Radon is the second leading cause of lung cancer after tobacco smoke. This gas is produced from the decay of naturally occurring uranium in soil and rocks. Inhaled radon and its progenies deposit alpha particle radiation on the bronchial epithelium. These alpha particles deliver high localized doses, inducing complex, clustered DNA damage that is challenging to repair. The World Health Organization recommends a national reference level for residential radon at 100 Bq/m³, wherever possible. However, co-exposure to other stressors, particularly tobacco smoke, complicates health risk estimates. Tobacco smoke contains over 60 carcinogens. Nicotine itself is not considered carcinogenic but has been shown to support a tumor-permissive environment. Epidemiological studies show that smoking amplifies radon-induced lung cancer risk, but the molecular mechanisms remain poorly understood. To address this, advanced in vitro approaches, particularly air-liquid interface (ALI) systems and whole-smoke exposure models, have been introduced for in vitro respiratory and toxicology research. These systems offer improved simulation of real-life conditions. Yet, these models require careful control of exposure parameters. This thesis presents studies conducted to improve the understanding of radon and cigarette smoke interactions, as well as to develop an in vitro system for studying combined pollutant exposure.

In Paper I, we demonstrate that nicotine modulates DNA repair following alpha particle exposure, promoting cell survival while increasing chromosomal instability and genomic alterations in human bronchial epithelial cells. In Paper II we used AI-based text mining (AOP-helpFinder) to systematically extract and organize existing literature on radon and tobacco smoke co-exposure, structuring the findings into Adverse Outcome Pathways (AOPs) and Aggregate Exposure Pathways (AEPs). The analysis showed that radon and tobacco interact both environmentally and biologically, yet important gaps remain in mechanistic understanding, particularly regarding epigenetics, and realistic exposure models. In Paper III we developed and validated a novel ALI exposure system that allows controlled, simultaneous exposure of bronchial cells to radon and cigarette smoke under physiologically relevant conditions. Using this system, we showed that while single exposures moderately reduced cell viability, combined exposure caused a significantly stronger reduction, demonstrating both the system’s reliability and the heightened toxicity of co-exposure. In Paper IV, we investigated how radon and cigarette smoke interact at the molecular and cellular levels using RNA-seq and functional assays. Combined exposure induced unique transcriptional and cellular changes when comparing with different single exposure groups, including alterations in inflammatory, oxidative stress, DNA damage response, and oncogenic pathways, as well as in genes related to cytoskeleton and cell adhesion, suggesting an environment that may promote early carcinogenic transformation.