Potassium silicate and zinc oxide nanoparticles modulate antioxidant system, membranous H+-ATPase and nitric oxide content in faba bean (Vicia faba) seedlings exposed to arsenic toxicity.

Current research focused on the potential role of zinc oxide nanoparticles (ZnONPs) and potassium (K+ ) in mitigation of arsenic (As) toxicity in Vicia faba L. seedlings. Faba bean seedlings were grown for 30days in potted soil. As stress curtailed root and shoot length, chlorophyll (Chl) content and net photosynthetic rate in V. faba seedlings. However, ZnONPs and K+ curtailed As stress in faba bean seedling through enhanced activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) enzyme. Furthermore, ZnONPs and K+ significantly enhanced cysteine (Cys) content and serine acetyletransferase (SAT) activity in faba bean seedling exposed to As-toxificated soil. Application of ZnONPs and K+ curtailed superoxide ionic content and hydrogen peroxide (H2 O2 ) accumulation in V. faba seedlings exposed to As-polluted soil. Nitric oxide (NO) content also increased in faba bean seedlings treated with ZnONPs and K+ in normal and As-polluted soil. As stress alleviation was credited to reduce As uptake in faba bean seedlings treated with synergistic application of ZnONPs and K+ . It is proposed that K+ interaction with nanoparticles can be exploited at molecular level to understand the mechanisms involved in abiotic stress tolerance.

Selenium seed priming enhanced the growth of salt-stressed Brassica rapa L. through improving plant nutrition and the antioxidant system.

Various abiotic stresses may affect the germination, growth, and yield of direct-seeded vegetable crops. Seed priming with effective antioxidant mediators may alleviate these environmental stresses by maintaining uniformity in seed germination and improving the subsequent health of developing seedlings. Salt-induced stress has become a limiting factor for the successful cultivation of Brassica rapa L., especially in Southeast Asian countries. The present study was performed to elucidate the efficacy of seed priming using selenium (Se) in mitigating salt-induced oxidative stress in turnip crops by reducing the uptake of Na+. In this study, we administered three different levels of Se (Se-1, 75 µmol L-1; Se-2, 100 µmol L-1; and Se-3, 125 µmol L-1) alone or in combination with NaCl (200 mM). Conspicuously, salinity and Se-2 modulated the expression levels of the antioxidant genes, including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX). The upregulated expression of stress-responsive genes alleviated salt stress by scavenging the higher reactive oxygen species (ROS) level. The stress ameliorative potential of Se (Se-2 = 100 µmol L-1) enhanced the final seed germination percentage, photosynthetic content, and seedling biomass production up to 48%, 56%, and 51%, respectively, under stress. The advantageous effects of Se were attributed to the alleviation of salinity stress through the reduction of the levels of malondialdehyde (MDA), proline, and H2O2. Generally, treatment with Se-2 (100 µmo L-1) was more effective in enhancing the growth attributes of B. rapa compared to Se-1 (75 µmo L-1) and Se-3 (125 µmo L-1) under salt-stressed and non-stressed conditions. The findings of the current study advocate the application of the Se seed priming technique as an economical and eco-friendly approach for salt stress mitigation in crops grown under saline conditions.

A Dual Approach of an Oil-Membrane Composite and Boron-Doped Diamond Electrode to Mitigate Biofluid Interferences.

Electrochemical biosensors promise a simple method to measure analytes for both point-of-care diagnostics and continuous, wearable biomarker monitors. In a liquid environment, detecting the analyte of interest must compete with other solutes that impact the background current, such as redox-active molecules, conductivity changes in the biofluid, water electrolysis, and electrode fouling. Multiple methods exist to overcome a few of these challenges, but not a comprehensive solution. Presented here is a combined boron-doped diamond electrode and oil-membrane protection approach that broadly mitigates the impact of biofluid interferents without a biorecognition element. The oil-membrane blocks the majority of interferents in biofluids that are hydrophilic while permitting passage of important hydrophobic analytes such as hormones and drugs. The boron-doped diamond then suppresses water electrolysis current and maintains peak electrochemical performance due to the foulant-mitigation benefits of the oil-membrane protection. Results show up to a 365-fold reduction in detection limits using the boron-doped diamond electrode material alone compared with traditional gold in the buffer. Combining the boron-doped diamond material with the oil-membrane protection scheme maintained these detection limits while exposed to human serum for 18 h.

