Effect of Surface Modification on the Pulmonary and Systemic Toxicity of Cellulose Nanofibrils.

Cellulose nanofibrils (CNFs) have emerged as sustainable options for a wide range of applications. However, the high aspect ratio and biopersistence of CNFs raise concerns about potential health effects. Here, we evaluated the in vivo pulmonary and systemic toxicity of unmodified (U-CNF), carboxymethylated (C-CNF), and TEMPO (2,2,6,6-tetramethyl-piperidin-1-oxyl)-oxidized (T-CNF) CNFs, fibrillated in the same way and administered to mice by repeated (3×) pharyngeal aspiration (14, 28, and 56 µg/mouse/aspiration). Toxic effects were assessed up to 90 days after the last administration. Some mice were treated with T-CNF samples spiked with lipopolysaccharide (LPS; 0.02-50 ng/mouse/aspiration) to assess the role of endotoxin contamination. The CNFs induced an acute inflammatory reaction that subsided within 90 days, except for T-CNF. At 90 days post-administration, an increased DNA damage was observed in bronchoalveolar lavage and hepatic cells after exposure to T-CNF and C-CNF, respectively. Besides, LPS contamination dose-dependently increased the hepatic genotoxic effects of T-CNF.

Surface functionalization and size modulate the formation of reactive oxygen species and genotoxic effects of cellulose nanofibrils.

BACKGROUND: Cellulose nanofibrils (CNFs) have emerged as a sustainable and environmentally friendly option for a broad range of applications. The fibrous nature and high biopersistence of CNFs call for a thorough toxicity assessment, but it is presently unclear which physico-chemical properties could play a role in determining the potential toxic response to CNF. Here, we assessed whether surface composition and size could modulate the genotoxicity of CNFs in human bronchial epithelial BEAS-2B cells. We examined three size fractions (fine, medium and coarse) of four CNFs with different surface chemistry: unmodified (U-CNF) and functionalized with 2,2,6,6-tetramethyl-piperidin-1-oxyl (TEMPO) (T-CNF), carboxymethyl (C-CNF) and epoxypropyltrimethylammonium chloride (EPTMAC) (E-CNF). In addition, the source fibre was also evaluated as a non-nanosized material. RESULTS: The presence of the surface charged groups in the functionalized CNF samples resulted in higher amounts of individual nanofibrils and less aggregation compared with the U-CNF. T-CNF was the most homogenous, in agreement with its high surface group density. However, the colloidal stability of all the CNF samples dropped when dispersed in cell culture medium, especially in the case of T-CNF. CNF was internalized by a minority of BEAS-2B cells. No remarkable cytotoxic effects were induced by any of the cellulosic materials. All cellulosic materials, except the medium fraction of U-CNF, induced a dose-dependent intracellular formation of reactive oxygen species (ROS). The fine fraction of E-CNF, which induced DNA damage (measured by the comet assay) and chromosome damage (measured by the micronucleus assay), and the coarse fraction of C-CNF, which produced chromosome damage, also showed the most effective induction of ROS in their respective size fractions. CONCLUSIONS: Surface chemistry and size modulate the in vitro intracellular ROS formation and the induction of genotoxic effects by fibrillated celluloses. One cationic (fine E-CNF) and one anionic (coarse C-CNF) CNF showed primary genotoxic effects, possibly partly through ROS generation. However, the conclusions cannot be generalized to all types of CNFs, as the synthesis process and the dispersion method used for testing affect their physico-chemical properties and, hence, their toxic effects.

Effects of physicochemical properties of TiO2 nanomaterials for pulmonary inflammation, acute phase response and alveolar proteinosis in intratracheally exposed mice.

Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO2 in hazard assessment might be challenging because of variation in physicochemical properties. We exposed C57BL/6 J mice to a single dose of four anatase TiO2 NMs with various sizes and shapes by intratracheal instillation and assessed the pulmonary toxicity 1, 3, 28, 90 or 180 days post-exposure. The quartz DQ12 was included as benchmark particle. Pulmonary responses were evaluated by histopathology, electron microscopy, bronchoalveolar lavage (BAL) fluid cell composition and acute phase response. Genotoxicity was evaluated by DNA strand break levels in BAL cells, lung and liver in the comet assay. Multiple regression analyses were applied to identify specific TiO2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO2 NMs induced similar inflammatory responses when surface area was used as dose metrics, although inflammatory and acute phase response was greatest and more persistent for the TiO2 tube. Similar histopathological changes were observed for the TiO2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO2 NMs indicated that rutile TiO2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO2 NMs are important predictors for the observed pulmonary effects of TiO2 NMs.

