Limits...
Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation.

Yoo KC, Yoon CH, Kwon D, Hyun KH, Woo SJ, Kim RK, Lim EJ, Suh Y, Kim MJ, Yoon TH, Lee SJ - Int J Nanomedicine (2012)

Bottom Line: In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO(2)-induced apoptotic cell death.These results indicate that sub-100 nm sized TiO(2) treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax.Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biochemistry, Department of Chemistry, Hanyang University, Seoul, Republic of Korea.

ABSTRACT

Background: Titanium dioxide (TiO(2)) has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO(2) particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO(2) has yet to be defined.

Methods and results: In this study, we demonstrated that combined treatment with TiO(2) nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO(2) nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO(2) (P25-70)) together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO(2)-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO(2) (P25-70) and ultraviolet A irradiation.

Conclusion: These results indicate that sub-100 nm sized TiO(2) treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax. Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials.

Show MeSH

Related in: MedlinePlus

Reactive oxygen species are critically involved in the TiO2P25–70-induced Fas upregulation, Bax activation, and apoptotic cell death. (A) Measurement of intracellular reactive oxygen species levels by FACS analysis. Combined treatment of Chang cells with TiO2P25–70 and ultraviolet A irradiation increased intracellular reactive oxygen species levels, whereas single treatment with either TiO2P25–70 or ultraviolet A had no effect on intracellular reactive oxygen species levels. (B) Pretreatment with the antioxidant, N-acetyl-L-cysteine, attenuated cell death induced by combined treatment with TiO2P25–70 and ultraviolet A irradiation. Cell death was measured by FACS analysis after double staining with propidium iodide and Annexin V. (C) Pretreatment with antioxidant, N-acetyl-L-cysteine attenuated FAS expression (upper) and suppressed the increase of proform, caspase-8 (lower). (D) Pretreatment with N-acetyl-L-cysteine attenuated oligomerization of Bax in mitochondria-enriched membrane fractions of Chang cells treated with combination of TiO2P25–70 and ultraviolet A. (E) Pretreatment with N-acetyl-L-cysteine suppressed mitochondrial membrane potential loss (upper), the release of cytochrome c and apoptosis-inducing factor to cytosol (lower). (F) Visualization of intracellular reactive oxygen species levels in fluorescence microscopy by staining with DCFA and mitochondria specific MitoSox Red. Chang cells treated with combination of TiO2P25–70 and ultraviolet A irradiation show increased intracellular reactive oxygen species levels on both staining with DCFA and MitoSox Red.Notes: *P < 0.005. Error bars represent the mean ± standard deviation of triplicate samples.Abbreviations: AIF, UVA, ultraviolet A; DMSO, dimethylsulfoxide; AIF, apoptosis-inducing factor; HSP, heat shock protein; NAC, N-acetyl-L-cysteine.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3298386&req=5

f6-ijn-7-1203: Reactive oxygen species are critically involved in the TiO2P25–70-induced Fas upregulation, Bax activation, and apoptotic cell death. (A) Measurement of intracellular reactive oxygen species levels by FACS analysis. Combined treatment of Chang cells with TiO2P25–70 and ultraviolet A irradiation increased intracellular reactive oxygen species levels, whereas single treatment with either TiO2P25–70 or ultraviolet A had no effect on intracellular reactive oxygen species levels. (B) Pretreatment with the antioxidant, N-acetyl-L-cysteine, attenuated cell death induced by combined treatment with TiO2P25–70 and ultraviolet A irradiation. Cell death was measured by FACS analysis after double staining with propidium iodide and Annexin V. (C) Pretreatment with antioxidant, N-acetyl-L-cysteine attenuated FAS expression (upper) and suppressed the increase of proform, caspase-8 (lower). (D) Pretreatment with N-acetyl-L-cysteine attenuated oligomerization of Bax in mitochondria-enriched membrane fractions of Chang cells treated with combination of TiO2P25–70 and ultraviolet A. (E) Pretreatment with N-acetyl-L-cysteine suppressed mitochondrial membrane potential loss (upper), the release of cytochrome c and apoptosis-inducing factor to cytosol (lower). (F) Visualization of intracellular reactive oxygen species levels in fluorescence microscopy by staining with DCFA and mitochondria specific MitoSox Red. Chang cells treated with combination of TiO2P25–70 and ultraviolet A irradiation show increased intracellular reactive oxygen species levels on both staining with DCFA and MitoSox Red.Notes: *P < 0.005. Error bars represent the mean ± standard deviation of triplicate samples.Abbreviations: AIF, UVA, ultraviolet A; DMSO, dimethylsulfoxide; AIF, apoptosis-inducing factor; HSP, heat shock protein; NAC, N-acetyl-L-cysteine.

