Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions

Indium compounds have been widely used in manufacturing displays of mobile phones, computers and televisions. However, inhalation exposure to indium compounds causes interstitial pneumonia in exposed workers and lung cancer in experimental animals. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed under inflammatory conditions and may participate in indium-induced carcinogenesis. In this study, we examined 8-nitroG formation in A549 cultured human lung epithelial cells treated with indium compounds, including nanoparticles of indium oxide (In2O3) and indium-tin oxide (ITO), and indium chloride (InCl3). We performed fluorescent immunocytochemistry to examine 8-nitroG formation in indium-exposed A549 cells. All indium compounds significantly increased 8-nitroG formation in A549 cells at 5 ng/ml after 4 h incubation. 8-NitroG formation was largely reduced by 1400 W, methyl-β-cyclodextrin (MBCD) and monodansylcadaverine (MDC), suggesting the involvement of nitric oxide synthase and endocytosis. 8-NitroG formation in A549 cells was also largely suppressed by small interfering RNA (siRNA) for high-mobility group box-1 (HMGB1), receptor for advanced glycation and end products (AGER, RAGE) and Toll-like receptor 9 (TLR9). These results suggest that indium compounds induce inflammation-mediated DNA damage in lung epithelial cells via the HMGB1-RAGE-TLR9 pathway. This mechanism may contribute to indium-induced genotoxicity in the respiratory system.

Accumulation of indium compounds in the lung tissue causes chronic inflammation 5 . Chronic inflammation is known to contribute to a substantial part of cancer cases worldwide 15 . In inflammation-related carcinogenesis, reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated from inflammatory and epithelial cells play a critical role. These reactive species form mutagenic DNA lesions, including 8-nitroguanine (8-nitroG), via the interaction with DNA bases 16,17 . 8-NitroG is formed by the interaction of guanine with peroxynitrite (ONOO − ), which is generated by the reaction of nitric oxide (NO) and superoxide (O 2 ·− ) 18 . We have reported that 8-nitroG is formed at the sites of carcinogenesis in a wide variety of animal models and clinical specimens of cancer-prone inflammatory diseases and proposed that this DNA lesion can be a potential biomarker of inflammation-related cancer 19,20 . The objective of this study was to determine the genotoxic effects of indium compounds on lung epithelial cells, and clarify the molecular mechanism. We have recently reported that In 2 O 3 induced 8-nitroG formation in mouse macrophages 21 . In this study, we used In 2 O 3 and ITO nanoparticles as representative indium compounds to examine 8-nitroG formation in human lung epithelial cells. Tabei et al. have demonstrated that intracellular accumulation of indium ions, which are released from indium-containing particles, contributes to DNA damage 22 . Therefore, we also used indium chloride (InCl 3 ) to examine the ability of ionic indium to cause nitrative DNA damage in this study.
High mobility group box-1 (HMGB1) is a nuclear protein, which is released from damaged or necrotic cells and associated with inflammatory diseases and cancer 23 . HMGB1 forms a complex with DNA, and the HMGB1-DNA complex binds to receptor for advanced glycation end products (RAGE), which is a multi-ligand receptor on cell membrane involved in cancer, sepsis and other diseases 24 . Toll-like receptor 9 (TLR9) is located on the lysosomal membrane and involved in cancer, sepsis and other diseases. This receptor mediates inflammatory responses against a wide variety of infectious and non-infectious agents via interaction with CpG DNA of exogenous and endogenous origin [24][25][26] . Our recent study has demonstrated that the HMGB1-RAGE-TLR9 signaling pathway was involved in nitrative DNA damage in human lung epithelial cells treated with multi-walled carbon nanotube (MWCNT) 27 . To clarify whether this pathway is involved in indium-induced DNA damage, we examined inhibitory effects of small interfering RNA (siRNA) for these molecules on 8-nitroG formation.

