Silica-coated magnetic nanoparticles activate microglia and induce neurotoxic D-serine secretion.

BACKGROUND: Nanoparticles have been studied for brain imaging, diagnosis, and drug delivery owing to their versatile properties due to their small sizes. However, there are growing concerns that nanoparticles may exert toxic effects in the brain. In this study, we assessed direct nanotoxicity on microglia, the resident macrophages of the central nervous system, and indirect toxicity on neuronal cells exerted by silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)]. METHODS: We investigated MNPs@SiO2(RITC)-induced biological changes in BV2 murine microglial cells via RNA-sequencing-based transcriptome analysis and gas chromatography-mass spectrometry-based intracellular and extracellular amino acid profiling. Morphological changes were analyzed by transmission electron microscopy. Indirect effects of MNPs@SiO2(RITC) on neuronal cells were assessed by Transwell-based coculture with MNPs@SiO2(RITC)-treated microglia. MNPs@SiO2(RITC)-induced biological changes in the mouse brain in vivo were examined by immunohistochemical analysis. RESULTS: BV2 murine microglial cells were morphologically activated and the expression of Iba1, an activation marker protein, was increased after MNPs@SiO2(RITC) treatment. Transmission electron microscopy analysis revealed lysosomal accumulation of MNPs@SiO2(RITC) and the formation of vesicle-like structures in MNPs@SiO2(RITC)-treated BV2 cells. The expression of several genes related to metabolism and inflammation were altered in 100 µg/ml MNPs@SiO2(RITC)-treated microglia when compared with that in non-treated (control) and 10 µg/ml MNPs@SiO2(RITC)-treated microglia. Combined transcriptome and amino acid profiling analyses revealed that the transport of serine family amino acids, including glycine, cysteine, and serine, was enhanced. However, only serine was increased in the growth medium of activated microglia; especially, excitotoxic D-serine secretion from primary rat microglia was the most strongly enhanced. Activated primary microglia reduced intracellular ATP levels and proteasome activity in cocultured neuronal cells, especially in primary cortical neurons, via D-serine secretion. Moreover, ubiquitinated proteins accumulated and inclusion bodies were increased in primary dopaminergic and cortical neurons cocultured with activated primary microglia. In vivo, MNPs@SiO2(RITC), D-serine, and ubiquitin aggresomes were distributed in the MNPs@SiO2(RITC)-treated mouse brain. CONCLUSIONS: MNPs@SiO2(RITC)-induced activation of microglia triggers excitotoxicity in neurons via D-serine secretion, highlighting the importance of neurotoxicity mechanisms incurred by nanoparticle-induced microglial activation.

Toxicity of orally administered food-grade titanium dioxide nanoparticles.

This year, France banned the application of titanium dioxide nanoparticles as a food additive (hereafter, E171) based on the insufficient oral toxicity data. Here, we investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. There were no dose-related changes in the Organisation for Economic Cooperation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the IgM and granulocyte-macrophage colony-stimulating factor levels were significantly lower in the blood from rats exposed to E171 compared with the control. The colonic antioxidant protein level decreased with increasing Ti accumulation. Additionally, after 24-h exposure, E171 located in the perinuclear region of AGS cells and affected expression of endoplasmic reticulum stress-related proteins. However, cell death was not observed up to the used maximum concentration. A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Collectively, we concluded that the NOAEL of E171 for 90 days repeated oral administration is between 100 and 1,000 mg/kg for both male and female rats. Additionally, further study is needed to clarify the possible carcinogenesis following the chronic accumulation in the colon.

Inhaled underground subway dusts may stimulate multiple pathways of cell death signals and disrupt immune balance.