CDKN2A copy number and p16 expression in malignant pleural mesothelioma in relation to asbestos exposure.

BACKGROUND: Deletion of the CDKN2A locus is centrally involved in the development of several malignancies. In malignant pleural mesothelioma (MPM), it is one of the most frequently reported genomic alteration. MPM is strongly associated with a patients' asbestos exposure. However, the status of CDKN2A and the expression of the corresponding protein, p16, in relation to MPM patient's asbestos exposure is poorly known. Copy number alterations in 2p16, 9q33.1 and 19p13 have earlier been shown to accumulate in lung cancer in relation to asbestos exposure but their status in MPM is unclear. METHODS: We studied DNA copy numbers for CDKN2A using fluorescence in situ hybridization (FISH) and p16 expression by immunohistochemistry (IHC) in 92 MPM patients, 75 of which with known asbestos exposure status. We also studied, in MPM, copy number alterations in 2p16, 9q33.1 and 19p13 by FISH. RESULTS: We were unable to detect an association between p16 expression and pulmonary asbestos fiber count in MPM tumor cells. However, significantly more MPM patients with high pulmonary asbestos fiber count (> 1 million fibers per gram [f/g]) had stromal p16 immunoreactivity than MPM of patients with low exposure (≤ 0.5 million f/g) (51.4% vs 16.7%; p = 0.035, Chi-Square). We found that an abnormal copy number of CDKN2A in MPM tumor cells associated with a high pulmonary asbestos fiber count (p = 0.044, Fisher's Exact test, two-tailed). In contrast to our earlier findings in asbestos associated lung cancer, DNA copy number changes in 2p16, 9q33 and 19p13 were not frequent in MPM although single cases with variable copy numbers on those regions were seen. CONCLUSIONS: We found two instances where the gene locus CDKN2A or its corresponding protein expression, is associated with high asbestos exposure levels. This suggests that there may be biological differences between the mesotheliomas with high pulmonary asbestos fiber count and those with low fiber count.

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 α, interleukins 1ß 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.

Characterization of particle exposure in ferrochromium and stainless steel production.

This study describes workers' exposure to fine and ultrafine particles in the production chain of ferrochromium and stainless steel during sintering, ferrochromium smelting, stainless steel melting, and hot and cold rolling operations. Workers' personal exposure to inhalable dust was assessed using IOM sampler with a cellulose acetate filter (AAWP, diameter 25 mm; Millipore, Bedford, MA). Filter sampling methods were used to measure particle mass concentrations in fixed locations. Particle number concentrations and size distributions were examined using an SMPS+C sequential mobile particle sizer and counter (series 5.400, Grimm Aerosol Technik, Ainring, Germany), and a hand-held condensation particle counter (CPC, model 3007, TSI Incorporated, MN). The structure and elemental composition of particles were analyzed using TEM-EDXA (TEM: JEM-1220, JEOL, Tokyo, Japan; EDXA: Noran System Six, Thermo Fisher Scientific Inc., Madison,WI). Workers' personal exposure to inhalable dust averaged 1.87, 1.40, 2.34, 0.30, and 0.17 mg m(-3) in sintering plant, ferrochromium smelter, stainless steel melting shop, hot rolling mill, and the cold rolling mill, respectively. Particle number concentrations measured using SMPS+C varied from 58 × 10(3) to 662 × 10(3) cm(-3) in the production areas, whereas concentrations measured using SMPS+C and CPC3007 in control rooms ranged from 24 × 10(3) to 243 × 10(3) cm(-3) and 5.1 × 10(3) to 97 × 10(3) cm(-3), respectively. The elemental composition and the structure of particles in different production phases varied. In the cold-rolling mill non-process particles were abundant. In other sites, chromium and iron originating from ore and recycled steel scrap were the most common elements in the particles studied. Particle mass concentrations were at the same level as that reported earlier. However, particle number measurements showed a high amount of ultrafine particles, especially in sintering, alloy smelting and melting, and tapping operations. Particle number concentration and size distribution measurements provide important information regarding exposure to ultrafine particles, which cannot be seen in particle mass measurements.

