RESUMEN
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.
Asunto(s)
Depsipéptidos/toxicidad , Membranas Intracelulares/efectos de los fármacos , Lisosomas/patología , Micotoxinas/toxicidad , Permeabilidad/efectos de los fármacos , Animales , Autofagia Mediada por Chaperones/efectos de los fármacos , Fibroblastos , Eliminación de Gen , Proteínas del Choque Térmico HSC70/efectos de los fármacos , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas HSP90 de Choque Térmico/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Proteína 2 de la Membrana Asociada a los Lisosomas/efectos de los fármacos , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Chaperonas Moleculares/efectos de los fármacos , Chaperonas Moleculares/metabolismoRESUMEN
Bentonites are commonly used as feed additives to reduce the bioavailability and thus the toxicity of aflatoxins by adsorbing the toxins in the gastrointestinal tract. Aflatoxins are particular harmful mycotoxins mainly found in areas with hot and humid climates. They occur in food and feedstuff as a result of fungal contamination before and after harvest. The aim of this study was to modify Brazilian bentonite clay by incorporation of zinc (Zn) ions in order to increase the adsorption capacity and consequently reduce the toxicity of aflatoxins. The significance of Zn intercalating conditions such as concentration, temperature and reaction time were investigated. Our results showed that the Zn treatment of the bentonite increased the aflatoxin B1 (AFB1) adsorption and that Zn concentration had a negative effect. Indeed, temperature and time had no significant effect in the binding capacity. The modified bentonite (Zn-Bent1) was not cytotoxic to either fibroblasts (3T3) nor epithelial colorectal adenocarcinoma cells (Caco-2) cell lines. Interestingly, Zn-Bent1 has higher protective effect against AFB1 induced cytotoxicity than the unmodified bentonite. In conclusion, the Zn modified bentonite, Zn-Bent1, represent an improved tool to prevent aflatoxicosis in animals fed on AFB1 contaminated feed.