p38 MAPK Activation, DNA Damage, Cell Cycle Arrest and Apoptosis

Oct 8, 2010 - p38 MAPK Activation, DNA Damage, Cell Cycle Arrest and Apoptosis As Mechanisms of Toxicity of Silver Nanoparticles in Jurkat T Cells...
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Environ. Sci. Technol. 2010, 44, 8337–8342

p38 MAPK Activation, DNA Damage, Cell Cycle Arrest and Apoptosis As Mechanisms of Toxicity of Silver Nanoparticles in Jurkat T Cells HYUN-JEONG EOM AND JINHEE CHOI* School of Environmental Engineering, College of Urban Science, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743, Korea

Received June 17, 2010. Revised manuscript received September 14, 2010. Accepted September 16, 2010.

To identify potential harmful effects of silver nanoparticles (AgNPs) on human health, a comprehensive toxicity assay was conducted on human Jurkat T cells, using oxidative stressrelated endpoint. The effect of Ag ions was also investigated and compared with that of AgNPs, as it is anticipated that Ag ions will be released from AgNPs, which may be responsible for their toxicity. Cell viability tests indicated high sensitivity of Jurkat T cells when exposed to AgNPs compared to Ag ions; however, both AgNPs and Ag ions induce similar levels of cellular reactive oxygen species during the initial exposure period and; after 24 h, they were increased on exposure to AgNPs compared to Ag ions, which suggest that oxidative stress may be an indirect cause of the observed cytotoxicity of AgNPs. AgNPs exposure activates p38 mitogen-activated protein kinase through nuclear factor-E2-related factor-2 and nuclear factor-kappaB signaling pathways, subsequently inducing DNA damage, cell cycle arrest and apoptosis. Selective toxicity of AgNPs on Jurkat T cells suggests that rigorous toxicity evaluation should be conducted using various different cell types and biological systems prior to the widespread use of AgNPs.

Introduction Various types of use of nanoparticles (NPs), including biomedical applications, involve deliberate, direct ingestion or injection of NPs into the body, and as these NPs interact with cells, it is important to ensure they do not cause any adverse effects. Therefore, understanding the properties of NPs, as well as their effects on human cells is crucial prior to their use (1). Of various NPs, silver nanoparticles (AgNPs) are most commonly used in numerous consumer and health care products. However, despite the widespread application of AgNPs, including therapeutic ones, which naturally cause easy entry into cells, there is a serious lack of information concerning their toxicity to humans at the molecular and cellular levels. The first step toward understanding how AgNPs will react in the body often involves in vitro studies, which are easier to control and reproduce compared to in vivo studies. Several in vitro studies, using rat liver cells (2), mouse germline stem cells (3), human fibroblasts (4), and rat adrenal cells (5), have been published on the toxicity of AgNPs; more recent publications have shown strong evidence * Corresponding Author phone: 82-2-2210-5622; fax: 82-2-22442245; e-mail: [email protected]. 10.1021/es1020668

 2010 American Chemical Society

Published on Web 10/08/2010

that AgNPs are toxic to various cultured cells (6-8). To draw general conclusions on the toxicity of AgNPs, it is advantageous to conduct multiple toxicity tests using various cell types. However, comprehensive in vitro studies on AgNPs covering the detailed mechanism of toxicity are still very limited. In this study, to investigate the potential harmful effects of AgNPs on human health, a comprehensive toxicity assay was conducted on immortalized human T lymphocyte cells, Jurkat, using oxidative stress-related endpoint, as oxidative stress has been reported as one of the most important toxicity mechanisms related to AgNP exposure (9-11). As AgNPs release Ag ions, which is believed to be partially responsible for the toxicity of AgNPs (8, 12, 13), the toxicity of Ag ions was also investigated. The upstream signaling mechanism responsible for regulating oxidative stress was studied, focusing on the mitogen-activated protein kinase (MAPK) cascades; extracellular signal-regulating kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), and redox-sensitive transcription factors, such as nuclear factor-kappaB (NF-κB) and nuclear factor-E2-related factor-2 (Nrf-2). Subsequent cellular consequences of oxidative stresses, such as DNA damage, cell cycles and apoptosis, were also investigated.

