Dose dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non cytotoxic levels
BackgroundInteractions between nanoparticles and cells are now the focus of a fast growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles including those composed of titanium dioxide (TiO2 NPs) may disrupt the intracellular process of macroautophagy. Autophagy plays a key role in human health and disease, particularly in cancer and neurodegenerative diseases. We herein investigated the in vitro biological effects of TiO2 NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non cytotoxic levels.ResultsTiO2 NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering techniques. Cellular uptake, as evaluated by TEM and NanoSIMS revealed that NPs internalization led to the formation of autophagosomes. TiO2 NPs treatment did not reduce cell viability of HaCaT cells nor increased oxidative stress. Cellular autophagy was additionally evaluated by confocal microscopy using eGFP LC3 keratinocytes, western blotting of autophagy marker LC3I/II, immunodetection of p62 and NBR1 proteins, and gene expression of LC3II, p62, NBR1, beclin1 and ATG5 by RT qPCR. We also confirmed the formation and accumulation of autophagosomes in NPs treated cells with LC3 II upregulation. Based on the lack of degradation of p62 and NBR1 proteins, autophagosomes accumulation at a high dose (25.0 mg/ml) is due to blockage while a low dose (0.16 mg/ml) promoted autophagy. Cellular viability was not affected in either case.ConclusionsThe uptake of TiO2 NPs led to a dose dependent increase in autophagic effect under non cytotoxic conditions. Our results suggest dose dependent autophagic effect over time as a cellular response to TiO2 NPs. Most importantly, these findings suggest that simple toxicity data are not enough to understand the full impact of TiO2 NPs and their effects on cellular pathways or function.
Journal of Nanobiotechnology