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.

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.

Comparison of biodistribution of cerium oxide nanoparticles after repeated oral administration by gavage or snack in Sprague Dawley rats.

The rate of translocation of ingested nanoparticles (NPs) and how the uptake is affected by a food matrix are key aspects of health risk assessment. In this study, female Sprague Dawley rats (N = 4/group) received 0, 1.4, or 13 mg of cerium oxide (CeO2 NM-212) NPs/rat/day by gavage or in a chocolate spread snack 5 days/week for 1 or 2 weeks followed by 2 weeks of recovery. A dose and time-dependent uptake in the liver and spleen of 0.1-0.3 and 0.004-0.005 parts per million (ng/mg) of the total administered dose was found, respectively. There was no statistically significant difference in cerium concentration in the liver or spleen after gavage compared to snack dosing. Microscopy revealed indications of necrotic changes in the liver and decreased cellularity in white pulp in the spleen. The snack provided precise administration and a more human-relevant exposure of NPs and could improve animal welfare as alternative to gavage.

Pulmonary toxicity of synthetic amorphous silica - effects of porosity and copper oxide doping.

Materials can be modified for improved functionality. Our aim was to test whether pulmonary toxicity of silica nanomaterials is increased by the introduction of: a) porosity; and b) surface doping with CuO; and whether c) these modifications act synergistically. Mice were exposed by intratracheal instillation and for some doses also oropharyngeal aspiration to: 1) solid silica 100 nm; 2) porous silica 100 nm; 3) porous silica 100 nm with CuO doping; 4) solid silica 300 nm; 5) porous silica 300 nm; 6) solid silica 300 nm with CuO doping; 7) porous silica 300 nm with CuO doping; 8) CuO nanoparticles 9.8 nm; or 9) carbon black Printex 90 as benchmark. Based on a pilot study, dose levels were between 0.5 and 162 µg/mouse (0.2 and 8.1 mg/kg bw). Endpoints included pulmonary inflammation (neutrophil numbers in bronchoalveolar fluid), acute phase response, histopathology, and genotoxicity assessed by the comet assay, micronucleus test, and the gamma-H2AX assay. The porous silica materials induced greater pulmonary inflammation than their solid counterparts. A similar pattern was seen for acute phase response induction and histologic changes. This could be explained by a higher specific surface area per mass unit for the most toxic particles. CuO doping further increased the acute phase response normalized according to the deposited surface area. We identified no consistent evidence of synergism between surface area and CuO doping. In conclusion, porosity and CuO doping each increased the toxicity of silica nanomaterials and there was no indication of synergy when the modifications co-occurred.

Prediction of Chronic Inflammation for Inhaled Particles: the Impact of Material Cycling and Quarantining in the Lung Epithelium.

On a daily basis, people are exposed to a multitude of health-hazardous airborne particulate matter with notable deposition in the fragile alveolar region of the lungs. Hence, there is a great need for identification and prediction of material-associated diseases, currently hindered due to the lack of in-depth understanding of causal relationships, in particular between acute exposures and chronic symptoms. By applying advanced microscopies and omics to in vitro and in vivo systems, together with in silico molecular modeling, it is determined herein that the long-lasting response to a single exposure can originate from the interplay between the newly discovered nanomaterial quarantining and nanomaterial cycling between different lung cell types. This new insight finally allows prediction of the spectrum of lung inflammation associated with materials of interest using only in vitro measurements and in silico modeling, potentially relating outcomes to material properties for a large number of materials, and thus boosting safe-by-design-based material development. Because of its profound implications for animal-free predictive toxicology, this work paves the way to a more efficient and hazard-free introduction of numerous new advanced materials into our lives.

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.

Fast and Robust Proteome Screening Platform Identifies Neutrophil Extracellular Trap Formation in the Lung in Response to Cobalt Ferrite Nanoparticles.

