Genotoxicity of nanomaterials: DNA damage and micronuclei induced by carbon nanotubes and graphite nanofibres in human bronchial epithelial cells in vitro

Despite the increasing industrial use of different nanomaterials, data on their genotoxicity are scant. In the present study, we examined the potential genotoxic effects of carbon nanotubes (CNTs; >50% single-walled, ∼40% other CNTs; 1.1 nm × 0.5–100 μm; Sigma–Aldrich) and graphite nanofibres (GNFs; 95%; outer diameter 80–200 nm, inner diameter 30–50 nm, length 5–20 μm; Sigma–Aldrich) in vitro. Genotoxicity was assessed by the single cell gel electrophoresis (comet) assay and the micronucleus assay (cytokinesis-block method) in human bronchial epithelial BEAS 2B cells cultured for 24 h, 48 h, or 72 h with various doses (1–100 μg/cm², corresponding to 3.8–380 μg/ml) of the carbon nanomaterials. In the comet assay, CNTs induced a dose-dependent increase in DNA damage at all treatment times, with a statistically significant effect starting at the lowest dose tested. GNFs increased DNA damage at all doses in the 24-h treatment, at two doses (40 and 100 μg/cm²) in the 48-h treatment (dose-dependent effect) and at four doses (lowest 10 μg/cm²) in the 72-h treatment. In the micronucleus assay, no increase in micronucleated cells was observed with either of the nanomaterials after the 24-h treatment or with CNTs after the 72-h treatment. The 48-h treatment caused a significant increase in micronucleated cells at three doses (lowest 10 μg/cm²) of CNTs and at two doses (5 and 10 μg/cm²) of GNFs. The 72-h treatment with GNFs increased micronucleated cells at four doses (lowest 10 μg/cm²). No dose-dependent effects were seen in the micronucleus assay. The presence of carbon nanomaterial on the microscopic slides disturbed the micronucleus analysis and made it impossible at levels higher than 20 μg/cm² of GNFs in the 24-h and 48-h treatments. In conclusion, our results suggest that both CNTs and GNFs are genotoxic in human bronchial epithelial BEAS 2B cells in vitro. This activity may be due to the fibrous nature of these carbon nanomaterials with a possible contribution by catalyst metals present in the materials—Co and Mo in CNTs (<5 wt.%) and Fe (<3 wt.%) in GNFs.     

Single wall and multiwall carbon nanotubes induce different toxicological responses in rat alveolar macrophages

Human exposure to airborne carbon nanotubes (CNT) is increasing because of their applications in different sectors; therefore, they constitute a biological hazard. Consequently, developing studies on CNT toxicity become a necessity. CNTs can have different properties in term of length, size and charge. Here, we compared the cellular effect of multiwall (MWCNTs) and single wall CNTs (SWCNTs). MWCNTs consist of multiple layers of graphene, while SWCNTs are monolayers. The effects of MWCNTs and SWCNTs were evaluated by the water‐soluble tetrazolium salt cell proliferation assay on NR8383 cells, rat alveolar macrophage cell line (NR8383). After 24 hours of exposure, MWCNTs showed higher toxicity (50% inhibitory concentration [IC₅₀] = 3.2 cm²/cm²) than SWCNTs (IC₅₀ = 44 cm²/cm²). Only SWCNTs have induced NR8383 cells apoptosis as assayed by flow cytometry using the annexin V/IP staining test. The expression of genes involved in oxidative burst (Ncf1), inflammation (Nfκb, Tnf‐α, Il‐6 and Il‐1β), mitochondrial damage (Opa) and apoptotic balance (Pdcd4, Bcl‐2 and Casp‐8) was determined. We found that MWCNT exposure predominantly induce inflammation, while SWCNTs induce apoptosis and impaired mitochondrial function. Our results clearly suggest that MWCNTs are ideal candidates for acute inflammation induction. In vivo studies are required to confirm this hypothesis. However, we conclude that toxicity of CNTs is dependent on their physical and chemical characteristics.     

