Mechanistic insights and putative role of menthol flavorings into E-cigarette-driven modulation of inflammatory and lung Epithelial–Mesenchymal Transition signaling

Introduced as harm-reduction tools intended to decrease tobacco dependence, electronic cigarettes (e-cigs) have been widely promoted as a safer alternative to conventional combustible cigarettes. However, accumulating evidence from recent studies indicates that e-cigs use is not devoid of health risks. We previously demonstrated that device-related settings including the variations in liquid (e-liquid) composition influence the generation of harmful by-products. Moreover, data from in vitro studies have raised concerns regarding the contribution of certain flavoring agents to the exacerbation of toxicological responses. The present study was conceived to explore the lung pro-inflammatory effects and the Epithelial–Mesenchymal Transition (EMT) signaling of a sub-chronic (28-days/3h per day) total-body exposure of adult male Sprague-Dawley rats to e-cigarette (e-cig) mainstream from nicotine-containing (18 mg/mL) flavorless or 10% v/v menthol-flavored e-liquids. Compared to controls, animals exposed to vapors generated by both e-liquid show an accumulation of multiple inflammatory mediators including TNF-α, IL-6, IL-5, along with the NF-κB activation as a central upstream regulator as typically observed in traditional tobacco cigarette smoke models. Furthermore, the up-regulation of cell adhesion intermediates such as I-CAM-1 and V-CAM-1—known NF-κB target genes—further supported the presence of an active inflammatory microenvironment pattern. However, no significant differences were detected between menthol-flavored and flavorless formulations in the magnitude of these inflammatory responses. In tobacco smoke models the release of pro-inflammatory cytokine such as TNF-α and IL-6 can promote epithelial plasticity and EMT-like changes contributing to the down-regulation of genes encoding for epithelial cell junction proteins such as E-cadherin and the overexpression of proteins that contribute to the mesenchymal phenotype, such as N-cadherin. Although our model reports an up-regulation of N-cadherin no significant changes were observed for E-cadherin expression through groups. Consistently, the lack of vimentin and MMP9 modulation as hallmarks of EMT and extracellular matrix degradation and tissue invasion respectively, along with no changes in TGF-β, Snail and ZO-1 signaling cascade, indicate that e-cig exposure may induce a mild epithelial destabilization with no progression to fully EMT. As a whole, data here presented seems indicate that the e-cig exposure affects some molecular pathways that could play a critical role in inflammatory lung disease. Although some EMT-like pattern raised, the changes here observed are mild and the signaling pathways considered do not show a unique trend. In contrast to some recent studies, the inclusion of menthol flavoring did not significantly modify the toxicological outcomes assessed in this model, suggesting that adhering to recommended formulation guidelines, menthol flavoring does not exacerbate e-cigarette–associated pulmonary toxicity.