Increased DNMT1 Involvement in the Activation of LO2 Cell Death Induced by Silver Nanoparticles via Promoting TFEB-Dependent Autophagy

The accumulation of exogenous silver nanoparticles (AgNPs) will terminally bring about liver injury, including cell death, where DNA methylation tends to be a crucial epigenetic modulator. The change in the cell autophagy level verified to be closely associated with hepatocyte death has been followed with wide interest. But the molecular toxicological mechanisms of AgNPs in relation to DNA methylation, autophagy, and cell death remain inconclusive. To address the issue above, in LO2 cells treated with increasing concentrations of AgNPs (0, 5, 10, and 20 mu g/mL), a cell cytotoxicity assay was performed to analyze the level of cell death, which also helped to choose an optimal concentration for next experiments. An immunofluorescence assay was used to determine the autophagic flux as well as TFEB translocation, with qRT-PCR and western blot being used to analyze the expression level of autophagy-related genes and proteins. According to our findings, in the determination of cell viability, 20 mu g/mL (AgNPs) was adopted as the best working concentration. LO2 cell death, autophagy, and TFEB nuclear translocation were induced by AgNPs, which could be inhibited by lysosome inhibitor chloroquine (CQ) or siRNA specific for TFEB. Moreover, AgNP exposure led to DNA hypermethylation, with DNMT1 taking part mainly, which could be obviously prevented by 5-Aza-2 '-deoxycytidine (5-AzaC) or trichostatin A (TSA) treatment or DNMT1 knockout in LO2 cells. Our studies suggest that through TFEB-dependent cell autophagy, increased DNMT1 may facilitate cell death induced by AgNPs.

Automated summary

The accumulation of exogenous silver nanoparticles (AgNPs) can cause liver injury, including cell death, which may be related to DNA methylation and cell autophagy. However, the molecular mechanisms of AgNPs in DNA methylation, autophagy, and cell death are still unclear. In this study, LO2 cells were treated with different concentrations of AgNPs, and it was found that AgNPs induced cell death, autophagy, and TFEB nuclear translocation, and led to DNA hypermethylation. Further experiments revealed that cell death induced by AgNPs may be facilitated through TFEB-dependent cell autophagy and increased DNMT1.