Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro

Oxidative stress and inflammatory responses induced by silica nanoparticles were evaluated both in mice and in RAW264.7 cell line. Single treatment of silica nanoparticles (50 mg/kg, i.p.) led to the activation of peritoneal macrophages, the increased blood level of IL-1β and TNF-α, and the increased level of nitric oxide released from the peritoneal macrophages. mRNA expressions of inflammation-related genes such as IL-1, IL-6, TNF-α, iNOS, and COX-2 were also elevated in the cultured peritoneal macrophages harvested from the treated mice. When the viability of splenocytes from the mice treated with silica nanoparticles (50 mg/kg, 100 mg/kg, and 250 mg/kg, i.p.) was measured, the viability of splenocytes was significantly decreased in the higher dose-treated groups (100 mg/kg, 200 mg/kg i.p.). However, cell proliferation without cytotoxicity was shown in group treated with relatively low dose of 50 mg/kg i.p. When leukocyte subtypes of mouse spleen were evaluated using flow cytometry analysis, it was found that the distributions of NK cells and T cells were increased to 184.8% and 115.1% of control, respectively, while that of B cells was decreased to 87.7%. To elucidate the pro-inflammatory mechanism of silica nanoparticles in vivo, in vitro study using RAW 264.7 cell line which is derived from mouse peritoneal macrophage was done. Treatment of silica nanoparticles to the cultured RAW264.7 cells led to the reactive oxygen species (ROS) generation with a decreased intracellular GSH. In accordance with ROS generation, silica nanoparticles increased the level of nitric oxide released from the cultured macrophage cell line. These results suggested that silica nanoparticles generate ROS and the generated ROS may trigger the pro-inflammatory responses both in vivo and in vitro.     

In vitro assessment of cellular responses to rod-shaped hydroxyapatite nanoparticles of varying lengths and surface areas

Abstract

Rod-shaped hydroxyapatite nanoparticles of varying dimensions (≈ 60 ± 10, 120 ± 15, 240 ± 30 nm in length, labeled respectively as nHA60, nHA120 and nHA240) with specific surface areas (47.02, 23.33, 46.12 nm², respectively), were synthesized and their effects on cell viability, reactive oxygen species generation and cellular interaction with BEAS-2B, RAW264.7 and HepG2 were investigated. In vitro exposure of these cell lines to rod shape nHA particles within a range of 10–300 μg/ml for 24 h did not significantly alter cell viability studied by the WST-8 assay. A significant increase in reactive oxygen species (ROS) generation was however observed with the dihydrofluorescein diacetate (DFDA) assay after 4 h incubation with these nanoparticles. The lowest level of ROS generation was observed with nHA120 (with the smallest specific surface area); whereas nHA60 and nHA240 exhibited comparable ROS generation. Subsequently, the Alizarin Red-S (ARS) assay indicated a weaker association of calcium with cells compared to nHA60 and nHA240. The results thus suggest that high surface area may increase cell-particle interaction, which in turn influenced ROS generation. The combined results from all the cell lines thus indicated high biocompatibility of rod-shaped nHA.     

Mechanistic studies of in vitro cytotoxicity of poly(amidoamine) dendrimers in mammalian cells

Poly(amidoamine) (PAMAM) dendrimer nanoparticles have been demonstrated to elicit a well defined cytotoxicological response from mammalian cell lines, the response increasing systematically with dendrimer generation and number of surface amino groups. In this work, using generation G4, G5, and G6 dendrimers, this systematic response is furthermore demonstrated for the generation of reactive oxygen species, lysosomal activity, and the onset of apoptosis and levels of DNA damage. The results are consistent with a pathway of localisation of PAMAM dendrimers in the mitochondria leading to ROS production causing oxidative stress, apoptosis and DNA damage. ROS production is co-located in the mitochondria, and both generated levels and timescales are systematically generation dependent (G4 < G5 < G6). Flow cytometry confirms that with increasing dose, the percentage of healthy and early apoptotic cells decreases, whereas the late apoptotic and necrotic cell populations increase. This process is again systematically generation dependent. DNA damage as measured using the TUNEL assay further demonstrates a systematic trend, G4, G5 and G6 showing 4.69%, 25.87% and 89.63% DNA breakage respectively. Increases in lysosomal activity at timescales of ~ 24 h are observed in HaCaT but not SW480 cells upon low concentration PAMAM exposure. Overall, significant differences are observed between the responses of the dermal cell line, HaCaT, and the colon cell line, SW480, and it is suggested that these can be understood in terms of differing intrinsic antioxidant levels.     