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 24 h. 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/cm² 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/cm² (corresponding to 60.8 μg/ml) and doubling of the percentage of DNA in tail at 48 μg/cm². 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.     

Macrophage receptor with collagenous structure (MARCO) is a dynamic adhesive molecule that enhances uptake of carbon nanotubes by CHO-K1 cells

The toxicity of carbon nanotubes (CNTs), a highly promising nanomaterial, is similar to that of asbestos because both types of particles have a fibrous shape and are biopersistent. Here, we investigated the characteristics of macrophage receptor with collagenous structure (MARCO), a membrane receptor expressed on macrophages that recognizes environmental or unopsonized particles, and we assessed whether and how MARCO was involved in cellular uptake of multi-walled CNTs (MWCNTs). MARCO-transfected Chinese hamster ovary (CHO-K1) cells took up polystyrene beads irrespective of the particle size (20 nm-1 μm). In the culture of MARCO-transfected CHO-K1 cells dendritic structures were observed on the bottom of culture dishes, and the edges of these dendritic structures were continually renewed as the cell body migrated along the dendritic structures. MWCNTs were first tethered to the dendritic structures and then taken up by the cell body. MWCNTs appeared to be taken up via membrane ruffling like macropinocytosis, rather than phagocytosis. The cytotoxic EC₅₀ value of MWCNTs in MARCO-transfected CHO-K1 cells was calculated to be 6.1 μg/mL and transmission electron microscopic observation indicated that the toxicity of MWCNTs may be due to the incomplete inclusion of MWCNTs by the membrane structure.     

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24?h after a single pharyngeal aspiration of MWCNT-S (1–200?μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8?mg/m³) for 4 d, 4?h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200?μg/mouse) and in MNPCEs after inhalation exposure (17.5?mg/m³). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10?μg/cm²), while MWCNT-T increased strand breakage only at 200?μg/cm². Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.     

In vivo genotoxicity evaluation of lung cells from Fischer 344 rats following 28 days of inhalation exposure to MWCNTs,plus 28 days and 90 days post-exposure

Abstract

Despite their useful physico-chemical properties, carbon nanotubes (CNTs) continue to cause concern over occupational and human health due to their structural similarity to asbestos. Thus, to evaluate the toxic and genotoxic effect of multi-wall carbon nanotubes (MWCNTs) on lung cells in vivo, eight-week-old rats were divided into four groups (each group?=?25 animals), a fresh air control (0?mg/m³), low (0.17?mg/m³), middle (0.49?mg/m³), and high (0.96?mg/m³) dose group, and exposed to MWCNTs via nose-only inhalation 6?h per day, 5 days per week for 28 days. The count median length and geometric standard deviation for the MWCNTs determined by TEM were 330.18 and 1.72?nm, respectively, and the MWCNT diameters ranged from 10 to 15?nm. Lung cells were isolated from five male and five female rats in each group on day 0, day 28 (only from males) and day 90 following the 28-day exposure. The total number of animals used was 15 male and 10 female rats for each concentration group. To determine the genotoxicity of the MWCNTs, a single cell gel electrophoresis assay (Comet assay) was conducted on the rat lung cells. As a result of the exposure, the olive tail moments were found to be significantly higher (p?<?0.05) in the male and female rats from all the exposed groups when compared with the fresh air control. In addition, the high-dose exposed male and middle and high-dose exposed female rats retained DNA damage, even 90 days post-exposure (p?<?0.05). To investigate the mode of genotoxicity, the intracellular reactive oxygen species (ROS) levels and inflammatory cytokine levels (TNF-α, TGF- β, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ) were also measured. For the male rats, the H₂O₂ levels were significantly higher in the middle (0 days post-exposure) and high- (0 days and 28 days post-exposure) dose groups (p?<?0.05). Conversely, the female rats showed no changes in the H₂O₂ levels. The inflammatory cytokine levels in the bronchoalveolar lavage (BAL) fluid did not show any statistically significant difference. Interestingly, the short-length MWCNTs deposited in the lung cells were persistent at 90 days post-exposure. Thus, exposing lung cells to MWCNTs with a short tube length may induce genotoxicity.     

