Article pubs.acs.org/est
Flow Cytometric Evaluation of Nanoparticles Using Side-Scattered Light and Reactive Oxygen Species-Mediated Fluorescence− Correlation with Genotoxicity Yousuke Toduka, Tatsushi Toyooka, and Yuko Ibuki* Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan S Supporting Information *
ABSTRACT: 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, Fe3O4, TiO2, 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 TiO2 than ZnO and CuO, whereas ROS production was higher for ZnO and CuO than TiO2, 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.
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INTRODUCTION With the rising use of nanosized materials due to the development of nanotechnology, harmful effects of nanoparticles are of increasing concern.1−4 This is due to their small size and unique physicochemical properties that are not present in conventional bulk materials, surface area, charge, shape, solubility, surface chemistry, and so forth. Many researchers have found that nanoparticles differed in toxicity to larger particles. For example, one of the most widely manufactured nanoparticles, TiO2, which is smaller than 29 nm, increased inflammation and altered macrophage chemotactic response in rat lungs, compared to 250 nm TiO2 particles.5 Smaller TiO2 could induce oxidative damage to human bronchial epithelial cells, brain microglia, and neurons.6,7 In contrast, some studies have shown that the toxicity of particles is independent of size. Nanosized TiO2 rods and dots produced inflammatory responses that were not different from the pulmonary effects of larger TiO2 particles.8 Also, in other nanoparticles, the toxicity and its mechanism are controversial, and no definite conclusion has been reached. One of the reasons for this is that simple and standardized screening methods for particle toxicity have not yet been established. This might be attributable to the difficulty in determining the physicochemical and biological properties of particles responsible for the toxicity. Therefore, new testing strategies have been discussed in the world.2,9,10 Previously, we have reported novel methods to evaluate the uptake potential of nanoparticles in mammalian cells using flow cytometry (FCM) .11 Uptake potential is an important factor mediating the toxicity of nanoparticles. Not only particle size, © 2012 American Chemical Society
but also surface condition, form, and so forth are attributable to the translocation potential of particles to the human body and cells. We used side-scattered light (SSC) in FCM as an indicator of uptake potential. When particles are taken up into cells, SSC intensity consequently increases because the intracellular density of cells is enhanced. This evaluation of the uptake potential of nanoparticles using SSC in FCM has the advantage that cumbersome treatments (staining, labeling, etc.) except for the preparation of a single cell suspension are not required. In addition, statistically valid information about cell populations is quickly obtained, since thousands of living cells are analyzed per second in FCM. Since our publication in 2007,11 some researchers have used SSC in FCM to evaluate intracellular uptake of nanoparticles.12−14 Although this method could indicate the uptake potential according to kinds of particles easily and rapidly, direct information leading to cellular and genetic toxicity could not be obtained. Many researchers have reported that oxidative stress, the production of reactive oxygen species (ROS), contributed to the cytotoxicity of nanoparticles.4,15 This would explain why even poorly soluble particles such as TiO2, carbon black, silica, and so forth show cytotoxicity.16 ROS can oxidize cellular membranes, DNA, and so forth activating a wide variety of cellular events, cell cycle arrest, apoptosis, and disruptions of cellular signal transduction. In vivo, Received: Revised: Accepted: Published: 7629
February 2, 2012 June 13, 2012 June 15, 2012 June 15, 2012 dx.doi.org/10.1021/es300433x | Environ. Sci. Technol. 2012, 46, 7629−7636
Environmental Science & Technology
Article
Figure 1. Time- and dose-dependent change of SSC and DCF intensity after treatment with several kinds of nanoparticles. CHO-K1 cells were incubated in the presence of DCFH-DA for 1 h. (A) They were treated with nanoparticles (100 μg/mL) for ∼8 h. (B) They were treated with several doses (10−300 μg/mL) of nanoparticles for 4 h. SSC and DCF intensity were analyzed by FCM. The lines were colored up with raised concentrations of nanoparticles or incubation time. The mean SSC and DCF (ratio: treated/control) were included as tables in histograms.
bases, DNA adducts, single strand breaks (SSBs) and cross-linking, and the repair of such damage.24,25 In addition to the advantage that γ-H2AX can be detected in response to many types of DNA damage, we and other researchers are convinced that γ-H2AX provides a considerably more sensitive measurement of DNA damage than other techniques such as pulse field gel electrophoresis and comet assays.26−28 Therefore, using γ-H2AX as a genotoxic marker is a good way to identify the genotoxic potential of nanoparticles and to add value to FCM analysis. In this study, we developed advanced FCM methods by simultaneous detection of SSC and fluorescence for ROS to analyze the toxicity of nanoparticles and examined whether the results of the FCM analysis correlate with the genotoxicity of nanoparticles using γ-H2AX.
the uptake by target cells such as macrophages or epithelial cells plays a central role in the production of ROS, which contributes to pulmonary inflammation.1,4,5 These ROS themselves and the accompanying inflammation cause cellular and genetic damage. We speculate that uptake of some kinds of nanoparticles results in an increase of ROS followed by genetic damage. Therefore, we considered that ROS production is a suitable indicator to analyze simultaneously with SSC intensity by FCM. The small size and large surface area of nanoparticles, coupled to other physicochemical features, may induce unpredictable genotoxic properties.17 We previously reported that phosphorylation of histone H2AX (γ-H2AX) is a sensitive marker of genotoxicity for several environmental chemicals.18−22 H2AX is a minor component of histone H2A, and its phosphorylation was originally identified as an early event after the direct formation of DNA double strand breaks (DSBs) by ionizing radiation.23 However, the generation of γ-H2AX is now considered to also occur after the indirect formation of DSBs caused by the collision of the replication forks at sites of DNA damage including oxidative
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EXPERIMENTAL SECTION Chemicals. All nanosized particles (ZnO (cat. no. 544906, vendor size