Nanoparticle (NP) uptake by type I alveolar epithelial cells and their oxidant stress response

Abstract

Mammalian cells take up nanoparticles (NPs) and some NPs increase ROS. We used imaging and measure ROS in parallel to evaluate NP-cell interactions with type I-like alveolar epithelial cells exposed to NPs at 1.2 µg/cm². Titanium dioxide (Ti0₂), gold (Au), silver (Ag), and manganese (Mn) were internalized by R3-1 cells; copper (Cu) NPs were observed at the cell surface only. TiO₂ and Au did not increase cell death but Mn and Cu did, with surviving cells recovering after initial Cu exposure. Ag NPs caused 80% of R3-1 cells to lift off the slides within 1 h. Amplex Red was used to report H₂O₂ production after exposure to 0.4 µg/cm² TiO₂, Au, Cu, Mn and Ag. TiO₂, Au, and Ag caused no significant increase in H₂O₂ while Cu and Mn increased H₂O₂. NPs that give up electrons, increase ROS production and cause cell death in R3-1 cells.     

Tephrosin-induced autophagic cell death in A549 non-small cell lung cancer cells

Anticancer effect of tephrosin (1) has been documented; however, the molecular mechanisms underlying the cytotoxicity of tephrosin in cancer cells remain unclear. In the present paper, the proliferation inhibition rate of several cancer cells was tested using the MTT assay; cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were determined by flow cytometry; poly(ADP-ribose) polymerase (PARP) cleavage and heat shock protein 90 (Hsp90) expression were evaluated by Western blotting; autophagy was examined by confocal microscopy and light chain 3 (LC3) conversion assay. The results showed that exposure of the cells to tephrosin induced significant proliferation inhibition in a dose-dependent manner, especially on A549 with G₂/M being arrested. Tephrosin was not found to induce cell apoptosis as PARP cleavage was not detected after 24 h treatment, but the formation of acidic vesicular organelle of autophagy character was found, and autophagy was further confirmed by the increase in the ratio of LC3-II to LC3-I. It was observed that tephrosin induced ROS generation and Hsp90 expression inhibition. These results indicate that tephrosin induces A549 cancer cell death via the autophagy pathway, and the roles of ROS generation and Hsp90 expression inhibition in this process need further study in the future.     

Effect of mesoporous silica nanoparticles on cell viability and markers of oxidative stress

Abstract

In the recent years, the use of mesoporous silica nanoparticles (MSNs) has been extended in biomedical fields such as cancer therapy, drug and gene delivery, biosensors, and enzyme immobilization. Although nanomaterials are currently being widely used in modern technology, there is a lack of information regarding to the health and environmental implications of manufactured nanomaterials. In the present study, the effects of MSNs and surface functionalized MSNs on cell viability, markers of oxidative damages (mainly intracellular reactive oxygen species (ROS) formation), and oxidative DNA damage were investigated in vitro in rat pheochromocytoma PC12 cells. Following exposure of these nanoparticles (1.95–1000?µg/mL) to PC12 cells for 12 and 24?h, no significant reduction of cell viability was observed compared with control. Moreover, ROS formation and oxidative DNA damage were not significantly changed by these nanoparticles even at high concentrations or prolong exposures. In conclusion, the results showed that neither MSNs nor functionalized MSNs exhibited any remarkable in vitro toxic properties in PC12 cells even at high concentration.     

Investigation on the mechanism of non‐photocatalytically TiO2‐induced reactive oxygen species and its significance on cell cycle and morphology