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO₂) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO₂ NPs. The purpose of this study is to evaluate the genotoxicity of TiO₂ NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO₂ NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200 mg/kg body weight every day for 30 days. The γ-H₂AX assay showed TiO₂ NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO₂ NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO₂ NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24 h and 48 h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24 h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2 h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO₂ NPs, and TiO₂ NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO₂ NPs induced genotoxic effects.     

An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part II: Oxidative damage to DNA

Ambient air particulate matter (atmospheric aerosol; PM) is an important factor in the development of various diseases. Oxidative stress is believed to be one of the mechanisms of action of PM on the human organism. The aim of our study was to investigate the ability of organic extracts of size segregated aerosol particles (EOM; three fractions of aerodynamic diameter 1–10 μm, 0.5–1 μm and 0.17–0.5 μm) to induce oxidative damage to DNA in an in vitro acellular system of calf thymus (CT) DNA with and without S9 metabolic activation. PM was collected in the Czech Republic at four places with different levels of air pollution. Levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) tended to increase with decreasing sizes of PM. S9 metabolic activation increased the oxidative capacity of PM; mean levels of 8-oxodG/10⁵ dG per 1000 m³ of air for samples with and without metabolic activation were 0.093 and 0.067, respectively (p < 0.05). When results of oxidative damage to DNA were normalized per microgram of aerosol mass, mean levels of 8-oxodG/10⁵ dG were 0.265 and 0.191, for incubation with and without S9 fraction, respectively (p < 0.05). We observed a significant positive association between concentrations of polycyclic aromatic hydrocarbons (c-PAHs) bound to PM and levels of 8-oxodG/10⁵ dG per 1000 m³ of air after metabolic activation of EOM samples (R = 0.695, p < 0.05). The correlation was weaker and non-significant for samples without metabolic activation (R = 0.523, p = 0.08). In conclusion, we showed that organic extracts of PM were able to induce oxidative damage to DNA in vitro; this ability was increased after S9 metabolic activation of EOM and with decreasing sizes of PM.     

Mutagenic and genotoxic effects of carbosulfan in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis

Carbosulfan insecticide is widely used in agriculture and was recently proposed for treatment against pyrethroid-resistant mosquitoes. The mutagenic and genotoxic effect of carbosulfan was carried out in fish Channa punctatus using micronucleus (MN) test and comet assay. The 96 h LC₅₀, estimated by probit analysis in a semi-static bioassay experiment, was 0.268 mg l⁻¹. Based on the LC₅₀ value, three sub-lethal concentrations of carbosulfan (1/4th LC₅₀ = ∼67 μg l⁻¹, 1/2nd LC₅₀ = ∼134 μg l⁻¹ and 3/4th LC₅₀ = ∼201 μg l⁻¹) were selected and fishes were exposed to the said concentrations for 96 h and the samplings were done at regular intervals of 24 h for assessment of the MN frequencies and DNA damage. In general, significant effects (P < 0.01) from both concentrations and time of exposure were observed in exposed fishes. The MN induction was highest on 96 h at all the concentrations in the peripheral blood. Similar trend was observed for the DNA damage measured in terms of the percentage of tail DNA in the erythrocyte and gill cells. This study confirmed that the comet and micronucleus assays are useful tools in determining potential genotoxicity of water pollutants and might be appropriate as a part of monitoring program.     

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

Abstract

We examined if three commercially available nanomaterials – short singlewall carbon nanotubes (SWCNTs), short multiwall carbon nanotubes (MWCNTs) and nanosized titanium dioxide anatase (TiO₂; 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 TiO₂ 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.     

