Ultrafine amorphous silica induces chronic epithelial cell stress and pro-fibrotic reprogramming in a particle size‑ and exposure duration‑dependent manner

Occupational exposure to respirable silica is recognised in industries such as stonemasonry, tunnelling, and mining, with emerging applications in drug delivery systems, cosmetics, and food additives. In the context of rising global silicosis cases, amorphous silica-containing materials are frequently marketed as “safer” alternatives to high crystalline silica engineered stone. However, the safety profile of amorphous silica remains incompletely characterised, particularly regarding particle size and exposure duration, potentially leaving workers vulnerable to silica-related disease. This study investigated the effects of continuous, low‑dose exposure to amorphous silica on lung epithelial cells across multiple respirable size ranges. By integrating size‑dependent comparisons with a chronic exposure paradigm, we provide new insight into how particle size and exposure duration drive epithelial reprogramming. Human bronchial epithelial BEAS-2B cells were exposed to amorphous silica for 3 days (acute exposure) or 17 days (chronic exposure). Particle sizes included ultrafine (11 nm), submicron (500 nm), and micron-scale particles (1 μm and 3 μm). Acute exposure to ultrafine silica resulted in marked cytotoxicity, an effect not observed with larger particle sizes. Across all sizes, acute exposure induced endoplasmic reticulum (ER) stress, oxidative stress, and DNA damage responses, with particularly pronounced effects in cells treated with 11 nm and 3 μm particles. In contrast, chronic exposure produced sustained cellular stress characterised by altered antioxidant defence, altered DNA repair mechanisms, and upregulation of heat shock proteins indicating persistent proteotoxicity. Importantly, chronic exposure to ultrafine silica triggered pro-fibrotic signalling, including increased expression of TGF-β1, MMP2, and β-Catenin. These effects were less pronounced in larger particle size groups. Exposure to amorphous silica induces persistent epithelial cell stress responses and genomic instability in a particle size- and time-dependent manner. Chronic ultrafine amorphous silica exposure promotes a pro-fibrotic phenotype, while larger respirable particles induce sustained proteotoxic stress, highlighting distinct but synergistic mechanisms of disease development during long-term occupational exposure. These findings challenge assumptions of low hazard and highlight gaps in current workplace health and safety frameworks that overlook particle size. Integration of chronic, size‑resolved models into toxicological assessment will aid in informing evidence-based exposure guidelines and strengthen measures to prevent occupational disease. Not applicable.