Satu Suhonen

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, approximately 40% other CNTs; 1.1 nm x 0.5-100 microm; Sigma-Aldrich) and graphite nanofibres (GNFs; 95%; outer diameter 80-200 nm, inner diameter 30-50 nm, length 5-20 microm; 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 24h, 48h, or 72h with various doses (1-100 microg/cm(2), corresponding to 3.8-380 microg/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 microg/cm(2)) in the 48-h treatment (dose-dependent effect) and at four doses (lowest 10 microg/cm(2)) 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 microg/cm(2)) of CNTs and at two doses (5 and 10 microg/cm(2)) of GNFs. The 72-h treatment with GNFs increased micronucleated cells at four doses (lowest 10 microg/cm(2)). 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 microg/cm(2) 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 (<5wt.%) and Fe (<3wt.%) in GNFs.

Genotoxic effects of nanosized and fine TiO2.

The in-vitro genotoxicity of nanosized TiO2 rutile and anatase was assessed in comparison with fine TiO2 rutile in human bronchial epithelial BEAS 2B cells using the single-cell gel electrophoresis (comet) assay and the cytokinesis-block micronucleus test. BEAS 2B cells were exposed to eight doses (1—100 μg/cm2) of titanium(IV) oxide nanosized rutile (>95%, <5% amorphous SiO2 coating; 10 × 40 nm), nanosized anatase (99.7%; <25 nm), or fine rutile (99.9%; <5 μm) for 24, 48, and 72 h. Fine rutile reduced cell viability at lower doses than nanosized anatase, which was more cytotoxic than nanosized rutile. In the comet assay, nanosized anatase and fine rutile induced DNA damage at several doses with all treatment times. Dose-dependent effects were seen after the 48- and 72-h treatments with nanosized anatase and after the 24-, 48- (in one out of two experiments), and 72-h treatments (one experiment) with fine rutile. The lowest doses inducing DNA damage were 1 μg/cm2 for fine rutile and 10 μg/cm 2 for nanosized anatase. Nanosized rutile showed a significant induction in DNA damage only at 80 μg/cm2 in the 24-h treatment and at 80 and 100 μg/ cm2 in the 72-h treatment (with a dose-dependent effect). Only nanosized anatase could elevate the frequency of micronucleated BEAS 2B cells, producing a significant increase at 10 and 60 μg/cm 2 after the 72-h treatment (no dose-dependency). At increasing doses of all the particles, MN analysis became difficult due to the presence of TiO2 on the microscopic slides. In conclusion, our studies in human bronchial epithelial BEAS 2B cells showed that uncoated nanosized anatase TiO2 and fine rutile TiO2 are more efficient than SiO 2-coated nanosized rutile TiO2 in inducing DNA damage, whereas only nanosized anatase is able to slightly induce micronuclei.

Genotoxicity of inhaled nanosized TiO2 in mice

In vitro studies have suggested that nanosized titanium dioxide (TiO(2)) is genotoxic. The significance of these findings with respect to in vivo effects is unclear, as few in vivo studies on TiO(2) genotoxicity exist. Recently, nanosized TiO(2) administered in drinking water was reported to increase, e.g., micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs) and DNA damage in leukocytes. Induction of micronuclei in mouse PCEs was earlier also described for pigment-grade TiO(2) administered intraperitoneally. The apparent systemic genotoxic effects have been suggested to reflect secondary genotoxicity of TiO(2) due to inflammation. However, a recent study suggested that induction of DNA damage in mouse bronchoalveolar lavage (BAL) cells after intratracheal instillation of nanosized or fine TiO(2) is independent of inflammation. We examined here, if inhalation of freshly generated nanosized TiO(2) (74% anatase, 26% brookite; 5 days, 4 h/day) at 0.8, 7.2, and (the highest concentration allowing stable aerosol production) 28.5 mg/m(3) could induce genotoxic effects in C57BL/6J mice locally in the lungs or systematically in peripheral PCEs. DNA damage was assessed by the comet assay in lung epithelial alveolar type II and Clara cells sampled immediately following the exposure. MN were analyzed by acridine orange staining in blood PCEs collected 48 h after the last exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level produced a clear increase in neutrophils in BAL fluid, indicating an inflammatory effect, no significant effect on the level of DNA damage in lung epithelial cells or micronuclei in PCEs was observed, suggesting no genotoxic effects by the 5-day inhalation exposure to nanosized TiO(2) anatase. Our inhalation exposure resulted in much lower systemic TiO(2) doses than the previous oral and intraperitoneal treatments, and lung epithelial cells probably received considerably less TiO(2) than BAL cells in the earlier intratracheal study.

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.