Mentions: Oxidative damage is often implicated in apoptotic cell death.30,31 We therefore subsequently examined changes in intracellular reactive oxygen species levels in Chang cells treated with TiO2P25–70. As shown in Figure 6A, treatment with TiO2P25–70 under ultraviolet A irradiation led to an approximately three-fold increase in mean 2′,7′-dichlorofluorescein fluorescence, indicating an increase of intracellular reactive oxygen species. To confirm further a link between elevation of intracellular reactive oxygen species and mitochondrial cell death, the cells were preincubated with N-acetyl-L-cysteine, an antioxidant, prior to TiO2P25–70 treatment. Treatment of TiO2P25–70 did not induce cell death in the presence of N-acetyl-L-cysteine (Figure 6B). Pretreatment of N-acetyl-L-cysteine also inhibited TiO2P25–70-induced Fas upregulation and caspase-8 activation (Figure 6C). Moreover, N-acetyl-L-cysteine clearly inhibited conformational change and mitochondrial redistribution of Bax induced by TiO2P25–70 (Figure 6D), mitochondrial membrane potential loss (Figure 6E), and apoptosis-inducing factor and cytochrome c release (Figure 6E). These observations suggest that an increase in intracellular reactive oxygen species is critically required for TiO2P25–70-induced Fas upregulation-mediated and Bax activation-mediated apoptotic cell death.


Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation.

Yoo KC, Yoon CH, Kwon D, Hyun KH, Woo SJ, Kim RK, Lim EJ, Suh Y, Kim MJ, Yoon TH, Lee SJ - Int J Nanomedicine (2012)

Reactive oxygen species are critically involved in the TiO2P25–70-induced Fas upregulation, Bax activation, and apoptotic cell death. (A) Measurement of intracellular reactive oxygen species levels by FACS analysis. Combined treatment of Chang cells with TiO2P25–70 and ultraviolet A irradiation increased intracellular reactive oxygen species levels, whereas single treatment with either TiO2P25–70 or ultraviolet A had no effect on intracellular reactive oxygen species levels. (B) Pretreatment with the antioxidant, N-acetyl-L-cysteine, attenuated cell death induced by combined treatment with TiO2P25–70 and ultraviolet A irradiation. Cell death was measured by FACS analysis after double staining with propidium iodide and Annexin V. (C) Pretreatment with antioxidant, N-acetyl-L-cysteine attenuated FAS expression (upper) and suppressed the increase of proform, caspase-8 (lower). (D) Pretreatment with N-acetyl-L-cysteine attenuated oligomerization of Bax in mitochondria-enriched membrane fractions of Chang cells treated with combination of TiO2P25–70 and ultraviolet A. (E) Pretreatment with N-acetyl-L-cysteine suppressed mitochondrial membrane potential loss (upper), the release of cytochrome c and apoptosis-inducing factor to cytosol (lower). (F) Visualization of intracellular reactive oxygen species levels in fluorescence microscopy by staining with DCFA and mitochondria specific MitoSox Red. Chang cells treated with combination of TiO2P25–70 and ultraviolet A irradiation show increased intracellular reactive oxygen species levels on both staining with DCFA and MitoSox Red.Notes: *P < 0.005. Error bars represent the mean ± standard deviation of triplicate samples.Abbreviations: AIF, UVA, ultraviolet A; DMSO, dimethylsulfoxide; AIF, apoptosis-inducing factor; HSP, heat shock protein; NAC, N-acetyl-L-cysteine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3298386&req=5

