Acute phase response following pulmonary exposure to soluble and insoluble metal oxide nanomaterials in mice.

BACKGROUND: Acute phase response (APR) is characterized by a change in concentration of different proteins, including C-reactive protein and serum amyloid A (SAA) that can be linked to both exposure to metal oxide nanomaterials and risk of cardiovascular diseases. In this study, we intratracheally exposed mice to ZnO, CuO, Al2O3, SnO2 and TiO2 and carbon black (Printex 90) nanomaterials with a wide range in phagolysosomal solubility. We subsequently assessed neutrophil numbers, protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, Saa3 and Saa1 mRNA levels in lung and liver tissue, respectively, and SAA3 and SAA1/2 in plasma. Endpoints were analyzed 1 and 28 days after exposure, including histopathology of lung and liver tissues. RESULTS: All nanomaterials induced pulmonary inflammation after 1 day, and exposure to ZnO, CuO, SnO2, TiO2 and Printex 90 increased Saa3 mRNA levels in lungs and Saa1 mRNA levels in liver. Additionally, CuO, SnO2, TiO2 and Printex 90 increased plasma levels of SAA3 and SAA1/2. Acute phase response was predicted by deposited surface area for insoluble metal oxides, 1 and 28 days post-exposure. CONCLUSION: Soluble and insoluble metal oxides induced dose-dependent APR with different time dependency. Neutrophil influx, Saa3 mRNA levels in lung tissue and plasma SAA3 levels correlated across all studied nanomaterials, suggesting that these endpoints can be used as biomarkers of acute phase response and cardiovascular disease risk following exposure to soluble and insoluble particles.

Pulmonary toxicity and translocation of gallium phosphide nanowires to secondary organs following pulmonary exposure in mice.

BACKGROUND: III-V semiconductor nanowires are envisioned as being integrated in optoelectronic devices in the near future. However, the perspective of mass production of these nanowires raises concern for human safety due to their asbestos- and carbon nanotube-like properties, including their high aspect ratio shape. Indeed, III-V nanowires have similar dimensions as Mitsui-7 multi-walled carbon nanotubes, which induce lung cancer by inhalation in rats. It is therefore urgent to investigate the toxicological effects following lung exposure to III-V nanowires prior to their use in industrial production, which entails risk of human exposure. Here, female C57BL/6J mice were exposed to 2, 6, and 18 µg (0.12, 0.35 and 1.1 mg/kg bw) of gallium phosphide (III-V) nanowires (99 nm diameter, 3.7 µm length) by intratracheal instillation and the toxicity was investigated 1, 3, 28 days and 3 months after exposure. Mitsui-7 multi-walled carbon nanotubes and carbon black Printex 90 nanoparticles were used as benchmark nanomaterials. RESULTS: Gallium phosphide nanowires induced genotoxicity in bronchoalveolar lavage cells and acute inflammation with eosinophilia observable both in bronchoalveolar lavage and lung tissue (1 and 3 days post-exposure). The inflammatory response was comparable to the response following exposure to Mitsui-7 multi-walled carbon nanotubes at similar dose levels. The nanowires underwent partial dissolution in the lung resulting in thinner nanowires, with an estimated in vivo half-life of 3 months. Despite the partial dissolution, nanowires were detected in lung, liver, spleen, kidney, uterus and brain 3 months after exposure. CONCLUSION: Pulmonary exposure to gallium phosphide nanowires caused similar toxicological effects as the multi-walled carbon nanotube Mitsui-7.

Identification of a Gene Signature Predicting (Nano)Particle-Induced Adverse Lung Outcome in Rats.

Nanoparticles are extensively used in industrial products or as food additives. However, despite their contribution to improving our quality of life, concerns have been raised regarding their potential impact on occupational and public health. To speed up research assessing nanoparticle-related hazards, this study was undertaken to identify early markers of harmful effects on the lungs. Female Sprague Dawley rats were either exposed to crystalline silica DQ-12 with instillation, or to titanium dioxide P25, carbon black Printex-90, or multi-walled carbon nanotube Mitsui-7 with nose-only inhalation. Tissues were collected at three post-exposure time points to assess short- and long-term effects. All particles induced lung inflammation. Histopathological and biochemical analyses revealed phospholipid accumulation, lipoproteinosis, and interstitial thickening with collagen deposition after exposure to DQ-12. Exposure to the highest dose of Printex-90 and Mitsui-7, but not P25, induced some phospholipid accumulation. Comparable histopathological changes were observed following exposure to P25, Printex-90, and Mitsui-7. Comparison of overall gene expression profiles identified 15 potential early markers of adverse lung outcomes induced by spherical particles. With Mitsui-7, a distinct gene expression signature was observed, suggesting that carbon nanotubes trigger different toxicity mechanisms to spherical particles.

