Predicting toxic effects observed in vivo after acute exposure to poorly soluble and inhalable nanomaterials by using more complex in vitro models

International audienceAnimal models are powerful tools to predict potential adverse effects in human after inhalation of Nanomaterials (NM), because of similar levels of complexity. Nevertheless, considering the number of poorly soluble NM used and their physicochemical diversity, it seems difficult to rely only on animal experimentation. In vitro studies represent alternatives to assess acute toxicity after inhalation. In vitro, cells in monoculture are usually exposed to suspensions of NM, to study mechanisms of toxicity or because they are cheaper and easier to implement than in vivo studies. However, using too simple experimental conditions does not allow mimicking accurately the complexity of interactions occurring between particles and lungs in the human body. Moreover, in submerged conditions, particles may interact with some components of the culture medium, resulting in the formation of a specific corona, which may modify cell-particle interactions. In this context, the general aim of our work was to assess if using more complex in vitro method, by exposing alveolar cells in monoculture or co-culture at the Air Liquid Interface (ALI) to aerosols of poorly soluble NM, better simulates adverse effects observed in vivo. Animal and cell models were exposed to TiO2 (NM105: 21 nm, NM101: 6 nm, NM100: 100 nm) and CeO2 (NM212: 28 nm) NM. In vitro, alveolar epithelial cells in monoculture (A549) or in co-culture with alveolar macrophages (A549 + THP-1) were exposed either at the ALI to aerosols, or in submerged condition to suspensions. The real mass deposited on cells in vitro was either assessed by direct dosage (ALI exposures) or estimated using the in vitro sedimentation diffusion and dosimetry (ISDD) model, after measuring the hydrodynamic diameter and the effective density of agglomerates. In vivo, rats were exposed by intratracheal instillation. Deposition on cells in vivo was assessed by dividing the lung charge by the alveolar surface. Biological activity (viability, oxidative stress and inflammation) was assessed at 24h, in vitro on cells and in vivo on bronchoalveolar lavage fluids. Doses were expressed in mass/surface to allow in vitro /in vivo comparisons. According to our results, pro-inflammatory effects were the most relevant biological markers, to compare in vitro and in vivo results after 24h exposure. In vitro, co-cultures appeared more sensitive than mono-cultures. We observed significant effects for all NM and at lower doses when cells were exposed at the ALI to aerosols compared to suspensions. Moreover in vitro, NM101 and NM105 appeared more toxic than NM100 and NM212. In vivo, we observed significant adverse effects for TiO2 NM101 and NM105 and CeO2 NM212, but not with TiO2 NM100. These effects were observed at lower doses than in vitro. In vivo results are in agreement with literature observation where toxic effects observed are dependent on NM size. Both in vivo and in vitro, TiO2 nanomaterials were ranked similarly in function of toxic effects observed and whatever the exposure method used. However, CeO2 was ranked differently in vivo compared to in vitro. In conclusion, we showed that in vitro methods could be used for the relative ranking of TiO2 NM, and we propose the hypothesis that in vitro methods could be used also for the ranking of poorly soluble NM of similar chemical form. We also showed that ALI method seems to better simulate in vivo adverse effects regarding biological activation levels