Fig 1.
Transmission Electron Microscopy images to characterize the crystal shape of the TiO2 nanoparticles as seen in DI water; (a) P25, (b) Anatase, 50 nm particle size and (c) Rutile, 50 nm particle size.
Table 1.
Characterization of TiO2 nanoparticles aggregates forming in hepatocyte culture medium using Dynamic Light Scattering (DLS) at 37°C and pH of 7.4.
Table 2.
Lethal Concentration (LC50) analysis of the different TiO2 nanoparticles treatment of primary rat hepatocytes.
Fig 2.
Scanning Electron Microscopy (SEM) images to visualize the morphology of primary hepatocytes when treated with TiO2 nanoparticles after 72 h of exposure.
Scale bar: 30 microns. Yellow arrows point to primary hepatocytes.
Fig 3.
MTT assay to quantify primary hepatocyte viability after treatment with different TiO2 nanoparticles at 20, 50 and 100 ppm after 72 h of exposure normalized to the untreated hepatocytes.
The values are the mean ± SD of five different samples, significant difference with respect to control is denoted as * p value < 0.001.
Fig 4.
Characterizing the effect of the different TiO2 nanoparticles treatment (50 ppm) on primary hepatocytes specific functions (A) Quantification of urea synthesized primary hepatocytes after 72 h of exposure normalized to the untreated cells and (B) Quantification of albumin synthesized by primary hepatocytes after 72 h of exposure normalized to the untreated cells.
The values are normalized with respect to loss in cell viability. The values are the mean ± SD of six different samples, significant difference with respect to control is denoted as * p value < 0.001, # p value< 0.05.
Fig 5.
Characterizing the state of oxidative stress in primary hepatocytes upon TiO2 nanoparticle treatment at a concentration of 50 ppm for a duration of 72 h (A) Quantification of Reactive Oxygen Species produced using H2DCFDA based fluorescence assay (B) In gel mitochondrial MnSOD enzyme activity assay normalized with respect to untreated cells (C) Fold change in the TMRM staining to quantify mitochondrial membrane potential using flow cytometry reported relative to the unstained cells.
The values are the mean ± SD of four different samples, significant difference with respect to control is denoted as * p value < 0.001, # p value< 0.05.
Fig 6.
Characterization of the effect of TiO2 nanoparticle treatment (50 ppm) for duration of 72 h on primary hepatocyte mitochondrial dynamics.
(A-B) Relative gene expressions of mitochondrial fusion markers through qPCR using double normalization with respect to total RNA and housekeeping gene (GAPDH). The values are the mean ± SD of four different samples, significant difference with respect to control is denoted as * p value < 0.001, # p value< 0.05. (C) Fluorescent imaging of the mitochondrial morphology in primary rat hepatocytes using Mitotracker green FM. Scale 20 microns. In the control image, long fiber-like mitochondrial morphology can be observed, as compared to fragmented and swollen mitochondria as seen in nanoparticle treated samples.
Fig 7.
Schematic representation of the possible damaging role of TiO2 on primary hepatocytes.
We propose that TiO2 induces loss in hepatocyte functions on primary hepatocytes through the induction of oxidative stress mediated by an increase of ROS production, loss in MnSOD enzyme function, loss in MMP and damage to mitochondria dynamics by down-regulating the fusion cycle in the mitochondrial dynamics.