Follow the time course of inflammation caused by intraperitoneal administration of multi-wall carbon nanotubes in mice.

OBJECTIVE: Understand the progress of inflammation over time caused by multi-walled carbon nanotubes (MWCNT). METHODS: Two types of MWCNTs were administered to C57BL/6N mice via intraperitoneal administration at low and high doses (0.05 and 1.0 mg/mouse, respectively). Inflammation was evaluated until 6 months after administration based on cytokine levels and pathological observations. The abdominal cavity lavage fluid was collected and analyzed 1 week, 1, 3, and 6 month(s) after administration. IL-6 expression markedly increased 3 months after the administration of high-dose MWCNT-7. RESULTS: Notable inflammation was observed in the groups administered with one of the MWCNT, MWCNT-7. On the other hand, inflammation in another MWCNT-treated group was milder than that in the MWCNT-7-treated group. MWCNT-7 induced pronounced inflammation but did not induce tumor formation during the experimental period. Inflammation reaction is one of the most important biological responses to MWCNT. CONCLUSION: Three months post-exposure becomes a turning point for the harmful effects of the intraperitoneally administered MWCNT-7.

Contaminant microorganisms in the in vitro evaluation of cellular responses of cellulose nanofibers and their microbial inactivation using gamma irradiation.

Cellulose nanofibers (CNFs) are fibrous nanomaterials produced from plants. Since some nanomaterials are toxic, toxicity evaluation, including in vitro examinations using cultured cells, is essential for the effective use of CNFs. On the other hand, microorganisms in the environment can contaminate CNF suspensions. The contamination of CNF samples and the effects of contaminating microorganisms on in vitro examinations were investigated in this study. Microorganism contamination in CNF samples was examined, and microbial inactivation of CNF suspensions using gamma irradiation was evaluated. After gamma-ray irradiation at absorbed doses of 0.5, 1, 5, and 10 kGy, the cellular effects of CNF suspensions were examined using 6 types of cultured cell, HaCaT, A549, Caco-2, MeT-5A, THP-1, and NR8383 cells. CNF samples were contaminated with bacteria and CNF suspensions exhibited endotoxin activity. Gamma irradiation effectively inactivated the microorganisms contained in the CNF suspensions. When the absorbed dose was 10 kGy, the fiber length of CNF was shortened, but the effect on CNF was small at 1.0 kGy or less. CNF suspensions showed lipopolysaccharides (LPS)-like cellular responses and strongly induced interleukin-8, especially in macrophages. Absorbed doses of at least 10 kGy did not affect the LPS-like activity. In this study, it was shown that the CNF suspension may be contaminated with microorganisms. Gamma irradiation was effective for microbial inactivation of suspension for invitor toxicity evaluation of CNF. In vitro evaluation of CNFs requires attention to the effects of contaminants such as LPS.

Toxicity of nanomaterials due to photochemical degradation and the release of heavy metal ions.

The increased production of semiconductor nanomaterials such as heavy metal quantum dots and perovskites for applications such as in energy harvesting, optoelectronic devices, bioanalysis, phototherapy and consumer health products raises concerns regarding nanotoxicity. After disposal, these materials degrade upon interaction with the environment, such as rain and surface waters, soil and oxygen, and solar irradiation, leading to the release of heavy metal ions in the environment with exposure to aquatic and terrestrial animals and plants, and humans. Researchers are in the early stages of understanding the potential toxicity of such nanomaterials by quantifying the amount of heavy metal ions released due to environmental or biological transformation. Here, we evaluate the toxicity of environmentally transformed nanomaterials by considering PbS quantum dots as a model system. Using metal ion sensors and steady-state fluorescence spectroscopy, we quantify the amount of Pb2+ released by the photochemical etching of quantum dots. Furthermore, with the help of cytotoxicity and comet assays, and DNA gel electrophoresis, we evaluate the adverse effects of the released metal ions into the cultured lung epithelial (H1650), and neuronal (PC12) cells. These studies reveal higher levels of cell proliferation and DNA damage to PC12 cells, suggesting the neurotoxicity of lead due to not only the downregulation of glutathione, elevated levels of reactive oxygen and nitrogen species, and a calcium influx but also the proactivation of activator protein 1 that is correlated with protein kinase c. This research shows the significance of molecular biology studies on different cells and animals to critically understand the health and environmental costs of heavy metal-based engineered nanomaterials.