Materials and Methods The details for the materials and methods are provided in the Supporting Information (SI).

Results and Discussion Cell Viability Screening. In vitro investigations of NPs often focus on one cell type from the organ or tissue of interest in order to gain an understanding of the implications of particle exposure and; thus, previous studies have principally concentrated on the penetration of AgNPs into the skin and intestine, pulmonary and hepato-toxicity of NPs (14). In this study, the sensitivities of different cells on exposure to AgNPs and Ag ions were screened using human cancerous cells derived from various organs, such as Jurkat T, NCI-H460, HeLa, HepG2, MCF-7 as well as normal human cells, Beas2B, in order to select a sensitive cell line to investigate the toxicity of AgNPs to human health (SI Figure S1). No statistically significant decreases in the cell viabilities were observed in Beas-2B, NCI-N460 and HeLa cells exposed to AgNPs or Ag ions. However, the cell viabilities were decreased by about 5-60% in MCF-7 and HepG2 cells on exposure to 1 mg/L of AgNPs; whereas, the decreases were less on exposure to Ag ions. Dramatically different susceptibilities were observed in Jurkat T cells on exposure to AgNPs and Ag ions. The cell viability of Jurkat T cells was less than 5% on exposure to 0.5 mg/L of AgNPs, but about 70% on exposure to the same concentration of Ag ions. Cells were not viable on exposure to 1 mg/L of AgNPs; however, they maintained about 60% viability on exposure to the same concentration of Ag ions. Variations in a cells’ responsiveness to chemical stress have often been reported, including exposure to NPs, depending on the cells tested; however, the mechanism has often not been clearly elucidated. Therefore, investigations are being conducted into why and how such different susceptibilities of cells to AgNPs exposure occur across tested cell lines. As dramatic decreases in the viabilities of Jurkat T cell were observed with between 0.1 and 0.5 mg/L of AgNPs, both the cell viability and morphology were investigated at an additional concentration between 0.1 and 0.5 mg/L (i.e., 0.2 mg/L) (SI Figure S2). A time course cell viability experiment VOL. 44, NO. 21, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Formation of ROS in Jurkat T cells exposed to 0.2 mg/L AgNPs and Ag ions for 1, 2, 4, 12, and 24 h. 100 µM H2O2 was used as the positive control. The cells were incubated with 25 µM DCFH-DA at 37 °C for 30 min, and Fluorescence intensity was quantified using flow cytometry. was also conducted at this exposure concentration, which revealed that after up to 12 h, more than 80% of the cells were viable on exposure to AgNPs, whereas, after 24 h exposure to 0.2 mg/L of AgNPs, Jurkat T cells showed about a 70% decrease in viability (SI Figure S2A). Cell morphology observations, using optical microscopy, indicated that the tendency for forming cell colonies decreased in the cells exposed to AgNPs, but not in those exposed to the same concentration of Ag ions. Lower confluency was the noticeable difference observed in the cells exposed to AgNPs compared to the control and Ag ion exposed cells (SI Figure S2B). Cell viability and morphology are crucial indicators for in vitro toxicity assays, which provide a first insight into the cellular response to a toxicant; therefore, Jurkat T cells were selected for a more detailed analysis, with 0.2 mg/L of AgNPs as the maximum exposure level for a mechanistic study, as about 70% of cells were viable at this concentration. Characterization of Silver Nanoparticles. As the physicochemical attributes of NPs are critical parameters in determining the degree of cytotoxicity, size, shape, dispersion, surface area, and surface chemistry of NPs have been evaluated in many studies (15-17). In this study, as Jurkat T cells were selected for a depth investigation of AgNPs toxicity, the shape, and size distribution of AgNPs in Jurkat T cell culture medium was determined under cell free conditions (SI Figure S3). The particle size provided by the manufacturer was below 100 nm; however, the TEM images (SI Figure S3A) indicated that AgNPs prepared via the THF method had an even distribution, with 5-10 nm individual particle sizes. However, AgNPs may tend to agglomerate when exposed to the cells, as the results of DLS (SI Figure S3B) indicated that the main NPs sizes distributed in the medium were about 28-35 nm. A previous characterization study on NPs, including various types of AgNPs (18), showed that among the tested NPs, only a few particles had zeta potential values larger than the absolute value of 30 mV for stable dispersion. Having these high values allowed them to disperse with smaller sizes, increasing their exposed surface area. In their study, most of AgNPs exhibited lower zeta potential values, which could cause them to agglomerate, which was also observed in our previous work on optimization of aqueous AgNPs dispersions (19). However, the DLS results showed that even AgNPs tended to form agglomerates, with sizes still mainly distributed in the nano scale range (less than 40 nm). Previously, Diaz et al. (20) studied the uptake of five types of NPs by human blood cells and tumorous cell lines, and observed the appearance of groups of NPs inside the human monocyte, as the NPs were rapidly taken in by the cells. The uptakes of AgNPs via micelles (21) and Ag ions via cationic channels/transporters have also been reported (22); however, how AgNPs and Ag ions enter the cells has not been clearly identified. Therefore, the incorporation of AgNPs inside the cells was investigated using FCM light scatter histograms, which demonstrated that AgNPs treatment resulted in higher 8338

