Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina.

Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve superresolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including three-dimensional-printed gel casting equipment. We show that in comparison with conventional immunostaining, IT-IF results in a higher signal-to-background ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal-processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative superresolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization-a prerequisite for studying intranuclear structural coregulation of cell function and fate.

The Effect of Severe Shot Peening on Fatigue Life of Laser Powder Bed Fusion Manufactured 316L Stainless Steel.

Severe shot peening (SSP) was used on additive manufactured 316L by laser powder bed fusion. The effect of the post processing on the surface features of the material was analyzed through residual stress measurements, tensile testing, hardness-depth profiles, and fatigue testing by flexural bending. The results showed that SSP can be utilized to form residual stresses up to -400 MPa 200 µm below the surface. At the same time, a clear improvement on the surface hardness was achieved from 275 HV to near 650 HV. These together resulted in a clear improvement on material strength which was recorded at 10% improvement in ultimate tensile strength. Most significantly, the fatigue limit of the material was tripled from 200 MPa to over 600 MPa and the overall fatigue strength raised similarly from a low to high cycle regime.

Sub-Surface Analysis of Grinding Burns with Barkhausen Noise Measurements.

Barkhausen noise (BN) measurements are commonly used for surface characterisation. However, often there is also a need to verify the sub-surface region because detrimental tensile stresses may be present after different manufacturing steps. Especially in a grinding burn, the surface stress may be compressive, but it changes quickly into tensile stress below the surface. The aim of this study was to find out whether regular surface-sensitive BN measurement is also sensitive to the stresses below the surface caused by grinding burns. More specifically, the aim was to study the relationship between BN features and sub-surface stresses and to identify a model that estimates sub-surface stresses. Real samples were collected from an actual process. The samples were cylindrical samples manufactured from commercial alloyed AISI/SAE L6 steel that was through-hardened prior to grinding. Barkhausen noise measurements were carried out for 42 grinding burn locations followed by X-ray diffraction-based residual stress surface measurements and residual stress depth profiles. Depth information was obtained through step-by-step electrolytic removal of thin layers. The stress profiles were pre-processed through interpolation and averaged stress was calculated as a function of depth below the surface. Correlation analysis was carried out to evaluate the relationships between BN features and stress at different depths and among BN features. The main outcome of the analysis was that BN measurement is dominated by the sub-surface tensile stresses rather than the compressive stress at the surface. It was also noticed that BN features form two groups, corresponding to average Barkhausen activity and magnetising field strength leading to maximum Barkhausen activity. Models for stress at different steps were identified systematically. The performance of the models for sub-surface stresses was reasonable with R2 values of around 0.85 and root mean squared error (RMSE) values of around 95 MPa. Based on the results, it is concluded that BN measurement provides information about sub-surface stresses and that stress can be evaluated through straightforward modelling, allowing fast detection of grinding burns.

Vanadia-Zirconia and Vanadia-Hafnia Catalysts for Utilization of Volatile Organic Compound Emissions.

Utilization is a sustainable and interesting alternative for the destructive treatment of volatile organic compounds due to avoided CO2 emission. This work concentrates on the development of active and sulfur-tolerant catalysts for the utilization of contaminated methanol. Impregnated and sol-gel prepared vanadia-zirconia and vanadia-hafnia catalysts were thoroughly characterized by N2 sorption, analytical (S)TEM, elemental analysis, XRD and Raman spectroscopy, and their performances were evaluated in formaldehyde production from methanol and methanethiol mixture. The results showed higher activity of the sol-gel prepared catalysts due to formation of mono- and polymeric vanadia species. Unfortunately, the most active vanadia sites were deactivated more easily than the metal-mixed oxide HfV2O7 and ZrV2O7 phases, as well as crystalline V2O5 observed in the impregnated catalysts. Metal-mixed oxide phases were formed in impregnated catalysts through formation of defects in HfO2 and ZrO2 structure during calcination at 600 °C, which was evidenced by Raman spectroscopy. The sol-gel prepared vanadia-zirconia and vanadia-hafnia catalysts were able to produce formaldehyde from contaminated methanol with high selectivity at temperature around 400 °C, while impregnated catalysts required 50-100 °C higher temperatures.

