Melanopsin DNA aptamers can regulate input signals of mammalian circadian rhythms by altering the phase of the molecular clock.

DNA aptamers can bind specifically to biomolecules to modify their function, potentially making them ideal oligonucleotide therapeutics. Herein, we screened for DNA aptamer of melanopsin (OPN4), a blue-light photopigment in the retina, which plays a key role using light signals to reset the phase of circadian rhythms in the central clock. Firstly, 15 DNA aptamers of melanopsin (Melapts) were identified following eight rounds of Cell-SELEX using cells expressing melanopsin on the cell membrane. Subsequent functional analysis of each Melapt was performed in a fibroblast cell line stably expressing both Period2:ELuc and melanopsin by determining the degree to which they reset the phase of mammalian circadian rhythms in response to blue-light stimulation. Period2 rhythmic expression over a 24-h period was monitored in Period2:ELuc stable cell line fibroblasts expressing melanopsin. At subjective dawn, four Melapts were observed to advance phase by >1.5 h, while seven Melapts delayed phase by >2 h. Some Melapts caused a phase shift of approximately 2 h, even in the absence of photostimulation, presumably because Melapts can only partially affect input signaling for phase shift. Additionally, some Melaps were able to induce phase shifts in Per1::luc transgenic (Tg) mice, suggesting that these DNA aptamers may have the capacity to affect melanopsin in vivo. In summary, Melapts can successfully regulate the input signal and shifting phase (both phase advance and phase delay) of mammalian circadian rhythms in vitro and in vivo.

Sedanolide Activates KEAP1-NRF2 Pathway and Ameliorates Hydrogen Peroxide-Induced Apoptotic Cell Death.

Sedanolide is a bioactive compound with anti-inflammatory and antitumor activities. Although it has been recently suggested that sedanolide activates the nuclear factor E2-related factor 2 (NRF2) pathway, there is little research on its effects on cellular resistance to oxidative stress. The objective of the present study was to investigate the function of sedanolide in suppressing hydrogen peroxide (H2O2)-induced oxidative damage and the underlying molecular mechanisms in human hepatoblastoma cell line HepG2 cells. We found that sedanolide activated the antioxidant response element (ARE)-dependent transcription mediated by the nuclear translocation of NRF2. Pathway enrichment analysis of RNA sequencing data revealed that sedanolide upregulated the transcription of antioxidant enzymes involved in the NRF2 pathway and glutathione metabolism. Then, we further investigated whether sedanolide exerts cytoprotective effects against H2O2-induced cell death. We showed that sedanolide significantly attenuated cytosolic and mitochondrial reactive oxygen species (ROS) generation induced by exposure to H2O2. Furthermore, we demonstrated that pretreatment with sedanolide conferred a significant cytoprotective effect against H2O2-induced cell death probably due to preventing the decrease in the mitochondrial membrane potential and the increase in caspase-3/7 activity. Our study demonstrated that sedanolide enhanced cellular resistance to oxidative damage via the activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 pathway.

The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

An international validation study of the interleukin-2 luciferase leukocyte toxicity test (IL-2 Luc LTT) to evaluate potential immunosuppressive chemicals and its performance after use with the interleukin-2 luciferase assay (IL-2 Luc assay).

We previously reported that the IL-2 Luc LTT can detect immunosuppressive effects of drugs that are attributed to their antimitotic activity. Here, we report an official validation study of the IL-2 Luc LTT. In the Phase I study that evaluated five coded chemicals, the within-laboratory reproducibility of three independent laboratories was 100.0%. In the combined results of the Phase I and II studies that evaluated 20 coded chemicals, the between-laboratory reproducibility was 92.0%. When compared with the reference data based on the previously-reported immunotoxicological information, the predictivity of the combined Phase I and II studies was 76.0% for Lab A and 72.0% for Labs B and C. In contrast, in the study in which the lead laboratory examined 37 non-pharmaceutical chemicals, the predictivity of the IL-2 Luc LTT and the IL-2 Luc assay was 48.6% and 64.9%, respectively, whereas that of the combined assays was 74.3%. It is clear that an integrated approach combining multiple assays is necessary for the development of in vitro immunosuppression testing. These data suggest that the IL-2 Luc LTT alone is not sufficient as a component of the integrated approach, but the combination of the IL-2 Luc assay and IL-2 Luc LTT is promising.