Characterization of exposure to carbon nanotubes in an industrial setting.

While production and use of carbon nanotubes (CNTs) is increasing, workers exposure to CNTs is expected to increase as well, with inhalation being potentially the main pathway for uptake. However, there have been few studies reporting results about workers' personal exposure to CNTs. In this study, worker exposure to single-walled CNTs (SWCNTs) during the production of conductive films in a modern up-scaling factory was assessed. Particulate matter concentrations (2.5-10 µm) and concentrations of CO and CO2 were monitored by using real-time instruments. Workers' exposure levels to SWCNTs were qualitatively estimated by analyzing particle samples by transmission electron microscopy (TEM). TEM samples identified high aspect ratio (length/width > 500) SWCNTs in workplace air. SWCNT concentrations estimated from micrographs varied during normal operation, reactor use without local exhaust ventilation (LEV), and cleaning between 1.7×10(-3), 5.6 and 6.0×10(-3) SWCNT cm(-3), respectively. However, during cleaning it was unclear whether the SWCNTs originated from the cleaning itself or from other reactor openings. We were unable to quantify the SWCNT emissions with online particle instrumentation due to the SWCNT low concentrations compared to background particle concentrations, which were on average 2.6±1.1×10(3)cm(-3). However, CO concentrations were verified as a good indicator of fugitive emissions of SWCNTs. During normal operation, exposure levels were well below proposed limit values (1.0×10(-2) fibers cm(-3) and 1 µg m(-3)) when LEV was used. Based on the results in this study, the analysis of TEM grids seems to be the only direct method to detect SWCNTs in workplace air.

Nanosized TiO2 caused minor airflow limitation in the murine airways.

The use of nanotechnology is increasing exponentially, whereas the possible adverse health effects of engineered nanoparticles (NPs) are so far less known. Standardized mouse bioassay was used to study sensory and pulmonary irritation, airflow limitation, and inflammation potency of nanosized TiO(2). Single exposure (0.5 h) to in situ generated TiO(2) (primary particle size 20 nm; geometric mean diameters of 91, 113, and 130 nm at mass concentrations of 8, 20, and 30 mg/m(3), respectively; crystal phase anatase + brookite (3:1)) caused airflow limitation in the conducting airways at each studied exposure concentration, which was shown as a reduction in expiratory flow, being at the lowest 73% of baseline. The response was not dose dependent. Repeated exposures (altogether 16 h, 1 h/day, 4 days/week for 4 weeks) to TiO(2) at mass concentration of 30 mg/m(3) caused as intense airflow limitation effect as the single exposures, and the extent of the responses stayed about the same along the exposure days. Sensory irritation was fairly minor. Pulmonary irritation was more pronounced during the latter part of the repeated exposures compared to the single exposures and the beginning of the repeated exposures. Sensory and pulmonary irritation were observed also in the control group, and, therefore, reaction by-products (NO(2) and C(3)H(6)) may have contributed to the irritation effects. TiO(2) NPs accumulated mainly in the pulmonary macrophages, and they did not cause nasal or pulmonary inflammation. In conclusion, the irritation and inflammation potencies of studied TiO(2) seemed to be low.

Molecular alterations at 9q33.1 and polyploidy in asbestos-related lung cancer.

