Polycyclic nitroaromatic compounds in HULIS as dominant pro-apoptotic agents in PM2.5: Biomass/coal combustion sources, structural insights, and biomarker discovery

Identifying the toxic components in PM_2.5 is crucial for effective air pollution control, yet it remains a challenge due to the presence of numerous unidentified organic compounds. In this study, correlation analysis between the chemical composition and apoptosis in human lung cells revealed that, within the same air volume, the water-soluble fraction (WSF) exhibits a stronger pro-apoptotic effect than the methanol extract fraction. Humic-like substances (HULIS) in the WSF were identified as the primary drivers of toxicity. We characterized the molecular composition and structural features of these toxic components. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and nuclear magnetic resonance (NMR) were used to identify condensed nitroaromatic ring structures in HULIS as the key functional groups contributing to toxicity. Ten molecular formulas were identified and listed as the potential risk drivers. Source apportionment indicated that HULIS, primarily originating from biomass burning and coal combustion, significantly contributed to apoptosis in human lung cell models. Gene sequencing and the benchmark dose (BMD) method were used to quantify the biomarkers of apoptosis linked to HULIS exposure. Methylation modifications of the AhR, CYP1B1, and DNMT3a genes were identified as candidate biomarkers for HULIS-induced apoptosis, as their uncertainty coefficients (calculated as the gene-specific benchmark dose lower confidence limit [BMDL] divided by the overall apoptosis BMDL) were all less than 10. This conclusion offers critical quantitative molecular evidence that holds promise for potentially enabling more reliable extrapolation of in vitro toxicity data to human health risk assessment. Our findings provide new insight into the toxicity mechanisms of previously unidentified polycyclic nitroaromatic compounds in the HULIS fraction of PM_2.5.