Pulmonary toxicity of Fe2O3, ZnFe2O4, NiFe2O4 and NiZnFe4O8 nanomaterials: Inflammation and DNA strand breaks.

Exposure to metal oxide nanomaterials potentially occurs at the workplace. We investigated the toxicity of two Fe-oxides: Fe2O3 nanoparticles and nanorods; and three MFe2O4 spinels: NiZnFe4O8, ZnFe2O4, and NiFe2O4 nanoparticles. Mice were dosed 14, 43 or 128 µg by intratracheal instillation. Recovery periods were 1, 3, or 28 days. Inflammation - neutrophil influx into bronchoalveolar lavage (BAL) fluid - occurred for Fe2O3 rods (1 day), ZnFe2O4 (1, 3 days), NiFe2O4 (1, 3, 28 days), Fe2O3 (28 days) and NiZnFe4O8 (28 days). Conversion of mass-dose into specific surface-area-dose showed that inflammation correlated with deposited surface area and consequently, all these nanomaterials belong to the so-called low-solubility, low-toxicity class. Increased levels of DNA strand breaks were observed for both Fe2O3 particles and rods, in BAL cells three days post-exposure. To our knowledge, this is, besides magnetite (Fe3O4), the first study of the pulmonary toxicity of MFe2O4 spinel nanomaterials.

Statistical Evaluation of Barkhausen Noise Testing (BNT) for Ground Samples.

Barkhausen noise testing (BNT) is a nondestructive method for investigating many properties of ferromagnetic materials. The most common application is the monitoring of grinding burns caused by introducing locally high temperatures while grinding. Other features, such as microstructure, residual stress changes, hardening depth, and so forth, can be monitored as well. Nevertheless, because BNT is a method based on a complex magnetoelectric phenomenon, it is not yet standardized. Therefore, there is a need to study the traceability and stability of the measurement method. This study aimed to carry out a statistical analysis of ferromagnetic samples after grinding processes by the use of BNT. The first part of the experiment was to grind samples in different facilities (Sweden and Finland) with similar grinding parameters, different grinding wheels, and different hardness values. The second part was to evaluate measured BNT parameters to determine significant factors affecting BNT signal value. The measurement data from the samples were divided into two different batches according to where they were manufactured. Both grinding batches contained measurement data from three different participants. The main feature for calculation was the root-mean-square (RMS) value. The first processing step was to normalize the RMS values for all the measurements. A standard analysis of variance (ANOVA) was applied for the normalized dataset. The ANOVA showed that the grinding parameters had a significant impact on the BNT signal value, while the other investigated factors (e.g., participant) were negligible. The reasons for this are discussed at the end of the paper.

The effect of inferior turbinate surgery on ciliated epithelium: A randomized, blinded study.

OBJECTIVES/HYPOTHESIS: The aim of this study was to evaluate statistically the effects of radiofrequency ablation, diode laser, and microdebrider-assisted inferior turbinoplasty techniques on ciliated epithelium and mucociliary function. STUDY DESIGN: Prospective randomized study. METHODS: A total of 66 consecutively randomized adult patients with enlarged inferior turbinates underwent either a radiofrequency ablation, diode laser, or microdebrider-assisted inferior turbinoplasty procedure. Assessments were conducted prior to surgery and 3 months subsequent to the surgery. The effect on ciliated epithelium was evaluated using a score based on the blinded grading of the preoperative and postoperative scanning electron microscopy images of mucosal samples. The effect on mucociliary function, in turn, was evaluated using saccharin transit time measurement. RESULTS: The score of the number of cilia increased statistically significantly in the radiofrequency ablation (P = .03) and microdebrider-assisted inferior turbinoplasty (P = .04) groups, but not in the diode laser group. The score of the squamous metaplasia increased statistically significantly in the diode laser group (P = .002), but not in the other two groups. There were no significant changes found between the preoperative and postoperative saccharin transit time values in any of the treatment groups. CONCLUSIONS: Radiofrequency ablation and microdebrider-assisted inferior turbinoplasty are more mucosal preserving techniques than the diode laser, which was found to increase the amount of squamous metaplasia at the 3-month follow-up. The number of cilia seemed to even increase after radiofrequency ablation and microdebrider-assisted inferior turbinoplasty procedures, but not after diode laser. Nevertheless, the mucociliary transport was equally preserved in all three groups. LEVEL OF EVIDENCE: 1b Laryngoscope, 129:18-24, 2019.