PURPOSE: Asbestos causes DNA damage and the fibers, together with tobacco smoke, have a synergistic effect on lung cancer risk. We recently identified 18 chromosomal regions that showed differences in DNA copy number between the lung tumors of asbestos-exposed and nonexposed patients. One of the previously identified asbestos-associated chromosomal regions at 9q was further analyzed for allelic imbalance and DNA copy number alterations (CNA) in the lung tumors of asbestos-exposed and nonexposed patients. In addition, the ploidy level of the tumors was studied. EXPERIMENTAL DESIGN: Allelic imbalance was analyzed at 9q31.3-34.3 with 15 microsatellite markers in 52 lung tumor samples from asbestos-exposed and nonexposed patients. CNA at 9q32-34.3 were characterized by fluorescent in situ hybridization (FISH) with six bacterial artificial chromosome probes in 95 lung tumors. The ploidy level was analyzed in 100 lung tumors with FISH using three to five centromere probes. RESULTS: Allelic imbalance at 9q31.3-q34.3 was found in all asbestos-exposed patient tumors (100%, 17 of 17) compared with 64% (14 of 22) in the nonexposed cases (P = 0.005). The most significant difference was detected at 9q33.1 (P = 0.002). FISH results showed that also CNA were more frequent at 9q33.1 in the three major histologic types of non-small-cell lung tumors of exposed patients, and the association showed a dose-dependent trend (P = 0.03). Furthermore, we detected more frequent polyploidy among the exposed (48%, 28 of 58) than among the nonexposed (29%, 12 of 42) patient tumors (P < 0.05). CONCLUSIONS: These results provide a basis for the development of a method to identify asbestos-related lung cancer on a molecular level.

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.

Asbestos fibers in para-aortic and mesenteric lymph nodes.

BACKGROUND: Asbestos fibers are known to accumulate in lung parenchyma and thoracic lymph nodes, but their presence and translocation into the extrapulmonary tissues need clarification. We assessed the presence of asbestos in the para-aortic (PA) and mesenteric (ME) lymph nodes. METHODS: PA and ME lymph nodes and lung tissue from 17 persons who underwent medicolegal autopsy for suspicion of asbestos-related disease and from five controls were analyzed for asbestos fibers using transmission electron microscopy. RESULTS: High concentrations of amphibole asbestos fibers were detected in several lung tissue samples and in the respective PA and ME lymph nodes. The mean concentration for the 10 persons with a lung asbestos content of >/=1 million fibers/g of dry tissue (f/g) was 0.85 (<0.05-4.36) million f/g in the PA lymph nodes and 0.55 (<0.02-2.86) million f/g in the ME lymph nodes. The respective mean values for the 12 persons with a lung asbestos concentration of <1 million f/g were 0.07 for the PA lymph nodes and 0.03 million f/g for the ME nodes. The lung asbestos burden that predicted the detection of asbestos in abdominal lymph nodes was 0.45 million f/g. CONCLUSIONS: In addition to their accumulation in lung tissue, asbestos fibers also collect in the retroperitoneal and the mesenteric lymph nodes. Even low-level occupational exposure results in the presence of crocidolite, amosite, anthophyllite, tremolite, or chrysotile in these abdominal lymph nodes. Our results support the hypothesis of lymph drainage as an important translocation mechanism for asbestos in the human body.

Identification of specific gene copy number changes in asbestos-related lung cancer.

Asbestos is a well-known lung cancer-causing mineral fiber. In vitro and in vivo experiments have shown that asbestos can cause chromosomal damage and aberrations. Lung tumors, in general, have several recurrently amplified and deleted chromosomal regions. To investigate whether a distinct chromosomal aberration profile could be detected in the lung tumors of heavily asbestos-exposed patients, we analyzed the copy number profiles of 14 lung tumors from highly asbestos-exposed patients and 14 matched tumors from nonexposed patients using classic comparative genomic hybridization (CGH). A specific profile could lead to identification of the underlying genes that may act as mediators of tumor formation and progression. In addition, array CGH analyses on cDNA microarrays (13,000 clones) were carried out on 20 of the same patients. Classic CGH showed, on average, more aberrations in asbestos-exposed than in nonexposed patients, and an altered region in chromosome 2 seemed to occur more frequently in the asbestos-exposed patients. Array CGH revealed aberrations in 18 regions that were significantly associated with either of the two groups. The most significant regions were 2p21-p16.3, 5q35.3, 9q33.3-q34.11, 9q34.13-q34.3, 11p15.5, 14q11.2, and 19p13.1-p13.3 (P < 0.005). Furthermore, 11 fragile sites coincided with the 18 asbestos-associated regions (P = 0.08), which may imply preferentially caused DNA damage at these sites. Our findings are the first evidence, indicating that asbestos exposure may produce a specific DNA damage profile.

