Air pollution is one of the leading risk factors for public health. Polycyclic aromatic hydrocarbons (PAHs) are among the most important pollutants, yet as a large family of related compounds, they remain relatively poorly characterized toxicologically. For the purpose of a comprehensive identification of their harmful effects, we developed a workflow using automated high-throughput fluorescence microscopy and quantitative image analysis. Using this approach, we studied potential genotoxicity (PARP1, γH2AX, pH3), DNA damage response (Gadd45α, p21, Ki67), and induction of oxidative stress (8-OxoG, Hmox1, SRXN1) in the HepG2 cell line. The methodological approach was validated in a 2D culture using model chemicals with known mechanisms of action (etoposide, colchicine, KBrO3) and benzo[a]pyrene (BAP) as a reference PAH, confirming high reliability and comparability of quantification between commercial software (Agilent BioTek Gen5) and the established software FIJI/ImageJ. Automated quantification achieved high correlation with manual analysis without systematic bias, while the throughput of analysis was comparable between 2D samples and paraffin sections of spheroids. In the second part, using the developed workflow,
we evaluated the toxicological profile of less studied PAHs – benzo[b]fluoranthene (BBF), benzo[ghi]perylene (BGP), and their binary mixture – and compared responses between the standard 2D model and an advanced 3DD cell model (mature HepG2 spheroids, cultured for 21 days under dynamic conditions). Data were analyzed using linear (mixed) models and multivariate approaches (PERMANOVA, PCA) in RStudio software. The results showed that BBF exhibits pronounced genotoxicity comparable to BAP, as it causes DNA double-strand breaks (γH2AX) and activates the DNA damage response (p21). BGP proved to be less genotoxic, but at higher concentrations primarily triggered oxidative stress (Hmox1,
SRXN1). In the 3D cell model, cellular responses to all tested xenobiotics were significantly altered and enhanced, with more pronounced aneugenic action (pH3), changes in proliferation (Ki67), and increased oxidative stress (8-OxoG, SRXN1). We conclude that the developed HTS microscopy approach is reliable, objective, and high-throughput, representing a robust platform for the comprehensive toxicological evaluation of air pollutants. The tested PAHs and their mixture are highly biologically active and harmful
to human cell models, acting through different predominant mechanisms (genotoxicity or oxidative stress), while the more complex 3D cell model significantly increases cell susceptibility to harmful effects.
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