In vitro and ex vivo toxicity screening for predicting toxicological effects of synthetic carbon black nanoparticles in humans

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.