Quantitative kinetics of intracellular singlet oxygen generation using a fluorescence probe.

Singlet oxygen (1O2) is a type of reactive oxygen species involved in numerous physiological activities. We previously reported that 1O2-specific oxidation products are increased in patients with prediabetes, suggesting that measurement of 1O2 may be an important indicator of physiological and pathological conditions. The turnover in the generation and quenching of 1O2 is extremely rapid during biological activities owing to it high reactivity and short lifetime in solution. However, the dynamic changes in 1O2 generation in living cells have not been fully explored. In this study, we investigated whether the kinetics of 1O2 generation can be quantified using a far-red fluorescent probe for mitochondrial 1O2, Si-DMA, following addition of the 1O2 generator, endoperoxide, to mammalian cells. The kinetics of Si-DMA fluorescence intensity dose-dependently increased following treatment of mammalian living cells with endoperoxide. Alternatively, treatment with 1O2 quenchers decreased the fluorescence intensities following endoperoxide treatment. Our results indicate that the kinetics of intracellular 1O2 can be readily obtained using Si-DMA and time-lapse imaging, which provides new insights into the mechanism of 1O2 generation in mammalian cells and the exploration of 1O2 generators and quenchers.

Comprehensive analysis of PPARγ agonist activities of stereo-, regio-, and enantio-isomers of hydroxyoctadecadienoic acids.

Hydroxyoctadecadienoic acids (HODEs) are produced by oxidation and reduction of linoleates. There are several regio- and stereo-isomers of HODE, and their concentrations in vivo are higher than those of other lipids. Although conformational isomers may have different biological activities, comparative analysis of intracellular function of HODE isomers has not yet been performed. We evaluated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ), a therapeutic target for diabetes, and analyzed PPARγ agonist activity of HODE isomers. The lowest scores for docking poses of 12 types of HODE isomers (9-, 10-, 12-, and 13-HODEs) were almost similar in docking simulation of HODEs into PPARγ ligand-binding domain (LBD). Direct binding of HODE isomers to PPARγ LBD was determined by water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiments. In contrast, there were differences in PPARγ agonist activities among 9- and 13-HODE stereo-isomers and 12- and 13-HODE enantio-isomers in a dual-luciferase reporter assay. Interestingly, the activity of 9-HODEs was less than that of other regio-isomers, and 9-(E,E)-HODE tended to decrease PPARγ-target gene expression during the maturation of 3T3-L1 cells. In addition, 10- and 12-(Z,E)-HODEs, which we previously proposed as biomarkers for early-stage diabetes, exerted PPARγ agonist activity. These results indicate that all HODE isomers have PPARγ-binding affinity; however, they have different PPARγ agonist activity. Our findings may help to understand the biological function of lipid peroxidation products.

Comparison of proinflammatory potential of needle-shaped materials: aragonite and potassium titanate whisker.

Among the crystal forms of calcium carbonate, aragonite has needle-like shape. Although materials with needle-shaped crystals are associated with pulmonary toxicity, the toxic activity of aragonite is unclear. Therefore, proinflammatory potential of aragonite, neutralized aragonite and potassium titanate whisker was evaluated. The cellular effects of aragonite were weaker than those of potassium titanate whisker. Aragonite treatment induced the expression of chemokines in A549 cells and macrophages. Although aragonite exhibited proinflammatory effects in vitro, pulmonary inflammation was not observed in vivo after intratracheal administration of aragonite in mice. We did not observe the induction of inflammatory cytokine secretion or tissue lesion in the lungs of mice after administration of aragonite. Potassium titanate whisker treatment induced chemokine secretion in vitro. An increase in the number of neutrophils was observed in the mice lung tissue after administration of potassium titanate whisker. Aragonite and neutralized aragonite both induced an increase in the levels of intracellular calcium, but the levels were significantly higher in cells treated with aragonite than in cells treated with neutralized aragonite. These results suggested that intracellular calcium release mediates the cellular effects of aragonite. The toxicity of aragonite based on its needle-like structure was also not observed.

