Abstract

Scientific progress and ethical considerations are increasingly shifting the toxicological focus from in vivo animal models to in vitro studies utilizing physiologically relevant cell cultures. Consequently, we evaluated and validated a three-dimensional (3D) model of the human lung using Calu-3 cells cultured at an air-liquid interface (ALI) for 28 days. Assessment of seven essential genes of differentiation and transepithelial electrical resistance (TEER) measurements, in conjunction with mucin (MUC5AC) staining, validated the model. We observed a time-dependent increase in TEER, genetic markers of mucus-producing cells (muc5ac, muc5b), basal cells (trp63), ciliated cells (foxj1), and tight junctions (tjp1). A decrease in basal cell marker krt5 levels was observed. Subsequently, we utilized this validated ALI-cultured Calu-3 model to investigate the adversity of the aerosols generated from three flavored electronic cigarette (EC) e-liquids: cinnamon, vanilla tobacco, and hazelnut. These aerosols were compared against traditional cigarette smoke (3R4F) to assess their relative toxicity. The aerosols generated from PG/VG vehicle control, hazelnut and cinnamon e-liquids, but not vanilla tobacco, significantly decreased TEER and increased lactate dehydrogenase (LDH) release compared to the incubator and air-only controls. Compared to 3R4F, there were no significant differences in TEER or LDH with the tested flavored EC aerosols other than vanilla tobacco. This starkly contrasted our expectations, given the common perception of e-liquids as a safer alternative to cigarettes. Our study suggests that these results depend on flavor type. Therefore, we strongly advocate for further research, increased user awareness regarding flavors in ECs, and rigorous regulatory scrutiny to protect public health.