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SS and lower FS intensities compared to the control (SI Figure S3C). Increased SS may reflect uptake of NPs by the cells. Previously, the use of FCM-SS analysis has been proposed as an initial screening index of “nanotoxicity”, by Suzuki et al. (23), and our previous study with ceria NPs in the Beas-2B cells also supported this hypothesis (24). Increased SS and decreased FS may also be an indication of apoptosis, as increased cell granularity and cellular shrinkage are modification encountered during the early phase of apoptosis (25, 26). Along with other evidence, this led us to study apoptosis as an endpoint of AgNPs toxicity. To check whether THF, which may remain in AgNPs solutions, would affect the cytotoxicity, the response of cells to THF solution was also examined (SI Figure S3D). THF did not provoke any significant effect on cell viability. ROS Formation and MAPK Signaling. Following the characterization of AgNPs, a mechanistic study was subsequently conducted on Jurkat T cells using oxidative stress parameters, as recent in vitro studies have indicated that oxidative stress is a potential toxic mechanism of AgNPs (27, 28). ROS production was investigated as an initial step of oxidative stress, which revealed an increase in ROS formation in the cells exposed to both AgNPs and Ag ions (0.2 mg/L) compared to unexposed cells (Figure 1 and SI Figure S4). No significant difference was observed in the ROS formation between cells exposed to AgNPs- and Ag ions until 24 h of exposure. After 24 h of exposure, the fluorescent signal emitted from the cells was significantly increased on exposure to AgNPs compared to those exposed to Ag ions. This result suggests that oxidative stress might be an indirect cause of the cytotoxicity of AgNPs, rather than the primary cause. Downstream events, such as oxidative stress signal transduction, subsequent cell damage and repair, and scavenging capacity, may be different between cells exposed to AgNPs and Ag ions, which would exacerbate the levels of ROS in the cells after 24 h and, ultimately, the different susceptibilities of cells. However, a variation may be found depending on the type of NPs. As Diaz et al. (20) reported that Jurkat cells showed low levels of ROS production in short time period experiments (5-30 min), but no production with longer periods of incubation (24, 48, and 72 h) on exposure to different types of NPs, such as iron or silica NPs. As the formation of ROS may not be a direct cause of the toxicity of AgNPs toward Jurkat T cells, the involvement of the oxidative stress responding signal transduction pathway was subsequently investigated by examining ERK, p38 and JNK MAPK, as well as the related transcription factors, Nrf-2 and NF-κB (Figure 2 and SI Figure S5). Of the MAPK tested, the expression of p-p38 MAPK protein increased on exposure to AgNPs at all concentrations tested (0.05, 0.1, and 0.2 mg/ L), with the most important increase in the cells exposed to 0.2 mg/L of AgNPs (Figure 2A). A temporal analysis revealed that p-p38 MAPK increased as early as 4 h after exposure, reaching a maximum after 12-24 h of exposure. An increase