Aligned Poly(ε-caprolactone) Nanofibers Guide the Orientation and Migration of Human Pluripotent Stem Cell-Derived Neurons, Astrocytes, and Oligodendrocyte Precursor Cells In Vitro.

Stem cell transplantations for spinal cord injury (SCI) have been studied extensively for the past decade in order to replace the damaged tissue with human pluripotent stem cell (hPSC)-derived neural cells. Transplanted cells may, however, benefit from supporting and guiding structures or scaffolds in order to remain viable and integrate into the host tissue. Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. All cell types are shown to efficiently spread over the nanofiber platform and orient according to the fiber alignment. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment. In this work, clinically relevant materials and substrates for nanofibers, fiber coatings, and hydrogel scaffolds are used and combined with cells suitable for developing functional cell grafts for SCI repair.

Effect of particle size and dispersion status on cytotoxicity and genotoxicity of zinc oxide in human bronchial epithelial cells.

Data available on the genotoxicity of zinc oxide (ZnO) nanoparticles (NPs) are controversial. Here, we examined the effects of particle size and dispersion status on the cytotoxicity and genotoxicity of nanosized and fine ZnO, in the presence and absence of bovine serum albumin (BSA; 0.06%) in human bronchial epithelial BEAS-2B cells. Dynamic light scattering analysis showed the most homogenous dispersions in water alone for nanosized ZnO and in water with BSA for fine ZnO. After a 48-h treatment, both types of ZnO were cytotoxic within a similar, narrow dose range (1.5-3.0µg/cm(2)) and induced micronuclei at a near toxic dose range (1.25-1.75µg/cm(2)), both with and without BSA. In the comet assay, nanosized ZnO (1.25-1.5µg/cm(2)), in the absence of BSA, caused a statistically significant increase in DNA damage after 3-h and 6-h treatments, while fine ZnO did not. Our findings may be explained by better uptake or faster intracellular dissolution of nanosized ZnO without BSA during short treatments (3-6h; the comet assay), with less differences between the two ZnO forms after longer treatments (>48h; the in vitro micronucleus test). As ZnO is genotoxic within a narrow dose range partly overlapping with cytotoxic doses, small experimental differences e.g. in the dispersion of ZnO particles may have a substantial effect on the genotoxicity of the nominal doses added to the cell culture.

Insight to Nanoparticle Size Analysis-Novel and Convenient Image Analysis Method Versus Conventional Techniques.

The aim of this paper is to introduce a new image analysis program "Nanoannotator" particularly developed for analyzing individual nanoparticles in transmission electron microscopy images. This paper describes the usefulness and efficiency of the program when analyzing nanoparticles, and at the same time, we compare it to more conventional nanoparticle analysis techniques. The techniques which we are concentrating here are transmission electron microscopy (TEM) linked with different image analysis methods and X-ray diffraction techniques. The developed program appeared as a good supplement to the field of particle analysis techniques, since the traditional image analysis programs suffer from the inability to separate the individual particles from agglomerates in the TEM images. The program is more efficient, and it offers more detailed morphological information of the particles than the manual technique. However, particle shapes that are very different from spherical proved to be problematic also for the novel program. When compared to X-ray techniques, the main advantage of the small-angle X-ray scattering (SAXS) method is the average data it provides from a very large amount of particles. However, the SAXS method does not provide any data about the shape or appearance of the sample.

Hydrothermal carbonization of pulp mill streams.

The progress of the conversion, the yield, the structure and the morphology of the produced carbonaceous materials as a function of time were systematically studied with pyrolysis-GC/FID and FESEM microscope. The conversion of galactoglucomannan, bleached kraft pulp and TEMPO oxidized cellulose nanofibrils followed the reaction route of glucose being slower though with fibrous material, higher molar mass and viscosity. The conversion of kraft lignin was minor following completely different reaction route. Carbonaceous particles of different shape and size were produced with yields between 23% and 73% after 4h with being higher for lignin than carbohydrates. According to the results, potential pulp mill streams represent lignocellulosic resources for generation of carbonaceous materials.

