Longer-term 3D lung epithelial responses to respirable mineral dust reveal phase-dependent iron-silica interactions

Respirable mineral dust generated during mining and mineral processing represents a widespread environmental particulate contaminant, yet the physicochemical factors that govern its long-term biological impacts remain poorly resolved. In particular, most studies assess acute responses to individual elemental or mineral phases, leaving the role of mineral phase interactions in complex particulate mixtures largely unexplored. Here, we investigated prolonged epithelial responses to respirable mineral dust using three-dimensional lung epithelial spheroids exposed for up to 30 days to 14 compositionally diverse mineral dust samples collected from coal mining regions in Queensland and New South Wales, Australia, under conditions designed to reflect sustained particle-cell interactions. Dust exposure induced heterogeneous and time-dependent changes in intracellular reactive oxygen species, connective tissue growth factor production, and cell fate distributions. Multivariate and interaction analyses revealed that biological variability was strongly associated with the combined presence of silica- and iron-bearing mineral phases. Quartz-associated responses were substantially amplified in the presence of specific iron phases, with pyrite and Fe(III)-silicate producing stronger remodeling-associated and cytotoxic outcomes than carbonate-associated iron (siderite), potentially reflecting differences in iron redox cycling and the persistence of reactive surface sites. Notably, these amplification effects emerged predominantly at later exposure stages, indicating that mineral phase interactions become increasingly important during prolonged particle-cell contact. Together, these findings demonstrate that the toxicity of respirable mineral particles cannot be inferred from individual phases alone but instead emerges from interactions between mineral components within complex particulate mixtures. Integrating longer-term epithelial models with phase-resolved mineral characterization therefore provides a framework for understanding how mineralogical interactions influence the hazard potential of respirable mineral dust contaminants.