Oil-Membrane Protection of Electrochemical Sensors for Fouling- and pH-Insensitive Detection of Lipophilic Analytes.

To take full advantage of the reagent- and label-free sensing capabilities of electrochemical sensors, a frequent and remaining challenge is interference and degradation of the sensors due to uncontrolled pH or salinity in the sample solution or foulants from the sample solution. Here, we present an oil-membrane sensor protection technique that allows for the permeation of hydrophobic (lipophilic) analytes into a sealed sensor compartment containing ideal salinity and pH conditions while simultaneously blocking common hydrophilic interferents (proteins, acids, bases, etc.) In this paper, we validate the oil-membrane sensor protection technique by demonstrating continuous cortisol detection via electrochemical aptamer-based (EAB) sensors. The encapsulated EAB cortisol sensor exhibits a 5 min concentration-on rise time and maintains a measurement signal of at least 7 h even in the extreme condition of an acidic solution of pH 3.

Examining cellular responses to reconstituted antibody protein liquids.

Protein ionic liquids (PIL) are a new class of biologic stabilizers designed to protect the functionality and extend the shelf-life of biotechnological and therapeutic agents making them more readily available, and resistant to austere environments. Protein biorecognition elements such as monoclonal antibodies are commonly utilized therapeutics that require the robust stabilization offered by PILs, but biocompatibility remains an important issue. This study has focused on characterizing the biocompatibility of an antibody based PIL by exposing multiple cells types to a cationized immunoglobulin suspended in an anionic liquid (IgG-IL). The IgG-IL caused no significant alterations in cellular health for all three cell types with treatments < 12.5 µg/mL. Concentrations ≥ 12.5 µg/mL resulted in significant necrotic cell death in A549 and HaCaT cells, and caspase associated cell death in HepG2 cells. In addition, all cells displayed evidence of oxidative stress and IL-8 induction in response to IgG-IL exposures. Therapeutic Ig can be utilized with a wide dose range that extends into concentrations we have found to exhibit cytotoxicity raising a toxicity concern and a need for more extensive understanding of the biocompatibility of IgG-ILs.

Engineered aluminum nanoparticle induces mitochondrial deformation and is predicated on cell phenotype.

The main role of mitochondria is to generate the energy necessary for the cell to survive and adapt to different environmental stresses. Energy demand varies depending on the phenotype of the cell. To efficiently meet metabolic demands, mitochondria require a specific proton homeostasis and defined membrane structures to facilitate adenosine triphosphate production. This homeostatic environment is constantly challenged as mitochondria are a major target for damage after exposure to environmental contaminants. Here we report changes in mitochondrial structure profiles in different cell types using electron microscopy in response to particle stress exposure in three different representative lung cell types. Endpoint analyses include nanoparticle intracellular uptake; quantitation of mitochondrial size, shape, and ultrastructure; and confirmation of autophagosome formation. Results show that low-dose aluminum nanoparticles exposure (1 ppm; 1 µg/mL; 1.6 × 1 0-7 µg/cell)) to primary and asthma cells incurred significant mitochondrial deformation and increases in mitophagy, while cancer cells exhibited only slight changes in mitochondrial morphology and an increase in lipid body formation. These results show low-dose aluminum nanoparticle exposure induces subtle changes in the mitochondria of specific lung cells that can be quantified with microscopy techniques. Furthermore, within the lung, cell type by the nature of origin (i.e. primary vs. cancer vs. asthma) dictates mitochondrial morphology, metabolic health, and the metabolic stress response of the cell.

Human Nontumorigenic Microglia Synthesize Strongly Fluorescent Au/Fe Nanoclusters, Retaining Bioavailability.