Hydroxy-ß-sanshool isolated from Zanthoxylum piperitum (Japanese pepper) shortens the period of the circadian clock.

We showed that an ethanol extract from Zanthoxylum piperitum can shorten the circadian rhythm at the cellular level and that this activity was due to hydroxy-ß-sanshool, a secondary metabolite in this plant. An ethanol extract of Z. piperitum was repeatedly fractionated using solid phase extraction and reverse-phase HPLC, then the circadian rhythms of cells to which the fractions were loaded were monitored using real-time reporter gene assays. We purified one HPLC peak and identified it as hydroxy-ß-sanshool using liquid chromatography (LC)-precision-mass spectrometry (MS). This compound shortened the period of Bmal1 and Per2 at the cellular level. Incubation cells for 24 h with hydroxy-ß-sanshool resulted in upregulated Per2 promoter activity. Hydroxy-ß-sanshool also dose-dependently upregulated expression of the clock genes Bmal1, Per1, Per2 and Cry1 and the clock-controlled oxidative stress responsive genes Gpx1and Sod2.

Panel of human cell lines with human/mouse artificial chromosomes.

Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. Recently, microcell-mediated chromosome transfer (MMCT) of HACs/MACs has been achieved in various human cells that include human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene introduction with HACs/MACs requires laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via MMCT. To overcome these limitations and accelerate chromosome vector-based functional assays in human cells, we established various human cell lines (HEK293, HT1080, hiMSCs, and hiPSCs) with HACs/MACs that harbour a gene-loading site via MMCT. Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into these preprepared HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors. The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using this HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs.

Brain-derived neurotrophic factor predominantly regulates the expression of synapse-related genes in the striatum: Insights from in vitro transcriptomics.

AIM: The striatum, a main component of the basal ganglia, is a critical part of the motor and reward systems of the brain. It consists of GABAergic and cholinergic neurons and receives projections of dopaminergic, glutamatergic, and serotonergic neurons from other brain regions. Brain-derived neurotrophic factor (BDNF) plays multiple roles in the central nervous system, and striatal BDNF has been suggested to be involved in psychiatric and neurodegenerative disorders. However, the transcriptomic impact of BDNF on the striatum remains largely unknown. In the present study, we performed transcriptomic profiling of striatal cells stimulated with BDNF to identify enriched gene sets (GSs) and their novel target genes in vitro. METHODS: We carried out RNA sequencing (RNA-Seq) of messenger RNA extracted from primary dissociated cultures of rat striatum stimulated with BDNF and conducted Generally Applicable Gene-set Enrichment (GAGE) analysis on 10599 genes. Significant differentially expressed genes (DEGs) were determined by differential expression analysis for sequence count data 2 (DESeq2). RESULTS: GAGE analysis identified significantly enriched GSs that included GSs related to regulation and dysregulation of synaptic functions, such as synaptic vesicle cycle and addiction to nicotine and morphine, respectively. It also detected GSs related to various types of synapses, including not only GABAergic and cholinergic synapses but also dopaminergic and glutamatergic synapses. DESeq2 revealed 72 significant DEGs, among which the highest significance was observed in the apolipoprotein L domain containing 1 (Apold1). CONCLUSIONS: The present study indicates that BDNF predominantly regulates the expression of synaptic-function-related genes and that BDNF promotes synaptogenesis in various subtypes of neurons in the developing striatum. Apold1 may represent a unique target gene of BDNF in the striatum.

Application of Micropore Device for Accurate, Easy, and Rapid Discrimination of Saccharomyces pastorianus from Dekkera spp.

