Many classes of nanoparticles have been synthesized and widely applied, however, there is a serious lack of information concerning their effects on human health and the environment. Considering that their use will increase, accurate and cost-effective measurement techniques for characterizing "nanotoxicity" are required. One major toxicological concern is that nanoparticles are easily taken up in the human body. In this study, we developed a method of evaluating the uptake potential of nanosized particles using flow cytometric light scatter. Suspended titanium dioxide (TiO2) particles (5, 23, or 5000 nm) were added to Chinese hamster ovary cells. Observation by confocal laser scanning microscopy showed that the TiO2 particles easily moved to the cytoplasm of the cultured mammalian cells, not to the nucleus. The intensity of the side-scattered light revealed that the particles were taken up in the cells dose-, time-, and size-dependently. In addition, surface-coating of TiO2 particles changed the uptake into the cells, which was accurately reflected in the intensity of the side-scattered light. The uptake of other nanoparticles such as silver (Ag) and iron oxide (Fe3O4) also could be detected. This method could be used for the initial screening of the uptake potential of nanoparticles as an index of "nanotoxicity".
We recently clarified that the side-scatter(ed) light (SSC) of flow cytometry (FCM) could be used as a guide to measure the uptake potential of nanoparticles [ Suzuki et al. Environ. Sci. Technol. 2007 , 41 , 3018 - 3024 ]. In this paper, the method was improved so as to be able to determine simultaneously the uptake potential of nanoparticles and the production of reactive oxygen species (ROS), and correlations with genotoxicity were evaluated. In the FCM analysis, SSC and fluorescence of 6-carboxy-2,7'-diclorodihydrofluorescein diacetate, di(acetoxy ester) based on ROS production were concurrently detected after treatments with ZnO, CuO, Fe(3)O(4), TiO(2), and Ag nanoparticles. The ZnO and CuO nanoparticles caused high ROS production, which was more significant in the cells with higher SSC intensity. The increase of SSC intensity was more significant for TiO(2) than ZnO and CuO, whereas ROS production was higher for ZnO and CuO than TiO(2), suggesting that the extent of ROS production based on the uptake of nanoparticles differed with each kind of nanoparticle. ROS production was correlated with generation of the phosphorylated histone H2AX (γ-H2AX), a marker of DNA damage, and an antioxidant, n-acetylcysteine, could partially suppress the γ-H2AX. This method makes it possible to predict not only uptake potential but also genotoxicity.
The uptake of nanoparticles by cells is an important factor to assess nanotoxicity. In general, the nanoparticles taken up by the cells have been identified by transmission electron microscope, inductively coupled plasma mass spectrometry, etc.; however, the methods required an immense amount of time and effort. Flow cytometry (FCM) has been used and developed in the fields of biochemistry and clinical hematology, and has advantages to analyze thousands of cells in seconds. We recently clarified that the side-scatter(ed) light of FCM could be used as a guide to measure uptake potential of nanoparticles. Here, we describe the protocol for screening of the uptake potential of nanoparticles using FCM.
Post-translational modifications in histones have been associated with cancer. Although cigarette sidestream smoke (CSS) as well as mainstream smoke are carcinogens, the relationship between carcinogenicity and histone modifications has not yet been clarified. Here, we demonstrated that CSS induced phosphorylation of histones, involving a carcinogenic process. Treatment with CSS markedly induced the phosphorylation of histone H3 at serine 10 and 28 residues (H3S10 and H3S28), which was independent from the cell cycle, in the human pulmonary epithelial cell model, A549 and normal human lung fibroblasts, MRC-5 and WI-38. Using specific inhibitors and small interfering RNA, the phosphorylation of H3S10 was found to be mediated by c-jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. These pathways were different from that of the CSS-induced phosphorylation of histone H2AX (γ-H2AX) mediated by Ataxia telangiectasia-mutated (ATM) and ATM-Rad3-related (ATR) protein kinases. A chromatin immunoprecipitation assay revealed that the phosphorylation of H3S10 was increased in the promoter sites of the proto-oncogenes, c-fos and c-jun, which indicated that CSS plays a role in tumor promotion. Because the phosphorylation of H3S10 was decreased in the aldehyde-removed CSS and was significantly induced by treatment with formaldehyde, aldehydes are suspected to partially contribute to this phosphorylation. These findings suggested that any chemicals in CSS, including aldehydes, phosphorylate H3S10 via JNK and PI3K/Akt pathways, which is different from the DNA damage response, resulting in tumor promotion.
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