Background: The respiratory health effects of inhalation exposure to e-cigarette flavoring chemicals are not well understood. We focused our study on the immuno-toxicological and the oxidative stress effects by these e-cigarette flavoring chemicals on two types of human monocytic cell lines, Mono Mac 6 (MM6) and U937. The potential to cause oxidative stress by these flavoring chemicals was assessed by measuring the production of reactive oxygen species (ROS). We hypothesized that the flavoring chemicals used in e-juices/e-liquids induce an inflammatory response, cellular toxicity, and ROS production.

Methods: Two monocytic cell types, MM6 and U937 were exposed to commonly used e-cigarette flavoring chemicals; diacetyl, cinnamaldehyde, acetoin, pentanedione, o-vanillin, maltol and coumarin at different doses between 10 and 1,000 μM. Cell viability and the concentrations of the secreted inflammatory cytokine interleukin 8 (IL-8) were measured in the conditioned media. Cell-free ROS produced by these commonly used flavoring chemicals were also measured using a 2′,7′dichlorofluorescein diacetate probe. These DCF fluorescence data were expressed as hydrogen peroxide (H₂O₂) equivalents. Cytotoxicity due to the exposure to selected e-liquids was assessed by cell viability and the IL-8 inflammatory cytokine response in the conditioned media.

Results: Treatment of the cells with flavoring chemicals and flavored e-liquid without nicotine caused cytotoxicity dose-dependently. The exposed monocytic cells secreted interleukin 8 (IL-8) chemokine in a dose-dependent manner compared to the unexposed cell groups depicting a biologically significant inflammatory response. The measurement of cell-free ROS by the flavoring chemicals and e-liquids showed significantly increased levels of H₂O₂ equivalents in a dose-dependent manner compared to the control reagents. Mixing a variety of flavors resulted in greater cytotoxicity and cell-free ROS levels compared to the treatments with individual flavors, suggesting that mixing of multiple flavors of e-liquids are more harmful to the users.

Conclusions: Our data suggest that the flavorings used in e-juices can trigger an inflammatory response in monocytes, mediated by ROS production, providing insights into potential pulmonary toxicity and tissue damage in e-cigarette users.