Assay integration for reproducible toxicity testing of particulate matter in a single well: cytotoxicity, oxidative stress, inflammatory response, and mRNA profiling

Airborne particulate matter (PM) is an important risk factor for mortality. Accordingly, toxicity testing is needed to identify potency determinants. As PM mass is often limited, especially for environmental samples, integrated assessment of multiple endpoints would increase data yield and throughput while minimizing sample use. We established an approach to assess multiple endpoints from a single well, and evaluated consistency and reproducibility of biological responses in two cell lines (A549, J774) 24 h post-exposure to standard reference PM (urban EHC-6802 (EHC), cristobalite (CRI, SRM-154b), SiO₂ (SRM-1879)) at multiple doses (0, 3, 10, 30, 60, 180 μg/cm²). Cell viability, metabolic activity, reactive oxygen species (ROS), mRNA profiling, and inflammatory mediators were assessed. PM effects included: 1) non-monotonic ROS response and little change in viability in A459; 2) dose-dependent ROS increase and reduced viability in J774; 3) ROS and mRNA sensitivity at the lowest dose assessed; 4) endpoint-specific potency differences (e.g. J774: CRI > EHC > TiO₂ for cell viability, EHC > CRI > TiO₂ for cytokines); 4) no evidence of artifacts in cell-free controls. Results were reproduced one year later, confirming the robustness of the approach. By monitoring key events in a single well, this approach minimises particle mass use and facilitates a more holistic assessment of PM toxicity.