Increased oxidative stress responses in murine macrophages exposed at the air-liquid interface to third- and fourth-generation electronic nicotine delivery system (ENDS) aerosols

BACKGROUND: New fourth generation electronic nicotine delivery system (ENDS) devices contain high levels of nicotine salt (up to 60 mg/mL), whose cellular and molecular effects on immune cells are currently unknown. Here, we used a physiologically-relevant in vitro air-liquid interface (ALI) exposure model to assess the toxicity of distinct ENDS, a 3rd-generation electronic-cigarette (e-cig) and two 4th-generation ENDS devices (JUUL and Posh Plus). METHODS: Murine macrophages (RAW 264.7) were exposed at the ALI to either air, Menthol or Crème Brûlée-flavored ENDS aerosols generated from those devices for 1-hour per day for 1 or 3 consecutive days. Cellular and molecular toxicity was evaluated 24 h post-exposure. RESULTS: 1-day of Menthol-flavored JUUL aerosol exposure significantly decreased cell viability and significantly increased lactate dehydrogenase (LDH) levels compared to air controls. Further, JUUL Menthol elicited significantly increased reactive oxygen species (ROS) and nitric oxide (NO) production compared to air controls. Posh Crème Brûlée-flavored aerosols displayed significant cytotoxicity – decreased cell viability and increased LDH levels –after 1- and 3-day exposures, while the Crème Brûlée-flavored aerosol produced by the 3rd-generation e-cig device only displayed significant cytotoxicity after 3 days compared to air controls. Further, both Posh and third-generation e-cig Crème Brûlée flavored-aerosols elicited significantly increased ROS plus high levels of 8-isoprostane after 1 and 3 days compared to air controls, indicating increased oxidative stress. Posh and third-generation e-cig Crème Brûlée flavored-aerosols elicited reduction in NO levels after one day, but elicited increase in NO after 3 days. Genes in common dysregulated by both devices after 1 day included α(7)nAChR, Cyp1a1, Ahr, Mmp12, and iNos. CONCLUSION: Our results suggest that ENDS Menthol and Crème Brûlée-flavored aerosol exposures from both 3rd- and 4th-generation ENDS devices are cytotoxic to macrophages and cause oxidative stress. This can translate into macrophage dysfunction. Although 4th-generation disposable ENDS devices have no adjustable operational settings and are considered low-powered ENDS devices, their aerosols can induce cellular toxicity compared to air-exposed control cells. This study provides scientific evidence for regulation of nicotine salt-based disposable ENDS products.