Traceability analysis, such as identification and discrimination of yeasts used for fermentation, is important for ensuring manufacturing efficiency and product safety during brewing. However, conventional methods based on morphological and physiological properties have disadvantages such as time consumption and low sensitivity. In this study, the resistive pulse method (RPM) was employed to discriminate between Saccharomyces pastorianus and Dekkera anomala and S. pastorianus and D. bruxellensis by measuring the ionic current response of cells flowing through a microsized pore. The height and shape of the pulse signal were used for the simultaneous measurement of the size, shape, and surface charge of individual cells. Accurate discrimination of S. pastorianus from Dekkera spp. was observed with a recall rate of 96.3 ± 0.8%. Furthermore, budding S. pastorianus was quantitatively detected by evaluating the shape of the waveform of the current ionic blockade. We showed a proof-of-concept demonstration of RPM for the detection of contamination of Dekkera spp. in S. pastorianus and for monitoring the fermentation of S. pastorianus through the quantitative detection of budding cells.

Bioluminescence Measurement of Time-Dependent Dynamic Changes of CYP-Mediated Cytotoxicity in CYP-Expressing Luminescent HepG2 Cells.

We sought to develop a cell-based cytotoxicity assay using human hepatocytes, which reflect the effects of drug-metabolizing enzymes on cytotoxicity. In this study, we generated luminescent human hepatoblastoma HepG2 cells using the mouse artificial chromosome vector, in which click beetle luciferase alone or luciferase and major drug-metabolizing enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are expressed, and monitored the time-dependent changes of CYP-mediated cytotoxicity expression by bioluminescence measurement. Real-time bioluminescence measurement revealed that compared with CYP-non-expressing cells, the luminescence intensity of CYP-expressing cells rapidly decreased when the cells were treated with low concentrations of aflatoxin B1 or primaquine, which exhibits cytotoxicity in the presence of CYP3A4 or CYP2D6, respectively. Using kinetics data obtained by the real-time bioluminescence measurement, we estimated the time-dependent changes of 50% inhibitory concentration (IC50) values in the aflatoxin B1- and primaquine-treated cell lines. The first IC50 value was detected much earlier and at a lower concentration in primaquine-treated CYP-expressing HepG2 cells than in primaquine-treated CYP-non-expressing cells, and the decrease of IC50 values was much faster in the former than the latter. Thus, we successfully monitored time- and concentration-dependent dynamic changes of CYP-mediated cytotoxicity expression in CYP-expressing luminescent HepG2 cells by means of real-time bioluminescence measurement.

Effect of Electrolyte Concentration on Cell Sensing by Measuring Ionic Current Waveform through Micropores.

Immunostaining has been widely used in cancer prognosis for the quantitative detection of cancer cells present in the bloodstream. However, conventional detection methods based on the target membrane protein expression exhibit the risk of missing cancer cells owing to variable protein expressions. In this study, the resistive pulse method (RPM) was employed to discriminate between cultured cancer cells (NCI-H1650) and T lymphoblastoid leukemia cells (CCRF-CEM) by measuring the ionic current response of cells flowing through a micro-space. The height and shape of a pulse signal were used for the simultaneous measurement of size, deformability, and surface charge of individual cells. An accurate discrimination of cancer cells could not be obtained using 1.0 × phosphate-buffered saline (PBS) as an electrolyte solution to compare the size measurements by a microscopic observation. However, an accurate discrimination of cancer cells with a discrimination error rate of 4.5 ± 0.5% was achieved using 0.5 × PBS containing 2.77% glucose as the electrolyte solution. The potential application of RPM for the accurate discrimination of cancer cells from leukocytes was demonstrated through the measurement of the individual cell size, deformability, and surface charge in a solution with a low electrolyte concentration.

Highly Sensitive and Rapid Quantitative Detection of Plasmodium falciparum Using an Image Cytometer.

