEF).