Nitrative DNA damage in cultured macrophages exposed to indium oxide

Abstract

Objectives:

Indium compounds are used in manufacturing displays of mobile phones and televisions. However, these materials cause interstitial pneumonia in exposed workers. Animal experiments demonstrated that indium compounds caused lung cancer. Chronic inflammation is considered to play a role in lung carcinogenesis and fibrosis induced by particulate matters. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation and may participate in carcinogenesis. To clarify the mechanism of carcinogenesis, we examined 8-nitroG formation in indium-exposed cultured cells.

Methods:

We treated RAW 264.7 mouse macrophages with indium oxide (In₂O₃) nanoparticles (primary diameter: 30-50 nm), and performed fluorescent immunocytochemistry to detect 8-nitroG. The extent of 8-nitroG formation was evaluated by quantitative image analysis. We measured the amount of nitric oxide (NO) in the culture supernatant of In₂O₃-treated cells by the Griess method. We also examined the effects of inhibitors of inducible NO synthase (iNOS) and endocytosis on In₂O₃-induced 8-nitroG formation.

Results:

In₂O₃ significantly increased the intensity of 8-nitroG formation in RAW 264.7 cells in a dose-dependent manner. In₂O₃-induced 8-nitroG formation was observed at 2 h and further increased at 4 h, and the amount of NO released from In₂O₃-exposed cells was significantly increased at 2-4 h compared with the control. 8-NitroG formation was suppressed by 1400W (an iNOS inhibitor), methyl-β-cyclodextrin and monodansylcadaverine (inhibitors of caveolae- and clathrin-mediated endocytosis, respectively).

Conclusions:

These results suggest that endocytosis and NO generation participate in indium-induced 8-nitroG formation. NO released from indium-exposed inflammatory cells may induce DNA damage in adjacent lung epithelial cells and contribute to carcinogenesis.

Introduction

Indium has been used as a metal, in alloys, and for electronics application. The worldwide demand for indium metal has increased noticeably in recent years due to growth of microelectronics industry. Indium compounds, especially indium-tin oxide (ITO), have been used for the production of liquid crystal displays, mobile phone displays and solar cells due to their characteristics of high electrical conductivity, transparency and mechanical resistance. The first case of indium-related interstitial pneumonia in a worker who had been working as a grinder in an ITO target manufacturing facility in Japan was reported in 2003¹⁾. An epidemiological study has demonstrated the relationship between indium exposure and interstitial changes in the lungs detected by high-resolution computed tomography²⁾. A similar causal relationship between indium exposure and interstitial lung disorders has been reported in Korea³⁾. In addition, two cases of pulmonary alveolar proteinosis in the United States and one case of pulmonary alveolar proteinosis in China have been reported^4,5). A recent 5-year follow-up study was conducted on indium-exposed workers and long-term adverse effects on emphysematous changes were observed⁶⁾. Lung carcinogenicity has been observed in long-term inhalation studies with indium compounds in experimental animals. A 2-year animal experiment of ITO inhalation revealed that there was a clear evidence of lung carcinogenicity in male and female rats⁷⁾. Recently, ITO has been evaluated as a Group 2B carcinogen (possibly carcinogenic to humans) by the International Agency for Research on Cancer (IARC)^8,9). In addition, indium phosphide has been classified as a Group 2A carcinogen (probably carcinogenic to human) by IARC¹⁰⁾. These findings raise a concern that lung cancer may occur in humans exposed to indium compounds in the future.

Inhalation exposure to particulate matters causes chronic inflammation in the respiratory systems, leading to lung carcinogenesis and fibrosis. Chronic inflammation, which is induced by various environmental factors such as infection, inflammatory diseases and physicochemical agents, is postulated to play a substantial part in human carcinogenesis^11,12). Under inflammatory conditions, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated in inflammatory and epithelial cells. These reactive species interact with DNA bases to form oxidative and nitrative DNA lesions^13,14). 8-Nitroguanine (8-nitroG) is a mutagenic nitrative DNA lesion formed during chronic inflammation. Nitric oxide (NO) reacts with superoxide (O₂^{・ -}) to form highly reactive peroxide (ONOO^-), which interacts with guanine to form 8-nitroG¹⁵⁾. 8-NitroG was formed at the sites of carcinogenesis in various animal models and clinical specimens of cancer-prone inflammatory diseases and is expected to be a potential biomarker of inflammation-related carcinogenesis^16,17). We have demonstrated that 8-nitroG was formed in cultured macrophage and lung epithelial cells by particulate matters, including multiwalled carbon nanotube^18,19) and carbon black²⁰⁾.

In this study, to investigate the mechanism of indium-induced genotoxicity and carcinogenicity, we performed immunocytochemical analysis to examine 8-nitroG formation in RAW 264.7 mouse macrophages treated with indium oxide (In₂O₃), which accounts for a large part (approximately 90%-95%) of ITO. We used RAW 264.7 cells in this study, because macrophages in airway and alveolar spaces play a substantial role in lung carcinogenesis via the interaction with epithelial cells^21,22). Macrophages initially contact and engulf inhaled particles, and then release ROS, RNS and inflammatory cytokines. These molecules secondarily induce the generation of reactive species and the expression of inflammatory molecules in adjacent epithelial cells, leading to lung carcinogenesis²¹⁾. We have previously reported that endocytosis plays a role in DNA damage caused by particulate matters^18-20). To clarify the mechanism of indium-induced genotoxicity, we examined the effects of endocytosis inhibitors on 8-nitroG formation.

Materials and Methods

Preparation of In₂O₃ particles

In₂O₃ nanoparticles (primary diameter: 30-50 nm) were obtained from Nanostructured and Amorphous Materials, Inc. (Houston, TX, USA). Purity was 99.99% and surface area was 15 m²/g (disclosed by the manufacturer). In₂O₃ was suspended in Dulbecco's Modified Eagles Medium (DMEM, Gibco/BRL, New York, NY, USA) containing 5% (v/v) heat-inactivated fetal bovine serum (FBS) and 100 mg/l kanamycin. The suspension was vortexed for 1 min and then sonicated for 20 min at 40 W with an ultrasonic homogenizer (Model 450 Branson Ultrasonic, Danbury, CT, USA) to disperse agglomerates as described previously¹⁹⁾. The suspensions of the agglomerates were stored at -80°C until use. We thawed and vortexed the suspensions to use for experiments, and measured the size distributions of the agglomerates with a Zetasizer Nano particle size analyzer (Malvern, Worcestershire, UK) as described previously^19,20).

Evaluation of indium-induced cytotoxicity

We evaluated In₂O₃-induced cytotoxicity by trypan blue exclusion assay. RAW 264.7 mouse macrophage cells (5 × 10⁵ cells/ml, RIKEN BioResource Center, Tsukuba, Japan) were seeded in 1.2 ml of DMEM containing 5% (v/v) FBS and 100 mg/l kanamycin in 6 Well Clear Multiwell Plates (BD Falcon, Franklin Lakes, NJ, USA). Immediately after seeding, the cells were incubated with 0-50 μg/ml of In₂O₃ for 24 h at 37°C in an atmosphere containing 5% CO₂. We employed these concentrations of In₂O₃, because we have previously demonstrated that other types of nanomaterials induced significant cytotoxic and/or genotoxic effects at similar concentrations^18,20). Then, the cell suspensions were mixed with trypan blue, and the viability was calculated with a TC20 Automated cell counter (Bio-Rad Laboratories, Hercules CA, USA).

Detection of 8-nitroG formation

Localization of 8-nitroG formation in In₂O₃-exposed cells was assessed by immunocytochemical analysis as described previously²⁰⁾. RAW 264.7 cells (5 × 10⁵ cells/ml) were seeded in 1.2 ml of DMEM containing 5% (v/v) FBS and 100 mg/l kanamycin in 6 Well Clear Multiwell Plates (BD Falcon). Then, the cells were incubated with In₂O₃ for 2 or 4 h at 37°C in an atmosphere containing 5% CO₂. In a certain experiment, RAW 264.7 cells were co-treated with 1 μM 1400 W [an inhibitor of inducible NO synthase (iNOS) ], 0.5 mM methyl-β-cyclodextrin (MBCD, an inhibitor of caveolae-mediated endocytosis), 50 μM monodansylcadaverine (MDC, an inhibitor of clathrin-mediated endocytosis). We employed these concentrations of the inhibitors, because they did not show significant cytotoxic effects as described in the Results section. These inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA).