Preparation of nanoparticle dispersions for in-vitro toxicity testing

Studies on potential toxicity of engineered nanoparticle (ENP) in biological systems require a proper and accurate particle characterization to ensure the reproducibility of the results and to understand biological effects of ENP. A full characterization of ENP should include various measurements such as particle size and size distribution, shape and morphology, crystallinity, composition, surface chemistry, and surface area of ENP. It is also important to characterize the state of ENP dispersions. In this study, four different ENPs, rutile and anatase titanium dioxides and short single- and multi-walled carbon nanotubes, were characterized in two dispersion media: bronchial epithelial growth medium, used for bronchial epithelial BEAS cells, and RPMI-1640 culture media with 10% of fetal calf serum (FCS) for human mesothelial (MeT-5A) cells. The purpose of this study was to determine the characteristics of ENPs and their dispersions as well as to compare dispersion additives suitable for toxicity tests and thus establish an appropriate way to prepare dispersions that performs well with the selected ENP. Dispersion additives studied in the media were bovine serum albumin (BSA) as a protein resource, dipalmitoyl phosphatidylcholine (DPPC) as a model lung surfactant, and combination of BSA and DPPC. Dispersions were characterized using optical microscopy and transmission electron microscopy. Our results showed that protein addition, BSA or FCS, in cell culture media generated small agglomerates of primary particles with narrow size variations and improved the stability of the dispersions and thus also the relevance of the in-vitro genotoxicity tests to be done.

Genotoxicity of short single-wall and multi-wall carbon nanotubes in human bronchial epithelial and mesothelial cells in vitro

Although some types of carbon nanotubes (CNTs) have been described to induce mesothelioma in rodents and genotoxic effects in various cell systems, there are few previous studies on the genotoxicity of CNTs in mesothelial cells. Here, we examined in vitro DNA damage induction by short multi-wall CNTs (MWCNTs; 10-30 nm × 1-2 μm) and single-wall CNTs (SWCNTs; >50% SWCNTs, ~40% other CNTs; <2 nm × 1-5 μm) in human mesothelial (MeT-5A) cells and bronchial epithelial (BEAS 2B) cells, using the single cell gel electrophoresis (comet) assay and the immunoslot blot assay for the detection of malondialdehyde (M1dG) DNA adducts. In BEAS 2B cells, we also studied the induction of micronuclei (MN) by the CNTs using the cytokinesis-block method. The cells were exposed to the CNTs (5-200 μg/cm(2), corresponding to 19-760 μg/ml) for 24 and 48h in the comet assay and for 48 and 72 h in the MN and M1dG assays. Transmission electron microscopy (TEM) showed more MWCNT fibres and SWCNT clusters in BEAS 2B than MeT-5A cells, but no significant differences were seen in intracellular dose expressed as area of SWCNT clusters between TEM sections of the cell lines. In MeT-5A cells, both CNTs caused a dose-dependent induction of DNA damage (% DNA in comet tail) in the 48-h treatment and SWCNTs additionally in the 24-h treatment, with a statistically significant increase at 40 μg/cm(2) of SWCNTs and (after 48 h) 80 μg/cm(2) of both CNTs. SWCNTs also elevated the level of M1dG DNA adducts at 1, 5, 10 and 40 μg/cm(2) after the 48-h treatment, but both CNTs decreased M1dG adduct level at several doses after the 72-h treatment. In BEAS 2B cells, SWCNTs induced a statistically significant increase in DNA damage at 80 and 120 μg/cm(2) after the 24-h treatment and in M1dG adduct level at 5 μg/cm(2) after 48 h and 10 and 40 μg/cm(2) after 72 h; MWCNTs did not affect the level of DNA damage but produced a decrease in M1dG adducts in the 72-h treatment. The CNTs did not affect the level of MN. In conclusion, MWCNTs and SWCNTs induced DNA damage in MeT-5A cells but showed a lower (SWCNTs) or no (MWCNTs) effect in BEAS 2B cells, suggesting that MeT-5A cells were more sensitive to the DNA-damaging effect of CNTs than BEAS 2B cells, despite the fact that more CNT fibres or clusters were seen in BEAS 2B than MeT-5A cells. M1dG DNA adducts were induced by SWCNTs but decreased after a 3-day exposure to MWCNTs and (in MeT-5A cells) SWCNTs, indicating that CNTs may lead to alterations in oxidative effects within the cells. Neither of the CNTs was able to produce chromosomal damage (MN).

