All nicotine forms are not created equal – Impact of free base and salt nicotine on pulmonary responses in juvenile mice

Fourth generation (4th-gen) electronic nicotine delivery systems (ENDS) are increasingly popular among U.S. youth, yet little is known about the pulmonary effects of nicotine-rich ENDS aerosols during adolescence. We investigated the long-term impact of ENDS aerosols from third generation (3rd-gen) and 4th-gen devices on lung structure, function, and biochemical responses in juvenile male and female mice. Beginning at 4-6 weeks of age, mice were whole-body exposed to either air, tobacco-flavored 3rd-gen ENDS aerosols containing 24 mg/mL free base nicotine, or tobacco-flavored 4th-gen ENDS aerosols containing 24 mg/mL nicotine salt, for 1 hr/day over 3 months. Exposures were performed at similar aerosol particle size distributions (∼274 nm median size) and total particulate matter concentrations (∼0.4 mg/puff). Despite comparable exposure levels, we found that free base nicotine (3rd-gen ENDS) induces more severe airway remodeling than nicotine salts (4th-gen ENDS) in juvenile mice, as evidenced in males by significantly increased collagen deposition, persistent restrictive lung function, pulmonary macrophage infiltration, and upregulation of fibrosis- and inflammation-related genes, corroborating lung structural and functional changes. Both ENDS exposures significantly increased mean linear intercepts, indicating altered lung architecture. In contrast, nicotine salts produce milder lung function effects despite the enhanced serum cotinine bioavailability. These findings demonstrate differential pulmonary toxicity of ENDS aerosols by nicotine chemical form. Also, sex-specific patterns emerged, with male mice showing greater adverse pulmonary responses to ENDS aerosols compared to female mice. Together, our results highlight that youth vaping may alter respiratory health trajectory and increase susceptibility for lung diseases.