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, we investigated whether an imaging cytometer was useful for the highly sensitive quantitative detection of parasites. Whole blood samples were prepared from uninfected individuals spiked with Plasmodium falciparum-infected erythrocytes. Thereafter, erythrocytes were purified using a push column comprising of a syringe filter unit with SiO2-nanofiber filters. After adding the erythrocytes, stained with nuclear stain, to a six-well plate, quantitative detection of the parasites was performed using an image cytometer, CQ1. Imaging of 2.6 × 106 erythrocytes was completed in 3 min, and the limit of detection indicated parasitemia of 0.00010% (≈5 parasites/µL of blood). In addition to rapid, highly sensitive, and quantitative detection, the ease of application and economic costs, image cytometry could be efficiently applied to diagnose submicroscopic parasites in infected people from endemic countries.

Meganuclease-Based Artificial Transcription Factors.

Embedding middle-scale artificial gene networks in live mammalian cells is one of the most important future goals for cell engineering. However, the applications of the highly orthogonal and conventional artificial transcription factors currently available are limited. In this study, we present a scalable pipeline to produce artificial transcription factors based on homing endonucleases, also known as meganucleases. The introduction of mutations at critical sites for nuclease activity renders these homing endonucleases a simple but highly specific DNA binding domain for their specific DNA target. The introduction of inactivated meganucleases linked to transcriptional activator domains strongly induced reporter gene expression, while their fusion to transcriptional repressor domains suppressed them. In addition, we show that inactivated meganuclease-based transcription factors could be embedded in the synthetic membrane receptor synNotch and used to construct synthetic circuits. These results suggest that inactivated meganucleases are useful DNA-binding domains for the construction of synthetic transcription factors in mammalian cells.

A new method of Bayesian causal inference in non-stationary environments.

Bayesian inference is the process of narrowing down the hypotheses (causes) to the one that best explains the observational data (effects). To accurately estimate a cause, a considerable amount of data is required to be observed for as long as possible. However, the object of inference is not always constant. In this case, a method such as exponential moving average (EMA) with a discounting rate is used to improve the ability to respond to a sudden change; it is also necessary to increase the discounting rate. That is, a trade-off is established in which the followability is improved by increasing the discounting rate, but the accuracy is reduced. Here, we propose an extended Bayesian inference (EBI), wherein human-like causal inference is incorporated. We show that both the learning and forgetting effects are introduced into Bayesian inference by incorporating the causal inference. We evaluate the estimation performance of the EBI through the learning task of a dynamically changing Gaussian mixture model. In the evaluation, the EBI performance is compared with those of the EMA and a sequential discounting expectation-maximization algorithm. The EBI was shown to modify the trade-off observed in the EMA.

An international validation study of the IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on T cells and a proposal for reference data for immunotoxic chemicals.

To evaluate the immunotoxic effects of xenobiotics, we have established the Multi-ImmunoTox assay, in which three stable reporter cell lines are used to evaluate the effects of chemicals on the IL-2, IFN-γ, IL-1ß and IL-8 promoters. Here, we report the official validation study of the IL-2 luciferase assay (IL-2 Luc assay). In the Phase I study that evaluated five coded chemicals in three sets of experiments, the average within-laboratory reproducibility was 86.7%. In the Phase II study, 20 coded chemicals were evaluated at multiple laboratories. In the combined results of the Phase I and II studies, the between-laboratory reproducibility was 80.0%. These results suggested that the IL-2 Luc assay was reproducible both between and within laboratories. To determine the predictivity, we collected immunotoxicological information and constructed the reference data by classifying the chemical into immunotoxic compounds targeting T cells or others according to previously reported criteria. When compared with the reference data, the average predictivity of the Phase I and II studies was 75.0%, while that of additional 60 chemicals examined by the lead laboratory was 82.5%. Although the IL-2 Luc assay alone is not sufficient to predict immunotoxicity, it will be a useful tool when combined with other immune tests.

PCR-based approach for site-specific conjugation of long double-stranded DNA to a single-domain VHH antibody.