A Single Aspiration of Rod-like Carbon Nanotubes Induces Asbestos-like Pulmonary Inflammation Mediated in Part by the IL-1 Receptor.

Carbon nanotubes (CNT) have been eagerly studied because of their multiple applications in product development and potential risks on health. We investigated the difference of two different CNT and asbestos in inducing proinflammatory reactions in C57BL/6 mice after single pharyngeal aspiration exposure. We used long tangled and long rod-like CNT, as well as crocidolite asbestos at a dose of 10 or 40 µg/mouse. The mice were sacrificed 4 and 16 h or 7, 14, and 28 days after the exposure. To find out the importance of a major inflammatory marker IL-1ß in CNT-induced pulmonary inflammation, we used etanercept and anakinra as antagonists as well as Interleukin 1 (IL-1) receptor (IL-1R-/-) mice. The results showed that rod-like CNT, and asbestos in lesser extent, induced strong pulmonary neutrophilia accompanied by the proinflammatory cytokines and chemokines 16 h after the exposure. Seven days after the exposure, neutrophilia had essentially disappeared but strong pulmonary eosinophilia peaked in rod-like CNT and asbestos-exposed groups. After 28 days, pulmonary granulomas, goblet cell hyperplasia, and Charcot-Leyden-like crystals containing acidophilic macrophages were observed especially in rod-like CNT-exposed mice. IL-1R-/- mice and antagonists-treated mice exhibited a significant decrease in neutrophilia and messenger ribonucleic acid (mRNA) levels of proinflammatory cytokines at 16 h. However, rod-like CNT-induced Th2-type inflammation evidenced by the expression of IL-13 and mucus production was unaffected in IL-1R-/- mice at 28 days. This study provides knowledge about the pulmonary effects induced by a single exposure to the CNT and contributes to hazard assessment of carbon nanomaterials on airway exposure.

In vitro platelet activation, aggregation and platelet-granulocyte complex formation induced by surface modified single-walled carbon nanotubes.

Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood.

A secretomics analysis reveals major differences in the macrophage responses towards different types of carbon nanotubes.

Certain types of carbon nanotubes (CNT) can evoke inflammation, fibrosis and mesothelioma in vivo, raising concerns about their potential health effects. It has been recently postulated that NLRP3 inflammasome activation is important in the CNT-induced toxicity. However, more comprehensive studies of the protein secretion induced by CNT can provide new information about their possible pathogenic mechanisms. Here, we studied protein secretion from human macrophages with a proteomic approach in an unbiased way. Human monocyte-derived macrophages (MDM) were exposed to tangled or rigid, long multi-walled CNT (MWCNT) or crocidolite asbestos for 6 h. The growth media was concentrated and secreted proteins were analyzed using 2D-DIGE and DeCyder software. Subsequently, significantly up- or down-regulated protein spots were in-gel digested and identified with an LC-MS/MS approach. Bioinformatics analysis was performed to reveal the different patterns of protein secretion induced by these materials. The results show that both long rigid MWCNT and asbestos elicited ample and highly similar protein secretion. In contrast, exposure to long tangled MWCNT induced weaker protein secretion with a more distinct profile. Secretion of lysosomal proteins followed the exposure to all materials, suggesting lysosomal damage. However, only long rigid MWCNT was associated with apoptosis. This analysis suggests that the CNT toxicity in human MDM is mediated via vigorous secretion of inflammation-related proteins and apoptosis. This study provides new insights into the mechanisms of toxicity of high aspect ratio nanomaterials and indicates that not all types of CNT are as hazardous as asbestos fibers.

Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

BACKGROUND: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma. METHODS: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation. RESULTS: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages. CONCLUSIONS: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

Topically applied ZnO nanoparticles suppress allergen induced skin inflammation but induce vigorous IgE production in the atopic dermatitis mouse model.