In this study, we aimed to identify a toxic mechanism and the potential health effects of ambient dusts in an underground subway station. At 24 h exposure to human bronchial epithelial (BEAS-2B) cells (0, 2.5, 10, and 40 µg/mL), dusts located within autophagosome-like vacuoles, whereas a series of autophagic processes appeared to be blocked. The volume, potential and activity of mitochondria decreased in consistent with a condensed configuration, and the percentage of late apoptotic cells increased accompanying S phase arrest. While production of reactive oxygen species, expression of ferritin (heavy chain) protein, secretion of IL-6, IL-8 and matrix metalloproteinases, and the released LDH level notably increased in dust-treated cells (40 µg/mL), intracellular calcium level decreased. At day 14 after a single instillation to mice (0, 12.5, 50, and 200 µg/head), the total number of cells increased in the lungs of dust-treated mice with no significant change in cell composition. The pulmonary levels of TGF-ß, GM-CSF, IL-12 and IL-13 clearly increased following exposure to dusts, whereas that of CXCL-1 was dose-dependently inhibited. Additionally, the population of cytotoxic T cells in T lymphocytes in the spleen increased relative to that of helper T cells, and the levels of IgA and IgM in the bloodstream were significantly reduced in the dust-treated mice. Subsequently, to improve the possibility of extrapolating our findings to humans, we repeatedly instilled dusts (1 time/week, 4 weeks, 0.25 and 1.0 mg/head) to monkeys. The total number of cells, the relative portion of neutrophils, the level of TNF-α significantly increased in the lungs of dust-treated monkeys, and the expression of cytochrome C was enhanced in the lung tissues. Meanwhile, the pulmonary level of MIP-α was clearly reduced, and the expression of caveolin-1 was inhibited in the lung tissues. More importantly, inflammatory lesions, such as granuloma, were seen in both mice and monkeys instilled with dusts. Taken together, we conclude that dusts may impair the host's immune function against foreign bodies by inhibiting the capacity for production of antibodies. In addition, iron metabolism may be closely associated with dust-induced cell death and inflammatory response.

Cigarette smoke extract may induce lysosomal storage disease-like adverse health effects.

Cigarette smoke is known to be associated with the incidence of a variety of pulmonary diseases, and alveolar macrophages are a key player in the defense mechanism against inhalable toxicants. Herein, we have found that a hydrophilic fraction in smoke extracts from 3R4F reference cigarettes (CSE) contains high concentrations of volatile substances compared to cigarette smoke condensate (amphoteric fraction). We also identified the toxic mechanism of CSE using MH-S, a mouse alveolar macrophage cell line. CSE decreased cell viability accompanying increased lactate dehydrogenase release. Additionally, mitochondrial volume and the potential increased along with enhanced expression of mitochondrial fusion proteins and decreased adenosine triphosphate production. Similarly, CSE clearly induced increase of catalase activity and intracellular calcium concentration and decrease of endoplasmic reticulum and lysosome volume at the highest dose. More interestingly, damaged organelles accumulated in the cytosol, and CSE-containing particles specifically penetrated to mitochondria. Meanwhile, any significant change in autophagy related protein expression was not found in CSE-treated cells. Subsequently, we evaluated the effects of CSE on secretion of inflammatory related cytokines and chemokines, considering the relationship between organelle damage and the disturbed immune response. Very importantly, we found that expression of innate and adaptive immunity related mediators is disrupted following CSE exposure. Taken together, we suggest that CSE may cause the accumulation of damaged organelles in the cytoplasm by impairing selective autophagic function. In addition, this accumulation is responsible for the inadequate ability of immune cells to repair the damage of lung tissue following exposure to CSE.

Comparison of subchronic immunotoxicity of four different types of aluminum-based nanoparticles.

