Investigation of in vitro and in vivo effects of ochratoxin a on autophagy and ubiquitin - proteasome system

Abstract

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium genera of fungi and contaminates food and feed. OTA deregulates various signaling pathways and provokes oxidative stress along with augmentation of oxidized proteins in di↵erent cell lines. There is no study examining the fate of proteins damaged by OTA to this date. In this Ph.D. study, we show that autophagy and ubiquitin-proteasome system (UPS) are activated sequentially upon OTA exposure in HK-2 cells. OTA stim ulated a transient autophagic activity at early hours of treatment, which was subsiding upon further treatment in HK-2 cells. OTA exposure led to more cellular death in WT MEF cells than autophagy halted Atg5-/- MEF cells suggesting, autophagy might have a pro-death e↵ect on OTA-induced cytotoxicity. Furthermore, prolonged OTA exposure decreased ubiquitinated protein levels implicating an increased proteasomal activity. Moreover, we verified enhanced activities of purified and cellular 26S protea some upon OTA exposure. Our results suggest that autophagy and UPS may be the culprits resulting in sustained activation of PI3K/AKT and MAPK/ERK1-2 pathways through regulating the levels of the phosphatases. Additionally, we tested the e↵ects of OTA exposure on the mutant Ataxin 3 aggresomes and observed that OTA success fully dispersed the aggresomes. Finally, we examined the in vivo e↵ects of OTA on the proteolytic and PI3K/AKT, MAPK/ERK1-2 signaling pathways in the kidneys, livers and spleens of mice. OTA exposure (1 mg/kg bw) for 72 hours still produces the early response observed in cell culture. In conclusion, our results shed further light to the possible mode of action of OTA and demonstrate that OTA with or without modifications may be used as a therapeutic agent to disperse protein aggregates.