Mie Suzuki

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.

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.

Evaluation of changes of cell-surface thiols as a new biomarker for in vitro sensitization test.

In order to find a novel biomarker for a simple assay to predict skin sensitization, we evaluated cell-surface thiols as a biomarker reflecting intracellular signaling in THP-1 cells (human monocytic cell line). First, we found that a decrease of cell-surface thiols on hapten-treated THP-1 cells was induced in parallel with phosphorylation of p38 MAPK. Next, we confirmed that 2-mercaptoethanol in the culture medium and the differentiation state of THP-1 cells did not affect the changes of cell-surface thiols by hapten. Changes of cell-surface thiols on THP-1 cells were detected after 2h treatment with most allergens (e.g., DNCB, NiSO(4)), as well as some non-allergens (e.g., Tween80, benzalkonium chloride), though other non-allergens (e.g., SDS, glycerol) had no effect. When either a significant decrease or increase of cell-surface thiols (more than 15% in each case) was used as a criterion, the results using 36 allergens and 16 non-allergens were in good accordance with those of in vivo assays. Finally, we confirmed that ATP, which is released as a consequence of cytotoxicity, did not affect the changes of cell-surface thiols. Our results suggest that changes of cell-surface thiols may be useful for an in vitro sensitization assay, which we designate as the SH test.

Oxidation of Cell Surface Thiol Groups by Contact Sensitizers Triggers the Maturation of Dendritic Cells

p38 mitogen-activated protein kinase (MAPK) has a crucial role in the maturation of dendritic cells (DCs) by sensitizers. Recently, it has been reported that the oxidation of cell surface thiols by an exogenous impermeant thiol oxidizer can phosphorylate p38 MAPK. In this study, we examined whether sensitizers oxidize cell surface thiols of monocyte-derived DCs (MoDCs). When cell surface thiols were quantified by flow cytometry using Alexa fluor maleimide, all the sensitizers that we examined decreased cell surface thiols on MoDCs. To examine the effects of decreased cell surface thiols by sensitizers on DC maturation, we analyzed the effects of an impermeant thiol oxidizer, o-phenanthroline copper complex (CuPhen). The treatment of MoDCs with CuPhen decreased cell surface thiols, phosphorylated p38 MAPK, and induced MoDC maturation, that is, the augmentation of CD83, CD86, HLA-DR, and IL-8 mRNA, as well as the downregulation of aquaporin-3 mRNA. The augmentation of CD86 was significantly suppressed when MoDCs were pretreated with N-acetyl-L-cystein or treated with SB203580. Finally, we showed that epicutaneous application of 2,4-dinitrochlorobenzene on mouse skin significantly decreased cell surface thiols of Langerhans cells in vivo. These data suggest that the oxidation of cell surface thiols has some role in triggering DC maturation by sensitizers.

Development of an in vitro photosensitization assay using human monocyte-derived cells

In this study, with the aim of developing a cell-based in vitro photosensitization assay, we examined whether changes of CD86 and CD54 expression on cells of a human monocytic cell line, THP-1, could be used to assess the photosensitizing potential of chemicals. First, we identified suitable conditions of UV-irradiation (irradiation dose; 5.0 J/cm(2), irradiation intensity; 1.7 mW/cm(2)) by investigating the effect of UV-irradiation on CD86 and CD54 expression on untreated or 6-methylcoumarin (a representative photoallergen)-treated THP-1 cells (irradiation method). However, acridine, a representative photo-irritant, augmented CD86 and CD54 expression on THP-1 cells, apparently via induction of reactive oxygen species (ROS). In order to abolish the effect of ROS, we examined CD86 and CD54 expression on THP-1 cells treated with pre-irradiated chemicals (pre-irradiation method). We found that UV-irradiated photoallergens, but not photo-irritants, enhanced CD86 and/or CD54 expression on the THP-1 cells. Finally, based on the results of irradiation, non-irradiation, and pre-irradiation with 18 test chemicals, we built a decision tree, which allows us to distinguish between photoallergens and photo-irritants. We suggest that this system may be useful for in vitro evaluation of the photoallergic potential of chemicals.