Comparison of the effects of multiwall carbon nanotubes on the epithelial cells and macrophages.

Effects of two kinds of multiwall carbon nanotubes (MWCNTs) on cells were examined. The effects of MWNT-7, which has been reported to be carcinogenic, and MWCNT-B, whose toxicity is unclear, were examined in both epithelial cells and macrophages. Human lung carcinoma A549 cells were used as representative epithelial cells and differentiated human monocyte THP-1 cells, as well as rat pulmonary macrophages NR8383, were employed to examine possible harmful effects of the MWCNTs. The MWCNTs induced the production of chemokines such as interleukin-8 (IL-8). MWCNTs were found to more strongly affect macrophages than epithelial cells. In addition, the toxicity was more pronounced in the MWNT-7 exposed cells than in those exposed to MWCNT-B. Cytochalasin D and amiloride treatment of differentiated THP-1 cells reduced cell-associated MWCNTs and IL-8 induction. To confirm these cellular influences in vivo, intratracheal administration of each type of MWCNT was performed by pharyngeal aspiration in the mouse lung. Analysis of bronchoalveolar lavage fluid (BALF) showed increase of inflammatory monocyte in MWNT-7 exposed animals at 1week after. In addition, neutrophils in the BALF were also significantly increased MWNT-7 exposed animals at 1 week and 1 month after. Aspiration of MWNT-7 caused formation of granulomas in the lung. Formation of the granulomas was not observed in the case of MWCNT-B. These results suggest that cellular uptake of the MWCNTs by phagocytosis and chemokine induction is important aspects of their toxicity.

Regional characteristics of Lactobacillus plantarum group strains isolated from two kinds of Japanese post-fermented teas, Ishizuchi-kurocha and Awa-bancha.

Properties of Lactobacillus plantarum group strains isolated from two kinds of Japanese post-fermented teas, Ishizuchi-kurocha and Awa-bancha, were compared. Although lactic acid bacteria isolated from the fermented teas were identified as L. plantarum via homology comparison of 16S ribosomal RNA gene sequences, classification of L. plantarum based on ribosomal proteins showed that the strains isolated from Ishizuchi-kurocha and Awa-bancha were different. According to classification by the ribosomal protein typing, Ishizuchi-kurocha-derived strains belong to the same group as L. plantarum subsp. plantarum JCM 1149T. Awa-bancha-derived strains were assigned to a different group. This pattern was also applicable to strains isolated more than 10 years ago. A further analysis based on recA and a dnaK gene showed that Awa-bancha-derived strains were closely related to L. pentosus. The interactions with cultured cells were different between strain JCM 1149T and the Ishizuchi-kurocha-derived strains. The Ishizuchi-kurocha-derived strains showed strong adhesion to Caco-2 cells. In contrast, strain JCM 1149T and the Awa-bancha-derived strains hardly adhered to Caco-2 cells. According to the ribosomal protein typing, sugar utilization, and interaction with Caco-2 cells, although these properties were dependent on the strain strictly speaking, the L. plantarum group strains in this study can be subdivided into two groups: (1) type strain JCM 1149T and Ishizuchi-kurocha-derived strains and (2) Awa-bancha-derived strains. A regionally unique microorganism may persist in each traditional fermented drink.

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.

Evaluation of probiotic and prebiotic-like effects of Bacillus subtilis BN on growth of lactobacilli.

