The role of synthetic coolants, WS-3 and WS-23, in modulating E-cigarette-induced reactive oxygen species (ROS) in lung epithelial cells.

There has been a substantial rise in e-cigarette (e-cig) use or vaping in the past decade, prompting growing concerns about their adverse health effects. Recently, e-cig manufacturers have been using synthetic cooling agents, like WS-23 and WS-3, to provide a cooling sensation without the "menthol taste". Studies have shown that aerosols/vapes generated by e-cigs can contain significant levels of reactive oxygen species (ROS). However, studies investigating the role of synthetic coolants in modulating ROS levels generated by e-cigs are lacking. This study seeks to understand how synthetic coolants, e-cig additives that have become increasingly prevalent in e-liquids sold in the United States (US), impact acellular ROS production from e-liquid aerosols as well as cellular ROS levels from pulmonary epithelial cells exposed to these e-liquids. To further explain, our study aims to understand whether the addition of WS-3 and WS-23 to e-liquid base and e-liquid base with nicotine significantly modifies generated acellular ROS levels within aerosolized e-liquids, as well as cellular ROS within BEAS-2B cells treated with these same e-liquids. Aerosols were generated from e-liquids with and without synthetic coolants through a single-puff aerosol generator; subsequently, acellular ROS was semi-quantified in H2O2 equivalents via fluorescence spectroscopy. Our acellular ROS data suggest that adding WS-3 to e-liquid base (PG:VG), regardless of nicotine content, has a minimal impact on modifying e-cig generated acellular ROS levels. Additionally, we also measured cellular ROS in lung epithelial cells using both e-liquids containing and not containing synthetic coolants via the CellROX Green fluorescent sensor. Similar comparable results were found in BEAS2B cells though ROS was increased by WS-3 and WS-23 treated in e-cig nicotine groups. Altogether, our data suggest that neither the addition of WS-23 nor WS-3 to e-liquid base solution, with and without nicotine, significantly modifies e-cig generated acellular ROS levels within aerosolized e-liquids and cellular ROS levels within treated BEAS-2B cells. Together, our data provide insight into whether synthetic coolants added to e-liquids could impact vaping-induced oxidative stress in the lungs.