Phototoxic effect of TPPS4 and MgTPPS4 on DNA fragmentation of HeLa cells

Photodynamic therapy (PDT) is an alternative method of tumour treatment. It is based on a photochemical reaction of a photosensitizer, irradiation, and O₂ which converts to cytotoxic ¹O₂ and other forms of reactive oxygen species (ROS). The comet assay (also called single-cell gel electrophoresis, SCGE) is a sensitive, simple and quantitative technique for detection of DNA damage. In our study we investigated the phototoxicity of the two porphyrin photosensitizers, TPPS4 and MgTPPS4, on HeLa cells. Three different radiation doses and six different concentrations of the photosensitizers were used. Our results show that the DNA of the cells treated with the TPPS₄ and MgTPPS₄ at the concentrations higher than 5 μM was highly fragmented indicating a strong phototoxic effect resulting in a cell apoptosis. On the base of our results we can hypothesize that even the irradiation dose of 1 J cm⁻² is sufficient enough to provoke the DNA fragmentation.     

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC₅₀ of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.     

Genotoxicity and carcinogenicity risk of carbon nanotubes

Novel materials are often commercialized without a complete assessment of the risks they pose to human health because such assessments are costly and time-consuming; additionally, sometimes the methodology needed for such an assessment does not exist. Carbon nanotubes have the potential for widespread application in engineering, materials science and medicine. However, due to the needle-like shape and high durability of multiwalled carbon nanotubes (MWCNTs), concerns have been raised that they may induce asbestos-like pathogenicity when inhaled. Indeed, experiments in rodents supported this hypothesis. Notably, the genetic alterations in MWCNT-induced rat malignant mesothelioma were similar to those induced by asbestos. Single-walled CNTs (SWCNTs) cause mitotic disturbances in cultured cells, but thus far, there has been no report that SWCNTs are carcinogenic. This review summarizes the recent noteworthy publications on the genotoxicity and carcinogenicity of CNTs and explains the possible molecular mechanisms responsible for this carcinogenicity. The nanoscale size and needle-like rigid structure of CNTs appear to be associated with their pathogenicity in mammalian cells, where carbon atoms are major components in the backbone of many biomolecules. Publishing adverse events associated with novel materials is critically important for alerting people exposed to such materials. CNTs still have a bright future with superb economic and medical merits. However, appropriate regulation of the production, distribution and secondary manufacturing processes is required, at least to protect the workers.     

Investigation on cobalt‐oxide nanoparticles cyto‐genotoxicity and inflammatory response in two types of respiratory cells

The increasing use of cobalt oxide (Co₃O₄) nanoparticles (NPs) in several applications and the suggested genotoxic potential of Co‐oxide highlight the importance of evaluating Co₃O₄ NPs toxicity. Cyto‐genotoxic and inflammatory effects induced by Co₃O₄ NPs were investigated in human alveolar (A549), and bronchial (BEAS‐2B) cells exposed to 1–40 µg ml^–1. The physicochemical properties of tested NPs were analysed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cytotoxicity was studied to analyze cell viability (WST1 test) and membrane damage (LDH assay), direct/oxidative DNA damage was assessed by the Formamido‐pyrimidine glycosylase (Fpg)‐modified comet assay and inflammation by interleukin (IL)‐6, IL‐8 and tumor necrosis factor‐alpha (TNF‐α) release (ELISA). In A549 cells, no cytotoxicity was found, whereas BEAS‐2B cells showed a viability reduction at 40 µg ml^–1 and early membrane damage at 1, 5 and 40 µg ml–1. In A549 cells, direct and oxidative DNA damage at 20 and 40 µg ml^–1 were detected without any effects on cytokine release. In BEAS‐2B cells, significant direct DNA damage at 40 µg ml^–1 and significant oxidative DNA damage with a peak at 5 µg ml^–1, that was associated with increased TNF‐α release at 1 µg ml^–1 after 2 h and increased IL‐8 release at 20 µg ml^–1 after 24 h, were detected. The findings show in the transformed alveolar cells no cytotoxicity and genotoxic/oxidative effects at 20 and 40 µg ml^–1. In normal bronchial cells, moderate cytotoxicity, direct DNA damage only at the highest concentration and significant oxidative‐inflammatory effects at lower concentrations were detected. The findings confirm the genotoxic‐oxidative potential of Co₃O₄ NPs and show greater sensitivity of BEAS‐2B cells to cytotoxic and oxidative‐inflammatory effects suggesting the use of different cell lines and multiple end‐points to elucidate Co₃O₄ NPs toxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