The aim of this study was to determine the probiotic and the prebiotic-like properties of Bacillus subtilis BN, a spore-forming bacterium, also known as "natto-kin", which is used for making the Japanese fermented food, natto. We used the spores and vegetative cells of this strain and compared their effects on the growth of lactobacilli. Culture supernatant from B. subtilis BN was added to a glucose-free MRS medium used to culture lactobacilli. When lactobacilli were cultured in the supernatant-containing medium, growth was improved. This effect resulted from the digestion of starch by amylase, which was secreted by B. subtilis. Moreover, improved amylase-independent growth was also observed. Co-culture with B. subtilis improved the growth of lactobacilli, and this effect was only observed with vegetative cells; spores did not improve the growth of lactobacilli. This effect on growth was lost upon heat treatment of the vegetative cells. These results suggest that the surface protein of B. subtilis BN vegetative cells participates in the improved growth effect of lactobacilli. It is possible that B. subtilis BN could improve the intestinal flora. In addition, B. subtilis BN inhibited the growth of Salmonella enterica. Thus, it was shown that B. subtilis BN has both a probiotic and prebiotic potential. This is the first study demonstrating the selective growth improvement of indigenous intestinal lactobacilli using B. subtilis BN.

Comparative analysis of the intestinal flora in type 2 diabetes and nondiabetic mice.

A relationship between type 2 diabetes mellitus (T2DM) and intestinal flora has been suggested since development of analysis technology for intestinal flora. An animal model of T2DM is important for investigation of T2DM. Although there are some animal models of T2DM, a comparison of the intestinal flora of healthy animals with that of T2DM animals has not yet been reported. The intestinal flora of Tsumura Suzuki Obese Diabetes (TSOD) mice was compared with that of Tsumura, Suzuki, Non Obesity (TSNO) mice in the present study. The TSOD mice showed typical type 2 diabetes symptoms, which were high-fat diet-independent. The TSOD and the TSNO mouse models were derived from the same strain, ddY. In this study, we compared the intestinal flora of TSOD mice with that if TSNO mice at 5 and 12 weeks of age. We determined that that the number of operational taxonomic units (OTUs) was significantly higher in the cecum of TSOD mice than in that of TSNO mice. The intestinal flora of the cecum and that of the feces were similar between the TSNO and the TSOD strains. The dominant bacteria in the cecum and feces were of the phyla Firmicutes and Bacteroidetes. However, the content of some bacterial species varied between the two strains. The percentage of Lactobacillus spp. within the general intestinal flora was higher in TSOD mice than in TSNO mice. In contrast, the percentages of order Bacteroidales and family Lachnospiraceae were higher in TSNO mice than in TSOD mice. Some species were observed only in TSOD mice, such as genera Turicibacter and SMB53 (family Clostridiaceae), the percentage of which were 3.8% and 2.0%, respectively. Although further analysis of the metabolism of the individual bacteria in the intestinal flora is essential, genera Turicibacter and SMB53 may be important for the abnormal metabolism of type 2 diabetes.

Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages.

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO3)-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO3 particles.

Comparison of antioxidant activities among four kinds of Japanese traditional fermented tea.

Antioxidant activities of four kinds of Japanese traditional fermented tea, Gishi-cha, Ishizuchi-kurocha, Awa-bancha, and Batabatacha, were compared. Antioxidant activity was evaluated by three parameters: copper ion reduction ability, radical trapping ability, and oxygen consumption rate. Processes of fermentation of these fermented teas are different. Goichi-cha and Ishizuchi-kurocha are produced by a two-stage fermentation process, aerobic fermentation and subsequent anaerobic fermentation. Awa-bancha is produced by anaerobic fermentation. And batabata-cha is produced by aerobic fermentation. Additionally, unfermented green tea was also employed as control. These tea leaves were extracted by boiling water and measured antioxidant activities. And concentrations of caffeine and catechins were measured in green tea and in the four kinds of fermented tea: Ishizuchi-kurocha, Goishi-cha, Awa-Bancha, and Batabata-cha. Concentrations of caffeine and catechins were lower in the fermented teas than in green tea. Among the fermented teas, epigallocatechin content was the highest in Ishizuchi-kurocha, whereas Batabata-cha hardly contained any epigallocatechin. Goichi-cha, Ishizuchi-kurocha, and Awa-bancha showed antioxidative activity regardless of measurement method. Batabatacha had hardly any antioxidative activity. Among the fermented teas, Ishizuchi-kurocha had the strongest antioxidant activity. The antioxidative activities of green tea and the four kinds of fermented tea were significantly different among each other (p < .01). Implication of this study is as follows: although contents of catechins were lower than that of green tea, three kinds of fermented tea showed antioxidative activity comparable to green tea. The results suggest that anaerobic fermentation process is beneficial at least for antioxidative activity.