FIGURE 2. The expressions of ERK, p38 and JNK MAPK, as well as the transcription factors, Nrf-2 and NF-KB, in Jurkat T cells exposed to 0.05, 0.1, and 0.2 mg/L AgNPs and Ag ions for 24 h (A) and 0.2 mg/L for 4, 12, and 24 h (B). The expressions of ERK were measured in cytosol; whereas, those of Nrf-2 and NF-KB were measured in the nuclear fractions. The densitometric values for the expressions of the proteins were normalized to that of Actin, and presented as relative units compared to the control. Data represent the mean ( standard error of the mean of three individual experiments. * p < 0.05 compared to the control group. in the expression of p-p38 was barely observed on exposure to Ag ions (Figure 2B). Subsequently, the expression of the downstream transcription factors, Nrf-2 and NF-κB proteins, were investigated in the nuclear fractions of both treated and untreated cells. Increased expressions of NF-κB were observed with exposure to 0.05, 0.1, and 0.2 mg/L of AgNPs (Figure 2A). A temporal analysis indicated that the expression of Nrf-2 increased in an exposure time dependent manner up to 24 h on exposure to AgNPs, but that of NF-κB increased after 4 h of exposure, which continued until the end of the experiment (Figure 2B). The expressions of Nrf-2 and NF-κB increased as early as 4 h after exposure, with the activation reaching a maximum after 24 h of exposure. Neither the expression of Nrf-2 nor NF-κB was affected by exposure to Ag ions. Although the roles of the MAPK pathways in various stress responses have already been widely investigated (29, 30) they have not been approached to any great extent in a nanotoxicological context. The activations of p38 and ERK in ceria and silica NPs exposed Beas-2B cells, respectively, have previously been reported (24, 31). MAPK signaling pathways are known to be evolutionally conserved, which was also proved in our previous study with C. elegans, where an increased expression of p38 was observed on exposure to AgNPs (32). Hsin et al. (28) reported that AgNPs act through ROS and JNK MAPK to induce apoptosis via the mitochondrial pathway in NIH3T3 fibroblast cells. The present study revealed increased expressions of p-p38, Nrf-2 and NF-κB, but only on exposure to AgNPs, not to Ag ions, which strongly suggest that p38 MAPK, via Nrf-2 and NF-κB pathways, may be involved in the selective toxicity of AgNPs over that of Ag ions in Jurkat T cells. Cell Cycle, DNA Damage and Apoptosis. The affect of AgNPs on the cell cycle was then investigated (Figure 3), as the activation of p38 MAPK, through the oxidative stress (Nrf2) and inflammation (NF-κB) related transcription factors, probably results in various types of cellular damages, such

