Arisa Miyauchi

PROTEIN ADSORPTION OF ULTRAFINE METAL OXIDE AND ITS INFLUENCE ON CYTOTOXICITY TOWARD CULTURED CELLS

Many investigations about the cellular response by metal oxide nanoparticles in vitro have been reported. However, the influence of the adsorption ability of metal oxide nanoparticles toward cells is unknown. The aim of this study is to understand the influence of adsorption by metal oxide nanoparticles on the cell viability in vitro. The adsorption abilities of six kinds of metal oxide nanoparticles, namely, NiO, ZnO, TiO2, CeO2, SiO2, and Fe2O3, to Dulbeccos modified Eagles medium supplemented with a 10% fetal bovine serum (DMEM-FBS) component such as serum proteins and Ca2) were estimated. All of the metal oxide nanoparticles adsorbed proteins and Ca2+ in the DMEM-FBS; in particular, TiO2, CeO2, and ZnO showed strong adsorption abilities. Furthermore, the influence of the depletion of medium components by adsorption to metal oxide nanoparticles on cell viability and proliferation was examined. The particles were removed from the dispersion by centrifugation, and the supernatant was applied to the cells. Both the cell viability and the proliferation of human keratinocyte HaCaT cells and human lung carcinoma A549 cells were affected by the supernatant. In particular, cell proliferation was strongly inhibited by the supernatant of TiO2 and CeO2 dispersions. The supernatant showed depletion of serum proteins and Ca2+ by adsorption to metal oxide nanoparticles. When the adsorption effect was blocked by the pretreatment of particles with FBS, the inhibitory effect was lost. However, in NiO and ZnO, which showed ion release, a decrease of inhibitory effect by pretreatment was not shown. Furthermore, the association of the primary particle size and adsorption ability was examined in TiO2. The adsorption ability of TiO2 depended on the primary particle size. The TiO2 nanoparticles were size dependently absorbed with proteins and Ca2+, thereby inducing cytotoxicity. In conclusion, the adsorption ability of metal oxide nanoparticles is an important factor for the estimation of cytotoxicity in vitro for low-toxicity materials.

Ultrafine NiO particles induce cytotoxicity in vitro by cellular uptake and subsequent Ni(II) release.

Nickel oxide (NiO) is one of the important industrial materials used in electronic substrates and for ceramic engineering. Advancements in industrial technology have enabled the manufacture of ultrafine NiO particles. On the other hand, it is well-known that nickel compounds exert toxic effects. The toxicity of nickel compounds is mainly caused by nickel ions (Ni(2+)). However, the ion release properties of ultrafine NiO particles are still unclear. In the present study, the influences of ultrafine NiO particles on cell viability were examined in vitro to obtain fundamental data for the biological effects of ultrafine green NiO and ultrafine black NiO. Ultrafine NiO particles showed higher cytotoxicities toward human keratinocyte HaCaT cells and human lung carcinoma A549 cells than fine NiO particles and also showed higher solubilities in culture medium (Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum) than fine NiO particles. In particular, the concentration of Ni(2+) released into the culture medium by ultrafine green NiO was 150-fold higher than that released by fine green NiO. The concentrations of Ni(2+) released by both types of NiO particles in an aqueous solution containing amino acids were remarkably higher than those released by NiO particles in water. Moreover, we prepared a uniform and stable dispersion of ultrafine black NiO in culture medium and examined its influence on cell viability in comparison with that of NiCl(2), a soluble nickel compound. A medium exchange after 6 h of exposure resulted in a loss of cytotoxicity in the cells exposed to NiCl(2), whereas cytotoxicity was retained in the cells exposed to NiO. Transmission electron microscope observations revealed uptake of both ultrafine and fine NiO particles into HaCaT cells. Taken together, the present results suggest that the intracellular Ni(2+) release could be an important factor that determines the cytotoxicity of NiO. Ultrafine NiO is more cytotoxic than fine NiO in vitro.