Does photocatalytic activity of TiO2 nanoparticles correspond to photo-cytotoxicity? Cellular uptake of TiO2 nanoparticles is important in their photo-cytotoxicity.

Titanium dioxide (TiO2) nanoparticles are important industrial nano-objects with wide applications, including as photocatalysts and sunscreen components. Recently, the phototoxicity of TiO2 nanoparticles has been a concern. However, phototoxicity caused by photocatalytic activity may differ between anatase and rutile nanoparticles. In the present study, we compared the phototoxicity of anatase and rutile nanoparticles. Human keratinocyte HaCaT cells were treated with stable TiO2 nanoparticle suspensions. Without UVA irradiation, TiO2 nanoparticles did not affect mitochondrial activity or cell membranes. However, exposure to rutile nanoparticle suspensions inhibited cell growth and induced HO-1 gene expression without UVA irradiation. These effects may be explained by the hydrophobic surface of rutile nanoparticles. Next, TiO2-exposed cells were irradiated with UVA for 4 h and effects of TiO2 nanoparticles on cells were examined. The rutile nanoparticles did not show any cellular effects after UVA irradiation. However, the anatase nanoparticles caused strong phototoxicity. Decreased mitochondrial activity, cell membrane damage and the induction of oxidative stress were observed in the cells exposed to anatase nanoparticles with UVA irradiation. Cellular uptake of the nanoparticles was observed in both anatase- and rutile-exposed cells. These results suggest that internalized anatase nanoparticles are important for phototoxicity. Additionally, the exposure of a 3D skin model to TiO2 nanoparticles did not result in significant toxicity. In conclusion, rutile nanoparticles used in sunscreen did not exhibit phototoxic activity. Despite the strong phototoxic activity of anatase nanoparticles in cell cultures, they demonstrated no phototoxicity using a 3D skin model.

Nanoparticles speckled by ready-to-conjugate lanthanide complexes for multimodal imaging.

Multimodal and multifunctional contrast agents receive enormous attention in the biomedical imaging field. Such contrast agents are routinely prepared by the incorporation of organic molecules and inorganic nanoparticles (NPs) into host materials such as gold NPs, silica NPs, polymer NPs, and liposomes. Despite their non-cytotoxic nature, the large size of these NPs limits the in vivo distribution and clearance and inflames complex pharmacokinetics, which hinder the regulatory approval for clinical applications. Herein, we report a unique method that combines magnetic resonance imaging (MRI) and fluorescence imaging modalities together in nanoscale entities by the simple, direct and stable conjugation of novel biotinylated coordination complexes of gadolinium(III) to CdSe/ZnS quantum dots (QD) and terbium(III) to super paramagnetic iron oxide NPs (SPION) but without any host material. Subsequently, we evaluate the potentials of such lanthanide-speckled fluorescent-magnetic NPs for bioimaging at single-molecule, cell and in vivo levels. The simple preparation and small size make such fluorescent-magnetic NPs promising contrast agents for biomedical imaging.

Photouncaging nanoparticles for MRI and fluorescence imaging in vitro and in vivo.