Titanium dioxide (TiO₂) nanoparticles are widely used in daily human life, and were reported to elicit biological effects such as oxidative stress either generating reactive oxygen species (ROS) or causing cell necrosis without generating ROS, whose underlying molecular mechanisms are not yet known. In this study, the role of dissolved oxygen in TiO₂ catalytic activity in dark environment, and long‐term cytotoxic effects of TiO₂ exposure were investigated. To determine the effect of dissolved oxygen, the anatase‐TiO₂ nanoparticle suspension was prepared both in deoxygenated and regular MilliQ water, and a ~ 9‐fold higher ROS in regular MilliQ samples was observed compared to deoxygenated samples while in the dark, which suggested dissolved oxygen as the driving agent behind the TiO₂ catalytic reaction. On the other hand, the differential cell viability and endogenous ROS activity was demonstrated through a sensitive macrophage‐based assay, on a dose‐ and time‐dependent manner. Both the cell number and endogenous ROS activity increased with increase in time till 48 h, followed by a reduction at 72 h exposure period. Long‐term exposures to these nanoparticles even at low concentrations were found detrimental to cells, where late apoptosis until 48 h and necrosis at 72 h leading to cell death were noted. Late apoptotic events and cell membrane cytoskeletal actin rearrangement observed were hypothesized to be induced by particle‐mediated cellular ROS. This in addition to radical generation ability of TiO₂ in the dark will help further in better understanding of the toxicity mechanism in cells beyond ROS generation. Copyright © 2016 John Wiley & Sons, Ltd.     

Investigating the toxic effects induced by iron oxide nanoparticles on neuroblastoma cell line: an integrative study combining cytotoxic,genotoxic and proteomic tools

Abstract

Nanomaterials have gained much attention for their use and benefit in several fields. Iron Oxide Nanoparticles (IONPs) have been used in Biomedicine as contrast agents for imaging cancer cells. However, several studies reported the potential toxicity of those nanoparticles in different models, especially in cells. Therefore, in our present study, we investigated the effects of IONPs on the SH-SY5Y neuroblastoma cell line. We carried out cytotoxic and genotoxic studies to evaluate the phenotypic effects, and proteomic investigation to evaluate the molecular effects and the mechanisms by which this kind of NPs could induce toxicity. Our results showed that the use of three different sizes of IONPs (14, 22 and 30?nm) induced cell detachment, cell morphological changes, size, and concentration-dependent IONP internalization and cell mortality. IONPs induced slight genotoxic damage assayed by modified comet assay without affecting cell cycle, mitochondrial function, membrane integrity, intracellular calcium level, and without inducing ROS generation. All the studies were performed to compare also the effects of IONPs to the ferric iron by incubating cells with equivalent concentration of FeCl₃. In all tests, the NPs exhibited more toxicity than the ferric iron. The proteomic analysis followed by gene ontology and pathway analysis evidenced the effects of IONPs on cytoskeleton, cell apoptosis, and cancer development. Our findings provided more information about IONP effects on human cells and especially on cancer cell line.     

Antitumor 1-nitroacridine derivative C-1748, induces apoptosis, necrosis or senescence in human colon carcinoma HCT8 and HT29 cells

C-1748 is a DNA-binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biologically relevant concentrations (EC₉₀ and their multiplicity). Cell cycle analysis showed gradual increase in HCT8 cells with sub-G1 DNA content (25% after 72 h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4× EC₉₀ concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction and caspase activation confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in the state of senescence, based on altered cell morphology and expression of a pH 6-dependent β-galactosidase. On the contrary, no β-galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168 h) resulted in massive detachment of cells from culture plates, which together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug.     

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.     

Cell Death by Polyvinylpyrrolidine-Coated Silver Nanoparticles is Mediated by ROS-Dependent Signaling

Silver nanoparticles (AgNPs) are widely used nanoparticles and they are mainly used in antibacterial and personal care products. In this study, we evaluated the effect of AgNPs on cell death induction in the murine dendritic cell line DC2.4. DC2.4 cells exposed to AgNPs showed a marked decrease in cell viability and an induction of lactate dehydrogenase (LDH) leakage in a time- and dose-dependent manner. In addition, AgNPs promoted reactive oxygen species (ROS)-dependent apoptosis and AgNP-induced ROS triggered a decrease in mitochondrial membrane potential. The activation of the intracellular signal transduction pathway was also observed in cells cultured with AgNPs. Taken together, our data demonstrate that AgNPs are able to induce a cytotoxic effect in DCs through ROS generation. This study provides important information about the safety of AgNPs that may help in guiding the development of nanotechnology applications.     

