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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.

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Temporal variations of hydrodynamic size distributions of TiO2 in media supplemented with fetal bovine serum. In the cell culture media, ie, RPMI with 10% fetal bovine serum, hydrodynamic sizes of TiO2 nanoparticles were monitored for 48 hours. The hydrodynamic size was increased in the unfractionated TiO2P25–300 (A) with time and caused micron-sized particles with heavy sedimentation, whereas the hydrodynamic size of TiO2P25–130 (B) and TiO2P25–70 (C) were not changed with time, indicating no aggregation.
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f2-ijn-7-1203: Temporal variations of hydrodynamic size distributions of TiO2 in media supplemented with fetal bovine serum. In the cell culture media, ie, RPMI with 10% fetal bovine serum, hydrodynamic sizes of TiO2 nanoparticles were monitored for 48 hours. The hydrodynamic size was increased in the unfractionated TiO2P25–300 (A) with time and caused micron-sized particles with heavy sedimentation, whereas the hydrodynamic size of TiO2P25–130 (B) and TiO2P25–70 (C) were not changed with time, indicating no aggregation.

Mentions: Based on our scanning electron microscopy and x-ray diffraction measurements, the P25 TiO2 powder had a primary particle size of approximately 21 nm and was composed mainly of anatase (87%) and rutile (13%) crystalline phase. The Brunauer-Emmett-Teller specific surface area was 57 m2/g and the point-of-zero charge was estimated to be in the vicinity of pH 5.8. These basic characteristics agree well with those provided by the manufacturer and by previous studies.20–22 In this study, we divided the P25 TiO2 suspension into three fractions with different hydrodynamic size distributions. To this end, the primary P25 TiO2 suspension was centrifuged using different speeds (0, 4000, 6000 rpm). TiO2 nanoparticles with different hydrodynamic size distributions were then obtained by taking the supernatant and designating it according to hydrodynamic sizes of 327 nm (without fractionation, TiO2P25–300), 133 nm (fractionated at 4000 rpm, TiO2P25–130), and 74 nm (fractionated at 6000 rpm, TiO2P25–70). The TiO2P25–300 fraction was mainly composed of heavily agglomerated/aggregated particles, while TiO2P25–130 and TiO2P25–70 were composed of much smaller agglomerates/aggregates (Figure 1). The sub-100 nm fraction (ie, TiO2P25–70) was found to be stable without agglomeration and sedimentation for more than a week, whereas the TiO2P25–300 and TiO2P25–130 fractions were easily aggregated and sedimented in slight acidic conditions (pH 4–5), However, exposure of TiO2P25–70 to typical in vitro cell culture medium (eg, RPMI 1640 or Dulbecco’s Modified Eagle’s Medium) instantly caused strong agglomeration and sedimentation, which has been previously reported to cause problems in performing accurate and reproducible toxicity assessments of nanoparticles.5,23–27 To overcome this problem of colloidal instability, nanoparticles are often stabilized with various surface-modifying ligands.19 In this study, rather than adding additional surface-modifying compounds, fetal bovine serum (a supplement used for in vitro cell culture) was used as an efficient stabilizing agent.17 By monitoring the hydrodynamic size distributions of all three fractions of TiO2 in RPMI media supplemented with fetal bovine serum for 48 hours, we observed that the unfractionated TiO2P25–300 increased in hydrodynamic size with aggregation over time and formed micron-sized particles with heavy sedimentation (Figure 2A), whereas the hydrodynamic size of TiO2P25–130 and TiO2P25–70 were not changed with time, indicating no aggregation of TiO2 (Figure 2B and C). Zeta potential measurements were also performed for all three types of TiO2 in RPMI media with fetal bovine serum, yielding values in the range of −9.42 to −10.4 mV. In addition, the impact of exposure to ultraviolet A irradiation on the physicochemical properties of TiO2 was also investigated. However, no significant changes in hydrodynamic size, zeta potential, or colloidal stability were observed after exposure to 0.75 J/hour ultraviolet A irradiation for 48 hours.