After the treatment with In₂O₃, the cells were fixed with 4% (v/v) formaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature and washed with PBS. Then, the cells were treated with 0.5% (v/v) Triton X100 for 3 min and incubated with 1% (w/v) skim milk for 1 h at room temperature. To detect 8-nitroG, the cells were incubated with rabbit polyclonal anti-8-nitroG antibody (1 μg/ml) produced by our group^23,24) overnight at room temperature. Then the cells were incubated with fluorescent secondary antibody [Alexa 594-labeled goat antibody against rabbit IgG (1:400, molecular probes, Eugene, OR, USA) ] for 3 h. The nuclei were stained with 5 μM Hoechst 33258 (Polysciences Inc., Warrington, PA, USA). The stained cells were examined under florescent microscope (BX53, Olympus, Tokyo, Japan). Staining intensity of 8-nitroG per cell was quantified by analyzing 3-5 randomly selected fields per sample, containing approximately 800 cells in average, with an ImageJ software.

Analysis of NO products

To analyze NO released from In₂O₃-treated cells, we measured the concentration of its products, nitrate (NO₃^-) and nitrite (NO₂^-), in the culture supernatant by using the Griess method. RAW 264.7 cells (5 × 10⁵ cells/ml) were seeded in 1.2 ml of phenol red-free DMEM (Gibco/BRL) containing 5% (v/v) FBS and 100 mg/l kanamycin in 6 Well Clear Multiwell Plates (BD Falcon). Then the cells were treated with 20 μg/mlIn₂O₃ for 2 or 4 h at 37°C. The culture supernatant was collected and centrifuged at 40,000 × g for 10 min at 4°C to remove In₂O₃ particles. To reduce NO₃^- to NO₂^-, the supernatant was incubated with 0.1 units/ml of nitrate reductase from Aspergillus niger (Sigma-Aldrich) in the presence of 1 mM glucose-6-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 0.3 units/ml of glucose-6-phosphate dehydrogenase and 20 μM NADPH (Oriental Yeast, Tokyo, Japan) for 30 min at room temperature. The reaction mixture was incubated with 0.25% (w/v) sulfanilamide (Griess reagent I, Wako) and 0.025% (w/v) naphthylethylenediamine (Griess reagent II, Sigma-Aldrich) in 0.625% (v/v) phosphoric acid for 10 min at room temperature. The absorbance was measured at 540 nm with a microplate reader (Model 680, Bio-Rad laboratories) and NO₂^- concentration was determined by comparison with a standard curve generated with sodium nitrate (NaNO₂, Wako).

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test or Student's t-test using an SPSS software (20.0 for Mac). Results were presented as means±SD. Probability values less than 0.05 were considered to be statistically significant.

Results

Dispersion and size distribution of In₂O₃

We suspended In₂O₃ in DMEM and dispersed agglomerates with an ultrasonic homogenizer. Fig. 1A shows the photographs of In₂O₃ agglomerates obtained before and after the sonication. Light microscopy revealed that most In₂O₃ agglomerates were dispersed into particles of a few micrometers in size, capable of reaching alveolus in human. The size distribution of In₂O₃ agglomerates showed that the diameters of most particles were distributed from approximately 100 to 500 nm as shown in Fig. 1B. The mean diameter of In₂O₃ agglomerates was 214.5 nm.

Fig. 1.

Dispersion of In₂O₃ agglomerates. In₂O₃ was suspended in DMEM and sonicated as described in Materials and Methods section. (A) In₂O₃ agglomerates before and after the sonication. The agglomerates were observed with a light microscope. Bar = 10 µm. (B) Size distribution of dispersed In₂O₃ particles. The size distribution was measured with a Zetasizer Nano particle size analyzer (Malvern Worcestershire UK).