Nanoparticles (NPs) have recently emerged as an inhalable pollutant, owing to their applications, aluminum-based NPs (Al-NPs) have been prioritized for toxicity testing. In the current study, we compared the pulmonary biopersistence and subsequent toxicity of four different types of Al-NPs (two rod-type aluminum oxide NPs [AlONPs] with different aspect ratios [short (S)- and long (L)-AlONPs], spherical aluminum cerium oxide NPs [AlCeO3 , AlCeONPs] and spherical γ-aluminum oxide hydroxide nanoparticles [AlOOHNPs]) 13weeks after a single intratracheal instillation, considering the importance of their properties in their toxicity. We found that the pulmonary biopersistence of Al-NPs was strengthened by a high aspect ratio in the rod-type AlONPs and by the presence of hydroxyl groups in the spherical-type Al-NPs. The highest toxicity was observed in the mice treated with AlOOHNPs, which showed low biostability. More importantly, we identified that the commercially available AlCeONPs were Al2 O3 -coated CeO2 NPs, but not AlCeO3 NPs, although they have been sold under the trade name of AlCeONPs. In conclusion, the aspect ratio and biostability may be important factors in the determination of the biopersistence of NPs and the subsequent biological response. In addition, the physicochemical properties of NPs should be examined in detail before their release into the market to prevent unexpected adverse health effects.

Production of Selenomethionine-Enriched Bifidobacterium bifidum BGN4 via Sodium Selenite Biocatalysis.

Selenium is a trace element essential for human health that has received considerable attention due to its nutritional value. Selenium's bioactivity and toxicity are closely related to its chemical form, and several studies have suggested that the organic form of selenium (i.e., selenomethionine) is more bioavailable and less toxic than its inorganic form (i.e., sodium selenite). Probiotics, especially Bifidobacteriium and Lactobacillus spp., have received increasing attention in recent years, due to their intestinal microbial balancing effects and nutraceutical benefits. Recently, the bioconversion (a.k.a biotransformation) of various bioactive molecules (e.g., minerals, primary and secondary metabolites) using probiotics has been investigated to improve substrate biofunctional properties. However, there have been few reports of inorganic selenium conversion into its organic form using Bifidobacterium and Lactobacillus spp. Here we report that the biosynthesis of organic selenium was accomplished using the whole cell bioconversion of sodium selenite under controlled Bifidobacterium bifidum BGN4 culture conditions. The total amount of organic and inorganic selenium was quantified using an inductively coupled plasma-atomic emission spectrometer (ICP-AES). The selenium species were separated via anion-exchange chromatography and analyzed with inductively coupled plasma-mass spectrometry (ICP-MS). Our findings indicated that the maximum level of organic selenium was 207.5 µg/g in selenium-enriched B. bifidum BGN4. Selenomethionine was the main organic selenium in selenium-enriched B. bifidum BGN4 (169.6 µg/g). Considering that B. bifidum BGN4 is a commercial probiotic strain used in the functional food industry with clinically proven beneficial effects, selenium-enriched B. bifidum BGN4 has the potential to provide dual healthy functions as a daily supplement of selenium and regulator of intestinal bacteria. This is the first report on the production of organic selenium using B. bifidum spp.

Ambient fine particulate matters induce cell death and inflammatory response by influencing mitochondria function in human corneal epithelial cells.

Ambient fine particulate matter (AFP) is a main risk factor for the cornea as ultraviolet light. However, the mechanism of corneal damage following exposure to AFP has been poorly understood. In this study, we first confirmed that AFP can penetrate the cornea of mice, considering that two-dimensional cell culture systems are limited in reflecting the situation in vivo. Then, we investigated the toxic mechanism using human corneal epithelial (HCET) cells. At 24h after exposure, AFP located within the autophagosome-like vacuoles, and cell proliferation was clearly inhibited in all the tested concentration. Production of ROS and NO and secretion of pro-inflammatory cytokines were elevated in a dose-dependent manner. Additionally, conversion of LC3B from I-type to II-type and activation of caspase cascade which show autophagic- and apoptotic cell death, respectively, were observed in cells exposed to AFP. Furthermore, AFP decreased mitochondrial volume, inhibited ATP production, and altered the expression of metabolism-related genes. Taken together, we suggest that AFP induces cell death and inflammatory response by influencing mitochondrial function in HCET cells. In addition, we recommend that stringent air quality regulations are needed for eye health.