The ability for cells to self-synthesize metal-core nanoclusters (mcNCs) offers increased imaging and identification opportunities. To date, much work has been done illustrating the ability for human tumorigenic cell lines to synthesize mcNCs; however, this has not been illustrated for nontumorigenic cell lines. Here, we present the ability for human nontumorigenic microglial cells, which are the major immune cells in the central nervous system, to self-synthesize gold (Au) and iron (Fe) core nanoclusters, following exposures to metallic salts. We also show the ability for cells to internalize presynthesized Au and Fe mcNCs. Cellular fluorescence increased in most exposures and in a dose dependent manner in the case of Au salt. Scanning transmission electron microscopic imaging confirmed the presence of the metal within cells, while transmission electron microscopy images confirmed nanocluster structures and self-synthesis. Interestingly, self-synthesized nanoclusters were of similar size and internal structure as presynthesized mcNCs. Toxicity assessment of both salts and presynthesized NCs illustrated a lack of toxicity from Au salt and presynthesized NCs. However, Fe salt was generally more toxic and stressful to cells at similar concentrations.

In Vitro Aerosol Exposure to Nanomaterials: From Laboratory to Environmental Field Toxicity Testing.

Exposure to nanomaterials (NMs) is inevitable, requiring robust toxicological assessment to understand potential environmental and human health effects. NMs are favored in many applications because of their small size; however, this allows them to easily aerosolize and, subsequently, expose humans via inhalation. Toxicological assessment of NMs by conventional methods in submerged cell culture is not a relevant way to assess inhalation toxicity of NMs because of particle interference with bioassays and changes in particokinetics when dispersed in medium. Therefore, an in vitro aerosol exposure chamber (AEC) was custom designed and used for direct deposition of NMs from aerosols in the environment to the air-liquid interface of lung cells. Human epithelial lung cell line, A549, was used to assess the toxicity of copper, nickel, and zinc oxide nanopowders aerosolized by acoustic agitation in laboratory study. Post optimization, the AEC was used in the field to expose the A549 cells to NM aerosols generated from firing a hand gun and rifle. Toxicity was assessed using nondestructive assays for cell viability and inflammatory response, comparing the biologic effect to the delivered mass dose measured by inductively coupled plasma-mass spectrometry. The nanopowder exposure to submerged and ALI cells resulted in dose-dependent toxicity. In the field, weapon exhaust from the M4 reduced cell viability greater than the M9, while the M9 stimulated inflammatory cytokine release of IL-8. This study highlights the use of a portable chamber with the capability to assess toxicity of NM aerosols exposed to air-liquid interface in vitro lung cell culture.

Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria.

Ras proteins are small GTPases localized to the plasma membrane (PM), which regulate cellular proliferation, apoptosis and differentiation. After a series of post-translational modifications, H-Ras and N-Ras traffic to the PM from the Golgi via the classical exocytic pathway, but the exact mechanism of K-Ras trafficking to the PM from the ER is not fully characterized. ATP5G1 (also known as ATP5MC1) is one of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that overexpression of the mitochondrial targeting sequence of ATP5G1 perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and redistributes phosphatidylserine (PtdSer) to mitochondria and other endomembranes, resulting in K-Ras translocation to mitochondria. Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. Glucose supplementation restores PtdSer and K-Ras PM localization and PI4P at the Golgi. We further show that inhibition of the Golgi-localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to mitochondria and endomembranes, respectively. We conclude that PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras.This article has an associated First Person interview with the first author of the paper.

In situ Synthesis of Fluorescent Gold Nanoclusters by Nontumorigenic Microglial Cells.

To date, the directed in situ synthesis of fluorescent gold nanoclusters (AuNCs) has only been demonstrated in cancerous cells, with the theorized synthesis mechanism prohibiting AuNC formation in nontumorigenic cell lines. This limitation hinders potential biostabilized AuNC-based technology in healthy cells involving both chemical and mechanical analysis, such as the direct sensing of protein function and the elucidation of local mechanical environments. Thus, new synthesis strategies are required to expand the application space of AuNCs beyond cancer-focused cellular studies. In this contribution, we have developed the methodology and demonstrated the direct in situ synthesis of AuNCs in the nontumorigenic neuronal microglial line, C8B4. The as-synthesized AuNCs form in situ and are stabilized by cellular proteins. The clusters exhibit bright green fluorescence and demonstrate low (<10%) toxicity. Interestingly, elevated ROS levels were not required for the in situ formation of AuNCs, although intracellular reductants such as glutamate were required for the synthesis of AuNCs in C8B4 cells. To our knowledge, this is the first-ever demonstration of AuNC synthesis in nontumorigenic cells and, as such, it considerably expands the application space of biostabilized fluorescent AuNCs.