Results
Dispersion of indium nanoparticles and size distribution. In 2 O 3 and ITO nanoparticles were suspended in Dulbecco's Modified Eagles Medium (DMEM) containing fetal bovine serum (FBS) and kanamycin, and agglomerates were dispersed with an ultrasonic homogenizer. Figure 1A shows In 2 O 3 and ITO agglomerates before and after sonication. After sonication, the particles were dispersed into submicron-sized particles, capable of reaching human alveolus. Figure 1B shows size distribution of dispersed In 2 O 3 and ITO agglomerates analyzed with a particle size analyzer. The values concerning size distribution [peak, Z-average and polydispersity indexes (PdI)] of these compounds were as follows: In 2 O 3 (peak, 214.5 nm; Z-average, 208.0 nm; PdI, 0.399); ITO (peak, 194.5 nm; Z-average, 149.0 nm; PdI, 0.281).  www.nature.com/scientificreports/ Cytotoxic effect of indium compounds. Cytotoxic effects of indium compounds on A549 human lung epithelial cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A549 cells were treated with 5-50 µg/ml of indium compounds (In 2 O 3 , ITO and InCl 3 ) for 24 h. These compounds did not significantly decrease cell viability and there was no significant difference in the viability among these compounds under the conditions used (two-way ANOVA, Supplementary Figure S1 online).

8-NitroG formation in indium-treated cells.
We performed immunocytochemical analysis for 8-nitroG formation in A549 cells treated with indium compounds. Figure 2A shows fluorescent images of 8-nitroG formation in indium-treated cells. Positive control shows 8-nitroG formation in A549 cells incubated in culture supernatant of MWCNT-exposed cells, which was prepared as reported previously 27 . Clear fluorescence was observed at 5 ng/ml (equivalent to 1.42 ng/cm 2 ) in In 2 O 3 -, ITO-and InCl 3 -treated cells. No or weak staining was observed in non-treated control. The staining pattern of 8-nitroG overlapped with that of Hoechst 33258, suggesting that 8-nitroG was formed mainly in the nucleus ( Fig. 2A). Image analysis revealed that the staining intensity of 8-nitroG in In 2 O 3 -, ITO-and InCl 3 -treated A549 cells was significantly increased at 5 ng/ ml compared with the non-treated control (p < 0.05, Fig. 2B). In 2 O 3 and InCl 3 induced 8-nitroG formation in a dose-dependent manner, whereas ITO caused 8-nitroG formation to a similar extent at 5-200 ng/ml. Figure 3A shows fluorescent images of 8-nitroG formation in A549 cells treated with indium compounds for different durations. All indium compounds induced clear 8-nitroG formation at 2, 4 and 8 h. The staining intensity of 8-nitroG formation in indium-treated cells was significantly greater after 2-8 h incubation than that in non-treated control (p < 0.05, Fig. 3B). Staining intensity of 8-nitroG tended to be weaker at 8 h than those at 2 and 4 h (Fig. 3B). The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 4-8 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control by ANOVA followed by Tukey's test. www.nature.com/scientificreports/

Effects of inducible nitric oxide synthase (iNOS) and endocytosis inhibitors on indium-induced 8-nitroG formation.
To clarify the roles of iNOS expression and endocytosis in indium-induced DNA damage, we examined the effects of inhibitors for these events on 8-nitroG formation in A549 cells. In 2 O 3 , ITO and InCl 3 induced clear 8-nitroG formation and its immunoreactivity was largely suppressed by the treatment with inhibitors of iNOS (1400 W) and its transcription factor NF-κB (Bay11-7082, Bay), suggesting that iNOS expression was involved in DNA damage. 8-NitroG formation was also suppressed by inhibitors of caveolaemediated endocytosis (methyl-β-cyclodextrin, MBCD), clathrin-mediated endocytosis (monodansylcadaverine, MDC) and actin polymerization (cytochalasin D, CytoD) (Fig. 4A). Image analysis revealed that these inhibitors significantly reduced indium-induced 8-nitroG formation (p < 0.05, Fig. 4B).

Effects of siRNAs on indium-induced 8-nitroG formation.
To clarify the mechanism of indiuminduced DNA damage, we examined the inhibitory effects of HMGB1, AGER and TLR9 siRNA on 8-nitroG formation. Western blotting revealed that transfection of siRNAs for these genes reduced their expression levels, and negative control siRNA had no or weak inhibitory effect (Fig. 5A). Image analysis shows these siRNAs significantly reduced the expression of the corresponding proteins compared with control and negative control siRNA (p < 0.05, Fig. 5B). Indium compounds induced clear 8-nitroG formation in A549 cells, and its immunoreactivity was largely reduced by the transfection with HMGB1, AGER and TLR9 siRNA (Fig. 5C). Image analysis revealed that negative control siRNA did not affect indium-induced 8-nitroG formation and that transfection of siRNAs for these genes significantly reduced 8-nitroG formation (p < 0.01, Fig. 5D).