Genotoxic effects of fumes from asphalt modified with waste plastic and tall oil pitch

As the use of recycled materials and industrial by-products in asphalt mixtures is increasing, we investigated if recycled additives modify the genotoxicity of fumes emitted from asphalt. Fumes were generated in the laboratory at paving temperature from stone-mastic asphalt (SMA) and from SMA modified with waste plastic (90% polyethylene, 10% polypropylene) and tall oil pitch (SMA-WPT). In addition, fumes from SMA, SMA-WPT, asphalt concrete (AC), and AC modified with waste plastic and tall oil pitch (AC-WPT) were collected at paving sites. The genotoxicity of the fumes was studied by analysis of DNA damage (measured in the comet assay) and micronucleus formation in human bronchial epithelial BEAS 2B cells in vitro and by counting mutations in Salmonella typhimurium strains TA98 and YG1024. DNA damage was also assessed in buccal leukocytes from road pavers before and after working with SMA, SMA-WPT, AC, and AC-WPT. The chemical composition of the emissions was analysed by gas chromatography/mass spectrometry. The SMA-WPT fume generated in the laboratory induced a clear increase in DNA damage in BEAS 2B cells without metabolic activation. The laboratory-generated SMA fume increased the frequency of micronucleated BEAS 2B cells without metabolic activation. None of the asphalt fumes collected at the paving sites produced DNA damage with or without metabolic activation. Fumes from SMA and SMA-WPT from the paving sites increased micronucleus frequency without metabolic activation. None of the asphalt fumes studied showed mutagenic activity in Salmonella. No statistically significant differences in DNA damage in buccal leukocytes were detected between the pre- and post-shift samples collected from the road pavers. However, a positive correlation was found between DNA damage and the urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) after work shift, which suggested an association between occupational exposures during road paving and genotoxic effects. Our results indicate that fumes from SMA and SMA-WPT contain direct-acting genotoxic components.

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

Abstract 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 vitro sensitivity of normal human mesothelial and malignant mesothelioma cell lines to four new chemotherapeutic agents.

In this study, we used four human mesothelioma cell lines (M14K, M24K, M25K and M38K), one transformed human mesothelial cell line (MeT-5A) and one primary mesothelial culture (UPL) to test for in vitro sensitivity to docetaxel, paclitaxel, SN-38 [an active metabolite of irinotecan (CPT-11)] and gemcitabine, as single agents. Subconfluent cell cultures were treated with 2×10−9, 5×10−9, 10−8, 2×10−8 and 5×10−8 M concentrations of each drug for 48 h. The sensitivity was measured in terms of cell viability using the Trypan blue exclusion method. All four drugs were potent inhibitors of mesothelioma cell growth, but cell lines from different patients diverged in their sensitivity to the individual agents. In most cases docetaxel, paclitaxel and SN-38 were more potent killers of mesothelioma cells than gemcitabine. The induction of DNA damage was investigated using the Comet assay; cells from two cell lines (M14K and M25K) were treated with subtoxic 10−8 M concentrations of each drug for 4, 24 and 48 h. Each of the agents caused a slight increase in DNA single-strand breaks at a concentration of 10−8 M.

Genotoxic and inflammatory effects of nanofibrillated cellulose in murine lungs.

Nanofibrillated cellulose (NFC) is a sustainable and renewable nanomaterial, with diverse potential applications in the paper and medical industries. As NFC consists of long fibres of high aspect ratio, we examined here whether TEMPO-(2,2,6,6-tetramethyl-piperidin-1-oxyl) oxidised NFC (length 300–1000nm, thickness 10–25nm), administrated by a single pharyngeal aspiration, could be genotoxic to mice, locally in the lungs or systemically in the bone marrow. Female C57Bl/6 mice were treated with four different doses of NFC (10, 40, 80 and 200 µg/mouse), and samples were collected 24h later. DNA damage was assessed by the comet assay in bronchoalveolar lavage (BAL) and lung cells, and chromosome damage by the bone marrow erythrocyte micronucleus assay. Inflammation was evaluated by BAL cell counts and analysis of cytokines and histopathological alterations in the lungs. A significant induction of DNA damage was observed at the two lower doses of NFC in lung cells, whereas no increase was seen in BAL cells. No effect was detected in the bone marrow micronucleus assay, either. NFC increased the recruitment of inflammatory cells to the lungs, together with a dose-dependent increase in mRNA expression of tumour necrosis factor &agr;, interleukins 1&bgr; and 6, and chemokine (C-X-C motif) ligand 5, although there was no effect on the levels of the respective proteins. The histological analysis showed a dose-related accumulation of NFC in the bronchi, the alveoli and some in the cytoplasm of macrophages. In addition, neutrophilic accumulation in the alveolar lung space was observed with increasing dose. Our findings showed that NFC administered by pharyngeal aspiration caused an acute inflammatory response and DNA damage in the lungs, but no systemic genotoxic effect in the bone marrow. The present experimental design did not, however, allow us to determine whether the responses were transient or could persist for a longer time.