as oxidative damage and inflammatory reactions, which would generally be reflected by an analysis of the cell cycle. The results indicated that, in the control, the major cell population was observed in the G1 phase; whereas, in AgNPs exposed cells, a decrease in the G1 population, accompanied by an increase in the G2/M population, was observed. Serious increases in the numbers of cells in the G2/M and S phases were observed in cells exposed to 0.1 and 0.2 mg/L of AgNPs, respectively (Figure 3). A temporal analysis revealed that after 12 and 24 h of exposure, AgNPs lead to serious decreases in cells in the G1 phase and increases in cells in the G2/M and S phases (Figure 3). However, Ag ions did not affect any of the cell cycle indicators either the exposure concentrationor time-course studies. An increase in the G2/M and S phases and a decrease in the G1 phase on exposure to AgNPs, suggests that AgNPs may cause serious cell damage. The number of cells with a subdiploid DNA content increased in cells exposed to AgNPs in exposure concentration- and exposure duration-dependent manners (Figure 3), which strongly suggests DNA damage and apoptosis may be involved in AgNPs toxicity. DNA damage is usually accompanied by cell cycle arrest, as cells with damaged DNA will accumulate in the G1, S, or G2/M phase, which lead us to investigated DNA damage (Figure 4). The expression of the DNA damage marker protein, p-H2AX increased dramatically after 12 and 24 h of exposure to AgNPs; however, no such increase was observed in the cells exposed to Ag ions. The Comet assay revealed that tail moments were statistically significantly increased on exposure to AgNPs, but to a lesser extent on exposure to Ag ions. Both these results on DNA damage suggest that DNA strand breaks occur on exposure to AgNPs, but not or much less on exposure to Ag ions in Jurkat T cells, which may also contribute to the different susceptibilities of cells to AgNPs and Ag ions. More importantly, these results suggest the potential genotoxicity of AgNPs in Jurkat T cell, which was also observed in our previous ecotoxicity study of AgNPs VOL. 44, NO. 21, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Cell cycle analysis in Jurkat T cells exposed to 0.05, 0.1, and 0.2 mg/L AgNPs and Ag ions for 24 h and 0.2 mg/L for 4, 12, and 24 h. Histograms were analyzed using the MultiCycle AV software for Windows. Jurkat T cells exposed to 0.05, 0.1, and 0.2 mg/L AgNPs and Ag ions for 24 h and 0.2 mg/L for 4, 12, and 24 h.

FIGURE 4. DNA damage measured using the expression of p-H2AX (A) and the Comet assay (B) in Jurkat T cells exposed to 0.2 mg/L AgNPs and Ag ions for 4, 12, and 24 h (B). The expression of p-H2AX was measured in the nuclear fraction by Western blot analysis. The tail moments from the Comet assay represent the mean ( standard error of the mean of three individual experiments. * p < 0.05 and ** p < 0.01 compared to the control group. using an aquatic organism (33). The potential genotoxic effects of emerging nanomaterials, such as AgNPs, should 8340

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be thoroughly investigated on various biological systems, including in vitro systems, to allow for their safe use.

FIGURE 5. Apoptosis investigated in the Jurkat T cells exposed to 0.2 mg/L AgNPs and Ag ions for 4, 12, and 24 h, using Annexin V/PI staining by flow cytometry. It is well-known that cells with irreversible damage will undergo apoptosis, giving rise to the accumulation of DNA damage and; thus, to cells in the subG1 phase. Apoptosis has been reported as a major mechanism of cell death on exposure to NPs (28). Therefore, to investigate whether AgNPs induce apoptosis and/or necrosis, staining with Annexin V/PI, Hoechst 33342 and TMRE were carried out on Jurkat T cells (Figure 5 and SI Figure S6). The data from the Annexin V/PI staining experiment indicated that increases in the apoptotic and necrosis cell populations were exposure time dependent with both AgNPs and Ag ions, but more importantly with AgNPs. After 24 h of exposure, only about 4 and 19% of the populations were in apoptosis and necrosis in the control and Ag ions treated cells, respectively; whereas, about 39% of cells were undergoing apoptosis and necrosis on exposure to AgNPs. AgNPs induced cell deaths were in order: late apoptosis > necrosis > early apoptosis (Figure 5). The fluorescent image of cells stained with Hoechst 33342 indicated the formation of apoptotic bodies in the cells exposed to AgNPs (12 and 24 h), which was not observed on exposure to Ag ions (SI Figure S6A). The mitochondrial membrane potential (∆Ψm) also assessed in Jurkat T cells loaded with TMRE shows that treatment with AgNPs led to a significant decrease in the numbers of cells stained with TMRE, indicative of mitochondrial membrane depolarization (SI Figure S6B). However, no such change was observed in Ag ions exposed cells. Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and suggested to be central to the apoptotic pathway (34). Diaz et al. (20) investigated the cytotoxicity of five different NPs using several blood cell types and tumoral cell lines, including Jurkat, and reported that the cytotoxicity of NPs depended on the cell type, but they found no direct correlation between ROS production and cell toxicity. Selective sensitivity across cell lines from screening tests may also suggest the potential use of AgNPs as chemotherapy agents. To this end, it would be interesting to note why other cancer cell lines, as well as normal human bronchial epithelial cells, were not as sensitive to AgNPs exposure as Jurkat T cells. Therefore, the higher sensitivity of Jurkat T cells, DNA damage and their arrest in the G2/M phase could be further explored to evaluate the potential use of AgNPs in chemotherapy, which was also suggested by AshaRani et al. (6) in their study conducted on human glioblastoma cells. However, to consider the possibility of AgNPs as a chemotherapy agent, the responses of a broad range of normal, as well as cancer cell lines on exposure to AgNPs should be thoroughly investigated. For AgNPs to be actively applied in the biomedical area, it is important that nanotoxicology research gains an understanding of how these multiple factors