Antiproliferative effect of pheophorbide a–mediated photodynamic therapy and its synergistic effect with doxorubicin on multiple drug-resistant uterine sarcoma cell MES-SA/Dx5

Abstract

Prolonged cancer chemotherapy is associated with the development of multidrug resistance (MDR), which is a major cause of treatment failure. Photodynamic therapy (PDT) has been applied as anticancer therapy and a means of circumventing MDR. The antiproliferative effect of pheophorbide a–mediated photodynamic therapy (Pa-PDT) has been demonstrated in several human cancer cell lines, including the uterine sarcoma cell line, MES-SA. This study set out to evaluate, first, the therapeutic potential of Pa-PDT on MES-SA/Dx5 uterine sarcoma cells and, subsequently, the effectiveness of combination therapy using Pa-PDT with doxorubicin (Dox). Our results showed that Pa-PDT was able to circumvent MDR in the P-glycoprotein (P-gp) overexpressing human uterine sarcoma cell line, MES-SA/Dx5. Intracellular accumulation of Pa and Pa-PDT-induced cell death was not abrogated by MDR phenotype, when compared to the parental cell line, MES-SA. Combined therapy using Pa-PDT and Dox, a common chemotherapeutic drug, was found to be synergistic in the cell line, MES-SA/Dx5. Both activity and expression of MDR1 and P-gp were reduced by Pa-PDT treatment and such reductions were attenuated by α-tocopherol, the scavenger of reactive oxygen species (ROS), suggesting that the effect of Pa-PDT was mediated by the generation of intracellular ROS. In conclusion, our findings demonstrated the therapeutic potential of Pa-PDT alone or in combination with Dox in combating multidrug-resistant malignancies.     

Zinc sulfide nanoparticles selectively induce cytotoxic and genotoxic effects on leukemic cells: involvement of reactive oxygen species and tumor necrosis factor alpha

The aim of the present study was to develop zinc sulfide nanoparticles (ZnS NPs) and to study their cytotoxicity against the KG‐1A (human acute myeloid leukemia) cell line. ZnS NPs were synthesized using the pyrolytic method and characterized by X‐ray diffraction, dynamic light scattering, surface zeta potential, scanning electron microscopy and atomic force microscopy. Cell viability study and flow cytometric analysis confirmed the potent cytotoxic effects of ZnS NPs on cancer cells in a dose‐dependent fashion. Successful uptakes of ZnS NPs by leukemic cells were confirmed by phase contrast fluorescence microscopy. pH‐dependent dissolution of ZnS NPs was done using atomic absorption microscopy to understand the cell‐specific internalization of Zn⁺. This internalization of NPs facilitated the generation of excess reactive oxygen species (ROS), followed by tumor necrosis factor alpha (TNF‐α) secretion which caused severe DNA damage as observed in the comet assay and altered the mitochondrial membrane potential (MMP) in leukemic cells. Surprisingly ZnS NPs had no toxic effects on normal lymphocytes at doses up to 50 µg ml^–1. Pre‐treatment with ROS and TNF‐α inhibitor confirmed that these nanoparticles were able to kill leukemic cells by generating an excess amount of ROS and thereby initiated TNF‐α mediated apoptosis pathway. These findings clarify the mechanism with which ZnS NPs induced anticancer activities in vitro. To elicit its utilities and its application to cancer treatment in vivo is under investigation. Copyright © 2014 John Wiley & Sons, Ltd.     

Doxorubicin-loaded solid lipid nanoparticles to overcome multidrug resistance in cancer therapy

In the present study we developed doxorubicin-loaded solid lipid nanoparticles (SLN-Dox) using biocompatible compounds, assessed the in vitro hemolytic effect, and examined their in vivo effects on drug retention and apoptosis intensity in P-glycoprotein-overexpressing MCF-7/ADR cells, a representative Dox-resistant breast cancer cell line. Our SLNs did not show hemolytic activity in human erythrocytes. In comparison with Dox, SLN-Dox efficiently enhanced apoptotic cell death through the higher accumulation of Dox in MCF-7/ADR cells. Therefore, SLN-Dox have potential to serve as a useful therapeutic approach to overcome the chemoresistance of adriamycin-resistant breast cancer.From the Clinical EditorDoxorubicin loaded solid lipid nanoparticles (SLN-Dox) were studied in a cell line representative of doxorubicin resistant breast cancer. The nanoparticles did not show hemolytic activity; furthermore, they efficiently enhanced apoptotic cell death through higher accumulation of doxorubicin in cancer cells. This approach may be viable in overcoming the chemoresistance of adriamycin resistant breast cancer.     