Low-dose, subchronic exposure to silver nanoparticles causes mitochondrial alterations in Sprague-Dawley rats.

AIM: Nanoparticles (NPs) have increasingly been studied due to their probable harmful effects to both humans and the environment. However, despite several indications of possible harmful effects, no long-term studies using a low dose of silver nanoparticles (AgNP) have been conducted in vivo. RESULTS: Our data demonstrate that the prolonged exposure to a very low dose of AgNP was sufficient to cause alterations in hepatic mitochondrial function. Mitochondrial function compromised by AgNPs is recovered by pretreatment with the antioxidant N-acetylcysteine, which highlights the crucial role of oxidative stress in AgNPs' toxicity. CONCLUSION: Our data show for the first time that even a very low dose of AgNP can cause harmful effects on mitochondrial function, thus compromising the normal function of the organ.

Assessment of Carbon- and Metal-Based Nanoparticle DNA Damage with Microfluidic Electrophoretic Separation Technology.

In this study, we examined the feasibility of extracting DNA from whole cell lysates exposed to nanoparticles using two different methodologies for evaluation of fragmentation with microfluidic electrophoretic separation. Human lung macrophages were exposed to five different carbon- and metal-based nanoparticles at two different time points (2 h, 24 h) and two different doses (5 µg/ml, 100 µg/ml). The primary difference in the banding patterns after 2 h of nanoparticle exposure is more DNA fragmentation at the higher NP concentration when examining cells exposed to nanoparticles of the same composition. However, higher doses of carbon and silver nanoparticles at both short and long dosing periods can contribute to erroneous or incomplete data with this technique. Also comparing DNA isolation methodologies, we recommend the centrifugation extraction technique, which provides more consistent banding patterns in the control samples compared to the spooling technique. Here we demonstrate that multi-walled carbon nanotubes, 15 nm silver nanoparticles and the positive control cadmium oxide cause similar DNA fragmentation at the short time point of 2 h with the centrifugation extraction technique. Therefore, the results of these studies contribute to elucidating the relationship between nanoparticle physicochemical properties and DNA fragmentation results while providing the pros and cons of altering the DNA isolation methodology. Overall, this technique provides a high throughput way to analyze subcellular alterations in DNA profiles of cells exposed to nanomaterials to aid in understanding the consequences of exposure and mechanistic effects. Future studies in microfluidic electrophoretic separation technologies should be investigated to determine the utility of protein or other assays applicable to cellular systems exposed to nanoparticles.

The role of biological fluid and dynamic flow in the behavior and cellular interactions of gold nanoparticles.

BACKGROUND: Due to their distinctive physicochemical properties, nanoparticles (NPs) have proven to be extremely advantageous for product and application development, but are also capable of inducing detrimental outcomes in biological systems. Standard in vitro methodologies are currently the primary means for evaluating NP safety, as vast quantities of particles exist that require appraisal. However, cell-based models are plagued by the fact that they are not representative of complex physiological systems. The need for a more accurate exposure model is highlighted by the fact that NP behavior and subsequent bioresponses are highly dependent upon their surroundings. Therefore, standard in vitro models will likely produce inaccurate NP behavioral analyses and erroneous safety results. As such, the goal of this study was to develop an enhanced in vitro model for NP evaluation that retained the advantages of cell culture, but implemented the key physiological variables of accurate biological fluid and dynamic flow. RESULTS: In this study, a cellular microenvironment was modeled and created after an inhalation exposure scenario. This system comprised of A549 lung epithelial cells, artificial alveolar fluid (AAF), and biologically accurate dynamic flow. Under the influence of microenvironment variables, tannic acid coated gold NPs (AuNPs) displayed modulated physicochemical characteristics, including increased agglomeration, disruption of the spectral signature, and decreased rate of ionic dissolution. Furthermore, AuNP deposition efficiency, internalization patterns, and the nano-cellular interface varied as a function of fluid composition and flow condition. AAF incubation simultaneously influenced both AuNPs and cellular behavior, through excessive NP agglomeration and alteration to A549 morphology. Dynamic flow targeted the nano-cellular interface, with differential responses including modified deposition, internalization patterns, and cellular elongation. Lastly, the biocompatibility of the system was verified to ensure cellular health following AAF exposure and fluid dynamics. CONCLUSIONS: This study confirmed the feasibility of improving standard in vitro models through the incorporation of physiological variables. Utilization of this enhanced system demonstrated that to elucidate true NP behavior and accurately gauge their cellular interactions, assessments should be carried out in a more complex and relevant biological exposure model.

