BackgroundRecently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO2 and one nano-CeO2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the air-liquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed.ResultsDeposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used.ConclusionsWe showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0171-3) contains supplementary material, which is available to authorized users.

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Background: Combustion processes, especially in aerospace and defense fields, can lead to complex aerosols emission containing gases and nanoparticles (NPs). Alumina (Al2O3) NPs and hydrogen chloride gas (HClg) are for instance present in high concentrations after solid composite propellants use. Exposure to these pollutants mixtures by inhalation is thus possible but literature data towards their pulmonary toxicity are missing. To specify hazards resulting from these combustion aerosols, an inhalation study was implemented.Male Wistar rats were exposed by nose-only to Al2O3 NPs (13 nm) and/or HClg aerosols for 4h (unique exposures ; UE) or 4h a day for 4 days (iterative exposures ; IE). Bronchoalveolar lavage fluids (BALF) content and lungs histopathology were analyzed 24h after exposures.Results: Iterative co-exposures (IE) increased total proteins and lactate dehydrogenases (LDH) concentrations in BALF indicating alveolar-capillary barrier permeabilization and cytolysis. Early pulmonary inflammation was induced after IE to Al2O3 NPs ± HClg resulting in polymorphonuclear neutrophils (PMN) and pro-inflammatory cytokines increases (TNF-α, IL-1β, GRO/KC) in BALF. Moreover, after exposure to Al2O3 NPs ± HClg aerosols, both exposure scenarios induced early pulmonary histopathological lesions, among which vascular congestions, bronchial pre-exfoliations, vascular and interalveolar septum edemas. Lung oxidative damages (8-hydroxy-2'-deoxyguanosine ; 8-OHdG) were observed in situ following UE in each experimental condition, suggesting early oxidative stress induction by aerosols inhalation. However, no 8-isoprostane concentration increase was simultaneously found in animals BALF.Conclusions: Biological effects of the studied aerosols are dependent on both aerosol content and exposure scenario. Results showed an important early pro-inflammatory effect of Al2O3 NPs/HClg mixtures on rats lungs following iterative inhalations (IE). Taken together these data raise concerns towards potential long term pulmonary toxicity of combustion mixtures aerosols, and highlight the importance for workers to wear individual protections.

The authors have withdrawn this preprint due to author disagreement.