BackgroundCarbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both.MethodsPrintex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea.ResultsAll particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 μg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. ConclusionsOur results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-017-0189-1) contains supplementary material, which is available to authorized users.

Carbon black nanoparticle (CBNP) applications in high doses have been shown to be harmful to the lung. It is postulated that even small, environmentally relevant concentrations induce changes on lung homeostasis. The present study determined the impact of low-dose single and multiple CBNP (Printex 90) applications on mouse alveolar cell metabolism, especially inflammatory and oxidative stress parameters. Nanoparticles were administered to mice by a single or 8 oropharyngeal aspirations at wk 1, 2, 3, 5, 7, 9, 11, and 12 using 7 μg Printex 90, 7 μg DQ12 quartz (positive control), with water vehicle and saline as negative controls. After 2 d or 3 mo lung function was analyzed. Further lung histology, bronchoalveolar lavage fluid (BALF) parameters, and mRNA expression of cytokines and antioxidants enzymes in type II pneumocytes were measured on d 3 or after 3 mo. Single low-dose Printex 90 application induced no marked alterations in lung functions or BALF phospholipid levels but significant decrease in superoxide dismutase 2 and numerically elevated glutathione peroxidase 3 mRNA expression levels in type II pneumocytes. Multiple CBNP applications produced reduced lung function, collagen accumulation, elevated phospholipid levels in BALF, and a massive infiltration of macrophages. Type II pneumocyte mRNA expression of antioxidative enzymes remained unchanged throughout the subchronic experiment, but showed a significant decrease in interleukin (IL)-6Rα mRNA expression. This study demonstrates that an environmentally relevant CBNP concentration induced an acute inflammatory response, an effect that is exacerbated throughout the subchronic duration.

The aim of this study was to compare the effects of synthetic carbon black nanoparticles (CBNP) with and without polycyclic aromatic hydrocarbons adsorbed to their surface. Printex®90 and acetylene soot particles as well as particles covered with either benzo[a]pyrene (BaP) or nitroanthracene (NA) were tested in human pulmonary cell lines (16HBE14o-, Calu-3, A549) and precision cut lung slices (PCLS) of mice, rats and humans using a wide concentration range. Particle size distribution in the cell culture medium was determined by dynamic light scattering. Viability assays were LIVE/DEAD® staining and WST-1 assay for PCLS and WST-8 and neutral red assay for cell lines. CBNP-induced formation of reactive oxygen species (ROS) was assessed in A549 and 16HBE14o- cells by flow cytometry using the DCFH-DA assay. Furthermore, the effect of CBNP exposure on the transepithelial electrical resistance (TEER) was investigated in Calu-3 cells after 24, 48 and 120h treatment with 10 and 50 µg/ml CBNP. With PCLS, the inflammatory response was assessed by measuring pro-inflammatory cytokines (i.e. IL-1alpha, TNF-alpha, IL-8). In human cell lines, significant cytotoxicity was observed in the WST-8 assay for all CBNP tested albeit at different dose levels, whereas no effects were found in the neutral red assay. The dose-response relationships observed in the WST-8 assay varied considerably between the cell lines. Surface modified CBNP with BaP or NA proved to be more toxic compared to Printex®90 or acetylene soot particles. This result was confirmed in PCLS studies, where BaP-CBNP were found to increase cytotoxicity in a dose-dependent manner, while NA-CBNP showed the same tendency. Increased ROS formation was observed with all CBNP tested after 24 and 48h. Interestingly acetylene soot particles caused significant TEER reduction at both dose levels and all time points tested whereas Printex®90 and BaP-CBNP reduced the TEER only after 120h at the high dose. Neither of the CBNP tested induced the secretion of proinflammatory cytokines in mouse and rat PCLS. In conclusion, cytotoxic effects of CBNP depend on their surface properties. Furthermore, this study demonstrated, that the combination of in vitro and ex vivo models provides a valuable tool to assess the acute effects of CBNP on lung tissue

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