Dynamic characteristics of silver nanoparticles in physiological fluids: toxicological implications.

The field of nanotoxicology has made tremendous progress identifying novel and potentially adverse biological effects following nanomaterial (NM) exposure. However, one facet yet to be satisfactorily explored is how a physiological environment modifies NM physicochemical properties, thus introducing novel complexities associated with solid phase material exposures. In this study, artificial alveolar, lysosomal, and interstitial fluids were used to identify environmental-specific modulations to the properties and behavior of hydrocarbon-coated (Ag-HC) and polysaccharide-coated (Ag-PS) silver NMs. As inhalation is a common route of exposure, an alveolar macrophage cell model with deposition dosages representing approximately 2.5 months and 10 years of occupational exposure (0.5 and 25 ng/mL, respectively) were employed. Following dispersion in the artificial fluids, the Ag-HC and Ag-PS NMs demonstrated significant alterations to morphology, aggregation patterns, and particle reactivity. However, the Ag-PS also demonstrated a loss of particle coating, which elicited increased cytotoxicity, phagocytosis, and inflammation not associated with the original Ag-PS. This study demonstrated that in a physiological system NMs undergo considerable modulation, introducing a scenario where the toxicity of NMs may increase over time due to internal bioconditions. These findings highlight the critical influence that the dynamic and insoluble nature of NMs have on bioeffects and the importance of characterizing this behavior.

Gold nanoparticles regulate the blimp1/pax5 pathway and enhance antibody secretion in B-cells.

Nanoparticles are potential threats to human health and the environment; however, their medical applications as drug carriers targeting cancer cells bring hope to contemporary cancer therapy. As a model drug carrier, gold nanoparticles (GNPs) have been investigated extensively for in vivo toxicity. The effect of GNPs on the immune system, however, has rarely been examined. Antibody-secreting cells were treated with GNPs with diameters ranging from 2 to 50 nm. The GNPs enhanced IgG secretion in a size-dependent manner, with a peak of efficacy at 10 nm. The immune-stimulatory effect reached a maximum at 12 h after treatment but returned to control levels 24 h after treatment. This enhancing effect was validated ex vivo using B-cells isolated from mouse spleen. Evidence from RT-PCR and western blot experiments indicates that GNP-treatment upregulated B-lymphocyte-induced maturation protein 1 (blimp1) and downregulated paired box 5 (pax5). Immunostaining for blimp1 and pax5 in B-cells confirmed that the GNPs stimulated IgG secretion through the blimp1/pax5 pathway. The immunization of mice using peptide-conjugated GNPs indicated that the GNPs were capable of enhancing humoral immunity in a size-dependent manner. This effect was consistent with the bio-distribution of the GNPs in mouse spleen. In conclusion, in vitro, ex vivo, and in vivo evidence supports our hypothesis that GNPs enhance humoral immunity in mouse. The effect on the immune system should be taken into account if nanoparticles are used as carriers for drug delivery. In addition to their toxicity, the immune-stimulatory activity of nanoparticles could play an important role in human health and could have an environmental impact.

Porcine brain microvessel endothelial cells show pro-inflammatory response to the size and composition of metallic nanoparticles.