Association of the physical and chemical properties and the cytotoxicity of metal oxide nanoparticles: metal ion release, adsorption ability and specific surface area

Association of cellular influences and physical and chemical properties were examined for 24 kinds of industrial metal oxide nanoparticles: ZnO, CuO, NiO, Sb(2)O(3), CoO, MoO(3), Y(2)O(3), MgO, Gd(2)O(3), SnO(2), WO(3), ZrO(2), Fe(2)O(3), TiO(2), CeO(2), Al(2)O(3), Bi(2)O(3), La(2)O(3), ITO, and cobalt blue pigments. We prepared a stable medium dispersion for each nanoparticle and examined the influence on cell viability and oxidative stress together with physical and chemical characterizations. ZnO, CuO, NiO, MgO, and WO(3) showed a large amount of metal ion release in the culture medium. The cellular influences of these soluble nanoparticles were larger than insoluble nanoparticles. TiO(2), SnO(2), and CeO(2) nanoparticles showed strong protein adsorption ability; however, cellular influences of these nanoparticles were small. The primary particle size and the specific surface area seemed unrelated to cellular influences. Cellular influences of metal oxide nanoparticles depended on the kind and concentrations of released metals in the solution. For insoluble nanoparticles, the adsorption property was involved in cellular influences. The primary particle size and specific surface area of metal oxide nanoparticles did not affect directly cellular influences. In conclusion the most important cytotoxic factor of metal oxide nanoparticles was metal ion release.

Cellular responses induced by cerium oxide nanoparticles: induction of intracellular calcium level and oxidative stress on culture cells

Cerium oxide (CeO(2)) is an important metal oxide used for industrial products. Many investigations about the cellular influence of CeO(2) nanoparticles have been done, but results are contradictory. It has been reported that CeO(2) nanoparticles have an anti-oxidative effect in cells, but it has also been reported that CeO(2) nanoparticles induce oxidative stress. We investigated the potential influence on cells and the mechanisms induced by CeO(2) nanoparticles in vitro. We prepared a stable CeO(2) culture medium dispersion. Cellular responses in CeO(2) medium-exposed cells were examined. Cellular uptake of CeO(2) nanoparticles was observed. After 24-h exposure, a high concentration of CeO(2) nanoparticles (∼200 mg/ml) induced an increase in the intracellular level of reactive oxygen species (ROS); a low concentration of CeO(2) nanoparticles induced a decrease in the intracellular ROS level. On the other hand, exposure of CeO(2) nanoparticle for 24 h had little influence on the cell viability. Exposure of CeO(2) nanoparticles increased the intracellular Ca(2+) concentration and also Calpain was activated. These results suggest that CeO(2) nanoparticles have a potential to induce intracellular oxidative stress and increase the intracellular Ca(2+) level, but these influences are small.

Evaluation of Acute Oxidative Stress Induced by NiO Nanoparticles In Vivo and In Vitro

Evaluation of Acute Oxidative Stress Induced by NiO Nanoparticles In Vivo and In Vitro: Masanori Horie, et al. Health Research Institute —

Chromium(III) oxide nanoparticles induced remarkable oxidative stress and apoptosis on culture cells.

Chromium(III) oxide (Cr2O3) is used for industrial applications such as catalysts and pigments. In the classical form, namely the fine particle, Cr2O3 is insoluble and chemically stable. It is classified as a low‐toxicity chromium compound. Recently, industrial application of nanoparticles (a new form composed of small particles with a diameter of ≤100 nm, in at least one dimension) has been increasing. Cellular effects induced by Cr2O3 nanoparticles are not known. To shed light upon this, the release of soluble chromium from Cr2O3 nano‐ and fine‐particles in culture medium was compared. Fine Cr2O3 particles were insoluble in the culture medium; on the contrary, Cr2O3 nanoparticles released soluble hexavalent chromium into the culture medium. Cr2O3 nanoparticles showed severe cytotoxicity. The effect of Cr2O3 nanoparticles on cell viability was higher than that of fine particles. Cr2O3 nanoparticles showed cytotoxicity equal to that of hexavalent chromium (K2Cr2O7). Human lung carcinoma A549 cells and human keratinocyte HaCaT cells showed an increase in intracellular reactive oxygen species (ROS) level and activation of antioxidant defense systems on exposure to Cr2O3 nanoparticles. Exposure of Cr2O3 nanoparticles led to caspase‐3 activation, showing that the decrease in cell viability by exposure to Cr2O3 nanoparticles was caused by apoptosis. Cellular responses were stronger in the Cr2O3 nanoparticles‐exposed cells than in fine Cr2O3‐ and CrCl3‐exposed cells. Cellular uptake of Cr2O3 particles were observed in nano‐ and fine‐particles. The cellular influence of the extracellular soluble trivalent chromium was lower than that of Cr2O3 nanoparticles. Cr2O3 nanoparticles showed cytotoxicity by hexavalent chromium released at outside and inside of cells. The cellular influences of Cr2O3 nanoparticles matched those of hexavalent chromium. In conclusion, Cr2O3 nanoparticles have a high cytotoxic potential.