Despite broad application of magnetic nanoparticles in biomedicine and electronics, only a few in vivo studies on biocompatibility are available. In this study, toxicity of magnetic metal oxide nanoparticles on the respiratory system was examined in vivo by single intratracheal instillation in mice. Bronchoalveolar lavage fluid (BALF) samples were collected for proteome analyses by LC-MS/MS, testing Fe3O4 nanoparticles doped with increasing amounts of cobalt (Fe3O4, CoFe2O4 with an iron to cobalt ratio 5:1, 3:1, 1:3, Co3O4) at two doses (54 µg, 162 µg per animal) and two time points (day 1 and 3 days postinstillation). In discovery phase, in-depth proteome profiling of a few representative samples allowed for comprehensive pathway analyses. Clustering of the 681 differentially expressed proteins (FDR < 0.05) revealed general as well as metal oxide specific responses with an overall strong induction of innate immunity and activation of the complement system. The highest expression increase could be found for a cluster of 39 proteins, which displayed strong dose-dependency to iron oxide and can be attributed to neutrophil extracellular trap (NET) formation. In-depth proteome analysis expanded the knowledge of in vivo NET formation. During screening, all BALF samples of the study (n = 166) were measured label-free as single-injections after a short gradient (21 min) LC separation using the Evosep One system, validating the findings from the discovery and defining protein signatures which enable discrimination of lung inflammation. We demonstrate a proteomics-based toxicity screening with high sample throughput easily transferrable to other nanoparticle types. Data are available via ProteomeXchange with identifier PXD016148.

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.

Increased surface area of halloysite nanotubes due to surface modification predicts lung inflammation and acute phase response after pulmonary exposure in mice.

The toxicological potential of halloysite nanotubes (HNTs) and variants after functional alterations to surface area are not clear. We assessed the toxicological response to HNTs (NaturalNano (NN)) before and after surface etching (NN-etched). Potential cytotoxicity of the two HNTs was screened in vitro in MutaTMMouse lung epithelial cells. Lung inflammation, acute phase response and genotoxicity were assessed 1, 3, and 28 days after a single intratracheal instillation of adult female C57BL/6 J BomTac mice. The doses were 6, 18 or 54 µg of HNTs, compared to vehicle controls and the Carbon black NP (Printex 90) of 162 µg/mouse. The cellular composition of bronchoalveolar lavage (BAL) fluid was determined as a measure of lung inflammation. The pulmonary and hepatic acute phase responses were assessed by Serumamyloida mRNA levels in lung and liver tissue by real-time quantitative PCR. Pulmonary and systemic genotoxicity were analyzed by the alkaline comet assay as DNA strand breaks in BAL cells, lung and liver tissue. The etched HNT (NN-etched) had 4-5 times larger BET surface area than the unmodified HNT (NN). Instillation of NN-etched at the highest dose induced influx of neutrophils into the lungs at all time points and increased Saa3 mRNA levels in lung tissue on day 1 and 3 after exposure. No genotoxicity was observed at any time point. In conclusion, functionalization by etching increased BET surface area of the studied NN and enhanced pulmonary inflammatory toxicity in mice.

Assessment of nanomaterial-induced hepatotoxicity using a 3D human primary multi-cellular microtissue exposed repeatedly over 21 days - the suitability of the in vitro system as an in vivo surrogate.

BACKGROUND: With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver's recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. RESULTS: The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. CONCLUSION: In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.

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.

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.

Pulmonary effects of nanofibrillated celluloses in mice suggest that carboxylation lowers the inflammatory and acute phase responses.

