title: Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels.
abstract: BACKGROUND
  Interactions 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.
  
  
  RESULTS
  TiO2-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 μg/ml) is due to blockage while a low dose (0.16 μg/ml) promoted autophagy. Cellular viability was not affected in either case.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Clinical and Experimental Medicine, Cell Biology, Faculty of Medicine, Linköping University, 581 83, Linköping, Sweden.
authors.forename: Viviana R
authors.initials: VR
authors.lastname: Lopes
authors.affiliation: Department of Clinical and Experimental Medicine, Medical Microbiology, Faculty of Medicine, Linköping University, 581 83, Linköping, Sweden.
authors.forename: Vesa
authors.initials: V
authors.lastname: Loitto
authors.affiliation: Material Research & Technology Department (MRT), Luxembourg Institute of Science and Technology (LIST), 4422, Belvaux, Luxembourg.
authors.forename: Jean-Nicolas
authors.initials: JN
authors.lastname: Audinot
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, 106 91, Stockholm, Sweden.
authors.forename: Narges
authors.initials: N
authors.lastname: Bayat
authors.affiliation: Environmental Research and Innovation (ERIN) Department 41, Luxembourg Institute of Science and Technology (LIST), 4422, Belvaux, Luxembourg.
authors.forename: Arno C
authors.initials: AC
authors.lastname: Gutleb
authors.affiliation: Department of Clinical and Experimental Medicine, Cell Biology, Faculty of Medicine, Linköping University, 581 83, Linköping, Sweden. Susana.Cristobal@liu.se.|IKERBASQUE, Basque Foundation for Science, 48013, Bilbao, Bizkaia, Spain. Susana.Cristobal@liu.se.|Department of Physiology, Faculty of Medicine and Dentistry of University of Basque Country UPV/EHU, 48940, Leioa, Bizkaia, Spain. Susana.Cristobal@liu.se.
authors.forename: Susana
authors.initials: S
authors.lastname: Cristobal
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects; D053768:Metal Nanoparticles / Q000008:administration & dosage*; D018384:Oxidative Stress / Q000187:drug effects; D010588:Phagosomes / Q000187:drug effects; D014025:Titanium / Q000494:pharmacology*
chemical_list: C009495:titanium dioxide; D014025:Titanium
keywords: Autophagy; Cell-nanoparticle interactions; Dose; Keratinocytes; Titanium dioxide nanoparticles