Effects of anti-HMGB1 and RAGE antibodies on indium-induced 8-nitroG formation.
To confirm the role of the HMGB1-RAGE-TLR9 pathway in indium-induced DNA damage, we examined the effect of antibodies against HMGB1 and RAGE. In A549 cells, indium compounds induced clear 8-nitroG formation and its immunoreactivity was largely decreased by the pretreatment with antibodies against HMGB1 and RAGE www.nature.com/scientificreports/ ( Fig. 6A). Image analysis shows that isotype control IgGs did not affect indium-induced 8-nitroG formation and antibodies against HMGB1 and RAGE significantly reduced 8-nitroG formation (p < 0.01, Fig. 6B).

Discussion
Indium-containing particles are used extensively in the microelectronic industry. However, interstitial pneumonia and lung cancer occurred after inhalation exposure of workers and experimental animals, respectively. In this study, to clarify the mechanism of indium-induced carcinogenesis, we investigated the genotoxic effects of In 2 O 3 and ITO nanoparticles and InCl 3 in A549 human lung epithelial cells. We observed that all indium compounds significantly induced the formation of 8-nitroG in the nucleus of A549 cells. Our group has previously reported that particulate materials, such as MWCNT 27,28 and carbon black 29 , induced 8-nitroG formation in lung epithelial cells. In this study, we have first demonstrated that not only In 2 O 3 and ITO particles but also InCl 3 induced clear 8-nitroG formation in lung epithelial cells. A previous study has shown that cellular uptake and solubilization of indium-containing particles, including ITO, via lysosomal acidification, leading to the release of indium ions, is needed for cytotoxicity 22,30 . Oxidative stress was induced by the accumulation of intracellular indium ions and mediated DNA damage evaluated by Comet assay 22 . These findings suggest that 8-nitroG formation caused by indium compounds can be accounted for by not only their particulate properties but also indium ions, derived from InCl 3 and released from In 2 O 3 and ITO particles in cell culture medium and/or intracellular compartments. Metal ions are known to interact with proteins to form aggregates, such as β-amyloid 31 . In the case of InCl 3 -exposed cells, the possibility that indium ions interact with proteins contained in FBS to form aggregates, which partially contribute to the genotoxicity, may not be neglected, but their contribution appears to be small under the conditions used. A549 cells have constitutively active Nrf-2 32 , which contributes to their protection against oxidative stress injury 33 . In this study, indium compounds caused clear nitrative DNA lesions in A549 cells, suggesting that indium-induced oxidative and nitrative stress overwhelmed their antioxidative potential.
In dose-response study, we demonstrated that indium compounds significantly increased 8-nitroG formation in A549 cells at an extremely low concentration of 5 ng/ml. We set 200 ng/ml as an optimal concentration Quantitive image analysis for the effects of iNOS and endocytosis inhibitors on indium-exposed A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 3-4 independent experiments. **p < 0.01 versus control and # p < 0.05, ## p < 0.01 versus indium-treated cells by ANOVA followed by Tukey's test.   