Cytotoxicity of In₂O₃

We examined a cytotoxic effect of In₂O₃ on RAW 264.7 cells by trypan blue exclusion assay. Cell viability tended to decrease with increasing In₂O₃ concentrations (Fig. 2). ANOVA plus Tukey's test revealed that In₂O₃ significantly decreased the cell viability at 50 μg/ml compared with the control (p<0.01, Fig. 2).

Fig. 2.

Cytotoxic effect of In₂O₃ on RAW 264.7 cells. Cells were incubated with indicated concentrations of In₂O₃ for 24 h and cell viability was evaluated by trypan blue exclusion assay. The data were expressed as means ± SD of 3 independent experiments. **p <0.01, compared with the control by ANOVA followed by Tukey’s test.

8-NitroG formation in In₂O₃ -treated cells

To investigate 8-nitroG formation in RAW 264.7 cells treated with In₂O₃, we performed immunocytochemical analysis. Fig. 3A shows the formation of 8-nitroG in In₂O₃-treated cells. No or weak staining was observed in non-treated control, and the immunoreactivity of 8-nitroG was increased in In₂O₃-treated cells. 8-NitroG was mainly formed in the nucleus, which was stained with Hoechst 33258. Quantitative image analysis revealed that the staining intensity of 8-nitroG in In₂O₃-treated cells was significantly greater at 20 and 50 μg/ml than that in non-treated control (p<0.05 and p<0.01, respectively, Fig. 3B). We examined the time course of In₂O₃-induced 8-nitroG formation in RAW 264.7 cells at 20 μg/ml, because In₂O₃ induced a clear and significant increase in the staining intensity of 8-nitroG at this concentration. Fig. 4A shows that In₂O₃ induced 8-nitroG formation at 2 h and its immunoreactivity was further increased at 4 h. Image analysis revealed that In₂O₃ significantly increased 8-nitroG formation at 2 and 4 h compared with the control (p<0.05 and p<0.001, respectively, Fig. 4B).

Fig. 3.

8-NitroG formation in In₂O₃-treated cells. (A) Immunofluorescent images of 8-nitroG formation in In₂O₃-treated cells. RAW 264.7 cells were treated with indicated concentrations of In₂O₃ for 4 h at 37°C. 8-NitroG formation was detected by immunocytochemistry as described in Materials and Methods section. The nucleus was stained with Hoechst 33258. Magnification, ×200. (B) Quantitative image analysis of 8-nitroG formation in In₂O₃-treated cells. The staining intensity per cell was quantified with an ImageJ software. The vertical axis shows the fold increase in the staining intensity compared with that of the control, which was set at 1. The data were expressed as means ± SD of 3-4 independent experiments. *p <0.05 and **p <0.01 compared with the control by ANOVA followed by Tukey’s test.

Fig. 4.

Time course of 8-nitroG formation in In₂O₃-treated cells. (A) Immunofluorescent images of 8-nitroG formation in In₂O₃-treated cells. RAW 264.7 cells were treated with 20 µg/ml of In₂O₃ for indicated durations at 37°C. 8-NitroG formation was detected by immunocytochemistry as described in Materials and Methods section. The nucleus was stained with Hoechst 33258. Magnification, ×200. (B) Quantitative image analysis of 8-nitroG formation in In₂O₃-treated cells. The staining intensity per cell was quantified with an ImageJ software. The vertical axis shows the fold increase in the staining intensity compared with that of the control at 2 h, which was set at 1. The data were expressed as means ± SD of 3-4 independent experiments. *p <0.05 and ***p <0.001 compared with the control by Student’s t-test.

NO release from In₂O₃-treated cells

We measured the levels of NO products, NO₂^- and NO₃^- in culture supernatant of In₂O₃-treated RAW 264.7 cells using nitrate reductase and Griess reagents as shown in Fig. 5. Compared with the control, In₂O₃ significantly increased NO release from the cells at 2 and 4 h (p<0.01, Fig. 5). The amount of NO released from In₂O₃-exposed cells was slightly decreased at 4 h compared to that at 2 h (Fig. 5).

Fig. 5.

NO release from In₂O₃-treated cells. RAW 264.7 cells were treated with 20 µg/ml of In₂O₃ for indicated durations at 37°C. NO₂^– and NO₃^– levels in the culture supernatant were measured by the Griess method as described in Materials and Methods section. The data were expressed as means ± SD of 4 independent experiments. **p <0.01 compared with control by Student’s t-test.