BACKGROUND: Metal oxide nanoparticles such as ZnO are used in sunscreens as they improve their optical properties against the UV-light that causes dermal damage and skin cancer. However, the hazardous properties of the particles used as UV-filters in the sunscreens and applied to the skin have remained uncharacterized. METHODS: Here we investigated whether different sized ZnO particles would be able to penetrate injured skin and injured allergic skin in the mouse atopic dermatitis model after repeated topical application of ZnO particles. Nano-sized ZnO (nZnO) and bulk-sized ZnO (bZnO) were applied to mechanically damaged mouse skin with or without allergen/superantigen sensitization. Allergen/superantigen sensitization evokes local inflammation and allergy in the skin and is used as a disease model of atopic dermatitis (AD). RESULTS: Our results demonstrate that only nZnO is able to reach into the deep layers of the allergic skin whereas bZnO stays in the upper layers of both damaged and allergic skin. In addition, both types of particles diminish the local skin inflammation induced in the mouse model of AD; however, nZnO has a higher potential to suppress the local effects. In addition, especially nZnO induces systemic production of IgE antibodies, evidence of allergy promoting adjuvant properties for topically applied nZnO. CONCLUSIONS: These results provide new hazard characterization data about the metal oxide nanoparticles commonly used in cosmetic products and provide new insights into the dermal exposure and hazard assessment of these materials in injured skin.

Carbon-based nanomaterials accelerate arteriolar thrombus formation in the murine microcirculation independently of their shape.

Although carbon-based nanomaterials (CBNs) have been shown to exert prothrombotic effects in microvessels, it is poorly understood whether CBNs also have the potential to interfere with the process of leukocyte-endothelial cell interactions and whether the shape of CBNs plays a role in these processes. Thus, the aim of this study was to compare the acute effects of two differently shaped CBNs, fiber-shaped single-walled carbon nanotubes (SWCNT) and spherical ultrafine carbon black (CB), on thrombus formation as well as on leukocyte-endothelial cell interactions and leukocyte transmigration in the murine microcirculation upon systemic administration in vivo. Systemic administration of both SWCNT and CB accelerated arteriolar thrombus formation at a dose of 1 mg kg(-1) body weight, whereas SWCNT exerted a prothrombotic effect also at a lower dose (0.1 mg kg(-1) body weight). In vitro, both CBNs induced P-selectin expression on human platelets and formation of platelet-granulocyte complexes. In contrast, injection of fiber-shaped SWCNT or of spherical CB did not induce leukocyte-endothelial cell interactions or leukocyte transmigration. In vitro, both CBNs slightly increased the expression of activation markers on human monocytes and granulocytes. These findings suggest that systemic administration of CBNs accelerates arteriolar thrombus formation independently of the CBNs' shape, but does not induce leukocyte-endothelial cell interactions or leukocyte transmigration.

Free radical scavenging and formation by multi-walled carbon nanotubes in cell free conditions and in human bronchial epithelial cells.

BACKGROUND: Certain multi-walled carbon nanotubes (MWCNTs) have been shown to elicit asbestos-like toxicological effects. To reduce needs for risk assessment it has been suggested that the physicochemical characteristics or reactivity of nanomaterials could be used to predict their hazard. Fibre-shape and ability to generate reactive oxygen species (ROS) are important indicators of high hazard materials. Asbestos is a known ROS generator, while MWCNTs may either produce or scavenge ROS. However, certain biomolecules, such as albumin - used as dispersants in nanomaterial preparation for toxicological testing in vivo and in vitro - may reduce the surface reactivity of nanomaterials. METHODS: Here, we investigated the effect of bovine serum albumin (BSA) and cell culture medium with and without BEAS 2B cells on radical formation/scavenging by five MWCNTs, Printex 90 carbon black, crocidolite asbestos, and glass wool, using electron spin resonance (ESR) spectroscopy and linked this to cytotoxic effects measured by trypan blue exclusion assay. In addition, the materials were characterized in the exposure medium (e.g. for hydrodynamic size-distribution and sedimentation rate). RESULTS: The test materials induced highly variable cytotoxic effects which could generally be related to the abundance and characteristics of agglomerates/aggregates and to the rate of sedimentation. All carbon nanomaterials were found to scavenge hydroxyl radicals (•OH) in at least one of the solutions tested. The effect of BSA was different among the materials. Two types of long, needle-like MWCNTs (average diameter >74 and 64.2 nm, average length 5.7 and 4.0 µm, respectively) induced, in addition to a scavenging effect, a dose-dependent formation of a unique, yet unidentified radical in both absence and presence of cells, which also coincided with cytotoxicity. CONCLUSIONS: Culture medium and BSA affects scavenging/production of •OH by MWCNTs, Printex 90 carbon black, asbestos and glass-wool. An unidentified radical is generated by two long, needle-like MWCNTs and these two CNTs were more cytotoxic than the other CNTs tested, suggesting that this radical could be related to the adverse effects of MWCNTs.