Extremely low-frequency electromagnetic field induces neural differentiation of hBM-MSCs through regulation of (Zn)-metallothionein-3.

Extremely low-frequency electromagnetic field (ELFEMF) can stimulate neural differentiation in human bone marrow-derived mesenchymal cells (hBM-MSCs), and this provides an opportunity for research on neurodegenerative diseases such as Alzheimer's disease (AD). Metallothionein-3 (MT3), an isoform of the metal-binding proteins, metallothioneins, involved in maintaining intracellular zinc (Zn) homeostasis and the deregulation of zinc homeostasis, has separately been implicated in AD. Here, we investigated the effect of ELFEMF-induced neural differentiation of hBM-MSCs on Zn-MT3 homeostatic interaction. Exposure to ELFEMF induced neural differentiation of hBM-MSCs, which was characterized by decreased proliferation and enhanced neural-like morphology. We observed expression of neuronal markers such as ß-tubulin3, pleiotrophin, and neurofilament-M at the mRNA level and MAP2 at the protein level. ELFEMF-induced neural differentiation correlated with decreased expression of metal-response element-transcription factor 1 and MT3, as well as decreased intracellular Zn concentration. In addition, upregulation of dihydropyrimidinase-related protein 2 was observed, but there was no change in γ-enolase expression. These data indicate a possible regulatory mechanism for MT3 during neural differentiation. Our findings provide considerable insight into molecular mechanisms involved in neural differentiation, which is useful for developing new treatments for neurodegenerative diseases. Bioelectromagnetics. 38:364-373, 2017. © 2017 Wiley Periodicals, Inc.

Tissue distribution following 28 day repeated oral administration of aluminum-based nanoparticles with different properties and the in vitro toxicity.

The tissue distribution and toxicity of nanoparticles (NPs) depend on their physical and chemical properties both in the manufactured condition and within the biological system. We characterized three types of commercially available aluminum-based NPs (Al-NPs), two rod-type aluminum oxide NPs (Al2 O3 , AlONPs), with different aspect ratios (short [S]- and long [L]-AlONPs), and spherical aluminum cerium oxide NPs (AlCeO3 , AlCeONPs). The surface area was in order of the S-AlONPs > L-AlONPs > AlCeONPs. Very importantly, we found that AlCeONPs is Al2 O3 -coated CeO2 NPs, but not AlCeO3 NPs, and that the Al level in AlCeONPs is approximately 20% of those in S- and L-AlONPs. All three types of Al-NPs were slightly ionized in gastric fluid and rapidly particlized in the intestinal fluid. There were no significant differences in the body weight gain following 28 days of repeated oral administration of the three different types of Al-NPs. All Al-NPs elevated Al level in the heart, spleen, kidney and blood at 24 hours after the final dose, accompanied by the altered tissue level of redox reaction-related trace elements. Subsequently, in four types of cells derived from the organs which Al-NPs are accumulated, H9C2 (heart), HEK-293 (kidney), splenocytes and RAW264.7 (blood), S-AlONPs showed a very low uptake level and did not exert significant cytotoxicity. Meanwhile, cytotoxicity and uptake level were the most remarkable in cells treated with AlCeONPs. In conclusion, we suggest that the physicochemical properties of NPs should be examined in detail before the release into the market to prevent unexpected adverse health effects.

Comparison of distribution and toxicity of different types of zinc-based nanoparticles.