f6-ijn-7-1203: Reactive oxygen species are critically involved in the TiO2P25–70-induced Fas upregulation, Bax activation, and apoptotic cell death. (A) Measurement of intracellular reactive oxygen species levels by FACS analysis. Combined treatment of Chang cells with TiO2P25–70 and ultraviolet A irradiation increased intracellular reactive oxygen species levels, whereas single treatment with either TiO2P25–70 or ultraviolet A had no effect on intracellular reactive oxygen species levels. (B) Pretreatment with the antioxidant, N-acetyl-L-cysteine, attenuated cell death induced by combined treatment with TiO2P25–70 and ultraviolet A irradiation. Cell death was measured by FACS analysis after double staining with propidium iodide and Annexin V. (C) Pretreatment with antioxidant, N-acetyl-L-cysteine attenuated FAS expression (upper) and suppressed the increase of proform, caspase-8 (lower). (D) Pretreatment with N-acetyl-L-cysteine attenuated oligomerization of Bax in mitochondria-enriched membrane fractions of Chang cells treated with combination of TiO2P25–70 and ultraviolet A. (E) Pretreatment with N-acetyl-L-cysteine suppressed mitochondrial membrane potential loss (upper), the release of cytochrome c and apoptosis-inducing factor to cytosol (lower). (F) Visualization of intracellular reactive oxygen species levels in fluorescence microscopy by staining with DCFA and mitochondria specific MitoSox Red. Chang cells treated with combination of TiO2P25–70 and ultraviolet A irradiation show increased intracellular reactive oxygen species levels on both staining with DCFA and MitoSox Red.Notes: *P < 0.005. Error bars represent the mean ± standard deviation of triplicate samples.Abbreviations: AIF, UVA, ultraviolet A; DMSO, dimethylsulfoxide; AIF, apoptosis-inducing factor; HSP, heat shock protein; NAC, N-acetyl-L-cysteine.
Mentions: Oxidative damage is often implicated in apoptotic cell death.30,31 We therefore subsequently examined changes in intracellular reactive oxygen species levels in Chang cells treated with TiO2P25–70. As shown in Figure 6A, treatment with TiO2P25–70 under ultraviolet A irradiation led to an approximately three-fold increase in mean 2′,7′-dichlorofluorescein fluorescence, indicating an increase of intracellular reactive oxygen species. To confirm further a link between elevation of intracellular reactive oxygen species and mitochondrial cell death, the cells were preincubated with N-acetyl-L-cysteine, an antioxidant, prior to TiO2P25–70 treatment. Treatment of TiO2P25–70 did not induce cell death in the presence of N-acetyl-L-cysteine (Figure 6B). Pretreatment of N-acetyl-L-cysteine also inhibited TiO2P25–70-induced Fas upregulation and caspase-8 activation (Figure 6C). Moreover, N-acetyl-L-cysteine clearly inhibited conformational change and mitochondrial redistribution of Bax induced by TiO2P25–70 (Figure 6D), mitochondrial membrane potential loss (Figure 6E), and apoptosis-inducing factor and cytochrome c release (Figure 6E). These observations suggest that an increase in intracellular reactive oxygen species is critically required for TiO2P25–70-induced Fas upregulation-mediated and Bax activation-mediated apoptotic cell death.

Bottom Line: In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO(2)-induced apoptotic cell death.These results indicate that sub-100 nm sized TiO(2) treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax.Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biochemistry, Department of Chemistry, Hanyang University, Seoul, Republic of Korea.

ABSTRACT

Background: Titanium dioxide (TiO(2)) has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO(2) particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO(2) has yet to be defined.

Methods and results: In this study, we demonstrated that combined treatment with TiO(2) nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO(2) nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO(2) (P25-70)) together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO(2)-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO(2) (P25-70) and ultraviolet A irradiation.

Conclusion: These results indicate that sub-100 nm sized TiO(2) treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein, Bax. Elucidating the molecular mechanisms by which nanosized particles induce activation of cell death signaling pathways would be critical for the development of prevention strategies to minimize the cytotoxicity of nanomaterials.

Show MeSH
Related in: MedlinePlus