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.

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.

Particle characterization and toxicity in C57BL/6 mice following instillation of five different diesel exhaust particles designed to differ in physicochemical properties.

BACKGROUND: Diesel exhaust is carcinogenic and exposure to diesel particles cause health effects. We investigated the toxicity of diesel exhaust particles designed to have varying physicochemical properties in order to attribute health effects to specific particle characteristics. Particles from three fuel types were compared at 13% engine intake O2 concentration: MK1 ultra low sulfur diesel (DEP13) and the two renewable diesel fuels hydrotreated vegetable oil (HVO13) and rapeseed methyl ester (RME13). Additionally, diesel particles from MK1 ultra low sulfur diesel were generated at 9.7% (DEP9.7) and 17% (DEP17) intake O2 concentration. We evaluated physicochemical properties and histopathological, inflammatory and genotoxic responses on day 1, 28, and 90 after single intratracheal instillation in mice compared to reference diesel particles and carbon black. RESULTS: Moderate variations were seen in physical properties for the five particles: primary particle diameter: 15-22 nm, specific surface area: 152-222 m2/g, and count median mobility diameter: 55-103 nm. Larger differences were found in chemical composition: organic carbon/total carbon ratio (0.12-0.60), polycyclic aromatic hydrocarbon content (1-27 µg/mg) and acid-extractable metal content (0.9-16 µg/mg). Intratracheal exposure to all five particles induced similar toxicological responses, with different potency. Lung particle retention was observed in DEP13 and HVO13 exposed mice on day 28 post-exposure, with less retention for the other fuel types. RME exposure induced limited response whereas the remaining particles induced dose-dependent inflammation and acute phase response on day 1. DEP13 induced acute phase response on day 28 and inflammation on day 90. DNA strand break levels were not increased as compared to vehicle, but were increased in lung and liver compared to blank filter extraction control. Neutrophil influx on day 1 correlated best with estimated deposited surface area, but also with elemental carbon, organic carbon and PAHs. DNA strand break levels in lung on day 28 and in liver on day 90 correlated with acellular particle-induced ROS. CONCLUSIONS: We studied diesel exhaust particles designed to differ in physicochemical properties. Our study highlights specific surface area, elemental carbon content, PAHs and ROS-generating potential as physicochemical predictors of diesel particle toxicity.

Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale.

Toxicity testing and regulation of advanced materials at the nanoscale, i.e. nanosafety, is challenged by the growing number of nanomaterials and their property variants requiring assessment for potential human health impacts. The existing animal-reliant toxicity testing tools are onerous in terms of time and resources and are less and less in line with the international effort to reduce animal experiments. Thus, there is a need for faster, cheaper, sensitive and effective animal alternatives that are supported by mechanistic evidence. More importantly, there is an urgency for developing alternative testing strategies that help justify the strategic prioritization of testing or targeting the most apparent adverse outcomes, selection of specific endpoints and assays and identifying nanomaterials of high concern. The Adverse Outcome Pathway (AOP) framework is a systematic process that uses the available mechanistic information concerning a toxicological response and describes causal or mechanistic linkages between a molecular initiating event, a series of intermediate key events and the adverse outcome. The AOP framework provides pragmatic insights to promote the development of alternative testing strategies. This review will detail a brief overview of the AOP framework and its application to nanotoxicology, tools for developing AOPs and the role of toxicogenomics, and summarize various AOPs of relevance to inhalation toxicity of nanomaterials that are currently under various stages of development. The review also presents a network of AOPs derived from connecting all AOPs, which shows that several adverse outcomes induced by nanomaterials originate from a molecular initiating event that describes the interaction of nanomaterials with lung cells and involve similar intermediate key events. Finally, using the example of an established AOP for lung fibrosis, the review will discuss various in vitro tests available for assessing lung fibrosis and how the information can be used to support a tiered testing strategy for lung fibrosis. The AOPs and AOP network enable deeper understanding of mechanisms involved in inhalation toxicity of nanomaterials and provide a strategy for the development of alternative test methods for hazard and risk assessment of nanomaterials.

Pulmonary toxicity of two different multi-walled carbon nanotubes in rat: Comparison between intratracheal instillation and inhalation exposure.

Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the "long and thick" NM-401 and the "short and thin" NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1-3 days and 28-30 days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area.

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.

Malignant pleural mesothelioma in Finland: regional and gender variation.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare occupational cancer with a poor prognosis. Even with a multimodality treatment approach, the treatment outcomes remain unsatisfactory. The use of asbestos has been banned in most developed countries, but MPM continues to be a significant occupational disease also in these countries. Aim of this study is to identify modern epidemiology and assess equality in care. METHODS: Our study cohort consists of 1010 patients diagnosed with MPM in Finland during 2000-2012. The data were collected from the Finnish Cancer Registry, the National Workers' Compensation Center Registry and the National Registry of Causes of Death, Statistics Finland. RESULTS: Women were diagnosed a mean of 4.5 years later than males (p = .001), but survival did not differ (overall median survival 9.7 months). A workers' compensation claim was more common in males (OR 11.0 [95% CI 7.5-16.2]) and in regions with a major asbestos industry (OR 1.7 [95% CI 1.3-2.2]). One-year and three-year survivals did not differ regionally. Patients without chemotherapy treatment had an inferior survival (RR 1.8 [95% CI 1.5-2.0]). The initial survival benefit gained with pemetrexed was diluted at 51 months. CONCLUSIONS: MPM is a disease with a poor prognosis, although chemotherapy appears to improve survival time. Significant gender and regional variation exists among patients, with notable differences in diagnostic and treatment practices. Long-term outcomes with pemetrexed remain indeterminate. IMPACT: Emphasize centralized consult services for the diagnosis, treatment and support that patients receive for MPM, facilitating equal outcomes and compensation.

Surface PEGylation suppresses pulmonary effects of CuO in allergen-induced lung inflammation.

BACKGROUND: Copper oxide (CuO) nanomaterials are used in a wide range of industrial and commercial applications. These materials can be hazardous, especially if they are inhaled. As a result, the pulmonary effects of CuO nanomaterials have been studied in healthy subjects but limited knowledge exists today about their effects on lungs with allergic airway inflammation (AAI). The objective of this study was to investigate how pristine CuO modulates allergic lung inflammation and whether surface modifications can influence its reactivity. CuO and its carboxylated (CuO COOH), methylaminated (CuO NH3) and PEGylated (CuO PEG) derivatives were administered here on four consecutive days via oropharyngeal aspiration in a mouse model of AAI. Standard genome-wide gene expression profiling as well as conventional histopathological and immunological methods were used to investigate the modulatory effects of the nanomaterials on both healthy and compromised immune system. RESULTS: Our data demonstrates that although CuO materials did not considerably influence hallmarks of allergic airway inflammation, the materials exacerbated the existing lung inflammation by eliciting dramatic pulmonary neutrophilia. Transcriptomic analysis showed that CuO, CuO COOH and CuO NH3 commonly enriched neutrophil-related biological processes, especially in healthy mice. In sharp contrast, CuO PEG had a significantly lower potential in triggering changes in lungs of healthy and allergic mice revealing that surface PEGylation suppresses the effects triggered by the pristine material. CONCLUSIONS: CuO as well as its functionalized forms worsen allergic airway inflammation by causing neutrophilia in the lungs, however, our results also show that surface PEGylation can be a promising approach for inhibiting the effects of pristine CuO. Our study provides information for health and safety assessment of modified CuO materials, and it can be useful in the development of nanomedical applications.

Airport emission particles: exposure characterization and toxicity following intratracheal instillation in mice.

