TiO₂ Nanoparticles Caused DNA Damage in Lung and Extra-Pulmonary Organs Through ROS-Activated FOXO3a Signaling Pathway After Intratracheal Administration in Rats

Figures & data

Figure 1 Characterization of Ti and the contents of Ti in organs. (A and B) Scanning electron microscopy and transmission electron microscopy images of Nano-TiO₂. The powder was deposited on 200-mesh copper grids. The particle primary structure was 10–30 nm and globular. (C) On the 7th day after instillation, the contents of Ti in tissues of rats in the 0, 0.2, and 1 g/kg Nano-TiO₂ treatment groups. N=5, *P<0.05, in comparison to the control group. (D) The contents of Ti in tissues of rats after 1 g/kg Nano-TiO₂ treatment for 1, 3, and 7 days. N=5, *P<0.05, in comparison to the 1-day group.

Figure 2 Effects of Nano-TiO₂ exposure on the histopathological changes in the lung, liver, and kidney at 7 days after treatment. (A) The representative images of lung tissue of rats stained with HE. (B) The representative images of liver tissue of rats stained with HE. (C) The representative images of kidney tissue of rats stained with HE. Scale bar represents 50 μm. The arrow in A₂ indicated irregular luminal inflammatory cell infiltration; The arrow in A₃indicated the alveolar wall thickening. The arrow in B₂ indicated cell disorder. The arrow in B₃ indicated the spotty necrosis. The arrow in C₂ indicated the gap increases. The arrow in C₃ indicated necrosis. 1: Control group, 2: 0.2 g/kg group, 3: 1 g/kg group.

Figure 3 The oxidative stress levels in the lung, liver, and kidney of rats after Nano-TiO₂ treatment. (A–C) The SOD, GSH-Px activities, and MDA content in lung of rats. (D–F) The SOD, GSH-Px activities, and MDA content in liver of rats. (G–I) The SOD, GSH-Px activities, and MDA content in kidney of rats. (J–L) ROS levels induced by Nano-TiO2 in organs of rats. N=5, *P<0.05, in comparison to the control group; ^#P<0.05, in comparison to the 0.2 g/kg group.

Abbreviations: SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malonaldehyde.

Figure 4 The DNA damage levels in the lung, liver, and kidney of rats after Nano-TiO₂ treatment. (A and B): The OTM value and Tail DNA% value changed in the lung after Nano-TiO₂ treatment at different times. (C and D) The OTM value and Tail DNA% value changed in the liver after Nano-TiO₂ treatment at different times. (E and F) The OTM value and Tail DNA% value changed in the lung after Nano-TiO₂ treatment at different times. (G) Representative images of DNA damage in the lung of rats after Nano-TiO₂ treatment for 7 days (400x). (G₁: control group; G₂: 0.2 g/kg Nano-TiO₂ treatment group; G₃: 1 g/kg Nano-TiO₂ treatment group). The result was the average of at least three independent experiments. N=5, *P<0.05: day 1 in comparison to the control group; ^#P<0.05: day 3 in comparison to the control group; ^○P<0.05: day 7 in comparison to the control group.

Figure 5 Activation of the PI3K-AKT-FOXO3a signal pathway in the lung, liver, and kidney of rats after Nano-TiO₂ treatment for 7 days. (A) The represented protein bands of PI3K, AKT, FOXO3a, p-AKT, and p-FOXO3a in the lung. (B–D) The fold changes of PI3K, p-AKT/AKT, and p-FOXO3a/FOXO3a levels in the lung. (E–H) The represented protein bands and fold changes of PI3K, AKT, FOXO3a, p-AKT, and p-FOXO3a in the liver. (I–L) The represented protein bands and fold changes of PI3K, AKT, FOXO3a, p-AKT, and p-FOXO3a in the kidney. N=5, *P<0.05, in comparison to the control group. ^#P<0.05, in comparison to the 0.2 g/kg group.

Abbreviations: PI3K, phosphatidyl inositol 3-kinase; AKT, protein kinase B; FOXO3a, the Fork head box class O 3a.

Figure 6 Changes of DNA repair proteins in the lung, liver, and kidney of rats exposed to Nano-TiO₂ for 7 days. (A–D) The represented protein bands and fold changes of XRCC1, ChK2, and GADD45α in the lung. (E–H) The represented protein bands and fold changes of XRCC1, ChK2, and GADD45α in the liver. (I–L) The represented protein bands and fold changes of XRCC1, ChK2, and GADD45α in the kidney. N=5, *P<0.05, in comparison to the control group. ^#P<0.05, in comparison to the 0.2 g/kg group.

Abbreviations: XRCC1, x-ray repair cross complementing gene 1; ChK2, checkpoint kinase 2; GADD45α, growth arrest and DNA damage α.

Figure 7 A proposed action model for the PI3K/AKT/FoxO3a pathway via ROS regulating DNA damage in the lung, liver, and kidney induced by Nano-TiO₂ intratracheal administration.