Zinc-based nanoparticles (Zn-NPs), mainly zinc oxide (ZnO) NPs, have promising application in a wide area, but their potential harmful effects on environment and human health have been continuously raised together with their high dissolution rate. In this study, we coated the surface of ZnO NPs with phosphate (ZnP NPs) and sulfide (ZnS NPs) which have very low solubility in water, administered orally (0.5 and 1 mg/kg) to mice for 28 days, and then compared their biodistribution and toxicity. As expected, ZnO NPs were rapidly ionized in an artificial gastric fluid. On the other hand, ZnO NPs were more particlized in an artificial intestinal fluid than ZnP and ZnS NPs. After repeated dosing, all three types of Zn-NPs the most distributed in the spleen and thymus and altered the level of redox reaction-related metal ions in the tissues. We also found that three types of Zn-NPs clearly disturb tissue ion homeostasis and influence immune regulation function. However, there were no remarkable difference in distribution and toxicity following repeated exposure of three types of Zn-NPs, although Na+ and K+ level in the spleen and thymus were notably higher in mice exposed to ZnO NPs compared to ZnP and ZnS NPs. Taken together, we suggest that all three types of Zn-NPs may influence human health by disrupting homeostasis of trace elements and ions in the tissues. In addition, the surface transformation of ZnO NPs with phosphate and sulfide may not attenuate toxicity due to the higher particlization rate of ZnO NPs in the intestine, at least in part. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1363-1374, 2017.

Comparison of distribution and toxicity following repeated oral dosing of different vanadium oxide nanoparticles in mice.

Vanadium is an important ultra-trace element derived from fuel product combustion. With the development of nanotechnology, vanadium oxide nanoparticles (VO NPs) have been considered for application in various fields, thus the possibility of release into the environment and human exposure is also increasing. Considering that verification of bioaccumulation and relevant biological responses are essential for safe application of products, in this study, we aimed to identify the physicochemical properties that determine their health effects by comparing the biological effects and tissue distribution of different types of VO NPs in mice. For this, we prepared five types of VO NPs, commercial (C)-VO2 and -V2O5 NPs and synthetic (S)-VO2, -V2O3, and -V2O5 NPs. While the hydrodynamic diameter of the two types of C-VO NPs was irregular and impossible to measure, those of the three types of S-VO NPs was in the range of 125-170nm. The S- and C-V2O5 NPs showed higher dissolution rates compared to other VO NPs. We orally dosed the five types of VO NPs (70 and 210µg/mouse, approximately 2 and 6mg/kg) to mice for 28 days and compared their biodistribution and toxic effects. We found that S-V2O5 and S-V2O3 NPs more accumulated in tissues compared to other three types of VO NPs, and the accumulated level was in order of heart>liver>kidney>spleen. Additionally, tissue levels of redox reaction-related elements and electrolytes (Na(+), K(+), and Ca(2+)) were most clearly altered in the heart of treated mice. Notably, all S- and C-VO NPs decreased the number of WBCs at the higher dose, while total protein and albumin levels were reduced at the higher dose of S-V2O5 and S-V2O3 NPs. Taken together, we conclude that the biodistribution and toxic effects of VO NPs depend on their dissolution rates and size (surface area). Additionally, we suggest that further studies are needed to clarify effects of VO NPs on functions of the heart and the immune system.

Single-walled carbon nanotubes disturbed the immune and metabolic regulation function 13-weeks after a single intratracheal instillation.

Due to their unique physicochemical properties, the potential health effects of single-walled carbon nanotubes (SWCNTs) have attracted continuous attention together with their extensive application. In this study, we aimed to identify local and systemic health effects following pulmonary persistence of SWCNTs. As expected, SWCNTs remained in the lung for 13 weeks after a single intratracheal instillation (50, 100, and 200µg/kg). In the lung, the total number of cells and the percentages of lymphocytes and neutrophils significantly increased at 200µg/kg compared to the control, and the Th1-polarized immune response was induced accompanying enhanced expression of tissue damage-related genes and increased release of chemokines. Additionally, SWCNTs enhanced the expression of antigen presentation-related proteins on the surface of antigen-presenting cells, however, maturation of dendritic cells was inhibited by their persistence. As compared to the control, a significant increase in the percentage of neutrophils and a remarkable decrease of BUN and potassium level were observed in the blood of mice treated with the highest dose. This was accompanied by the down-regulation of the expression of antigen presentation-related proteins on splenocytes. Moreover, protein and glucose metabolism were disturbed with an up-regulation of fatty acid ß-oxidation. Taken together, we conclude that SWCNTs may induce adverse health effects by disturbing immune and metabolic regulation functions in the body. Therefore, careful application of SWCNTs is necessary for the enforcement of safety in nano-industries.

