Characterization of engineered stone dust-induced reactive oxygen species generation and cytotoxicity in vitro

Engineered stone (ES) fabrication generates respirable dust containing crystalline silica (CS), linked to accelerated silicosis outbreaks. Mechanisms underlying this toxicity, particularly the role of particle aging, remain unclear. In the occupational setting, workers are exposed to engineered stone dust (ESD) upon generation by cutting and grinding ES; however, ESD-initiated toxicity is frequently studied in labs using aged particles. This study aimed to compare radical generation and in vitro cytotoxicity of fresh versus aged ESD. Three different respirable ES types (ES A: 60% CS; B: 20%; C: 0%), granite (30%), and Min-u-Sil 5 (MS5, 99.5%) were generated using an automated cutting system and analyzed either freshly stored under N₂ at -80°C or after aging in air at room temperature for 2 weeks. RAW 264.7 macrophages were exposed to particles (10 µg/well, 100 µg/ml, 31.25 µg/cm², 24 hr), and viability, apoptosis, necrosis, and intracellular reactive oxygen species (ROS) were measured. Fresh ESD/granite exhibited significantly higher electron paramagnetic resonance (EPR) radical signals than aged counterparts and MS5. Fresh ES/granite reduced macrophage viability, while aged materials/MS5 did not. Apoptosis increased with all particles where fresh/aged difference occurred only in ES B. Necrosis rose markedly with fresh ES A. Intracellular ROS was elevated by some materials, but N-acetylcysteine (NAC) antioxidant failed to prevent cytotoxicity induced by fresh particles. In conclusion, freshly generated ESD displayed greater radical-generating capacity and distinct cytotoxic effects compared to aged ESD, influenced by factors beyond CS content. ROS-independent mechanisms appear crucial for acute cytotoxicity. These findings indicate particle aging as a critical factor in ESD toxicological assessment.