Contributions of reactive oxygen species and mitogen-activated protein kinase signaling in arsenite-stimulated hemeoxygenase-1 production

Hemeoxygenase-1 (HO-1) is an oxidative stress responsive gene upregulated by various physiological and exogenous stimuli. HO-1 has cytoprotective activities and arsenite is a potent inducer of HO-1 in many cell types and tissues, including epidermal keratinocytes. We investigated the potential contributions of reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) activation to arsenite-dependent regulation of HO-1 in HaCaT cells, an immortalized human keratinocyte line. Both epidermal growth factor (EGF) and arsenite stimulated ROS production was detected by dihydroethidium (DHE) staining and fluorescence microscopy. Arsenite induced HO-1 in a time- and concentration-dependent manner, while HO-1 expression in response to EGF was modest and evident at extended time points (48-72 h). Inhibition of EGF receptor, MEK I/II or Src decreased arsenite-stimulated HO-1 expression by 20-30%. In contrast, addition of a superoxide scavenger or inhibition of p38 activity decreased the arsenite-dependent response by 80-90% suggesting that ROS and p38 are required for HO-1 induction. However, ROS generation alone was insufficient for the observed arsenite-dependent response as use of a xanthine/xanthine oxidase system to generate ROS did not produce an equivalent upregulation of HO-1. Cooperation between ERK signaling and ROS generation was demonstrated by synergistic induction of HO-1 in cells co-treated with EGF and xanthine/xanthine oxidase resulting in a response nearly equivalent to that observed with arsenite. These findings suggest that the ERK/MAPK activation is necessary but not sufficient for optimal arsenite-stimulated HO-1 induction. The robust and persistent upregulation of HO-1 may have a role in cellular adaptation to chronic arsenic exposure.     

乙醇诱导肝癌细胞凋亡的实验研究

目的研究低浓度乙醇对肝癌细胞的影响及作用机制。方法采用噻唑蓝比色法观察乙醇对细胞增殖的影响；采用流式细胞仪法观察乙醇对肝癌细胞内活性氧的影响；采用琼脂凝胶电泳法观察DNA片断化条带。结果低浓度乙醇可明显抑制细胞增殖；乙醇作用于细胞后2h内引起活性氧产生，24h后细胞出现典型的凋亡表现。结论乙醇能够通过诱导细胞内活性氧的产生引起细胞凋亡，呈明显的剂量依赖性。     

Oxidative stress mediated apoptosis induced by nickel ferrite nanoparticles in cultured A549 cells

Due to the interesting magnetic and electrical properties with good chemical and thermal stabilities, nickel ferrite nanoparticles are being utilized in many applications including magnetic resonance imaging, drug delivery and hyperthermia. Recent studies have shown that nickel ferrite nanoparticles produce cytotoxicity in mammalian cells. However, there is very limited information concerning the toxicity of nickel ferrite nanoparticles at the cellular and molecular level. The aim of this study was to investigate the cytotoxicity, oxidative stress and apoptosis induction by well-characterized nickel ferrite nanoparticles (size 26 nm) in human lung epithelial (A549) cells. Nickel ferrite nanoparticles induced dose-dependent cytotoxicity in A549 cells demonstrated by MTT, NRU and LDH assays. Nickel ferrite nanoparticles were also found to induce oxidative stress evidenced by generation of reactive oxygen species (ROS) and depletion of antioxidant glutathione (GSH). Further, co-treatment with the antioxidant L-ascorbic acid mitigated the ROS generation and GSH depletion due to nickel ferrite nanoparticles suggesting the potential mechanism of oxidative stress. Quantitative real-time PCR analysis demonstrated that following the exposure of A549 cells to nickel ferrite nanoparticles, the level of mRNA expressions of cell cycle checkpoint protein p53 and apoptotic proteins (bax, caspase-3 and caspase-9) were significantly up-regulated, whereas the expression of anti-apoptotic proteins (survivin and bcl-2) were down-regulated. Moreover, activities of caspase-3 and caspase-9 enzymes were also significantly higher in nickel ferrite nanoparticles exposed cells. To the best of our knowledge this is the first report showing that nickel ferrite nanoparticles induced apoptosis in A549 cells through ROS generation and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways.     