Role of air pollution Particulate Matter (PM2.5) in the occurrence of loss of heterozygosity in multiple critical regions of 3p chromosome in human epithelial lung cells (L132)

Lung cancer still remains the most frequent type of cancer all around the world and the leading cause of cancer-related death. Even if tobacco use takes a major part in etiology of lung cancer, other explanations like genetic and lifestyle factors, and occupational and/or environmental exposure to carcinogens have to be considered. Hence, in this study, we were interested in the ability of in vitro short-term exposure to air pollution Particulate Matter (PM) to induce genomic alterations in Dunkerque City's PM_2.5-exposed human epithelial lung cells (L132). The occurrence of MicroSatellite (MS) alterations in 3p multiple critical regions (i.e. 3p14.1, 3p14.2, 3p14.3, 3p21.1, 3p21.31, and 3p21.32) identified as showing frequent allelic losses in benign or malignant lung diseases, was also studied in Dunkerque City's PM_2.5-exposed L132 cells. Negative (i.e. TiO₂; desorbed PM, dPM), and positive (i.e. benzo[a]pyrene, B[a]P) controls were also included in the experimental design. Loss Of Heterozygosity (LOH) and/or MicroSatellite Instability (MSI) were reported 72 h after L132 cell exposure to dPM (i.e. 61.71 μg dPM/mL or 12.34 μg dPM/cm²), PM (i.e. 75.36 μg PM/mL or 15.07 μg PM/cm²), or B[a]P (i.e. 1 μM). In agreement with the current literature, such MS alterations might rely on the ability of dPM, PM or B[a]P to induce oxidative stress conditions, thereby altering DNA polymerase enzymes, enhancing DNA recombination rates, and inhibiting DNA repair enzymes. Hence, we concluded that the occurrence of dramatic MS alterations in 3p chromosome multiple critical regions could be a crucial underlying mechanism, which proceeded the lung toxicity in air pollution PM-exposed target L132 cells.     

Repeated exposure to carbon nanotube-based aerosols does not affect the functional properties of a 3D human epithelial airway model

Abstract

Carbon nanotubes (CNTs) represent one of the most promising engineered nanomaterials, with possible applications in advanced engineering and biomedical technologies. During their production, human exposure to CNTs may occur via inhalation. Therefore, the aim of this study was to mimic inhalation of multi-walled CNTs (MWCNTs) in vitro as realistically as possible, by producing MWCNTs aerosols via an Air–Liquid Interface Cell Exposure System (ALICE), combined with a 3D epithelial airway barrier model cultivated at the air–liquid interface (ALI). To address the consequences of an extended exposure period, repeated exposures of MWCNTs (total deposition 1.15?μg/cm²) were applied to the co-culture system, either over one day (one day repeated exposure) or three days (three day repeated exposure scenario). Although in both repeated exposure scenarios MWCNTs were found to interact with the different cell types, they did not induce any cytotoxicity or alterations in cell morphology, nor did they elucidate any significant increase in pro-inflammatory markers compared to control cultures. Similar results were also observed following single MWCNTs exposures at deposited concentrations of 0.14, 0.20 and 0.39?µg/cm². Cells exposed repeatedly to MWCNTs for three days, however did show a decrease in reduced glutathione levels, although not significant (p?>?0.05). In conclusion, we have presented a realistic in vitro alternative to mimic occupational exposure of MWCNTs and by applying this approach it was shown that repeated MWCNT exposures to lung cell cultures at the ALI elicit a limited biological impact over a three day period.     