BACKGROUND: Little is known about the exposure levels and adverse health effects of occupational exposure to airplane emissions. Diesel exhaust particles are classified as carcinogenic to humans and jet engines produce potentially similar soot particles. Here, we evaluated the potential occupational exposure risk by analyzing particles from a non-commercial airfield and from the apron of a commercial airport. Toxicity of the collected particles was evaluated alongside NIST standard reference diesel exhaust particles (NIST2975) in terms of acute phase response, pulmonary inflammation, and genotoxicity after single intratracheal instillation in mice. RESULTS: Particle exposure levels were up to 1 mg/m3 at the non-commercial airfield. Particulate matter from the non-commercial airfield air consisted of primary and aggregated soot particles, whereas commercial airport sampling resulted in a more heterogeneous mixture of organic compounds including salt, pollen and soot, reflecting the complex occupational exposure at an apron. The particle contents of polycyclic aromatic hydrocarbons and metals were similar to the content in NIST2975. Mice were exposed to doses 6, 18 and 54 µg alongside carbon black (Printex 90) and NIST2975 and euthanized after 1, 28 or 90 days. Dose-dependent increases in total number of cells, neutrophils, and eosinophils in bronchoalveolar lavage fluid were observed on day 1 post-exposure for all particles. Lymphocytes were increased for all four particle types on 28 days post-exposure as well as for neutrophil influx for jet engine particles and carbon black nanoparticles. Increased Saa3 mRNA levels in lung tissue and increased SAA3 protein levels in plasma were observed on day 1 post-exposure. Increased levels of DNA strand breaks in bronchoalveolar lavage cells and liver tissue were observed for both particles, at single dose levels across doses and time points. CONCLUSIONS: Pulmonary exposure of mice to particles collected at two airports induced acute phase response, inflammation, and genotoxicity similar to standard diesel exhaust particles and carbon black nanoparticles, suggesting similar physicochemical properties and toxicity of jet engine particles and diesel exhaust particles. Given this resemblance as well as the dose-response relationship between diesel exhaust exposure and lung cancer, occupational exposure to jet engine emissions at the two airports should be minimized.

Effectiveness of a New Single-Needle Single-Pump Dialysis System with Simultaneous Monitoring of Dialysis Dose.

To date, single-needle (SN) hemodialysis (HD) requires a dialysis machine equipped with two blood pumps-one controlling arterial blood flow (Qb) and one controlling venous Qb. B. Braun has developed an innovative single-pump SN HD system. Therefore, usability is improved by reducing complexity. The aim of this study was to compare dialysis parameters of the new single-pump SN HD system with a double-pump SN HD system available on the market (Fresenius Medical Care [FMC] 5008). In this two-armed crossover study, patients were randomized into two groups (B. Braun - FMC/FMC - B. Braun). Study period was 2 weeks (6 HD sessions) for each SN HD system. Both B. Braun and FMC dialysis machines were operated in the single-needle auto mode. With the FMC dialysis machines, Qb was optimized manually, whereas for B. Braun machines it was optimized automatically using the auto-mode functionality. A phase volume of 25 mL, treatment time, needle type and size, and dialyzer type and size were kept constant per patient throughout the study. Due to technical prerequisites in the SN mode, online dialysis adequacy (Kt/V: K - dialyzer clearance of urea; t - dialysis time; V - volume of distribution of urea) monitoring could only be performed in the B. Braun group. Twelve HD patients (5 male/7 female, mean age 75.5 ± 8.8 years, mean time on dialysis 4.97 ± 3.86 years, 3× weekly HD) were enrolled. Total number of treatments performed: n = 132 (65 B. Braun, 67 FMC) and the mean online Kt/V value in the B. Braun group was 1.26 ± 0.29 (n = 63). Mean dialysis time per session: B. Braun 253.4 ± 19.9 min, FMC 251.6 ± 18.8 min. Mean phase volume: B. Braun 25.1 ± 0.2 mL, FMC 25.4 ± 3.1 mL. Mean cumulated blood volume (CBV): B. Braun 55.0 ± 5.5 L, FMC 40.5 ± 5.9 L (P < 0.0001). Mean Qb: B. Braun 217.8 ± 12.9 mL/min, FMC 178.6 ± 14.9 mL/min (effective Qb) (P < 0.0001), which corresponds to a difference of 39.3 mL/min (22.0%). Higher Qb has an influence on the CBV. To evaluate this effect, CBV was corrected for the difference in Qb by calculating the CBV/Qb rate. The mean CBV/Qb rate was 252.2 ± 19.4 min (B. Braun) and 226.8 ± 27.6 min (FMC) (P < 0.0001) per session. This represents a highly significant difference of 11.4%. To support the in vivo data the dead time for opening/closure of the clamps of the FMC 5008 was measured, resulting in 364 milliseconds. Over a 240 min dialysis session, with a blood flow rate of 250 mL/min and a phase volume of 25 mL, it was estimated at about 14.56 min (6.1% of the session). Similarly, it was estimated that the dead time of the pumps of the FMC 5008 during 240 min dialysis session was 4.7 min (1.9% of the session). In case single needle therapy is the only practical option for a patient, the advantages of the new single-pump single needle system-namely the proven higher cumulative blood volume, the alarm-free auto-regulation of the blood flow and the easier handling for the nursing staff-ensure higher treatment efficiency than conventional double-pump single needle systems.

Asbestos-associated genome-wide DNA methylation changes in lung cancer.