Site-specific conjugation of double-stranded DNA using antibodies enables the development of unique applications for antibody-drug conjugates utilizing recent advances in nucleic acid medicines. Here, we describe a novel method to conjugate a camelid-derived single-domain VHH (variable domain of a heavy chain antibody) antibody with arbitrarily sized double-stranded DNA by PCR. Cysteine in anti-human epidermal growth factor receptor (EGFR) VHH was replaced by alanine, and an unpaired cysteine was introduced at the carboxyl terminus. These modifications enabled site-specific labelling with a maleimide-modified DNA oligo via thioether bond formation; the ensuing product-single-stranded DNA conjugated at the carboxyl terminus of VHH-retained its affinity for EGFR. To investigate whether this VHH-single-stranded DNA conjugate might be used as a forward primer, we subjected it to PCR, producing 100-500 bp DNA. We confirmed the amplification of the VHH-double-stranded DNA conjugate by examining its mobility on acrylamide gel; retention of the binding affinity of the conjugate for EGFR was identified by immuno-PCR.

Extended Bayesian inference incorporating symmetry bias.

We start by proposing a causal induction model that incorporates symmetry bias. This model has two parameters that control the strength of symmetry bias and includes conditional probability and conventional models of causal induction as special cases. We calculated the determination coefficients between assessments by participants in eight types of causal induction experiments and the estimated values using the proposed model. The mean coefficient of determination was 0.93. Thus, it can reproduce causal induction of human judgment with high accuracy. We further propose a human-like Bayesian inference method to replace the conditional probability in Bayesian inference with the aforementioned causal induction model. In this method, two components coexist: the component of Bayesian inference, which updates the degree of confidence for each hypothesis, and the component of inverse Bayesian inference that modifies the model of each hypothesis. In other words, this method allows not only inference but also simultaneous learning. Our study demonstrates that the method addresses unsteady situations where the target of inference occasionally changes not only by making inferences based on knowledge (model) and observation data, but also by modifying the model itself.

Novel and Stable Dual-Color IL-6 and IL-10 Reporters Derived from RAW 264.7 for Anti-Inflammation Screening of Natural Products.

Our previous study suggested that the interleukin (IL)-6 and IL-10 could serve as good biomarkers for chronic inflammatory disease. We previously established an IL-6 and IL-10 reporters assay that could examine reporter activity along with the reference gene in LPS-induced RAW 264.7 cells. In this study, we described new and stable RAW 264.7 derived dual-color IL-6/gapdh and IL-10/gapdh reporters. This assay allowed us to easily determine relative IL-6 and IL-10 levels with 96-well plate within one step. We evaluated the relative IL-6 and IL-10 levels in the LPS-induced stable cells testing 52 natural products by real-time bioluminescence monitoring and time-point determination using a microplate luminometer. The relative IL-6 and IL-6/IL-10 values decreased by the crude ethanol extracts from nutmeg and by 1'S-1'-acetoxychavicol from greater galangal using real-time bioluminescence monitoring. At the same time, the relative IL-10 was induced. The relative IL-6 and IL-6/IL-10 decreased by crude ethanol extracts from nutmeg and 1'S-1'-acetoxychavicol acetate at 6 h. Only crude ethanol extract from nutmeg induced IL-10 at 6 h. We suggested that the use of these stable cells by real-time monitoring could serve as a screening assay for anti-inflammatory activity and may be used to discover new drugs against chronic inflammatory disease.

The performance of an in vitro skin sensitisation test, IL-8 Luc assay (OECD442E), and the integrated approach with direct peptide reactive assay (DPRA).

