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.

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.

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.

Pulmonary glass particles may persist in the lung suppressing function of immune cells.

The health effects of silica may depend on the inherent properties of crystalline silica or on external factors affecting the biological activity or distribution of its polymorphs. Inhaled crystalline silica is classified as a Group I carcinogen, however, information on the health effects of amorphous silica is still insufficient. Considering that alveolar macrophages play a key role in both innate and adaptive immune responses for removal of foreign bodies that enter via the respiratory system, we treated sheet-like glass particles (SGPs), a type of noncrystalline amorphous silica, to MH-S cells, an alveolar macrophage cell line. SGPs reduced the generation of ROS and NO and induced cell death via multiple pathways. Although the expression of CD80, CD86, and CD40, increased by exposure to SGPs, the expression of MHC class II molecules had not notably changed. Additionally, expression of ICAM-1 tended to decrease. In mice, SGPs were distributed in the interstitial region of the lung without notable pathological lesion on day 14 after a single intratracheal instillation. Pulmonary total cell number increased significantly with the highest dose, but the levels of all measured inflammatory cytokines and chemokines, except IL-1, were lower in BAL fluid from SGP-treated mice compared to control. More interestingly, the expression of antigen presentation-related proteins was enhanced in the lungs of SGP-exposed mice concomitant with an increase in the number of mature dendritic cells, whereas the expression of ICAM-1, an important adhesion molecule for helper T cell recruitment, was suppressed. Taken together, we suggest that SGPs may induce adverse health effects by down-regulating function of immune cells in the lungs. Furthermore, ICAM-1 may play a key role in immune response to remove pulmonary SGPs.

A higher aspect ratio enhanced bioaccumulation and altered immune responses due to intravenously-injected aluminum oxide nanoparticles.

Aluminum oxide nanoparticles (AlO NP) have been widely utilized in a variety of areas, including in the optical, biomedical and electronic fields and in the overall development of nanotechnologies. However, their toxicological profiles are still not fully developed. This study compared the distribution and immunotoxicity of two rod-types of AlO NP. As reported previously, the two types of AlO NP had different aspect ratios (long-type: 6.2 ± 0.6, short-type: 2.1 ± 0.4), but the size and surface charge were very similar. On Day 14 after a single intravenous (IV) injection (1.25 or 5 mg/kg), both AlO NP accumulated primarily in the liver and spleen and altered the levels of redox response-related elements. The accumulated level was higher in mice exposed to the long-type AlO NP compared to the short-type. Additionally, it was noted that the levels of IL-1ß, IL-8 and MCP-1 were enhanced in the blood of mice exposed to both types of AlO NP and the percentages of neutrophils and monocytes among all white blood cells were increased only in mice injected with the long-type AlO NP (5 mg/kg). In addition, as compared to the control, co-expression of CD80 and CD86 (necessary for antigen presentation) on splenocytes together with a decreased expression of chemotaxis-related marker (CD195) was attenuated by exposure to the AlO NP, especially the long-type. Taken together, the data suggest that accumulation following a single IV injection with rod-types of AlO NP is strengthened by a high aspect ratio and, subsequently, this accumulation has the potential to influence immune functions in an exposed host.

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.

Toxic response of graphene nanoplatelets in vivo and in vitro.

With the development of nanotechnology, myriad types of novel materials have been discovered at the nanoscale, among which the most interesting material is graphene. However, the toxicity data available on graphene are extremely limited. In this study, we explored toxic response of commercially available graphene nanoplatelets (GNPs) in vivo and in vitro. The GNPs used in this study had a high surface area and feature considerably few defects. In mice, GNPs (2.5 and 5 mg/kg) remained in the lung until 28 days after a single instillation, and the secretion of inflammatory cytokines reached the maximal level at Day 14 and then decreased over time. In vitro study using BEAS-2B cells, a human bronchial epithelial cell line, GNPs located within autophagosome-like vacuoles 24 h after exposure. The GNPs (2.5, 5, 10, and 20 µg/mL) also dose-dependently reduced cell viability, which was accompanied by an increase in the portion of cells in the subG1 and S phases. Moreover, the GNPs down-regulated the generation of reactive oxygen species, suppressed ATP production, caused mitochondria damage, and elevated the levels of autophagy-related proteins. Based on these results, we suggest that GNPs provoked a subchronic inflammatory response in mice and that GNPs induced autophagy accompanying apoptosis via mitochondria damage in vitro.

Comparison of the toxicity of aluminum oxide nanorods with different aspect ratio.

