In vitro chromosome damage induced by quaternary mixtures of polycyclic aromatic hydrocarbon derivatives using Doehlert experimental design

Humans are continuously exposed to complex mixtures of environmental contaminants. This kind of multiple exposure occurs when reclaimed asphalt pavements are recycled, since the fumes contain polycyclic aromatic hydrocarbon derivatives (PAHs+) that are primarily been investigated as single compounds. In this study, we assessed the genotoxic potential of an environmentally relevant quaternary mixture composed of 1-nitropyrene, quinoline, 2-naphthol, and 6H-benzo[c,d]pyrene-6-one (6H-BPO) using the human bronchial epithelial BEAS-2B cell line. Cells were exposed according to a Doehlert experimental design and chromosomal damage was evaluated using the cytokinesis-block micronucleus assay. Response surface methodology was applied to model both cytostasis and micronucleus induction and their values were calculated in the whole domain. All quaternary exposure conditions produced acceptable cytostasis (<55%), enabling reliable genotoxicity assessment. The models revealed that 1-nitropyrene was the main driver of cytostatic effects, while oxygenated PAHs, 2-naphthol and 6H-BPO, mainly contributed to chromosomal damage. Moreover, significant response surfaces showed concentration-related interactions, demonstrating that genotoxic responses could not be explained by additive effects. By combining in vitro genotoxicity testing with innovative experimental design, this study provides a robust framework for investigating complex contaminant mixtures while significantly reducing the number of required experimental conditions. These findings also improve the mechanistic understanding of PAHs + mixture toxicity and support the integration of mixture-based approaches into environmental hazard assessment and human health risk evaluation.