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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.

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 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.

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 increased100-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.

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 TiO

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. Electronic supplementary material The online version of this article (10.1186/s12989-019-0305-5) contains supplementary material, which is available to authorized users.

We studied if the pulmonary and systemic toxicity of nanofibrillated celluloses can be reduced by carboxylation. Nanofibrillated celluloses administered at 6 or 18 mu g to mice by intratracheal instillation were: 1) FINE NFC, 2-20 mu m in length, 2-15 nm in width, 2) AS (-COOH), carboxylated, 0.5-10 mu m 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.

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.

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.

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.

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.

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.

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.