活性氧介导5, 7- 二甲氧基黄酮诱导结肠癌HT- 29 细胞凋亡

目的研究5,7-二甲氧基黄酮（5,7-dimethoxyflavone,DMF）诱导体外培养人结肠癌HT-29细胞凋亡作用及其机制。方法以HT-29细胞为研究对象,利用ELISA法测定细胞DNA断裂,流式细胞术检测细胞凋亡率和活性氧（ROS）水平。结果DMF激活HT-29细胞ROS生成（P〈0．05）,呈浓度依赖性。DMF以浓度依赖方式促进HT-29细胞DNA断裂（P〈0．05）,同时,诱导细胞凋亡率增高（P〈0．05）;10μmol．L^-1N-乙酰半胱氨酸预孵育30min,能有效阻断DMF诱导ROS生成和细胞凋亡作用（P〈0．05）。结论DFM通过促进结肠癌HT-29细胞ROS生成诱导细胞凋亡。     

Involvement of activation of NADPH oxidase and extracellular signal-regulated kinase (ERK) in renal cell injury induced by zinc

Zinc is employed as a supplement; however, zinc-related nephropathy is not generally known. In this study, we investigated zinc-induced renal cell injury using a pig kidney-derived cultured renal epithelial cell line, LLC-PK(1), with proximal kidney tubule-like features, and examined the involvement of free radicals and extracellular signal-regulated kinase (ERK) in the cell injury. The LLC-PK(1) cells showed early uptake of zinc (30 microM), and the release of lactate dehydrogenase (LDH), an index of cell injury, was observed 24 hr after uptake. Three hours after zinc exposure, generation of reactive oxygen species (ROS) was increased. An antioxidant, N, N'-diphenyl-p-phenylenediamine (DPPD), inhibited a zinc-related increase in ROS generation and zinc-induced renal cell injury. An NADPH oxidase inhibitor, diphenyleneiodonium (DPI), inhibited a zinc-related increase in ROS generation and cell injury. We investigated translocation from the cytosol fraction of the p67(phox) subunit, which is involved in the activation of NADPH oxidase, to the membrane fraction, and translocation was induced 3 hr after zinc exposure. We examined the involvement of ERK1/2 in the deterioration of zinc-induced renal cell injury, and the association between ERK1/2 and an increase in ROS generation. Six hours after zinc exposure, the activation (phosphorylation) of ERK1/2 was observed. An antioxidant, DPPD, inhibited the zinc-related activation of ERK1/2. An MAPK/ERK kinase (MEK1/2) inhibitor, U0126, almost completely inhibited zinc-related cell injury (the release of LDH), but did not influence ROS generation. These results suggest that early intracellular uptake of zinc by LLC-PK(1) cells causes the activation of NADPH oxidase, and that ROS generation by the activation of the enzyme leads to the deterioration of renal cell injury via the activation of ERK1/2.     

In vitro assessment of cellular responses to rod-shaped hydroxyapatite nanoparticles of varying lengths and surface areas

Rod-shaped hydroxyapatite nanoparticles of varying dimensions (≈ 60 ± 10, 120 ± 15, 240 ± 30 nm in length, labeled respectively as nHA60, nHA120 and nHA240) with specific surface areas (47.02, 23.33, 46.12 nm(2), respectively), were synthesized and their effects on cell viability, reactive oxygen species generation and cellular interaction with BEAS-2B, RAW264.7 and HepG2 were investigated. In vitro exposure of these cell lines to rod shape nHA particles within a range of 10-300 μg/ml for 24 h did not significantly alter cell viability studied by the WST-8 assay. A significant increase in reactive oxygen species (ROS) generation was however observed with the dihydrofluorescein diacetate (DFDA) assay after 4 h incubation with these nanoparticles. The lowest level of ROS generation was observed with nHA120 (with the smallest specific surface area); whereas nHA60 and nHA240 exhibited comparable ROS generation. Subsequently, the Alizarin Red-S (ARS) assay indicated a weaker association of calcium with cells compared to nHA60 and nHA240. The results thus suggest that high surface area may increase cell-particle interaction, which in turn influenced ROS generation. The combined results from all the cell lines thus indicated high biocompatibility of rod-shaped nHA.