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

El-Said KS, Ali EM, Kanehira K, Taniguchi A - J Nanobiotechnology (2014)

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

View Article: PubMed Central - PubMed

Affiliation: Cell-Material Interaction Group, Biomaterial Unit, Nano-Bio Field, Interaction Center for Material Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), Tsukuba, Japan. SAMYELSAIDALI.Karim@nims.go.jp.

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.

No MeSH data available.


Related in: MedlinePlus

Confocal laser scanning microscopic images of HepG2 cells, with and without TLR4 transfection, treated with TiO2NPs. (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.
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Fig7: Confocal laser scanning microscopic images of HepG2 cells, with and without TLR4 transfection, treated with TiO2NPs. (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.

Mentions: To confirm the enhancement of apoptosis in HepG2 cells, we used Hoechst DNA staining to observe nuclear fragmentation as an indication of apoptosis. As shown in Figure 7, morphological changes consistent with cellular apoptosis, including condensation of chromatin and nuclear fragmentation, were observed in the cells exposed to TiO2 NPs. Again, expression of TLR4 (Figure 7C) appeared to enhance the effects of TiO2 NPs (Figure 7B) on apoptosis. Microscopy analysis confirmed that cells exposed to TiO2 NPs and transfected with TLR4 undergo programmed cell death (apoptosis) because of DNA damage. HepG2 cells that did not express TLRs and that were not exposed to TiO2 NPs also underwent apoptosis: by counting the number of apoptotic cells, we determined that 28% of untreated, untransfected cells had fragmented nuclei (Figure 7A), 55% of HepG2 cells exposed to TiO2 NPs were apoptotic (Figure 7B), and 75% of cells over-expressing TLR4 and exposed to TiO2 NPs were apoptotic (Figure 7C).Figure 7


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

El-Said KS, Ali EM, Kanehira K, Taniguchi A - J Nanobiotechnology (2014)

Confocal laser scanning microscopic images of HepG2 cells, with and without TLR4 transfection, treated with TiO2NPs. (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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260178&req=5

Fig7: Confocal laser scanning microscopic images of HepG2 cells, with and without TLR4 transfection, treated with TiO2NPs. (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.
Mentions: To confirm the enhancement of apoptosis in HepG2 cells, we used Hoechst DNA staining to observe nuclear fragmentation as an indication of apoptosis. As shown in Figure 7, morphological changes consistent with cellular apoptosis, including condensation of chromatin and nuclear fragmentation, were observed in the cells exposed to TiO2 NPs. Again, expression of TLR4 (Figure 7C) appeared to enhance the effects of TiO2 NPs (Figure 7B) on apoptosis. Microscopy analysis confirmed that cells exposed to TiO2 NPs and transfected with TLR4 undergo programmed cell death (apoptosis) because of DNA damage. HepG2 cells that did not express TLRs and that were not exposed to TiO2 NPs also underwent apoptosis: by counting the number of apoptotic cells, we determined that 28% of untreated, untransfected cells had fragmented nuclei (Figure 7A), 55% of HepG2 cells exposed to TiO2 NPs were apoptotic (Figure 7B), and 75% of cells over-expressing TLR4 and exposed to TiO2 NPs were apoptotic (Figure 7C).Figure 7

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

View Article: PubMed Central - PubMed

Affiliation: Cell-Material Interaction Group, Biomaterial Unit, Nano-Bio Field, Interaction Center for Material Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), Tsukuba, Japan. SAMYELSAIDALI.Karim@nims.go.jp.

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.

No MeSH data available.


Related in: MedlinePlus