Table 1. According to recent study on personal indium exposure level in ITO workers (highest level: 24.0 µg/m 3 ) 34 and a particle deposition model 35 , we estimated that the level of In 2 O 3 and ITO deposition in human alveoli can reach 1.42 ng/cm 2 (equivalent to 5 ng/ml) in 0.225 and 0.195 years, respectively. This estimation was made by assuming that indium particles are evenly distributed on the alveolar surface and the clearance of these particles does not occur. Indium-induced 8-nitroG formation was inhibited by 1400 W and Bay, suggesting that iNOS expression was essential for DNA damage. NF-κB regulates expression of various genes involved in inflammatory responses, including iNOS 36,37 . Indium-induced 8-nitroG formation was also suppressed by MBCD, MDC and CytoD, suggesting that caveolae-and clathrin-mediated endocytosis was involved in DNA damage. Nanoparticles up to approximately 500 nm and 200 nm are primarily internalized by caveolae-and calthrin-mediated endocytosis respectively 38,39 . Regarding size distribution of indium compounds, the peaks for In 2 O 3 and ITO were 214.5 and 194.5 nm, and Z-averages for In 2 O 3 and ITO were 208.0 and 149.0 nm, respectively. Therefore, they are likely to be internalized via these types of endocytosis and cause DNA damage. We have previously reported that these endocytosis inhibitors largely reduced cellular uptake of nanoparticles into cultured cells as demonstrated by light and electron microscopy and flow cytometry, resulting in the reduction in 8-nitroG formation [27][28][29] . Therefore, 8-nitroG formation induced by indium compounds appears to be largely accounted for by cellular uptake of their particles. (B) Quantitative image analysis for the effects of antbodies on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control was set at 1. The data were expressed as means ± SD of 3-4 independent experiments. **p < 0.01, ***p < 0.001 versus control and ## p < 0.01 versus negative control by ANOVA followed by Tukey's test.  45 . In this study, we focused on the role of the HMGB1-RAGE-TLR9 pathway in indium-induced DNA damage, as demonstrated in DNA damage in human lung epithelial cells treated with MWCNT 27 . HMGB1 is a nuclear protein released from damaged or necrotic cells and interacts with DNA to form the HMGB1-DNA complex 26 . In this study, MTT assay revealed that even high concentrations (up to 50 µg/ml) of indium compounds did not show significant cytotoxicity. Therefore, it is speculated that HMGB1 and DNA were released from damaged cells that are not deadly. RAGE is a multi-ligand transmembrane receptor on the cell membrane and constitutively expressed in the lung throughout the life 24 . TLR9 resides in endosomes and lysosomes and activates inflammatory responses via interaction with CpG DNA of exogenous and endogenous origin 24,25 . The HMGB1-DNA complex binds to RAGE and then activates TLR9-mediated inflammatory responses. In this study, indium-induced 8-nitroG formation in A549 cells was largely reduced by the transfection with siRNA for HMGB1, AGER and TLR9 and the pretreatment with antibodies against HMGB1 and RAGE. This finding indicates that the HMGB1-RAGE-TLR9 signaling pathway plays a key role in indium-induced DNA damage. The transfection with negative control siRNA and treatment with isotype control IgGs did not affect 8-nitroG formation, confirming the involvement of this pathway in DNA damage. Figure 7 shows the proposed mechanism of indium-induced DNA damage. In 2 O 3 and ITO particles are taken up by the cell via endocytosis and InCl 3 may enter the cell via diffusion, leading to cell injury. Cell injury caused by indium compounds may be accounted for by both their particulate properties and indium ions, derived from not only InCl 3 but also the release from In 2 O 3 and ITO particles. The HMGB1-DNA complex released from damaged cells is captured by RAGE on the surface of neighboring cells. This receptor is internalized into endosome and/or lysosome, where CpG is recognized by TLR9. TLR9 mediates NF-κB activation and iNOS expression, resulting in nitrative DNA damage. This molecular mechanism may contribute to indium-induced carcinogenesis. Endocytosis inhibitors reduced 8-nitroG formation in cells treated with indium compounds. This result may be explained by the inhibition of cellular uptake of the HMGB1-DNA complex into the cells.

