Molecular mechanism of DNA damage induced by titanium dioxide nanoparticles in toll-like receptor 3 or 4 expressing human hepatocarcinoma cell lines

Abstract

Background: Titanium dioxide nanoparticles (TiO2 NPs) are widely used in the biological sciences. The increasing use of TiO2 NPs increases the risk of humans and the environment being exposed to NPs. We previously showed that toll-like receptors (TLRs) play an important role in the interactions between NPs and cells. Our previous results indicated that TLR4 increased the DNA damage response induced by TiO2 NPs, due to enhanced NP uptake into the cytoplasm, whereas TLR3 expression decreased the DNA damage response induced by TiO2 NPs because of NP retention in the endosome. In this study, we explored the molecular mechanism of the DNA damage response induced by TiO2 NPs using TLR3 or TLR4 transfected cells. We examined the effect of TLR3 or TLR4 over-expression on oxidative stress and the effect of DNA damage induced by TiO2 NPs on gene expression levels.

Results: Our results showed evidence for elevated oxidative stress, including the generation of reactive oxygen species (ROS), with increased hydrogen peroxide levels, decreased glutathione peroxidase, and reduced glutathione and activated caspase-3 levels in cells exposed for 48 h to 10 μg/ml TiO2 NPs. These effects were enhanced by TLR4 and reduced by TLR3 over-expression. Seventeen genes related to DNA double-strand breaks and apoptosis were induced, particularly IP6K3 and ATM.

Conclusion: Our results indicated that TiO2 NPs induced ROS, and the above molecules are implicated in the genotoxicity induced by TiO2 NPs.

Figure 1

Reactive oxygen species (ROS) generation in TiO ₂ NP-exposed HepG2 cells with and without TLR3 or TLR4 transfection. The transfected cells were exposed to 10 μg/ml TiO₂ NPs for 48 h. Each plot was produced from at least 3 replicate measurements. All values are presented as mean ± S.D. (n ≥3), (*P <0.05).

Figure 2

Hydrogen peroxide (H ₂ O ₂ ) levels in TiO ₂ NP-exposed HepG2 cells with and without TLR3 or TLR4 transfection. The transfected cells were exposed to 10 μg/ml TiO₂ NPs for 48 h. Each plot was produced from at least 3 replicate measurements. All values are presented as mean ± S.D. (n ≥3), (*P <0.05).

Figure 3

Glutathione peroxidase (GPx) activities in TiO ₂ NP-exposed HepG2 cells with and without TLR3 or TLR4 transfection. The transfected cells were exposed to 10 μg/ml TiO₂ NPs for 48 h. Each plot was produced from at least 3 replicate measurements. All values are presented as mean ± S.D. (n ≥3), (*P <0.05).

Figure 4

Reduced glutathione (GSH) levels in TiO ₂ NP-exposed HepG2 cells with and without TLR3 or TLR4 transfection. The transfected cells were exposed to 10 μg/ml TiO₂ NPs for 48 h. Each plot was produced from at least 3 replicate measurements. All values are presented as mean ± S.D. (n ≥3), (*P <0.05).

Figure 5

Caspase-3 activities in TiO ₂ NP-exposed HepG2 cells with and without TLR3 or TLR4 transfection. The transfected cells were exposed to 10 μg/ml TiO₂ NPs for 48 h. Each plot was produced from at least 3 replicate measurements. All values are presented as mean ± S.D. (n ≥3), (*P <0.05).

Figure 6

Expression of DNA damage marker mRNAs in TiO ₂ NP-exposed HepG2 cells. Cells were exposed to 10 μg/ml TiO₂ NPs for 48 h. Results are shown as the mean ± SD, n ≥3 for each marker, (*P <0.05).

Figure 7

Confocal laser scanning microscopic images of HepG2 cells, with and without TLR4 transfection, treated with TiO ₂ NPs. (A) HepG2 cells without transfection and without TiO₂ NP exposure, (B) Cells exposed to TiO₂ NPs only, (C) Cells transfected with TLR4 expression vector and exposed to 10 μg/ml TiO₂ NPs for 48 h. The white arrows show the apoptotic, nuclear fragmented cells. The confocal microscopic images show condensation of chromatin and nuclear fragmentation in HepG2 cells transfected with TLR4 expression vector and exposed to TiO₂ NPs.

Figure 8

A schematic representation of mitochondrial ROS implicated in DNA damage and apoptosis induced by TiO ₂ NP exposure, with and without TLR4 over-expression. The figure shows the site of induction and inhibition of respiratory complexes and oxidative stress molecules involved in ROS generation in HepG2 cells exposed to TiO₂ NPs with and without TLR4 over-expression. (+) indicate the sites of activation and (-) show the sites of inhibition.