Multimodal and multifunctional nanomaterials are promising candidates for bioimaging and therapeutic applications in the nanomedicine settings. Here we report the preparation of photouncaging nanoparticles with fluorescence and magnetic modalities and evaluation of their potentials for in vitro and in vivo bioimaging. Photoactivation of such bimodal nanoparticles prepared using photouncaging ligands, CdSe/ZnS quantum dots, and super paramagnetic iron oxide nanoparticles results in the systematic uncaging of the particles, which is correlated with continuous changes in the absorption, mass and NMR spectra of the ligands. Fluorescence and magnetic components of the bimodal nanoparticles are characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and elemental analyses using energy dispersive X-ray (EDX) spectroscopy and X-ray photoelectron spectroscopy (XPS). Bioconjugation of the nanoparticles with peptide hormones renders them with biocompatibility and efficient intracellular transport as seen in the fluorescence and MRI images of mouse melanoma cells (B16) or human lung epithelial adenocarcinoma cells (H1650). Biocompatibility of the nanoparticles is evaluated using MTT cytotoxicity assays, which show cell viability over 90%. Further, we combine MRI and NIR fluorescence imaging in C57BL/6 (B6) mice subcutaneously or intravenously injected with the photouncaging nanoparticles and follow the in vivo fate of the nanoparticles. Interestingly, the intravenously injected nanoparticles initially accumulate in the liver within 30 min post injection and subsequently clear by the renal excretion within 48 h as seen in the time-dependent MRI and fluorescence images of the liver, urinary bladder, and urine samples. Photouncaging ligands such as the ones reported in this article are promising candidates for not only the site-specific delivery of nanomaterials-based contrast agents and drugs but also the systematic uncaging and renal clearance of nanomaterials after the desired in vivo application.

Singlet-oxygen-sensitizing near-infrared-fluorescent multimodal nanoparticles.

Nanoprobes based on quantum clusters (QC) with near-infrared fluorescence, magnetic-resonance-imaging contrast, and singlet-oxygen-sensitized intracellular fluorescence are studied. The generation of singlet oxygen and singlet-oxygen-sensitized fluorescence uncaging by magnetic and NIR-emitting nanoparticles are exploited for multimodal bioimaging in vitro.

Impairments of cells and genomic DNA by environmentally transformed engineered nanomaterials.

Enormous increase in the production of nanomaterials and their growing applications in the device technology, biotechnology and biomedical areas suggest the need for developing models for predicting the environmental health and safety (EHS) risks posed by such nanomaterials. We hypothesize that CdSe quantum dots (QDs) and ZnO nanoparticles (NPs) encompassed in liposomes or not and transformed by simulated solar UV light can be model systems for studying the environmental toxicity of engineered nanomaterials. In this study, human lung epithelial adenocarcinoma cells (H1650) are exposed to photoirradiated CdSe QDs or ZnO nanopowder included or not in liposomes. The release of cadmium and zinc ions from the nanomaterials exposed to solar simulated UV radiation is detected and quantified by measuring the steady-state and time resolved fluorescence of the metal ion sensor tetracarboxyphenylporphyrin (TCPP) or the commercial Measure iT Pd/Cd sensor. Viability of cells treated with nanomaterials exposed to solar simulated UV radiation for different durations is measured by MTT assay. Enhanced etching of the nanoparticles exposed to solar simulated UV radiation results in the release of toxic levels of heavy metal ions, which considerably lower the viability of H1650 cells is due to the deactivation of DNA repair enzymes as evidenced by the pinching off of nuclear DNA in comet assays and DNA samples in electrophoresis. Results from this study highlight the need to obtain not only quantitative information about the environmental risks posed by engineered nanomaterials but also environment friendly nanomaterials for practical applications.

Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria.

Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.

Sodium-dependent phosphate cotransporters: lessons from gene knockout and mutation studies.

Inorganic phosphate (Pi) is an essential physiological compound, highlighted by the syndromes caused by hypo or hyperphosphatemic states. Hyperphosphatemia is associated with ectopic calcification, cardiovascular disease, and increased mortality in patients with chronic kidney disease (CKD). As phosphate control is not efficient with diet or dialysis, oral Pi binders are used in over 90% of patients with renal failure. However, achieving tight control of serum Pi is difficult, and lower levels of serum Pi (severe hypophosphatemia) do not lead to better outcomes. The inhibition of sodium-dependent Pi (NaPi) transporter would be a preferable method to control serum Pi levels in patients with CKD or patients undergoing dialysis. Three types of NaPi transporters (types I-III) have been identified: solute carrier series SLC17A1 (NPT1/NaPi-I/OATv1), SLC34 (NaPi-IIa, NaPi-IIb, NaPi-IIc), and SLC20 (PiT1, PiT2), respectively. Knockout mice have been created for types I-III NaPi transporters. In this review, we discuss the roles of the NaPi transporters in Pi homeostasis.