Effects of iNOS and endocytosis inhibitors on In₂O₃-induced 8-nitroG formation

We examined the effects of iNOS and endocytosis inhibitors on In₂O₃-induced 8-nitroG formation in RAW 264.7 cells by immunocytochemistry. In₂O₃ induced clear 8-nitroG formation, and its immunoreactivity was largely suppressed by the treatment with 1400 W (an iNOS inhibitor), MBCD (an inhibitor of caveolae-mediated endocytosis) and MDC (an inhibitor of clathrin-mediated endocytosis) as shown in Fig. 6A. We examined the viability of RAW 264.7 cells treated with In₂O₃ and these inhibitors to confirm that they do not exhibit cytotoxicity. In₂O₃ alone and In₂O₃ plus each inhibitor induced no or slight decrease in the cell viability compared with the control under the conditions used (Fig. 6B), indicating that the reduction in 8-nitroG formation by the inhibitors was not due to their cytotoxic effects. In addition, these inhibitors alone (without In₂O₃) did not show cytotoxic effects (Fig. S1).

Fig. 6.

Effects of iNOS and endocytosis inhibitors on In₂O₃-induced DNA damage and their cytotoxicity. (A) Effects of iNOS and endocytosis inhibitors on In₂O₃-induced 8-nitroG formation. RAW 264.7 cells were treated with 20 µg/ml of In₂O₃ for 4 h at 37°C. The cells were co-treated with 1 µM 1400W, 0.5 mM MBCD or 50 µM MDC. 8-NitroG formation was detected by immunocytochemistry as described in Materials and Methods section. The nucleus was stained with Hoechst 33258. Magnification, ×200. (B) Cytotoxicity of In₂O₃ plus inhibitors for iNOS and endocytosis. RAW 264.7 cells were treated with 20 µg/ml In₂O₃ plus 1 µM 1400W, 0.5 mM MBCD or 50 µM MDC for 4 h at 37°C. Cell viability was evaluated by trypan blue exclusion assay. The data were expressed as means ± SD of 4 independent experiments.

Discussion

In this study, we demonstrated that In₂O₃ induced the formation of 8-nitroG, a nitrative DNA lesion, in the nucleus of RAW 264.7 cells. We have previously reported that 8-nitroG was formed at the sites of inflammation-related carcinogenesis in animal models and clinical specimens^16,17,25). In the lung tissues of asbestos-exposed mice, 8-nitroG was formed in bronchial epithelial cells²⁶⁾. The extent of 8-nitroG formation was significantly correlated with asbestos contents in human lung tissues²⁷⁾. Multiwalled carbon nanotube and carbon black induced 8-nitroG formation in human lung epithelial cells under occupationally relevant conditions^18-20). In this study, immunocytochemistry and quantitative image analysis revealed that In₂O₃ significantly increased 8-nitroG formation in RAW 264.7 cells. The concentrations of In₂O₃ in this study appear to be higher than occupationally relevant concentrations according to the estimation of alveolar deposition of other types of nanomaterials^19,20), but In₂O₃ caused 8-nitroG formation in a short incubation time of 2 h. Therefore, it is supposed that long-time exposure to indium compounds causes genotoxicity in the respiratory systems even under occupationally relevant conditions. A recent study has demonstrated that indium compounds induced DNA damage in lung epithelial cells, but their concentrations were one or two orders of magnitude greater than those in this study²⁸⁾. Therefore, the experimental conditions in this study appear to be more relevant to occupational settings. NO generation was also significantly increased in In₂O₃-treated RAW 264.7 cells. In₂O₃-induced 8-nitroG formation was inhibited by 1400 W, suggesting that 8-nitroG formation is dependent on iNOS expression. The immunoreactivity of 8-nitroG was increased in In₂O₃-exposed cells at 4 h to a greater extent than at 2 h, whereas the amount of NO in the culture supernatant at 4 h was slightly lower than that at 2 h. These results indicate that In₂O₃ initially induced NO generation, which preceded 8-nitroG formation, as demonstrated in previous studies on nanoparticle-induced 8-nitroG formation^19,20). Nuclear factor-κB (NF-κB) is a transcription factor that regulates expression of various genes involved in inflammatory responses, including iNOS, and ROS and RNS can induce NF-κB activation²⁹⁾. These studies raise the possibility that reactive species and NF-κB positively regulate each other and contribute to DNA damage.