Previous studies have revealed a robust association between exposure to asbestos and human lung cancer. Accumulating evidence has highlighted the role of epigenome deregulation in the mechanism of carcinogen-induced malignancies. We examined the impact of asbestos on DNA methylation. Our genome-wide studies (using Illumina HumanMethylation450K BeadChip) of lung cancer tissue and paired normal lung from 28 asbestos-exposed or non-exposed patients, mostly smokers, revealed distinctive DNA methylation changes. We identified a number of differentially methylated regions (DMR) and differentially variable, differentially methylated CpGs (DVMC), with individual CpGs further validated by pyrosequencing in an independent series of 91 non-small cell lung cancer and paired normal lung. We discovered and validated BEND4, ZSCAN31 and GPR135 as significantly hypermethylated in lung cancer. DMRs in genes such as RARB (FDR 1.1 × 10-19 , mean change in beta [Δ] -0.09), GPR135 (FDR 1.87 × 10-8 , mean Δ -0.09) and TPO (FDR 8.58 × 10-5 , mean Δ -0.11), and DVMCs in NPTN, NRG2, GLT25D2 and TRPC3 (all with p <0.05, t-test) were significantly associated with asbestos exposure status in exposed versus non-exposed lung tumors. Hypomethylation was characteristic to DVMCs in lung cancer tissue from asbestos-exposed subjects. When DVMCs related to asbestos or smoking were analyzed, 96% of the elements were unique to either of the exposures, consistent with the concept that the methylation changes in tumors may be specific for risk factors. In conclusion, we identified novel DNA methylation changes associated with lung tumors and asbestos exposure, suggesting that changes may be present in causal pathway from asbestos exposure to lung cancer.

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.

Dectin-1 pathway activates robust autophagy-dependent unconventional protein secretion in human macrophages.

Dectin-1 is a membrane-bound pattern recognition receptor for ß-glucans, which are the main constituents of fungal cell walls. Detection of ß-glucans by dectin-1 triggers an effective innate immune response. In this study, we have used a systems biology approach to provide the first comprehensive characterization of the secretome and associated intracellular signaling pathways involved in activation of dectin-1/Syk in human macrophages. Transcriptome and secretome analysis revealed that the dectin-1 pathway induced significant gene expression changes and robust protein secretion in macrophages. The enhanced protein secretion correlated only partly with increased gene expression. Bioinformatics combined with functional studies revealed that the dectin-1/Syk pathway activates both conventional and unconventional, vesicle-mediated, protein secretion. The unconventional protein secretion triggered by the dectin-1 pathway is dependent on inflammasome activity and an active autophagic process. In conclusion, our results reveal that unconventional protein secretion has an important role in the innate immune response against fungal infections.

Driver Gene and Novel Mutations in Asbestos-Exposed Lung Adenocarcinoma and Malignant Mesothelioma Detected by Exome Sequencing.

BACKGROUND: Asbestos is a carcinogen linked to malignant mesothelioma (MM) and lung cancer. Some gene aberrations related to asbestos exposure are recognized, but many associated mutations remain obscure. We performed exome sequencing to determine the association of previously known mutations (driver gene mutations) with asbestos and to identify novel mutations related to asbestos exposure in lung adenocarcinoma (LAC) and MM. METHODS: Exome sequencing was performed on DNA from 47 tumor tissues of MM (21) and LAC (26) patients, 27 of whom had been asbestos-exposed (18 MM, 9 LAC). In addition, 9 normal lung/blood samples of LAC were sequenced. Novel mutations identified from exome data were validated by amplicon-based deep sequencing. Driver gene mutations in BRAF, EGFR, ERBB2, HRAS, KRAS, MET, NRAS, PIK3CA, STK11, and ephrin receptor genes (EPHA1-8, 10 and EPHB1-4, 6) were studied for both LAC and MM, and in BAP1, CUL1, CDKN2A, and NF2 for MM. RESULTS: In asbestos-exposed MM patients, previously non-described NF2 frameshift mutation (one) and BAP1 mutations (four) were detected. Exome data mining revealed some genes potentially associated with asbestos exposure, such as MRPL1 and SDK1. BAP1 and COPG1 mutations were seen exclusively in MM. Pathogenic KRAS mutations were common in LAC patients (42 %), both in non-exposed (n = 5) and exposed patients (n = 6). Pathogenic BRAF mutations were found in two LACs. CONCLUSION: BAP1 mutations occurred in asbestos-exposed MM. MRPL1, SDK1, SEMA5B, and INPP4A could possibly serve as candidate genes for alterations associated with asbestos exposure. KRAS mutations in LAC were not associated with asbestos exposure.