Genotoxicity of polyvinylpyrrolidone-coated silver nanoparticles in BEAS 2B cells.

Silver nanoparticles (AgNPs) are widely utilized in various consumer products and medical devices, especially due to their antimicrobial properties. However, several studies have associated these particles with toxic effects, such as inflammation and oxidative stress in vivo and cytotoxic and genotoxic effects in vitro. Here, we assessed the genotoxic effects of AgNPs coated with polyvinylpyrrolidone (PVP) (average diameter 42.5±14.5 nm) on human bronchial epithelial BEAS 2B cells in vitro. AgNPs were dispersed in bronchial epithelial growth medium (BEGM) with 0.6 mg/ml bovine serum albumin (BSA). The AgNP were partially well-dispersed in the medium and only limited amounts (ca. 0.02 µg Ag(+) ion/l) could be dissolved after 24h. The zeta-potential of the AgNPs was found to be highly negative in pure water but was at least partially neutralized in BEGM with 0.6 mg BSA/ml. Cytotoxicity was measured by cell number count utilizing Trypan Blue exclusion and by an ATP-based luminescence cell viability assay. Genotoxicity was assessed by the alkaline single cell gel electrophoresis (comet) assay, the cytokinesis-block micronucleus (MN) assay, and the chromosomal aberration (CA) assay. The cells were exposed to various doses (0.5-48 µg/cm(2) corresponding to 2.5-240 µg/ml) of AgNPs for 4 and 24 h in the comet assay, for 48 h in the MN assay, and for 24 and 48 h in the CA assay. DNA damage measured by the percent of DNA in comet tail was induced in a dose-dependent manner after both the 4-h and the 24-h exposures to AgNPs, with a statistically significant increase starting at 16 µg/cm(2) (corresponding to 60.8 µg/ml) and doubling of the percentage of DNA in tail at 48 µg/cm(2). However, no induction of MN or CAs was observed at any of the doses or time points. The lack of induction of chromosome damage by the PVP-coated AgNPs is possibly due to the coating which may protect the cells from direct interaction with the AgNPs, either by reducing ion leaching from the particles or by causing extensive agglomeration of the nanoparticles, with a possible reduction of the cellular uptake.

Induction of chromosomal aberrations by carbon nanotubes and titanium dioxide nanoparticles in human lymphocytes in vitro.

We examined if three commercially available nanomaterials - short singlewall carbon nanotubes (SWCNTs), short multiwall carbon nanotubes (MWCNTs) and nanosized titanium dioxide anatase (TiO(2); primary particle size <25 nm) - can induce structural chromosomal aberrations (CAs) in cultures of isolated human lymphocytes. To find a suitable sampling time, the cells were treated with 6.25-300 µg/ml of the nanomaterials for 24, 48 and 72 h. The 48-h treatment was the most effective, inducing a dose-dependent increase in chromosome-type CAs (all materials) and chromatid-type CAs (SWCNTs and TiO(2) anatase). The 72-h treatment yielded a positive result with SWCNTs. None of the treatments significantly affected cell count or the mitotic index. Our results suggest that with nanomaterials a continuous treatment for about two cell cycles is needed for CA induction, possibly reflecting access of nanomaterials to the nucleus during the first mitosis or delayed secondary genotoxic effect associated with the inflammatory process.