Biodistribution and toxicity of spherical aluminum oxide nanoparticles.

With the rapid development of the nano-industry, concerns about their potential adverse health effects have been raised. Thus, ranking accurately their toxicity and prioritizing for in vivo testing through in vitro toxicity test is needed. In this study, we used three types of synthesized aluminum oxide nanoparticles (AlONPs): γ-aluminum oxide hydroxide nanoparticles (γ-AlOHNPs), γ- and α-AlONPs. All three AlONPs were spherical, and the surface area was the greatest for γ-AlONPs, followed by the α-AlONPs and γ-AlOHNPs. In mice, γ-AlOHNPs accumulated the most 24 h after a single oral dose. Additionally, the decreased number of white blood cells (WBC), the increased ratio of neutrophils and the enhanced secretion of interleukin (IL)-8 were observed in the blood of mice dosed with γ-AlOHNPs (10 mg kg(-1)). We also compared their toxicity using four different in vitro test methods using six cell lines, which were derived from their potential target organs, BEAS-2B (lung), Chang (liver), HACAT (skin), H9C2 (heart), T98G (brain) and HEK-293 (kidney). The results showed γ-AlOHNPs induced the greatest toxicity. Moreover, separation of particles was observed in a transmission electron microscope (TEM) image of cells treated with γ-AlOHNPs, but not γ-AlONPs or α-AlONPs. In conclusion, our results suggest that the accumulation and toxicity of AlONPs are stronger in γ-AlOHNPs compared with γ-AlONPs and α-AlONPs owing their low stability within biological system, and the presence of hydroxyl group may be an important factor in determining the distribution and toxicity of spherical AlONPs.

Distribution and immunotoxicity by intravenous injection of iron nanoparticles in a murine model.

With the increased application of iron oxide nanoparticles (FeNPs) for biomedical imaging purposes, concerns regarding the onset of the unexpected adverse health effects following exposure have been rapidly raised. In this study, we investigated the tissue distribution and immunotoxicity of FeNPs (2 and 4 mg kg(-1)) over time (2, 4 and 13 weeks) after single intravenous injection. At 13 weeks after a single injection, the iron levels increased in all measured tissues compared to the control, and iron accumulation was notable in the liver, spleen and thymus. These changes were accompanied by changes in levels of redox reaction-related elements, including copper, manganese, zinc and cobalt. In addition, as compared to the control, the number of white blood cells and percentage of neutrophils significantly increased in the treated groups, and the interleukin-8 secretion and lactate dehydrogenase release were clearly elevated in the treated groups along with enhanced expressions of chemotaxis-related proteins. However, expression of antigen presenting related proteins attenuated following accumulation of FeNPs. Taken together, we suggest that FeNPs may primarily induce toxicity in the liver and immune system, and immunotoxicological evaluation should be considered to predict adverse health effects following exposure to NPs.

Experimental approach to the fundamental limit of the extinction coefficients of ultra-smooth and highly spherical gold nanoparticles.

The theoretical extinction coefficients of gold nanoparticles (AuNPs) have been mainly verified by the analytical solving of the Maxwell equation for an ideal sphere, which was firstly founded by Mie (generally referred to as Mie theory). However, in principle, it has not been directly feasible with experimental verification especially for relatively large AuNPs (i.e., >40 nm), as conventionally proposed synthetic methods have inevitably resulted in a polygonal shaped, non-ideal Au nanosphere. Here, mono-crystalline, ultra-smooth, and highly spherical AuNPs of 40-100 nm were prepared by the procedure reported in our recent work (ACS Nano, 2013, 7, 11064). The extinction coefficients of the ideally spherical AuNPs of 40-100 nm were empirically extracted using the Beer-Lambert law, and were then compared with the theoretical limits obtained by the analytical and numerical methods. The obtained extinction coefficients of the ideally spherical AuNPs herein agree much more closely with the theoretical limits, compared with those of the faceted or polygonal shaped AuNPs. In addition, in order to further elucidate the importance of being spherical, we systematically compared our ideally spherical AuNPs with the polygonal counterparts; effectively addressing the role of the surface morphology on the spectral responses in both theoretical and experimental manners.

