Divergent Mechanisms of Menthol and Tobacco E-Cigarette Toxicity in a 3D Airway Organoid Model: Metabolic Disruption and Immunosuppression versus Th2-Allergic Inflammation and Apoptotic Stress.

The increasing global use of e-cigarettes has raised concerns about their potential respiratory toxicity. While flavor additives such as menthol are widely used to enhance product appeal, their specific contributions to pulmonary injury remain poorly understood. In this study, we established a three-dimensional mouse airway organoid (MAO) model that recapitulates the structural and functional complexity of native airway epithelium. Using this physiologically relevant platform, we systematically compared the toxicological effects of aerosol extracts from tobacco- and menthol-flavored e-cigarettes. Both extracts concentration-dependently reduced organoid viability and growth, and similarly induced epithelial remodeling characteristic of chronic airway disease, including goblet cell hyperplasia, ciliated cell loss, and basal cell expansion. However, their underlying mechanisms were distinct. Menthol-flavored aerosol induced substantially higher oxidative stress and DNA damage, and elicited a broad immunosuppressive response. In contrast, tobacco-flavored aerosol exhibited more potent pro-apoptotic effects and induced a Th2-skewed allergic inflammation. Transcriptomic analysis corroborated these flavor-specific mechanisms, revealing that tobacco flavor primarily activated cellular stress and apoptotic pathways, whereas menthol flavor selectively disrupted lipid metabolism and metabolic homeostasis. Collectively, our study delineates distinct mechanistic pathways by which different e-cigarette flavors induce airway injury and underscores the critical role of flavor chemistry in shaping specific toxicological outcomes. This study establishes the value of organoid models for flavor-specific risk assessment and provides mechanistic insights to support evidence-based regulation of e-cigarette flavorings.