8-NitroG is considered to be a potentially mutagenic DNA lesion. The glycoside bond between 8-nitroG and deoxyribose is chemically unstable and thus 8-nitroG can be spontaneously released to form an apurinic site³⁰⁾. An apurinic site forms a pair with adenine during DNA replication, leading to G→T transversion³¹⁾. 8-NitroG also forms a pair with adenine and causes a similar type of base substitution³²⁾. Although recent in vitro studies have demonstrated that indium compounds induce cytotoxicity³³⁾, inflammatory responses³⁴⁾ and DNA damage²⁸⁾, this is the first study showing that indium compounds cause NO-dependent DNA damage in cultured cells.

8-NitroG formation induced by In₂O₃ was suppressed by MBCD and MDC, inhibitors of caveolae- and clathrin-mediated endocytosis, respectively. These findings raise a possibility that In₂O₃ particles were initially taken up into cells via endocytosis, resulting in induction of inflammatory responses and DNA damage. The average diameter of In₂O₃ agglomerates was 214.5 nm and the size of most agglomerates was distributed from 100 to 500 nm. Submicron-sized particles are internalized into cells by caveolae- and clathrin-mediated endocytosis^35,36), and inhibitors of clathrin-mediated endocytosis reduced cellular uptake of particles up to 200 nm³⁵⁾. We have recently reported that carbon black agglomerates of similar size caused DNA damage in macrophages, which involved clathrin-mediated endocytosis²⁰⁾. Therefore, according to the size distribution, In₂O₃-induced DNA damage appears to be, at least in part, accounted for by clathrin-mediated endocytosis. Nanoparticles are considered to induce cell death via the cellular internalization and lysosomal dysfunction³⁷⁾. In this study, high dose (50 μg/ml) of In₂O₃ significantly decreased the cell viability. These results suggest that In₂O₃ causes cell death in an endocytosis-dependent manner, although the precise mechanism remains to be clarified.

The molecular mechanisms of inflammatory responses induced by particulate matters have been investigated but not well understood. Some studies have demonstrated that fibrous and particulate materials cause inflammatory responses in the respiratory systems via NLRP3 inflammasome in in vivo experiments³⁸⁾. It has been reported that ITO induced pro-inflammatory responses in a cultured macrophage cell line, which are partially accounted for by inflammasome activation³⁴⁾. Recently, we proposed a new mechanism of 8-nitroG formation induced by particulate matters in lung epithelial cell lines, which involves the activation of Toll-like receptor 9 (TLR9) in lysosomes. Particulate matters cause cell injury or necrosis, and the nuclear protein HMGB1 and DNA are released from the cells. Then, the HMGB1-DNA complex interacts with receptor for advanced glycation end-products (RAGE) and is internalized into lysosomes. CpG DNA is recognized by TLR9, leading to NO generation and resulting DNA damage¹⁹⁾. Recent studies have demonstrated that HMGB1 is internalized into macrophages via endocytosis³⁹⁾ and that extracellular HMGB1 plus DNA induce TLR9-mediated macrophage activation⁴⁰⁾. Therefore, this pathway may play a substantial role in DNA damage in indium-exposed macrophages in addition to lung epithelial cells. There is a possibility that the reduction in In₂O₃-induced 8-nitroG formation by endocytosis inhibitors is partially accounted for by suppression of cellular uptake of the HMGB1-DNA-RAGE complex.

In conclusion, we have demonstrated that In₂O₃ induced nitrative DNA damage in cultured cells, and this event involves endocytosis and iNOS-dependent NO generation. NO released from indium-exposed inflammatory cells, including macrophages, may induce DNA damage not only in these cells but also in adjacent lung epithelial cells, which may contribute to indium-induced carcinogenesis.

Acknowledgments: This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (Grant numbers 25670313 and 15H04784).

Conflicts of interest: None declared.