The purpose of the current studies was to determine if systemic exposure of various metallic nanoparticles differing in size and composition [silver (Ag-NPs, 25, 40 and 80 nm), copper-oxide (Cu-NPs, 40 and 60 nm) or gold (Au-NPs, 3 and 5 nm)] can induce the release of pro-inflammatory mediators that influence the restrictive nature of the blood-brain barrier (BBB) in vitro. Confluent porcine brain microvessel endothelial cells (pBMECs) (8-12 days) were treated with various metallic nanoparticles (15 µg/ml). Extracellular concentrations of pro-inflammatory mediators (IL-1ß, TNFα and PGE2) were evaluated using ELISA. pBMECs were cultured in standard 12-well Transwell® inserts, and permeability was evaluated by measuring the transport of fluorescein across the pBMEC monolayers. PGE2 release following Cu-NP exposure was significantly increased when compared to the control. Similar results were observed for Ag-NPs but not Au-NPs. The secretion of TNFα and IL-1ß was observed for both Cu-NPs and Ag-NPs but not in response to Au-NPs. The post-treatment time profiles of TNFα and IL-1ß revealed that the IL-1ß response was more persistent. The permeability ratios (exposure/control) were significantly greater following exposure to Cu-NPs or Ag-NPs, compared to Au-NPs. Together, these data suggest that the composition and size of NPs can cause significant pro-inflammatory response that can influence the integrity of the BBB.

Hyperspectral microscopy for characterization of gold nanoparticles in biological media and cells for toxicity assessment.

Nanoparticles (NPs) are being implemented in a wide range of applications, and it is critical to proactively investigate their toxicity. Due to the extensive range of NPs being produced, in vitro studies are a valuable approach for toxicity screening. Key information required to support in vitro toxicity assessments include NP stability in biologically relevant media and fate once exposed to cells. Hyperspectral microscopy is a sensitive, real-time technique that combines the use of microscopy and spectroscopy for the measurement of the reflectance spectrum at individual pixels in a micrograph. This method has been used extensively for molecular imaging with plasmonic NPs as contrast agents (Aaron et al., Opt Express 16:2153-2167, 2008; Kumar et al., Nano Lett 7:1338-1343, 2007; Wax and Sokolov, Laser Photon Rev 3:146-158, 2009; Curry et al., Opt Express 14:6535-6542, 2006; Curry et al., J Biomed Opt 13:014022, 2008; Cognet et al., Proc Natl Acad Sci U S A 100:11350-11355, 2003; Sokolov et al., Cancer Res 63:1999-2004, 2003; Sönnichsen et al., Nat Biotechnol 23:741-745, 2005; Nusz et al., Anal Chem 80:984-989, 2008) and/or sensors (Nusz et al., Anal Chem 80:984-989, 2008; Ungureanu et al., Sens Actuators B 150:529-536, 2010; McFarland and Van Duyne, Nano Lett 3:1057-1062, 2003; Galush et al., Nano Lett 9:2077-2082, 2009; El-Sayed et al., Nano Lett 5:829-834, 2005). Here we describe an approach for using hyperspectral microscopy to characterize the agglomeration and stability of plasmonic NPs in biological media and their interactions with cells.

Gold nanoparticles induce transcriptional activity of NF-κB in a B-lymphocyte cell line.