Nanofibrillated cellulose causes acute pulmonary inflammation that subsides within a month.

Nanofibrillated cellulose (NFC) is a renewable nanomaterial that has beneficial uses in various applications such as packaging materials and paper. Like carbon nanotubes (CNT), NFCs have high aspect ratio and favorable mechanical properties. The aspect ratio also rises a concern whether NFC could pose a health risk and induce pathologies, similar to those triggered by multi-walled CNT. In this study, we explored the immunomodulatory properties of four NFCs in vitro and in vivo, and compared the results with data on bulk-sized cellulose fibrils and rigid multi-walled CNT (rCNT). Two of the NFCs were non-functionalized and two were carboxymethylated or carboxylated. We investigated the production of pro-inflammatory cytokines in differentiated THP-1 cells, and studied the pulmonary effects and biopersistence of the materials in mice. Our results demonstrate that one of the non-functionalized NFCs tested reduced cell viability and triggered pro-inflammatory reactions in vitro. In contrast, all cellulose materials induced innate immunity response in vivo 24 h after oropharyngeal aspiration, and the non-functionalized NFCs additionally caused features of Th2-type inflammation. Modest immune reactions were also seen after 28 days, however, the effects were markedly attenuated as compared with the ones after 24 h. Cellulose materials were not cleared within 1 month, as demonstrated by their presence in the exposed lungs. All effects of NFC were modest as compared with those induced by rCNT. NFC-induced responses were similar or exceeded those triggered by bulk-sized cellulose. These data provide new information about the biodurability and pulmonary effects of different NFCs; this knowledge can be useful in the risk assessment of cellulose materials.

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/m(3)) 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/m(3)). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 µg/cm(2)), while MWCNT-T increased strand breakage only at 200 µg/cm(2). 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.

Genotoxic and immunotoxic effects of cellulose nanocrystals in vitro.

Nanocellulosics are among the most promising innovations for a wide-variety of applications in materials science. Although nanocellulose is presently produced only on a small scale, its possible toxic effects should be investigated at this early stage. The aim of the present study was to examine the potential genotoxicity and immunotoxicity of two celluloses in vitro - cellulose nanocrystals (CNC; mean fibril length 135 nm, mean width 7.3 nm) and a commercially available microcrystalline (non-nanoscale) cellulose (MCC; particle size ∼50 µm). Both celluloses showed 55% cytotoxicity at approximately 100 µg/ml after 4-h, 24-h, and 48-h treatment of human bronchial epithelial BEAS 2B cells, as determined by luminometric detection of ATP and cell count (dead cells identified by propidium iodide). Neither of the materials was able to induce micronuclei (MN) in binucleate or mononucleate BEAS 2B cells after a 48-h treatment (2.5-100 µg/ml). In human monocyte-derived macrophages, MCC induced a release (measured by enzyme-linked immunosorbent assay; ELISA) of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and (after lipopolysaccharide-priming) interleukin 1ß (IL-1ß) after a 6-h exposure to a dose of 300 µg/ml, but CNC (30-300 µg/ml) did not. In conclusion, our results show that nanosized CNC is neither genotoxic nor immunotoxic under the conditions tested, whereas non-nanosized MCC is able to induce an inflammatory response. More studies are needed, especially in vivo, to further assess if CNC and other nanocelluloses induce secondary genotoxic effects mediated by inflammation.

Exposure to airborne particles and volatile organic compounds from polyurethane molding, spray painting, lacquering, and gluing in a workshop.

Due to the health risk related to occupational air pollution exposure, we assessed concentrations and identified sources of particles and volatile organic compounds (VOCs) in a handcraft workshop producing fishing lures. The work processes in the site included polyurethane molding, spray painting, lacquering, and gluing. We measured total VOC (TVOC) concentrations and particle size distributions at three locations representing the various phases of the manufacturing and assembly process. The mean working-hour TVOC concentrations in three locations studied were 41, 37, and 24 ppm according to photo-ionization detector measurements. The mean working-hour particle number concentration varied between locations from 3000 to 36,000 cm-3. Analysis of temporal and spatial variations of TVOC concentrations revealed that there were at least four substantial VOC sources: spray gluing, mold-release agent spraying, continuous evaporation from various lacquer and paint containers, and either spray painting or lacquering (probably both). The mold-release agent spray was indirectly also a major source of ultrafine particles. The workers' exposure can be reduced by improving the local exhaust ventilation at the known sources and by increasing the ventilation rate in the area with the continuous source.