influence the toxicity of AgNPs for their useful properties to be maximized and undesirable properties avoided. To interpret the overall results, it is also worthwhile to mention that the concentrations used in this study (less than 0.2 mg/L) were much lower than those applied in most other studies (up to 400 mg/L, for example in refs 35-37). To reveal the relevancy of the finding, the importance of NPs concentrations in toxicity testing has been emphasized, as the exposure concentration may influence the toxicity (14). At low levels of oxidative stress, a protective response may be initiated (38), and progress to a damaging response with increasing NPs concentration (2, 39). The present study suggests that AgNPs induce significant toxicity toward Jurkat T cells at relatively low exposure concentrations, which is closer to what would occur under real exposure conditions. High doses of AgNPs are unlikely to be encountered by humans within consumer and occupational settings, or upon directly entering cells during their applications, but might remain in the cells of target organs at low doses for a long time. The cell viability test revealed that Jurkat T cells exhibited higher sensitivity to AgNPs than Ag ions exposure. As the cellular ROS levels were similar with both AgNPs and Ag ions treatments during the early exposure period, but increased with AgNPs exposure during late exposure period, the oxidative stress was indirectly involved via p38 MAPK through the Nrf-2 and NF-κB signaling pathways, further inducing DNA damage. Accumulation of DNA damage; in turn, gives rise to cell cycle arrest and apoptosis, which may be important toxic phenomena of AgNPs, because such toxic symptoms did not occur in cells exposed to Ag ions. However, the potential to release Ag ions is believed to play a pivotal role in the toxicity of AgNPs; thus, it is still unclear why Jurkat T cell showed such a different sensitivity to AgNPs over that to Ag ions. To identify the underlying mechanism of the toxicity induced by AgNPs in Jurkat T cells over that induced by Ag ions, a microarray was conducted (data not shown); however, surprisingly enough, gene expression profiling revealed that only small numbers of genes were affected by AgNPs, suggesting AgNPs toxicity may be regulated at the post-transcriptional level. Further analysis on the interpretation of microarray is being conducted to better understand the AgNPs-induced cytotoxicity in Jurkat T cells.

Acknowledgments This work was supported by the Midcareer Researcher Program through National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0027722) and by the 2009 high-tech laboratory equipments for academic research of the University of Seoul.

Supporting Information Available The details for materials and methods. Culture condition of cells was presented in Table S1. Comparison of the cytotoxicity on various cell lines exposed to AgNPs and Ag ions was presented in Figure S1. Temporal analysis on cell viability and morphologies of Jurkat T cells exposed to AgNPs and Ag ions were presented in Figure S2. Characterization of AgNPs was presented in Figure S3. Formation of ROS in Jurkat T cells was presented in Figure S4. Western blot band image of expressed proteins was presented in Figure S5. Apoptosis detection by Hoechst 33342 staining and TMRE staining were presented in Figure S6. This material is available free of charge via the Internet at http://pubs.acs.org.

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