A 13-week repeated-dose oral toxicity and bioaccumulation of aluminum oxide nanoparticles in mice.

Because of an increase in the commercial applications of manufactured nanoparticles, the issue of potential adverse health effects of nanoparticles following intended or unintended exposure is rapidly gaining attention. In this study, we evaluated the toxicity of aluminum oxide nanoparticles (AlNPs, rod-type, 1.5, 3, and 6 mg/kg) after oral administration to mice for 13 weeks. Compared with the control group, the consumption of diet and drinking water and body weight gain decreased in the group treated with AlNPs. The group treated with 6 mg/kg AlNPs also showed a marked elevation in the count of white blood cells that associated with a significant decrease and increase to the proportion of eosinophils and lymphocytes, respectively. In addition, the secretion of IL-6 and monocyte chemotactic protein-1 increased in a dose-dependent manner in the treated groups. Furthermore, AlNPs showed the highest accumulation in the liver and kidneys compared with the control group, increased the lactate dehydrogenase level in the blood, and induced the development of a pathological lesion in the liver and kidneys. Taken together, we suggest that the target organs of rod-type AlNPs may be the liver, kidneys and the immune system, and the not-observed adverse effect level may be lower than 6 mg/kg.

Time-dependent bioaccumulation of distinct rod-type TiO2 nanoparticles: comparison by crystalline phase.

A complete understanding of the interaction between nanoparticles and biological systems, including nanoparticle uptake and distribution and the biological responses, could guide the design of safer and more effective nanoparticles than those currently available. In this study, we compared the distribution in mice over time of two rod-type titanium dioxide nanoparticles (TiNPs) that feature distinct phases, anatase (ATO) and brookite (BTO). Surface areas of BTO and ATO were estimated to be 102 and 268 m(2) g(-1) , respectively, and negative charge on the surface of ATO was higher than that of BTO in deionized water. Both TiNPs were rapidly distributed into tissues after injection. At 4 weeks after injection, both TiNPs were maximally accumulated in the spleen, followed by the liver, but the total accumulation of ATO in tissues measured in this study was more than that of BTO. Moreover, the cellular antioxidant function was similar although the levels of Ti measured in tissues were distinct between the two TiNPs. Based on these results, we suggest that the fate of TiNPs in the body may differ according to the size and surface charge of the TiNPs even when their shape is the same.

Differential susceptibility to arsenic in glutathione S-transferase omega 2 (GST-O2)-targeted freshwater water flea Daphnia magna mutants.

To examine the role of glutathione S-transferase omega class (GST-O2) genes in the biotransformation and detoxification in Daphnia magna, various responses such as in vivo endpoints, arsenic speciation, enzymatic activities, and gene expression pathways related to arsenic metabolism were investigated in wild-type (WT) and GST-O2-mutant-type (MT) fleas produced by CRISPR/Cas9. Sensitivity to arsenic in MT fleas was higher than in WT fleas. Also, the reduction rate of arsenate (AsV) to arsenite (AsIII) in the MT group was significantly lower and led to accumulation of higher arsenic concentrations, resulting in decreased protection against arsenic toxicity. Relative mRNA expression of other GST genes in the GST-O2-targeted MT group generally increased but the enzymatic activity of GST decreased compared with the WT group. Oxidative stress on arsenic exposure was more strongly induced in the MT group compared with the WT group, resulting in a decrease in the ability to defend against toxicity in GST-O2-targeted mutant D. magna. Our results suggest that GST-O2 plays an important role in arsenic biotransformation and detoxification functions in D. magna.