In all current in vitro skin sensitisation assays, DMSO is used to dissolve water-insoluble chemicals. However, our previous study suggested the superiority of the modified IL-8 Luc assay (mIL-8 Luc), in which X-VIVOTM 15 is used to dissolve chemicals, over the original assay using DMSO (oIL-8 Luc). In this study, to confirm the superiority of the mIL-8 Luc, we first increased the number of chemicals examined and demonstrated the superiority of the mIL-8 Luc, in which the mIL-8 Luc provided 87.6% of sensitivity, 74.2% of specificity, and 84.6% of accuracy. Next, to clarify the cause of false negative judgment by the mIL-8 Luc, we examined the effects of physical properties of chemicals on judgment. The results demonstrated that high molecular weight, high LogKo/w, or poor water solubility, did not cause false negative judgment. When it was accepted as an OECD test guideline, the criteria of the mIL-8 Luc to determine sensitisers were modified to further decrease false negative judgment by poor solubility. By applying the new criteria, the test guideline IL-8 Luc assay (tgIL-8 Luc) improved sensitivity but decreased specificity and increased the number of chemicals that cannot be judged. To overcome this problem, we examined a simple combination of the tgIL-8 Luc with direct peptide reactive assay (DPRA), which could improve specificity and decrease the number of the chemicals that cannot be judged. These data suggest that the tgIL-8 Luc is a promising in vitro skin sensitisation assay in combination with other in vitro or in chemico methods.

Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells.

In previous study, we suggested that the interleukin (IL)-6 and IL-10 could serve as a good biomarker for anti-inflammation that related to chronic inflammatory disease. Recently, we are finding new anti-inflammation compounds from natural products by screening of IL-6 and IL-10 levels. Although, we could measure IL-6 and IL-10 levels by several methods. However, all methods could not measure continuous kinetic of IL-6 and IL-10 levels. Most methods have multiple steps and take a long time. Therefore, there is no a suitable method for screening. To this end, we established IL-6 and IL-10 promoter assay which can monitor with reference gene as Glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter in living single cell. It could determine IL-6 and IL-10 levels continuously in real-time within two steps. We evaluated IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells with well-known anti-inflammatory compounds such as quercetin, xanthones, ß-D-glucan and dexamethasone. As the results, the expression of IL-6 and IL-10 reporters were strongly induced by LPS. The expression of IL-6 reporter was inhibited by all anti-inflammation compounds in LPS-induced RAW 264.7 cells. The expression of IL-10 reporter was inhibited by quercetin, xanthones and dexamethasone in LPS-induced RAW 264.7 cells. While, expression of IL-10 reporter was induced by ß-D-glucan. These results indicated that this assay could use for determination of IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells for anti-inflammation activity. Moreover, the results showed that natural compounds have an effect on the time course of IL-6 and IL-10 expressions. Therefore, real-time monitoring has a merit for natural compounds screening. We suggested that this assay could serve as a compound screening assay for anti-inflammation activity.

Reactive oxygen species independent genotoxicity of indium tin oxide nanoparticles triggered by intracellular degradation.

Indium tin oxide (ITO) is widely used as a transparent conducting electrode in photoelectron devices. Because ITO production has soared, the potential health hazards caused by occupational exposure to this material have attracted much attention. However, little is known about the mechanisms of the toxic action of ITO nanoparticles (NPs). The present study was designed to examine the genotoxic mechanisms of ITO NPs using human lung epithelial A549 cells. We found that exposing A549 cells to ITO NPs triggered the intracellular accumulation of ITO NPs, the generation of reactive oxygen species (ROS), and the induction of DNA damage. Treatment of the cells with N-acetyl-l-cysteine (NAC), an ROS quenching agent, decreased intracellular ROS levels but not DNA damage, indicating that the genotoxic effect of ITO NPs is not mediated by intracellular ROS. Interestingly, treatment with ammonium chloride, a lysosomotropic agent, decreased intracellular solubility of ITO NPs and attenuated DNA damage. Nuclear accumulation of indium ions in ITO-NP-exposed cells was confirmed by inductively coupled plasma-mass spectrometry. Our results indicate that the ITO-NP-mediated genotoxicity is caused by indium ions that are solubilized in the acidic lysosomal condition and accumulated in the nucleus where they damage DNA, without the involvement of ROS.