Aluminum oxide nanoparticles are listed among 14 high-priority nanomaterials published by the Organization for Economic Co-operation and Development, but limited information is available on their potential hazards. In this study, we compared the toxicity of two different aluminum oxide nanorods (AlNRs) commercially available in vivo and in vitro. Considering aspect ratio, one was 6.2 ± 0.6 (long-AlNRs) and the other was 2.1 ± 0.4 (short-AlNRs). In mice, long-AlNRs induced longer and stronger inflammatory responses than short-AlNRs, and the degree reached the maximum on day 7 for both types and decreased with time. In addition, in vitro tests were performed on six cell lines derived from potential target organs for AlNPs, HEK-293 (kidney), HACAT (skin), Chang (liver), BEAS-2B (lung), T98G (brain), and H9C2 (heart), using MTT assay, ATP assay, LDH release, and xCELLigence system. Long-AlNRs generally produced stronger toxicity than short-AlNRs, and HEK-293 cells were the most sensitive for both AlNRs, followed by BEAS-2B cells, although results from 4 kinds of toxicity tests conflicted among the cell lines. Based on these results, we suggest that toxicity of AlNRs may be related to aspect ratio (and resultant surface area). Furthermore, novel in vitro toxicity testing methods are needed to resolve questionable results caused by the unique properties of nanoparticles.

Sheet-type titania, but not P25, induced paraptosis accompanying apoptosis in murine alveolar macrophage cells.

In this study, we identified the toxic effects of sheet-type titania (TNS), which are being developed as a material for UV-blocking glass, comparing with P25, a benchmark control for titania, in MH-S cells, a mouse alveolar macrophage cell line. After 24 h exposure, the TNS-exposed cells formed large vacuoles while the P25-exposed ones did not. The decreased levels of cell viability were similar between the P25 and TNS groups, but ATP production was clearly lower in cells exposed to the TNS. P25 decreased the expression of calnexin protein, an endoplasmic reticulum (ER) membrane marker, and increased the number of cells generating ROS in a dose dependent manner. Meanwhile, TNS dilated the ER and mitochondria and increased the secretion of NO and pro-inflammatory cytokines, but not of ROS. Subsequently, we studied the molecular response following TNS-induced vacuolization. TNS started to form vacuoles in the cytosol since 20 min after exposure, and the expression of the mitochondria function-related genes were down-regulated the most in the cells exposed for 1 h. After 24 h exposure, the number of apoptotic cells and the relative levels of BAX to Bcl-2 increased. The expression of SOD1 protein, but not of SOD2, also dose-dependently increased with an increase in caspase-8 activity. Additionally, the MAPK pathway was significantly activated, even though the expression of p-EGFR did not change significantly. Furthermore, the number of apoptotic cells increased rapidly with time and with the inhibition of vacuole formation. Taken together, we suggest that P25 and TNS may target different organelles. In addition, TNS, but not P25, induced paraptosis accompanied by apoptosis in MH-S cells, and the formation of the cytoplasmic vacuoles allowed delay apoptosis following TNS exposure.

Comparison of toxicity between the different-type TiO2 nanowires in vivo and in vitro.

In this study, we compared their toxicity in vivo and in vitro based on the physicochemical properties of three different types of TiO2 nanowires, H2Ti3O7 nanowires (1HTO), hydrothermal treatment (2HTO), and calcination (3HTO) of 1HTO. The surface of 1HTO was smooth, and the surface of 2HTO was much rougher. The negative charge on the surface increased in the order of 2HTO, 3HTO, and 1HTO, whereas the surface area increased in the order of 3HTO, 1HTO, and 2HTO. The lung is a main exposure route of nanoparticles. On day 28 after a single instillation (1 mg/kg), three nanowires induced a Th2-type inflammatory response together with the relative increase in CD4⁺ T cells, especially by 2HTO. In vitro, three TiO2 nanowires (10 µg/ml) commonly induced the generation of cell debris in eight cell lines which may be the potential target organ of nanoparticles, especially by 2HTO. It seemed that the generation of cell debris coincides with the increase in autophagosome-like vacuoles in the cytosol. In further study using BEAS-2B cells originated from the lung, the protein amount from cells exposed to 2HTO decreased more clearly although the generation of reactive oxygen species (ROS) was less compared to 1HTO and 3HTO. Based on these results, we suggest that surface area may act as an important factor depends on the biological response by TiO2 nanowires. Furthermore, the increase in autophagosome-like vacuoles may be an important cause of cell death by nanoparticles with ROS.

Involvement of type I protein kinase A in the differentiation of L6 myoblast in conjunction with phosphatidylinositol 3-kinase.

To investigate the role of protein kinase A (PKA) (EC 2.7.1.37) in myogenesis, PKA activity was closely monitored during the differentiation of L6 rat skeletal myoblasts. As the differentiation proceeded, total PKA activity increased about 2-3 fold, and the protein levels of PKA RIalpha and Calpha subunits increased about 3-4 fold. We then looked at the effect of the specific inhibitor for PKA, N-[2-(p-bromocinnamy-lamino)-ethyl]-5-isoquinoline-sulfonamide (H89), on the differentiation of L6 myoblasts. H89 completely blocked the myotube formation and abolished the up-regulation of RIalpha and Ca. This inhibitory effect of H89 was dose-dependent and could be reversed upon removal of H89 from the culture medium. Furthermore, we demonstrated that specific inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin, and LY294002 blocked the myotube formation and abolished the increase of PKA activity, which normally accompanied the differentiation of myoblasts. These results suggest that type I PKA may play a functional role(s) in the differentiation of myoblast as a putative downstream effector of the PI3K signaling pathway.