Menthol in electronic cigarettes: A contributor to respiratory disease?
Graphical abstract
Mechanism of action of menthol on human bronchial epithelium. Three in vitro platforms were used to study the effect of menthol on bronchial epithelium. In submerged culture (using BEAS-2B cells), menthol produced rapid calcium influx followed by an increase in oxidative stress and inflammatory cytokines. ALI exposure of BEAS-2B cells to unheated menthol in a cloud chamber caused activation of an inflammatory transcription factor (NF-κB) and oxidative stress. Proteomics analysis of human EpiAirway tissues exposed at the ALI to heated menthol EC aerosols identified changes in the expression of proteins involved in oxidative stress and in an inflammatory response.
Introduction
Flavor chemicals are widely used in tobacco products, including electronic cigarettes (ECs) (Behar et al., 2018; Hua et al., 2019; Lisko et al., 2014; Tierney et al., 2016), and numerous attractive flavors have contributed to the rapid rise in the popularity of ECs in the US (U.S. Department of Health and Human Services, 2016; Miech et al., 2019; U.S. Department of Health and Human Services, 2016). While most flavor chemicals in consumer products are “generally regarded as safe” (GRAS), the Flavor and Extracts Manufacturers Association's (FEMA) GRAS designation pertains only to ingestion, not inhalation (Hallagan, 2014). Because the data on flavor chemical ingestion cannot be directly translated to inhalation, the health consequences of short-and long-term inhalation of flavor chemicals in ECs remain largely uncharacterized. This problem is compounded by the lack of validated methods for determining the effects of EC flavor chemicals and their reaction products on the respiratory system.
Menthol is often used in ECs (Behar et al., 2018; Hua et al., 2019) and is the only flavor chemical permitted in tobacco cigarettes under the Family Smoking Prevention and Tobacco Control Act (2009). EC refill fluids and conventional cigarettes sometimes contain menthol, even when they are sold as non-mentholated (Behar et al., 2018; Henderson, 2019; Omaiye et al., 2018). Menthol produces a cooling effect upon binding to the TRPM8 receptor (Transient Receptor Potential Melastatin 8), a cation channel with selectivity for calcium (Peier et al., 2002). Menthol is used in tobacco products to impart flavor and to reduce the harshness of tobacco smoke, making inhalation easier for novices (DeVito et al., 2019; Willis et al., 2011). Mentholated ECs may facilitate the initiation of smoking, increase nicotine dependence, and increase progression to conventional cigarette smoking (Food and Drug Administration, 2011; Nonnemaker et al., 2013; Villanti et al., 2017). Mentholated tobacco cigarettes also reduce cessation rates when compared to non-mentholated tobacco cigarettes (Delnevo et al., 2011). Mentholated tobacco cigarettes are widely distributed among African American and adolescent smokers, and are used more often by women than men (Food and Drug Administration, 2011).
In a weight of evidence analysis on conventional cigarettes, it was concluded that menthol is not associated with a disease risk to the user (Food and Drug Administration, 2011). However, this conclusion was based on comparisons of mentholated and non-mentholated conventional cigarettes, and it may not pertain to ECs, which often have much higher concentrations of menthol than those in food and other consumer products, including tobacco cigarettes (Hua et al., 2019; Tierney et al., 2016). As examples, in mentholated tobacco cigarettes the concentration of menthol ranges between 0.52 and 4.19 mg/cigarette (Ai et al., 2016) and averages 4.75 mg/cigarette (Paschke et al., 2017). In contrast, menthol concentration in one EC refill fluid was 85 mg/mL (Behar et al., 2017) and 15 mg/mL in mint flavored JUUL pods (Omaiye et al., 2018), a brand popular with adolescents (Barrington-Trimis and Leventhal, 2018).