Uptake and toxicity of cerium dioxide nanoparticles with different aspect ratio.

Cerium dioxide nanoparticles (CeONPs) have been extensively applied in research for future energy development due to two common oxidation states on their surface. Considering that shape (aspect ratio) is a key determinant of NPs-induced toxicity, we compared the toxicity of hexagonal (H)- and rod-shaped (R)-CeONPs in mice. At 24 h after pharyngeal aspiration, both types of CeONPs recruited surrounding immune cells (monocytes and neutrophils) into the lung, and R-CeONPs induced a more severe pulmonary inflammatory response compared with H-CeONPs. To identify an indicator to predict pulmonary inflammatory responses at the cellular level, we also investigated their responses in alveolar macrophage cells. At 24 h after treatment, both types of CeONPs were mainly located within the vacuoles (partially, in the lysosome) in the cytoplasm. Mitochondrial damage, intracellular calcium accumulation, and increased NO production were observed in cells exposed to both types of CeONPs, ultimately resulting in a decrease in cell viability. More interestingly, both types of CeONPs formed multinucleated giant cells. Meanwhile, contrary to when suspended in deionized water, R-CeONPs were strongly aggregated with a negative charge in cell culture media, whereas H-CeONPs were relatively well-dispersed with a positive charge. R-CeONPs-induced lysosomal extension was also recovered by premix with negatively charged DNA, and even NPs suspended in cell culture media without cells were detected under the FACS system, suggesting interference by protein corona. Therefore, we suggest that shape (aspect ratio) is an important factor determining inhaled NPs-induced pathology and that the effect of the surface charge and protein corona should be carefully considered in interpreting results derived from in vitro tests. Furthermore, we propose that the relationship between the formation of multinucleated giant cells and the inflammatory response of inhaled CeONPs should be further studied.

Subchronic pulmonary toxicity of ambient particles containing cement production-related elements.

Chronic respiratory disease is among the most common non-communicable diseases, and particulate materials (PM) are a major risk factor. Meanwhile, evidence of the relationship between the physicochemical characteristics of PM and pulmonary toxicity mechanism is still limited. Here, we collected particles (CPM) from the air of a port city adjacent to a cement factory, and we found that the CPM contained various elements, including heavy metals (such as arsenic, thallium, barium, and zirconium) which are predicted to have originated from a cement plant adjacent to the sampling site. We also delivered the CPM intratracheally to mice for 13 weeks to investigate the pulmonary toxicity of inhaled CPM. CPM-induced chronic inflammatory lesions with an increased total number of cells in the lung of mice. Meanwhile, among inflammatory mediators measured in this study, levels of IL-1ß, TNF-α, CXCL-1, and IFN-γ were elevated in the treated group compared with the controls. Considering that the alveolar macrophage (known as dust cell) is a professional phagocyte that is responsible for the clearance of PM from the respiratory surfaces, we also investigated cellular responses following exposure to CPM in MH-S cells, a mouse alveolar macrophage cell line. CPM inhibited cell proliferation and formed autophagosome-like vacuoles. Intracellular calcium accumulation and oxidative stress, and altered expression of pyrimidine metabolism- and olfactory transduction-related genes were observed in CPM-treated cells. More interestingly, type I-LC3B and full-length PARP proteins were not replenished in CPM-treated cells, and cell cycle changes, apoptotic and necrotic cell death, and caspase-3 cleavage were not significantly detected in cells exposed to CPM. Taken together, we conclude that dysfunction of alveolar macrophages may contribute to CPM-induced pulmonary inflammation. In addition, given the possible transformation of heart tissue observed in CPM-treated mice, we suggest that further study is needed to clarify the systemic pathological changes and the molecular mechanisms following chronic exposure to CPM.