Shinichi Kinugasa

Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung

Zinc oxide (ZnO) nanoparticles are one of the important industrial nanoparticles. The production of ZnO nanoparticles is increasing every year. On the other hand, it is known that ZnO nanoparticles have strong cytotoxicity. In vitro studies using culture cells revealed that ZnO nanoparticles induce severe oxidative stress. However, the in vivo influence of ZnO nanoparticles is still unclear. In the present study, rat lung was exposed to ZnO nanoparticles by intratracheal instillation, and the influences of ZnO nanoparticles to the lung in the acute phase, particularly oxidative stress, were examined. Additionally, in vitro cellular influences of ZnO nanoparticles were examined using lung carcinoma A549 cells and compared to in vivo examinations. The ZnO nanoparticles used in this study released zinc ion in both dispersions. In the in vivo examinations, ZnO dispersion induced strong oxidative stress in the lung in the acute phase. The oxidative stress induced by the ZnO nanoparticles was stronger than that of a ZnCl(2) solution. Intratracheal instillation of ZnO nanoparticles induced an increase of lipid peroxide, HO-1 and alpha-tocopherol in the lung. The ZnO nanoparticles also induced strong oxidative stress and cell death in culture cells. Intracellular zinc level and reactive oxygen species were increased. These results suggest that ZnO nanoparticles induce oxidative stress in the lung in the acute phase. Intracellular ROS level had a high correlation with intracellular Zn(2+) level. ZnO nanoparticles will stay in the lung and continually release zinc ion, and thus stronger oxidative stress is induced.

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.

Reliable size determination of nanoparticles using dynamic light scattering method for in vitro toxicology assessment.

Dynamic light scattering (DLS) is widely used for the evaluation of the particle size in the toxicity assessment of nanoparticles. However, the many types of DLS instruments and analytical procedures sometimes give different apparent sizes of particles and make it complicated to understand the size dependence on particles for the toxicity assay. In this study, we established an evaluation method of secondary nanoparticle sizes using a DLS analysis. First, we established a practical method for determining size with an appropriate evaluation of uncertainties. This proposed method could be a universal protocol for toxicity assessment that would allow researchers to achieve some degree of concordance on the size of nanoparticles for an assessment. Second, we investigated the processes associated with particles in suspension by examining the changes in the size and the light scattering intensity of secondary nanoparticles during an in vitro toxicity assessment, since the transport mode of particles to cells is significant in understanding in vitro nano-toxicity. In this study, these two points were investigated on TiO(2) nanoparticles suspension as an example. The secondary particles of TiO(2) with a light scattering intensity-averaged diameter (d(l)) of 150-250 nm were characterized with appropriate uncertainties. The sizes were found to be comparable with values determined using other analytical procedures and other instruments. It is suggested that d(l) could be an effective size parameter for toxicity assessments. Furthermore, TiO(2) secondary nanoparticle suspensions are well dispersed with slow gravity settling, no agglomeration, with the diffusion process as the primary transport mode of particles to cells.

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.

Practical guide for accurate quantitative solution state NMR analysis

A practical guide to experimental conditions such as statistical variation of signal intensity on quantitative solution state nuclear magnetic resonanace (NMR) analysis is discussed and presented. Statistical analysis showed that there is a relationship between a targeted precision and practical pulse intervals. The bandwidth of the audio filter needs to be set so that all the signals of interest fall into 80% of the centre part of the filter. When fulfilling these conditions, the relative standard deviation (RSD) of a signal area in repeated experiments can be estimated by determining the signal-to-noise ratio (SN) of a single spectrum. When the SN reached 1000, the RSD became constant with increasing SN. With such a condition, accuracy better than 1% should be obtained with quantitative NMR.

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 —

Quantitative comparison of a corona-charged aerosol detector and an evaporative light-scattering detector for the analysis of a synthetic polymer by supercritical fluid chromatography

A corona-charged aerosol detector (CAD) was developed to improve the sensitivity, reproducibility and quantitativeness of detection as compared to evaporative light-scattering detector (ELSD) for liquid chromatography. Our laboratory used the corona CAD as a detector for supercritical fluid chromatography (SFC) and evaluated its performance compared to the ELSD by using a certified reference material of poly(ethylene glycol) (PEG) and a well-defined equimass mixture of uniform PEG oligomers. The corona CAD was able to detect a 10 times more dilute solution of uniform oligomers compared to the ELSD. Although the original data of molecular mass by ELSD was 4.6% smaller than the certified value of PEG 1000, molecular mass distribution obtained by corona CAD was virtually almost the same as the certified value without any calibrations.

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.

Dispersion characteristics of various metal oxide secondary nanoparticles in culture medium for in vitro toxicology assessment.

The aim of this study is to characterize the dispersion characteristics of various metal oxide nanoparticles and secondary nanoparticle formation in culture medium. Many studies have already investigated the in vitro toxicities of various metal oxide nanoparticles; however, there have been few discussions about the particle transport mode to cells during a period of toxicity assessment. The particle transport mode would strongly affect the amount of uptake by cells; therefore, estimation of the transport mode for various metal oxide particles is important. Fourteen different metal oxide nanoparticle dispersions in a culture medium were examined. The sizes of the secondary nanoparticles were observed to be larger than 100 nm by dynamic light scattering (DLS). According to Stokes law and the Stokes-Einstein assumption, pure metal oxide particles with such sizes should gravitationally settle faster than diffusion processes; however, the secondary metal oxide particles examined in this study exhibited unexpectedly slower gravitational settling rates. The slow gravitational settling kinetics of particles was estimated to be caused by the inclusion of protein into the secondary nanoparticles, which resulted in lower densities than the pure metal oxide particles. The ratios of metal oxide to protein in secondary particles could be affected by the protein adsorption ability of the corresponding metal oxide primary particles. To the best of our knowledge, it was clarified for the first time that stably dispersed secondary metal oxide nanoparticles with slow gravitational settling kinetics are induced by secondary nanoparticles consisting of small amounts of metal oxide particles and large amounts of protein, which results in lower particle densities than the pure metal oxide particles. The estimation of particle dynamics in culture medium using this method would be significant to recognize the inherent toxicity of nanoparticles.