IN VITRO 2D AND 3D HUMAN LUNG MICROENVIRONMENT MODELS TO EVALUATE THE BIOLOGICAL POTENTIAL IMPACT OF ASBESTOS AND ASBESTOS-LIKE MINERAL FIBRES

The mineral fibres have always been widespread both as natural contaminants and in the man-made environment because they are exploited in industry due to their wide versatility of use. Among these, asbestos fibres are naturally occurring silicates (SiO4) which were extensively used in the past in construction materials, automotive applications and in many textiles. The generic commercial term of asbestos includes five amphibole minerals (Crocidolite, Amosite, Antophyllite, Tremolite and Actinolite) and the serpentine mineral Chrysotile. The Erionite indicating a fibrous/asbestiform mineral, is a naturally occurring fibrous zeolite. All six asbestos species and Erionite are considered by the IARC as “carcinogens to humans” (Group 1). After several decades of research about these fibres and related risks, it is generally accepted today that the physical-chemical characteristics of mineral fibres and morphometric parameters (e.g., length, width, aspect ratio) play a key role in carcinogenesis. The PRIN project n°20173X8WA4, to which this thesis work refers, aimed to examine the so-called "fibre toxicity paradigm", which considers only long and thin asbestos and asbestos-like mineral fibres as a human health hazard because they can reach the alveolar and pleural/peritoneal spaces where they induce adverse effects and chronic inflammation. In this multidisciplinary project different crystal-chemical-physical fibre parameters were assessed, including the role of surface iron species, biodurability, the release of ion toxic cargo after fibre dissolution in cellular environment and ion exchange of zeolite fibres. To date, in vivo models have been the most widely used to observe the mechanisms of triggering chronic inflammatory states following fibre inhalation. Since the issue of fibre toxicity arose, various animal models have been applied to research, although interspecies differences leading to different responses to inhalation agents due to different physiology were not considered, resulting in a non-predictive tool for the human alveolar microenvironment. Moreover, animal tests were expensive and posed ethical problems. On the other hand, traditional 2D in vitro lung epithelial models were not able to reproduce the complexity of the lung environment as they were maintained under static and submerged conditions. For these reasons, this research project planned to use in vitro 3D models that can more closely reproduce the in vivo situation. Firstly, the research work focused on the development of an advanced in vitro model. The A549 tumor cells, requiring differentiation into normal phenotype, were cultured in Air-Liquid Interface (ALI) conditions by seeding on a porous membrane within an insert system. However, the alveolar epithelial barrier setup was concluded during the last months of the PhD program, with the addition of a commercial surfactant model (Curosurf®) to the air-exposed surface, in which fibres were diluted in order to evaluate cell viability. On the other hand, several biological analyses were performed on two different cell lines involved in the damage response within the lung environment with the aim of investigating their role in early cytotoxic and genotoxic fibre damage mechanisms. The THP-1 circulating monocytic-like cells and HECV endothelial vein cells were exposed to three carcinogenic fibres (UICC Crocidolite, Chrysotile from Balangero and Erionite-Na), in indirect and direct contact respectively, up to 72 h. THP-1 cells placed on basolateral side of a well equipped with an insert system were divided by the fibre solutions, added to the apical side of a porous membrane (0.4 µm), in order to expose them only to the ions released by the fibres in culture medium. Cytotoxicity tests indicated that they were significantly affected by all three fibres. Even though Erionite-treated cells showed a higher prolonged release of reactive oxygen species, Chrysotile from Balangero resulted the most pro-inflammatory stimulus and genotoxic fibre compared with the others. In HECV cells directly treated with the three fibres, Crocidolite and Chrysotile affected the cell health balance via different pathways, without no significant cell activation from Erionite fibre. Subsequently, the early biological effects of a Russian commercial Chrysotile fibre, whose effects were unknown, but which is still used and commercialized in several countries today, in two different lengths (< 5 and > 5 µm, according to WHO risk guidelines) were analyzed. The tests were conducted directly on the HECV cell line to evaluate the early cytotoxic and proinflammatory effects. Moreover, a further investigation of conditioning of fibre - treated HECV cell media was performed in human HFFF2 fibroblasts to observe their indirect activation after endothelial cell exposure. Finally, the short- and long-term effects of the Russian Chrysotile fibre at the two different lengths were observed in an in vitro 3D respiratory epithelial model tissue named EpiAirway™ mimicking the tracheobronchial tract. The exposure with the fibrous mineral was monitored up to 12 days by viability testing, barrier integrity analysis, pro-inflammatory activation, and genotoxicity. However, only short-duration fibre treatments affected cell viability and showed early release of pro-inflammatory cytokines. In contrast, Crocidolite, used as a positive control, maintained a high cellular response even for long times, confirming its biodurable aspect. This type of long-term study in an advanced in vitro model represents the first pioneering attempt to investigate the onset and establishment of a chronic condition following inhalation of asbestos fibres. To conclude, the development of new advanced 3D cellular models and the refinement of existing ones could help to bridge the gap between in vivo and in vitro asbestos studies, overcoming some of the shortcomings of traditional in vitro models in order to obtain more reliable data on the toxicity and risks associated with exposure to mineral fibres.