Airway region-specific effects of carbon black nanoparticles (CBNP)

Abstract

CBNP are found in industrially produced carbon black which is preferably used for reinforcement of elastomers (e.g. in tyres) but are also present in paints, toner and batteries. CBNP may have lung cytotoxic and pro-inflammatory effects. The commercially available CBNP Printex 90 (P90) as well as the quartz DQ12 were shown to possess cytotoxic and pro-inflammatory properties in mice and human epithelial cell lines. We sought to investigate whether these particles exert similar effects in distal versus proximal airway explants of mice. Further, we investigated whether surface modifications with cancerogenic PAHs on CBNP lead to increase or decrease in the biological effects of CBNP. The impact of CBNP on different airway regions was assessed by microdissection of proximal and distal airways from mouse lungs and ex vivo stimulation for 0.5 - 48h. Our study could not show any cytotoxic effects (LDH release) on microdissected airways after incubation of up to 48h with DQ12, acetylene soot which displays various PAHs on its surface, Printex 90, as well as Printex 90 displaying the PAH 9-Nitroanthracene (NA-CBNP) or the PAH Benzo(a)pyrene (BaP-CBNP) on its surface. RT-QPCR results demonstrated that levels of IL-1-beta, KC, IFN-gamma, TNF-alpha, TGF-beta remained unchanged in mice and epithelial cell lines after 4 and 24h of particle incubation. However, IL-6 levels increased with particle concentration of any particle tested. Interestingly, this could only be observed in proximal airways. However, high variations between animals were observed. IL-6 protein did not exhibit any change after 24h of incubation. 2',7'-dichlorfluorescein-diacetate (DCFH) was used to examine oxidative stress levels. All particles showed an effect in proximal and distal airways at the highest concentration used. RT-QPCR results confirmed the induction of Glutathione peroxidase 3 (GPx-3) expression for detoxification of hydrogen peroxide and partly Glutathione reductase (GR). We can conclude that all particles tested did not result in an acute cytotoxic effect and did only slightly induce gene expression of the proinflammatory cytokine IL-6 in proximal airways. Oxidative stress was observed at the highest concentration used (50 µg/ml) whereas anti-oxidative gene expression was already induced from the lowest concentration used (0.1 µg/ml). Supported by the BMBF: joint project Carbon Black (03X0093A