Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions

Loret et al. Particle and Fibre Toxicology (2016) 13:58 DOI 10.1186/s12989-016-0171-3 RESEARCH Open Access Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions Thomas Loret1,2, Emmanuel Peyret1, Marielle Dubreuil1, Olivier Aguerre-Chariol3, Christophe Bressot3, Olivier le Bihan3, Tanguy Amodeo3, Bénédicte Trouiller1, Anne Braun1, Christophe Egles2,4 and Ghislaine Lacroix1* Abstract Background: Recently, 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 airliquid 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. Results: Deposited 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. Conclusions: We 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. Keywords: Nanomaterials, In vitro, Alveolar cells, Co-culture, Air-liquid interface, Submerged conditions, Toxicity * Correspondence: 1 Institut National de l’Environnement Industriel et des Risques (INERIS), (DRC/ VIVA/TOXI), Parc Technologique ALATA—BP 2, Verneuil-en-Halatte F-60550, France Full list of author information is available at the end of the article © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Loret et al. Particle and Fibre Toxicology (2016) 13:58 Background The growing utilization of nanomaterials (NMs) in nanotechnologies leads to an increased risk of human exposure [1], raising concerns about public health and safety [2–4]. Metallic and poorly soluble NMs are among the most widely used [5] and a major exposure route for these NMs is inhalation [6]. Nevertheless, occupational and environmental atmospheres have not been well characterized in terms of NMs [7], which partly explains the lack of epidemiological data on the relationship between exposure to airborne NMs and potential adverse human health effects. However, based on epidemiological studies showing an association between exposure to environmental ultrafine particles and adverse health effects [8], the potential toxicity of NM has been taken into consideration and been widely studied in cell cultures and animal models [9, 10]. Results from animal experimentations remain the most reliable [11, 12], especially because of the similar level of complexity compared with the human body. Besides ethical considerations, in vitro studies are widely used to study mechanisms of toxicity because they are usually cheaper, faster and easier to implement than in vivo studies [13]. Nevertheless, the relevance of in vitro studies to predict in vivo effects needs to be carefully assessed. In vivo, inhaled NMs can deposit in the alveolar region [14, 15] and interact with components of the alveolar barrier at the air-liquid interface (ALI) [15]. At the apical side of the barrier, insoluble NMs first interact with the thin layer of surfactant secreted by pneumocytes [16]. This layer covers the entire alveolar surface and transport of NMs occurs from the air to the aqueous surfactant phase [15]. NMs can then be taken up by circulating macrophages to be eliminated or interact directly with pneumocytes [15, 17]. If NMs cross the alveolar barrier [18, 19], they can interact with other components of the barrier such as endothelial cells or immune cells and be transferred to the blood and other organs [19, 20]. As a consequence of the particle-cell interactions, mechanisms of defense can become activated and cell damages can occur such as cell function impairment, release of proand anti-inflammatory cytokines, production of intracellular Reactive Oxygen Species (ROS) and anti-oxidant species, and genotoxicity [4, 6, 21]. In vitro, monocultures of pulmonary cells are usually exposed in submerged conditions to suspensions of NMs to determine mechanisms of toxicity [21] or high throughput screening of novel compounds [13]. However, these experimental conditions do not reflect cellcell communications and cell-particle interactions occurring in vivo in the lung, making in vitro results difficult to interpret [11, 12, 22–24]. Moreover, in submerged conditions, cell-particle interactions are dependent on the medium composition [25, 26]. NMs can interact Page 2 of 21 with components of the culture medium, resulting in the formation of a medium specific corona [26, 27] and can (...truncated)