Osteoclast activators are elevated in intervertebral disks with Modic changes among patients operated for herniated nucleus pulposus.

PURPOSE: Modic changes (MC) are associated with low back pain (LBP). Inflammation is considered as a key factor that triggers symptoms in especially type I MC, but so far of the potential inflammatory candidates only TNFα has been linked to MC. The objective of the study was to analyze a set of inflammatory mediators in human surgical disk samples and quantify their association with MC in the adjacent vertebral bodies. METHODS: The study sample consisted of 51 intervertebral disk tissue specimens; 20 'No MC' disks, 19 'Type I MC' disks, and 12 'Type II MC' disks. mRNA expression of 46 cytokines was quantified from isolated RNA. Tissue samples were stained using hematoxylin and eosin, toluidine blue, Herovici, CD68 and CD163. RESULTS: No significant differences were found in the amount of macrophages or presence of chondrocyte conglomerates between the MC groups. Of the multiple genes tested, statistically significant associations were observed for M-CSF1 (p = 0.028), RANKL (p = 0.035), RUNX1 (p = 0.032), and RUNX2 (p = 0.047) that were increased in 'Type II MC,' while OSCAR (p = 0.042) was increased in 'Type I MC' group compared to 'No MC.' CONCLUSIONS: Since these cytokines are related to differentiation and proliferation of osteoclasts, our data suggest that the stimulation of vertebral osteoclasts by factors secreted by disk tissue is involved in the pathophysiology of MC.

Visualization of Nanofibrillar Cellulose in Biological Tissues Using a Biotinylated Carbohydrate Binding Module of ß-1,4-Glycanase.

Nanofibrillar cellulose is a very promising innovation with diverse potential applications including high quality paper, coatings, and drug delivery carriers. The production of nanofibrillar cellulose on an industrial scale may lead to increased exposure to nanofibrillar cellulose both in the working environment and the general environment. Assessment of the potential health effects following exposure to nanofibrillar cellulose is therefore required. However, as nanofibrillar cellulose primarily consists of glucose moieties, detection of nanofibrillar cellulose in biological tissues is difficult. We have developed a simple and robust method for specific and sensitive detection of cellulose fibers, including nanofibrillar cellulose, in biological tissue, using a biotinylated carbohydrate binding module (CBM) of ß-1,4-glycanase (EXG:CBM) from the bacterium Cellulomonas fimi. EXG:CBM was expressed in Eschericia coli, purified, and biotinylated. EXG:CBM was shown to bind quantitatively to five different cellulose fibers including four different nanofibrillar celluloses. Biotinylated EXG:CBM was used to visualize cellulose fibers by either fluorescence- or horse radish peroxidase (HRP)-tagged avidin labeling. The HRP-EXG:CBM complex was used to visualize cellulose fibers in both cryopreserved and paraffin embedded lung tissue from mice dosed by pharyngeal aspiration with 10-200 µg/mouse. Detection was shown to be highly specific, and the assay appeared very robust. The present method represents a novel concept for the design of simple, robust, and highly specific detection methods for the detection of nanomaterials, which are otherwise difficult to visualize.

Negligible respiratory irritation and inflammation potency of pigmentary TiO2 in mice.

Titanium dioxide (TiO2) is manufactured in millions of tons yearly, and it is used widely as pigment in various applications. Until recently, TiO2 was considered toxicologically harmless and without adverse health effects. In this study, respiratory irritation and inflammation potencies of commercially available pigmentary TiO2 particles (<5 µm, rutile) were studied. Single head-only exposures (30 min) of male Crl:OF1 mice at mass concentrations 6, 11, 21, and 37 mg/m3, and repeated exposures (altogether 16 h, 1 h/day, 4 days/week for 4 weeks) of female BALB/c/Sca mice at mass concentration of 16 mg/m3 to pigmentary TiO2 were conducted. Minor sensory irritation was observed during acute and repeated exposures seen as elongation of the break after the inhalation, which is typical in sensory irritation, and caused by closure of the glottis inhibiting airflow from the lungs after inspiration. No pulmonary irritation, airflow limitation, nasal or pulmonary inflammation was observed. In conclusion, the respiratory irritation and inflammation potencies of the studied pigmentary TiO2 particles seemed to be low and thus can serve as an ideal control exposure agent in short-term studies in mice.