We studied if the pulmonary and systemic toxicity of nanofibrillated celluloses can be reduced by carboxylation. Nanofibrillated celluloses administered at 6 or 18 µg to mice by intratracheal instillation were: 1) FINE NFC, 2-20 µm in length, 2-15 nm in width, 2) AS (-COOH), carboxylated, 0.5-10 µm in length, 4-10 nm in width, containing the biocide BIM MC4901 and 3) BIOCID FINE NFC: as (1) but containing BIM MC4901. FINE NFC administration increased neutrophil influx in BAL and induced SAA3 in plasma. AS (-COOH) produced lower neutrophil influx and systemic SAA3 levels than FINE NFC. Results obtained with BIOCID FINE NFC suggested that BIM MC4901 biocide did not explain the lowered response. Increased DNA damage levels were observed across materials, doses and time points. In conclusion, carboxylation of nanofibrillated cellulose was associated with reduced pulmonary and systemic toxicity, suggesting involvement of OH groups in the inflammatory and acute phase responses.

Physicochemical predictors of Multi-Walled Carbon Nanotube-induced pulmonary histopathology and toxicity one year after pulmonary deposition of 11 different Multi-Walled Carbon Nanotubes in mice.

Multi-walled carbon nanotubes (MWCNT) are widely used nanomaterials that cause pulmonary toxicity upon inhalation. The physicochemical properties of MWCNT vary greatly, which makes general safety evaluation challenging to conduct. Identification of the toxicity-inducing physicochemical properties of MWCNT is therefore of great importance. We have evaluated histological changes in lung tissue 1 year after a single intratracheal instillation of 11 well-characterized MWCNT in female C57BL/6N BomTac mice. Genotoxicity in liver and spleen was evaluated by the comet assay. The dose of 54 µg MWCNT corresponds to three times the estimated dose accumulated during a work life at a NIOSH recommended exposure limit (0.001 mg/m3 ). Short and thin MWCNT were observed as agglomerates in lung tissue 1 year after exposure, whereas thicker and longer MWCNT were detected as single fibres, suggesting biopersistence of both types of MWCNT. The thin and entangled MWCNT induced varying degree of pulmonary inflammation, in terms of lymphocytic aggregates, granulomas and macrophage infiltration, whereas two thick and straight MWCNT did not. By multiple regression analysis, larger diameter and higher content of iron predicted less histopathological changes, whereas higher cobalt content significantly predicted more histopathological changes. No MWCNT-related fibrosis or tumours in the lungs or pleura was found. One thin and entangled MWCNT induced increased levels of DNA strand breaks in liver; however, no physicochemical properties could be related to genotoxicity. This study reveals physicochemical-dependent difference in MWCNT-induced long-term, pulmonary histopathological changes. Identification of diameter size and cobalt content as important for MWCNT toxicity provides clues for designing MWCNT, which cause reduced human health effects following pulmonary exposure.

In vivo-induced size transformation of cerium oxide nanoparticles in both lung and liver does not affect long-term hepatic accumulation following pulmonary exposure.

Recent findings show that cerium oxide (CeO2) nanoparticles may undergo in vivo-induced size transformation with the formation of smaller particles that could result in a higher translocation following pulmonary exposure compared to virtually insoluble particles, like titanium dioxide (TiO2). Therefore, we compared liver deposition of CeO2 and TiO2 nanoparticles of similar primary sizes 1, 28 or 180 days after intratracheal instillation of 162 µg of NPs in female C57BL/6 mice. Mice exposed to 162 µg CeO2 or TiO2 nanoparticles by intravenous injection or oral gavage were included as reference groups to assess the amount of NPs that reach the liver bypassing the lungs and the translocation of NPs from the gastrointestinal tract to the liver, respectively. Pulmonary deposited CeO2 nanoparticles were detected in the liver 28 and 180 days post-exposure and TiO2 nanoparticles 180 days post-exposure as determined by darkfield imaging and by the quantification of Ce and Ti mass concentration by inductively coupled plasma-mass spectrometry (ICP-MS). Ce and Ti concentrations increased over time and 180 days post-exposure the translocation to the liver was 2.87 ± 3.37% and 1.24 ± 1.98% of the initial pulmonary dose, respectively. Single particle ICP-MS showed that the size of CeO2 nanoparticles in both lung and liver tissue decreased over time. No nanoparticles were detected in the liver following oral gavage. Our results suggest that pulmonary deposited CeO2 and TiO2 nanoparticles translocate to the liver with similar calculated translocation rates despite their different chemical composition and shape. The observed particle size distributions of CeO2 nanoparticles indicate in vivo processing over time both in lung and liver. The fact that no particles were detected in the liver following oral exposure showed that direct translocation of nanoparticles from lung to the systemic circulation was the most important route of translocation for pulmonary deposited particles.

