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Mycotoxin Res. 2020 Feb;36(1):23-30. doi: 10.1007/s12550-019-00366-8. Epub 2019 Jul 1.

Enniatin B1-induced lysosomal membrane permeabilization in mouse embryonic fibroblasts.

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Department of Food Engineering, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, SP, 13635-900, Brazil.
Chemistry Section, Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106, Oslo, Norway.
Toxinology Research Group, Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106, Oslo, Norway.


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.


Chaperone-mediated autophagy (CMA); Cytotoxic activity; Enniatin B1 (ENN B1); Lysosomal pH; Mouse embryonic fibroblasts (MEF)


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