Comparison of acute oxidative stress on rat lung induced by nano and fine-scale, soluble and insoluble metal oxide particles: NiO and TiO2

The aim of the present study is to understand the association between metal ion release from nickel oxide (NiO) nanoparticles and induction of oxidative stress in the lung. NiO nanoparticles have cytotoxic activity through nickel ion release and subsequent oxidative stress. However, the interaction of oxidative stress and nickel ion release in vivo is still unclear. In the present study, we examined the effect of metal ion release on oxidative stress induced by NiO nanoparticles. Additionally, nano and fine TiO2 particles as insoluble particles were also examined. Rat lung was exposed to NiO and TiO2 nanoparticles by intratracheal instillation. The NiO nanoparticles released Ni2+ in dispersion. Bronchoalveolar lavage fluid (BALF) was collected at 1, 24, 72 h and 1 week after instillation. The lactate dehydrogenase (LDH) and HO-1 levels were elevated at 24 and 72 h after instillation in the animals exposed to the NiO nanoparticles. On the other hand, total hydroxyoctadecadienoic acid (tHODE), which is an oxidative product of linoleic acid, as well as SP-D and α-tochopherol levels were increased at 72 h and 1 week after instillation. Fine NiO particles, and nano and fine TiO2 particles did not show lung injury or oxidative stress from 1 h to 1 week after instillation. These results suggest that Ni2+ release is involved in the induction of oxidative stress by NiO nanoparticles in the lung. Ni2+ release from NiO nanoparticles is an important factor inoxidative stress-related toxicity, not only in vitro but also in vivo.

The Expression of Inflammatory Cytokine and Heme Oxygenase-1 Genes in THP-1 Cells Exposed to Metal Oxide Nanoparticles

The effect of manufactured nanoparticles on the expression of proinflammatory cytokine genes was examined. THP-1 cells differentiated into macrophage cells were exposed to TiO2 and NiO medium dispersions. After 2, 6, 12, or 24 hours exposure, the expression of IL-1β, IL-6, IL-8, TNF-α and HO-1 genes was determined by real-time PCR. TiO2 nanoparticles did not affect cytokine production. In addition, TiO2 nanoparticles did not dissolve in the dispersion. On the other hand, NiO nanoparticles enhanced the expression of all the genes tested. NiO dispersions were composed of 58.3 μg/mL of NiO nanoparticles and 45.8 μg/mL of Ni2+. The release of metal ions from the nanoparticles is associated with their cytotoxicity. Therefore, the effect of an NiCl2 solution containing 45.8 μg/mL of Ni2+ on the expression of cytokine genes was also examined. The effects of NiCl2 were similar to those of the NiO nanoparticles. Furthermore, the effect of ZnO, SiO2-coated ZnO, Sb2O3, and Cr2O3 nanoparticles on the expression of IL-1β, IL-8 and TNF-α genes was examined. Soluble nanoparticles, such as ZnO, SiO2-coated ZnO, and Cr2O3 enhanced the gene expression of cytokines. Sb2O3 nanoparticles showed poor solubility and did not affect the expression of cytokine genes. In conclusion, these results suggest that nanoparticle solubility plays an important role in regulating the expression of proinflammatory cytokines.