Gold nanoparticles (Au-NPs) have been designated as superior tools for biological applications owing to their characteristic surface plasmon absorption/scattering and amperometric (electron transfer) properties, in conjunction with low or no immediate toxicity towards biological systems. Many studies have shown the ease of designing application-based tools using Au-NPs but the interaction of this nanosized material with biomolecules in a physiological environment is an area requiring deeper investigation. Immune cells such as lymphocytes circulate through the blood and lymph and therefore are likely cellular components to come in contact with Au-NPs. The main aim of this study was to mechanistically determine the functional impact of Au-NPs on B-lymphocytes. Using a murine B-lymphocyte cell line (CH12.LX), treatment with citrate-stabilized 10 nm Au-NPs induced activation of an NF-κB-regulated luciferase reporter, which correlated with altered B lymphocyte function (i.e. increased antibody expression). TEM imaging demonstrated that Au-NPs can pass through the cellular membrane and therefore could interact with intracellular components of the NF-κB signaling pathway. Based on the inherent property of Au-NPs to bind to -thiol groups and the presence of cysteine residues on the NF-κB signal transduction proteins IκB kinases (IKK), proteins specifically bound to Au-NPs were extracted from CH12.LX cellular lysate exposed to 10 nm Au-NPs. Electrophoresis identified several bands, of which IKKα and IKKß were immunoreactive. Further evaluation revealed activation of the canonical NF-κB signaling pathway as evidenced by IκBα phosphorylation at serine residues 32 and 36 followed by IκBα degradation and increased nuclear RelA. Additionally, expression of an IκBα super-repressor (resistant to proteasomal degradation) reversed Au-NP-induced NF-κB activation. Altered NF-κB signaling and cellular function in B-lymphocytes suggests a potential for off-target effects with in vivo applications of gold nanomaterials and underscores the need for more studies evaluating the interactions of nanomaterials with biomolecules and cellular components.

Effects of copper nanoparticles on rat cerebral microvessel endothelial cells.

AIM: The purpose of the current study was to determine whether copper nanoparticles (Cu-NPs) can induce the release of proinflammatory mediators that influence the restrictive characteristics of the blood-brain barrier. MATERIAL & METHODS: Confluent rat brain microvessel endothelial cells (rBMECs) were treated with well-characterized Cu-NPs (40 or 60 nm). Cytotoxicity of the Cu-NPs was evaluated by cell proliferation assay (1.5-50 µg/ml). The extracellular concentrations of proinflammatory mediators (IL-1ß, IL-2, TNF-α and prostaglandin E(2)) were evaluated by ELISA. RESULTS: The exposure of Cu-NPs at low concentrations increases cellular proliferation of rBMECs, by contrast, high concentrations induce toxicity. Prostaglandin E(2) release was significantly increased (threefold; 8 h) for Cu-NPs (40 and 60 nm). The extracellular levels of both TNF-α and IL-1ß were significantly elevated following exposure to Cu-NPs. The P-apparent ratio, as an indicator of increased permeability of rBMEC was approximately twofold for Cu-NPs (40 and 60 nm). CONCLUSION: These data suggest that Cu-NPs can induce rBMEC, proliferation at low concentrations and/or induce blood-brain barrier toxicity and potential neurotoxicity at high concentrations.

Interference of silver, gold, and iron oxide nanoparticles on epidermal growth factor signal transduction in epithelial cells.

Metallic nanomaterials, including silver, gold, and iron oxide, are being utilized in an increasing number of fields and specialties. The use of nanosilver as an antimicrobial agent is becoming ever-more common, whereas gold and iron oxide nanomaterials are frequently utilized in the medical field due to their recognized "biocompatibility". Numerous reports have examined the general toxicity of these nanomaterials; however, little data exists on how the introduction of these nanomaterials, at nontoxic levels, affects normal cellular processes. In the present study the impact of low levels of 10 nm silver (Ag-NP), gold (Au-NP), and iron oxide nanoparticles (SPION) on epidermal growth factor (EGF) signal transduction within the human epithelial cell line, A-431, was investigated. Following a biocompatibility assessment, the nanoparticle-induced interference at four specific targets within the EGF signaling process was evaluated: (1) nanoparticle-EGF association, (2) Akt and Erk phosphorylation, (3) Akt activity, and (4) EGF-dependent gene regulation. For all tested nanoparticles, following cellular exposure, a disruption in the EGF signaling response transpired; however, the metallic composition determined the mechanism of alteration. In addition to inducing high quantities of ROS, Ag-NPs attenuated levels of Akt and Erk phosphorylation. Au-NPs were found to decrease EGF-dependent Akt and Erk phosphorylation as well as inhibit Akt activity. Lastly, SPIONs produced a strong alteration in EGF activated gene transcription, with targeted genes influencing cell proliferation, migration, and receptor expression. These results demonstrate that even at low doses, introduction of Ag-NPs, Au-NPs, and SPIONs impaired the A-431 cell line's response to EGF.