conclusion
In this study, we first demonstrated that indium compounds induced 8-nitroG formation in human lung epithelial cells. It is noteworthy that both particles of indium compounds (In 2 O 3 and ITO) and InCl 3 caused clear 8-nitroG formation at extremely low doses regardless their chemical and physical properties. In addition, we found that the HMGB1-RAGE-TLR9 pathway plays a key role in indium-induced DNA damage. These finding would provide an insight into the molecular mechanism of genotoxicity induced by a wide variety of industrial chemicals. Table 1. Estimation of alveolar In 2 O 3 and ITO deposition in exposed individuals. The calculation was performed on the assumption that particles are evenly distributed in the alveoli, and actually, particles may be accumulated in particular sites and the concentration will exceed the above value in a shorter duration. *(G) 8-NitroG formation was significantly increased at 5 ng/ml = 1 ng/0.

Detection of 8-nitroG formation in A549 cells.
To investigate the mechanism of indium-induced carcinogenesis, we performed an immunocytochemical analysis to detect the formation of 8-nitroG in A549 cells. A549 cells (0.2 × 10 6 cells/ml) were cultured in DMEM containing 5% (v/v) FBS and 100 mg/l kanamycin in 8-well culture slides (BD Falcon, Franklin Lakes, NJ, USA) and incubated overnight at 37 °C. Then cells were treated with the indicated doses (5-200 ng/ml) of In 2 O 3 , ITO and InCl 3 for indicated durations (2-8 h). Positive control for 8-nitroG formation was prepared by incubating A549 cells for 2 h at 37 °C with culture supernatant of MWCNT-exposed cells prepared as reported previously 27 . The culture supernatant was obtained after A549 cells were treated with 1 μg/ml of MWCNT for 8 h, and centrifuged to remove MWCNT. Then, the supernatant was used for the experiment.
To examine the effects of inhibitors of iNOS and endocytosis on indium-induced 8-nitroG formation, cells were co-treated with 1 µM 1400 W (an inhibitor of iNOS), 10 µM Bay (an inhibitor of NF-κB), 2 mM MBCD (an inhibitor of caveolae-mediated endocytosis), 50 µM MDC (an inhibitor of clathrin-mediated endocytosis) or 1 µM CytoD (an inhibitor of actin polymerization). We used these concentrations of inhibitors, because they did not show any significant cytotoxic effects 27 . These inhibitors were purchased from Sigma-Aldrich (St, Louis, MO, USA). After incubation, we dried the culture slides at 37 °C and treated with 4% (v/v) formaldehyde for 10 min. The cells were treated with 0.5% (v/v) Triton-X100 in phosphate-buffered saline (PBS, pH 7.4) for 3 min and then treated with 2 N HCl for 30 min to denature DNA so that the antibody can easily detect DNA lesions as described previously 46 . Then the cells were incubated with 1% (w/v) skim milk dissolved in PBS for 1 h at room temperature. We treated the cells with rabbit polyclonal anti-8-nitroG antibody (1 µg/ml) produced by our group 47,48 overnight, followed by the incubation with fluorescent Alexa 594-anti-rabbit IgG antibody (1:400, Molecular Probes, Eugene, OR, USA) for 3 h. To stain the nucleus, cells were treated with 5 µM Hoechst 33258. The stained cells were examined under a fluorescence microscope (BX53, Olympus, Tokyo, Japan) with the exposure time of 600 and 40 ms for red and blue fluorescence, respectively. We employed these conditions because the difference and linearity in fluorescence intensity between control and indium-exposed samples were clearly observed. Staining intensity of 8-nitroG was quantified by analyzing 5 randomly selected fields per sample with an image J software as follows. The total fluorescence intensity of 8-nitroG in the image was quantified and the intensity of the background, where no cells exist, was subtracted. Then the image of the cell nuclei, stained with Hoechst 33258, was converted to a binary image and the area of the nuclei was quantified. Finally, the fluorescence intensity of 8-nitroG was divided by the area of nuclei.  To confirm the inhibitory effects of siRNA on gene expression, we performed Western blotting as described previously 27,29 . A549 cells transfected with siRNA were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) and centrifuged at 14,000g for 10 min. The protein concentration in the supernatant was measured with Coomasie Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL, USA). Proteins were separated by 5-20% SDS-PAGE (SuperSep Ace, Wako Pure Chemical Industries, Osaka, Japan), and blotted onto a polyvinylidene difluoride membrane. The membrane was treated with 5% (w/v) skim milk in Tris-buffered saline (pH 7.4) containing 0.1% (v/v) Tween 20. Then the membrane was incubated with anti-HMGB1 mouse monoclonal antibody (1:500, ab77302, Abcam, Cambridge, UK), anti-RAGE mouse monoclonal antibody (1:500, ab54741, Abcam) or anti-TLR9 rabbit polyclonal antibody (1:500, ab37154, Abcam) along with anti-GAPDH rabbit polyclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The membrane was then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10,000; Santa Cruz Biotechnology) and/or anti-mouse IgG antibody (1:2,000, Santa Cruz Biotechnology) for 30 min. Finally, we treated the membrane with ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK) and analyzed with a LAS-4000 mini imager (Fujifilm, Tokyo, Japan). We measured the band intensity with an image J software and normalized with GAPDH.

Blocking of 8-nitroG formation in A549 cells using anti-HMGB1 and RAGE antibodies.
To examine the role of HMGB1 and RAGE in 8-nitroG formation, A549 cells were pretreated with 10 μg/ml anti-HMGB1 (ab77302, Abcam Cambridge, UK) and 10 μg/ml anti-RAGE (ab54741, Abcam, Cambridge, UK) antibodies. We also used the corresponding isotype control IgGs [mouse IgG 1 (ab18447, Abcam) for anti-HMGB1 antibody and IgG 2a (ab18414, Abcam) for anti-RAGE antibody] to confirm the specificity of these antibodies. Then the cells were incubated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 for 4 h. Then 8-nitroG formation was examined by immunocytochemistry as described above.
Statistical analysis. Statistical analysis was performed by analysis of variance (ANOVA) followed by Tukey's multiple comparison test using an SPSS software (20.0 for Mac) as described previously 27 . Results were presented as means ± SD. P values less than 0.05 were considered to be statistically significant.

Data availability
Correspondence and requests for further data should be available address to Y.H.