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.

Exposure to Aspergillus fumigatus spores induces chemokine expression in mouse macrophages.

Inhalation of fungal spores may cause inflammation and respiratory diseases, such as bronchitis, allergic alveolitis, and asthma. Alveolar macrophages provide the first line of defense in the respiratory tract. To examine the cellular mechanisms involved in Aspergillus fumigatus-induced airway inflammation, mouse macrophage cell line (RAW 264.7) cells were exposed for 2 h or 6 h to graded doses of A. fumigatus spores that were either alive or heat-killed. Furthermore, the ability of the cells to phagocytize the spores was visualized by electron microscopy. Expression of selected cytokines and chemokines was assessed by a real time quantitative PCR method and by enzyme-linked immunoabsorbent assay (ELISA) after exposure. A significant increase in mRNA expression of TNF-alpha, MIP-1alpha, MIP-1beta, and MCP-1 was observed with a maximal induction at 6h after exposure to the highest (1 x 10(7)) concentration of live spores. Similar response was not detected with heat-killed spores in the expression of chemokines and cytokines, even though there were no differences between the phagocytosis of live and heat-killed spores. These results suggest that exposure to live spores of A. fumigatus can modulate the expression of proinflammatory cytokines and chemokines in mouse macrophages and thus influence the development of inflammatory processes in the airways.

Co-exposure with fullerene may strengthen health effects of organic industrial chemicals.

In vitro toxicological studies together with atomistic molecular dynamics simulations show that occupational co-exposure with C60 fullerene may strengthen the health effects of organic industrial chemicals. The chemicals studied are acetophenone, benzaldehyde, benzyl alcohol, m-cresol, and toluene which can be used with fullerene as reagents or solvents in industrial processes. Potential co-exposure scenarios include a fullerene dust and organic chemical vapor, or a fullerene solution aerosolized in workplace air. Unfiltered and filtered mixtures of C60 and organic chemicals represent different co-exposure scenarios in in vitro studies where acute cytotoxicity and immunotoxicity of C60 and organic chemicals are tested together and alone by using human THP-1-derived macrophages. Statistically significant co-effects are observed for an unfiltered mixture of benzaldehyde and C60 that is more cytotoxic than benzaldehyde alone, and for a filtered mixture of m-cresol and C60 that is slightly less cytotoxic than m-cresol. Hydrophobicity of chemicals correlates with co-effects when secretion of pro-inflammatory cytokines IL-1ß and TNF-α is considered. Complementary atomistic molecular dynamics simulations reveal that C60 co-aggregates with all chemicals in aqueous environment. Stable aggregates have a fullerene-rich core and a chemical-rich surface layer, and while essentially all C60 molecules aggregate together, a portion of organic molecules remains in water.

Accumulation of genomic alterations in 2p16, 9q33.1 and 19p13 in lung tumours of asbestos-exposed patients.

We have previously demonstrated an association between genomic alterations in 19p13, 2p16, and 9q33.1 and asbestos exposure in patients' lung tumours. This study detected allelic imbalance (AI) in these regions in asbestos-exposed lung cancer (LC) patients' histologically normal pulmonary epithelium. We extended the analyses of tumour tissue to cover a large LC patient cohort and studied DNA copy number alteration (CNA) and AI in 19p13, 2p16, and 9q33.1 for the first time in combination. We found both CNA and AI in ≥2/3 of the regions to be significantly and dose-dependently (P < 0.001) associated with pulmonary asbestos fibre count. Twenty percent of the exposed patients' LC showed CNA in ≥2/3 of the regions, whereas none of the non-exposed patients' LC showed CNA in more than one region. AI was evident in 89% of the exposed and in only 26% of the non-exposed patients' LC. The genomic alterations in 19p13, 2p16, and 9q33.1 in compilation identified asbestos-exposed patients' lung tumours better than each of the regions alone. These alterations form the basis for the development of a combinatorial molecular assay that could be used to identify asbestos-related LC.