Defining early carcinogenic markers for the prediction of the hazard potential of mineral fibres in an in vitro human 3D bronchial model.

This study aimed to identify early carcinogenic markers to predict the hazard potential of mineral fibres using a human 3D bronchial model. Traditional 2D cell models inadequately mimic lung complexity, prompting the use of more physiologically relevant 3D systems. We investigated the effects of crocidolite (CRO) and two size fractions of chrysotile (≤ and > 5 μm length: CHR S and CHR L) to elucidate early toxicological mechanisms and relate them to the IARC key characteristics (KCs) of carcinogens. Initial analyses (MTT, TEER, and histology) at 24-48 h showed a transient acute toxicity and tissue resilience, indicating limited predictive value at this time point. However, after 12 d of exposure, fibre-treated tissues exhibited persistent alterations corresponding to established IARC KCs, suggesting the early onset of cell transformation pathways. Key findings included: sustained inflammatory signalling, characterized by prolonged overexpression of IL-1β, IL-6, and IL-8; ii) persistent genotoxicity, marked by the prolonged nuclear positivity of γH2AX, indicating DNA double-strand breaks without significant cell death; and iii) induction of fibrotic and epithelial-to-mesenchymal transition (EMT) signals, including the increased expression of key markers such as TGF-β, VEGF, ZEB-2, N-Cadherin, Vimentin, and Mesothelin. These alterations correspond to IARC KCs 2, 6, and 8. They were observed within a short time frame of tissue treatment and could serve as reliable, early predictive endpoints for in vitro toxicological tests. We propose that integrating these specific KC-based biosignatures in 3D lung models with physicochemical fibre characterization may provide a robust framework for predictive assessment of inhalable fibre carcinogenicity.