Gentiana asclepiadea exerts antioxidant activity and enhances DNA repair of hydrogen peroxide- and silver nanoparticles-induced DNA damage

Exposure to high levels of different environmental pollutants is known to be associated with induction of DNA damage in humans. Thus DNA repair is of great importance in preventing mutations and contributes crucially to the prevention of cancer. In our study we have focused on quantitative analysis of Gentiana asclepiadea aqueous or methanolic extracts obtained from flower and haulm, their antioxidant potency in ABTS post-column derivatisation, and their potential ability to enhance DNA repair in human lymphocytes after hydrogen peroxide (H₂O₂) treatment (250 μM, 5 min). We also studied DNA repair in human kidney HEK 293 cells after exposure to 20 nm silver nanoparticles (AgNPs) (100 μg/ml, 30 min) in the presence and absence of the plant extract. We have found that mangiferin along with unidentified polar compounds are the most pronounced antioxidants in the studied extracts. Extract from haulm exhibited slightly stronger antioxidant properties compared to flower extracts. However, all four extracts showed significant ability to enhance DNA repair in both cell types after H₂O₂ and AgNP treatments.     

Antioxidant and cytotoxic effect of oxidized lutein in human cervical carcinoma cells (HeLa)

This study investigated the antioxidant and cytotoxic effect of oxidized lutein using human cervical carcinoma cell lines (HeLa). Liposome contained phosphatidylcholine (20 μmol) in Tris–HCl buffer and lutein (200 μmol) was exposed to sunlight for 100 min. Photo-oxidized lutein products were characterized by LC–MS (APCI⁺) and studied for their antioxidant property and apoptosis in terms of cell viability, glutathione and malondialdehyde (MDA) levels. Photo-oxidized lutein fragmented ions were identified as 523 (M⁺+H⁺-3CH₃), 476 (M⁺+H⁺-6CH₃), 551 (M⁺+H⁺-H₂O) and its isomers as 13-Z lutein, 13′-Z lutein, 13-Z zeaxanthin, all-E zeaxanthin, 9-Z lutein, 9′-Z lutein. Free radical scavenging activity of oxidized lutein was higher by 45.9% (IC₅₀, 3.71 μg) than lutein (IC₅₀, 5.28 μg). Oxidized lutein lowered the lipid peroxidation by 20.7% than lutein. The viability of HeLa cells, glutathione and MDA levels were decreased by 64%, 40% and 18% than lutein. To conclude, oxidized lutein may be highly reactive, since oxidation results in radical ions, which can combine with similar reactive oxidative species that could lead to higher antioxidant effect. This may be true in this study that antioxidant property of oxidized lutein was higher than lutein that correlates with free radical scavenging activity and cytotoxic effects on HeLa cells.     

Pt-MWCNT modified carbon electrode strip for rapid and quantitative detection of H2O2 in food

A single-use screen-printed carbon electrode strip was designed and fabricated. Nanohybrids, prepared by deposition of platinum (Pt) nanoparticles on multi-wall carbon nanotube (MWCNT), was modified on the surface of screen-printed carbon electrode for the development of a fast, sensitive and cost-effective hydrogen peroxide (H₂O₂) detection amperometric sensor strip. With Pt-MWCNT nanohybrids surface modification, current generated in response to H₂O₂ by the screen-printed carbon electrode strip was enhanced 100 fold with an applied potential of 300 mV. Quality of as-prepared electrode strip was assured by the low coefficient of variation (CV) (<5%) of currents measured at 5 s. Three linear detection ranges with sensitivity of 75.2, 120.7, and 142.8 μA mM^?1 cm^?2 were observed for H₂O₂ concentration in the range of 1–15 mM, 0.1–1 mM, and 10–100 μM, respectively. The lowest H₂O₂ concentration could be measured by the as-prepared strip was 10 μM. H₂O₂ levels in green tea infusion and pressed Tofu could be rapidly detected with results comparable to that measured by ferrous oxidation xylenol orange (FOX) assay and peroxidase colorimetric method.