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)

Temporal variations of hydrodynamic size distributions of TiO2 in media supplemented with fetal bovine serum. In the cell culture media, ie, RPMI with 10% fetal bovine serum, hydrodynamic sizes of TiO2 nanoparticles were monitored for 48 hours. The hydrodynamic size was increased in the unfractionated TiO2P25–300 (A) with time and caused micron-sized particles with heavy sedimentation, whereas the hydrodynamic size of TiO2P25–130 (B) and TiO2P25–70 (C) were not changed with time, indicating no aggregation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3298386&req=5

f2-ijn-7-1203: Temporal variations of hydrodynamic size distributions of TiO2 in media supplemented with fetal bovine serum. In the cell culture media, ie, RPMI with 10% fetal bovine serum, hydrodynamic sizes of TiO2 nanoparticles were monitored for 48 hours. The hydrodynamic size was increased in the unfractionated TiO2P25–300 (A) with time and caused micron-sized particles with heavy sedimentation, whereas the hydrodynamic size of TiO2P25–130 (B) and TiO2P25–70 (C) were not changed with time, indicating no aggregation.
Mentions: Based on our scanning electron microscopy and x-ray diffraction measurements, the P25 TiO2 powder had a primary particle size of approximately 21 nm and was composed mainly of anatase (87%) and rutile (13%) crystalline phase. The Brunauer-Emmett-Teller specific surface area was 57 m2/g and the point-of-zero charge was estimated to be in the vicinity of pH 5.8. These basic characteristics agree well with those provided by the manufacturer and by previous studies.20–22 In this study, we divided the P25 TiO2 suspension into three fractions with different hydrodynamic size distributions. To this end, the primary P25 TiO2 suspension was centrifuged using different speeds (0, 4000, 6000 rpm). TiO2 nanoparticles with different hydrodynamic size distributions were then obtained by taking the supernatant and designating it according to hydrodynamic sizes of 327 nm (without fractionation, TiO2P25–300), 133 nm (fractionated at 4000 rpm, TiO2P25–130), and 74 nm (fractionated at 6000 rpm, TiO2P25–70). The TiO2P25–300 fraction was mainly composed of heavily agglomerated/aggregated particles, while TiO2P25–130 and TiO2P25–70 were composed of much smaller agglomerates/aggregates (Figure 1). The sub-100 nm fraction (ie, TiO2P25–70) was found to be stable without agglomeration and sedimentation for more than a week, whereas the TiO2P25–300 and TiO2P25–130 fractions were easily aggregated and sedimented in slight acidic conditions (pH 4–5), However, exposure of TiO2P25–70 to typical in vitro cell culture medium (eg, RPMI 1640 or Dulbecco’s Modified Eagle’s Medium) instantly caused strong agglomeration and sedimentation, which has been previously reported to cause problems in performing accurate and reproducible toxicity assessments of nanoparticles.5,23–27 To overcome this problem of colloidal instability, nanoparticles are often stabilized with various surface-modifying ligands.19 In this study, rather than adding additional surface-modifying compounds, fetal bovine serum (a supplement used for in vitro cell culture) was used as an efficient stabilizing agent.17 By monitoring the hydrodynamic size distributions of all three fractions of TiO2 in RPMI media supplemented with fetal bovine serum for 48 hours, we observed that the unfractionated TiO2P25–300 increased in hydrodynamic size with aggregation over time and formed micron-sized particles with heavy sedimentation (Figure 2A), whereas the hydrodynamic size of TiO2P25–130 and TiO2P25–70 were not changed with time, indicating no aggregation of TiO2 (Figure 2B and C). Zeta potential measurements were also performed for all three types of TiO2 in RPMI media with fetal bovine serum, yielding values in the range of −9.42 to −10.4 mV. In addition, the impact of exposure to ultraviolet A irradiation on the physicochemical properties of TiO2 was also investigated. However, no significant changes in hydrodynamic size, zeta potential, or colloidal stability were observed after exposure to 0.75 J/hour ultraviolet A irradiation for 48 hours.

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