Primary genotoxicity in the liver following pulmonary exposure to carbon black nanoparticles in mice.

BACKGROUND: Little is known about the mechanism underlying the genotoxicity observed in the liver following pulmonary exposure to carbon black (CB) nanoparticles (NPs). The genotoxicity could be caused by the presence of translocated particles or by circulating inflammatory mediators released during pulmonary inflammation and acute-phase response. To address this, we evaluated induction of pulmonary inflammation, pulmonary and hepatic acute-phase response and genotoxicity following exposure to titanium dioxide (TiO2), cerium oxide (CeO2) or CB NPs. Female C57BL/6 mice were exposed by intratracheal instillation, intravenous injection or oral gavage to a single dose of 162 µg NPs/mouse and terminated 1, 28 or 180 days post-exposure alongside vehicle control. RESULTS: Liver DNA damage assessed by the Comet Assay was observed after intravenous injection and intratracheal instillation of CB NPs but not after exposure to TiO2 or CeO2. Intratracheal exposure to NPs resulted in pulmonary inflammation in terms of increased neutrophils influx for all NPs 1 and 28 days post-exposure. Persistent pulmonary acute phase response was detected for all NPs at all three time points while only a transient induction of hepatic acute phase response was observed. All 3 materials were detected in the liver by enhanced darkfield microscopy up to 180 days post-exposure. In contrast to TiO2 and CeO2 NPs, CB NPs generated ROS in an acellular assay. CONCLUSIONS: Our results suggest that the observed hepatic DNA damage following intravenous and intratracheal dosing with CB NPs was caused by the presence of translocated, ROS-generating, particles detected in the liver rather than by the secondary effects of pulmonary inflammation or hepatic acute phase response.

Differences in inflammation and acute phase response but similar genotoxicity in mice following pulmonary exposure to graphene oxide and reduced graphene oxide.

We investigated toxicity of 2-3 layered >1 µm sized graphene oxide (GO) and reduced graphene oxide (rGO) in mice following single intratracheal exposure with respect to pulmonary inflammation, acute phase response (biomarker for risk of cardiovascular disease) and genotoxicity. In addition, we assessed exposure levels of particulate matter emitted during production of graphene in a clean room and in a normal industrial environment using chemical vapour deposition. Toxicity was evaluated at day 1, 3, 28 and 90 days (18, 54 and 162 µg/mouse), except for GO exposed mice at day 28 and 90 where only the lowest dose was evaluated. GO induced a strong acute inflammatory response together with a pulmonary (Serum-Amyloid A, Saa3) and hepatic (Saa1) acute phase response. rGO induced less acute, but a constant and prolonged inflammation up to day 90. Lung histopathology showed particle agglomerates at day 90 without signs of fibrosis. In addition, DNA damage in BAL cells was observed across time points and doses for both GO and rGO. In conclusion, pulmonary exposure to GO and rGO induced inflammation, acute phase response and genotoxicity but no fibrosis.

Identification of Gene Transcription Start Sites and Enhancers Responding to Pulmonary Carbon Nanotube Exposure in Vivo.

Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube (MWCNT) Mitsui-7. Our results revealed a massive gene regulatory response, where expression of key inflammatory genes (e.g., Csf3, Il24, and Fgf23) was increased >100-fold 24 h after Mitsui-7 exposure. Many of the Mitsui-7-responsive transcription start sites were alternative transcription start sites for known genes, and the number of alternative transcription start sites used in a given gene was correlated with overall Mitsui-7 response. Strikingly, genes that were up-regulated after Mitsui-7 exposure only through their main annotated transcription start site were linked to inflammatory and defense responses, while genes up-regulated only through alternative transcription start sites were functionally heterogeneous and not inflammation-associated. Furthermore, we identified almost 12 000 active enhancers, many of which were Mitsui-7-responsive, and we identified similarly responding putative target genes. Overall, our study provides the location and activity of Mitsui-7-induced enhancers and transcription start sites, providing a useful resource for targeted experiments elucidating the biological effects of nanomaterials and the identification of biomarkers for early detection of MWCNT-induced inflammation.

Multi-walled carbon nanotube physicochemical properties predict pulmonary inflammation and genotoxicity.

Lung deposition of multi-walled carbon nanotubes (MWCNT) induces pulmonary toxicity. Commercial MWCNT vary greatly in physicochemical properties and consequently in biological effects. To identify determinants of MWCNT-induced toxicity, we analyzed the effects of pulmonary exposure to 10 commercial MWCNT (supplied in three groups of different dimensions, with one pristine and two/three surface modified in each group). We characterized morphology, chemical composition, surface area and functionalization levels. MWCNT were deposited in lungs of female C57BL/6J mice by intratracheal instillation of 0, 6, 18 or 54 µg/mouse. Pulmonary inflammation (neutrophil influx in bronchoalveolar lavage (BAL)) and genotoxicity were determined on day 1, 28 or 92. Histopathology of the lungs was performed on day 28 and 92. All MWCNT induced similar histological changes. Lymphocytic aggregates were detected for all MWCNT on day 28 and 92. Using adjusted, multiple regression analyses, inflammation and genotoxicity were related to dose, time and physicochemical properties. The specific surface area (BET) was identified as a positive predictor of pulmonary inflammation on all post-exposure days. In addition, length significantly predicted pulmonary inflammation, whereas surface oxidation (-OH and -COOH) was predictor of lowered inflammation on day 28. BET surface area, and therefore diameter, significantly predicted genotoxicity in BAL fluid cells and lung tissue such that lower BET surface area or correspondingly larger diameter was associated with increased genotoxicity. This study provides information on possible toxicity-driving physicochemical properties of MWCNT. The results may contribute to safe-by-design manufacturing of MWCNT, thereby minimizing adverse effects.

Exploring arrays of vertical one-dimensional nanostructures for cellular investigations.

The endeavor of exploiting arrays of vertical one-dimensional (1D) nanostructures (NSs) for cellular applications has recently been experiencing a pronounced surge of activity. The interest is rooted in the intrinsic properties of high-aspect-ratio NSs. With a height comparable to a mammalian cell, and a diameter 100-1000 times smaller, NSs should intuitively reach far into a cell and, due to their small diameter, do so without compromising cell health. Single NSs would thus be expedient for measuring and modifying cell response. Further organization of these structures into arrays can provide up-scaled and detailed spatiotemporal information on cell activity, an achievement that would entail a massive leap forward in disease understanding and drug discovery. Numerous proofs-of-principle published recently have expanded the large toolbox that is currently being established in this rapidly advancing field of research. Encouragingly, despite the diversity of NS platforms and experimental conditions used thus far, general trends and conclusions from combining cells with NSs are beginning to crystallize. This review covers the broad spectrum of NS materials and dimensions used; the observed cellular responses with specific focus on adhesion, morphology, viability, proliferation, and migration; compares the different approaches used in the field to provide NSs with the often crucial cytosolic access; covers the progress toward biological applications; and finally, envisions the future of this technology. By maintaining the impressive rate and quality of recent progress, it is conceivable that the use of vertical 1D NSs may soon be established as a superior choice over other current techniques, with all the further benefits that may entail.