Existing studies indicate a need for further work on the potential for high menthol concentrations in ECs to be associated with disease. For example, in submerged 2-dimensional (2D) cell cultures, EC refill fluids and aerosols had cytotoxic effects on adult and embryonic cells, and these were often associated with flavor chemical concentrations (Bahl et al., 2012; Behar et al., 2017; Hua et al., 2019). Pure menthol was cytotoxic to bronchial epithelium at the concentrations found in EC products when tested in vitro with the MTT assay using 2D submerged cell cultures (Behar et al., 2017; Hua et al., 2019). Lin et al. (2018) showed that subchronic exposure of mice to mentholated cigarette smoke induced more inflammation in lungs than smoke from non-mentholated cigarettes. Recently, serious respiratory illness and death have been attributed to EC use, and patients requiring hospitalization have been reported to have “e-cigarette or vaping product use-associated lung injury” (EVALI) (Centers for Disease Control and Prevention, 2019). The etiology of EVALI is not understood, but EC products with high concentrations of flavor chemicals should be investigated as possible causative agents.
The purpose of this study was to determine how menthol affects human bronchial epithelium and to compare responses to menthol across three in vitro platforms. In all protocols, the concentrations tested produced no effect in the MTT assay (referred to as the MTT NOAEL – no observed adverse effect level). In the first protocol, human bronchial epithelium cells (BEAS-2B) were exposed to pure menthol using submerged 2D cultures and oxidative stress and cell proliferation were examined. This protocol also defined the MTT NOEAL and was valuable as an initial screen. In the second approach, BEAS-2B cells were exposed at the air liquid interface (ALI) to pure menthol aerosols produced in a cloud chamber without heat-generated reaction products or the use of solvents (propylene glycol or PG). Endpoints related to oxidative stress and cytokine signaling were examined. In the third protocol, 3D human respiratory epithelium (EpiAirway tissues) was exposed at the ALI to aerosol created by heating e-fluid in an EC, and tissue responses were analyzed using proteomics. This aerosol contained menthol, PG, and reaction products formed during heating and would be equivalent to aerosol inhaled by an EC user. Data were compared across the three platforms and evaluated for their potential to contribute to respiratory diseases. To give relevance to our data in the context of ECs, all tested menthol concentrations were within the range found in EC products (Behar et al., 2017; Hua et al., 2019).
Section snippets
Chemicals
Menthol (catalog number 63660-1G; Lot: BCBW5590), BCTC (N-(4-tert.-butyl-phenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide) and siRNA oligonucleotide against TRPM8 were purchased from Sigma (St. Louis, MO). We performed GC/MS on the menthol, and none of the other 11 known TRPM8 agonists (icilin, linalool, geraniol, hydroxy-citronellal, WS-3, WS-23, Frescolat MGA, Frescolat ML, PMD 38, Coolact P, M8-Ag and Cooling Agent 10) were present. Bronchial epithelial growth medium
Expression of TRPM8 receptor
Menthol mediates signal transduction through the TRPM8 receptor, a ligand-gated cation channel with moderate to high selectivity for calcium ions (Peier et al., 2002). The expression of the TRPM8 receptor in human lung epithelial cells and lung fibroblasts was evaluated using western blotting and immunofluorescence microscopy (Fig. 1A–C). Immunoreactivity of the TRPM8 receptor in BEAS-2B cells was intermediate between A549 cancer cells and human pulmonary fibroblasts (hPFs) (Fig. 1A). The
Protein pathway Interactome analysis using DAVID
Menthol and PG aerosol exposure data were analyzed using DAVID to show the pathway clusters affected by each treatment group (Fig. 7, Purple circle: PG, Green circle: Menthol). Menthol aerosol treated cells expressed proteins related to xenobiotic stress, oxidative stress, and inflammation among others, including cytoskeletal activity. Mitochondrial pathway clusters were affected both by menthol and PG aerosols.
Cell signaling pathways affected by menthol aerosol exposure using IPA
IPA pathway enrichment analysis was used to identify canonical pathways significantly impacted by menthol aerosol exposure (Fig. 8A). A positive z-score (>2) represents an increase in a cellular process, while a negative z-score (<−2) indicates a decrease. Enrichment of proteins related to oxidative stress (NRF2 mediated oxidative stress response, EIF2 signaling), inflammatory cytokine signaling (IL8 signaling), metabolic pathways (oxidative phosphorylation and gluconeogenesis) among other
Discussion
This is the first study to compare the toxic effects of MTT NOAEL concentrations of menthol on human respiratory epithelium using submerged cultures and ALI exposures with and without solvents and with and without heating the aerosols. In most assays, there was excellent agreement of results across the three in vitro platforms. At menthol concentrations that did not produce an effect in the MTT assay, oxidative stress was observed with all three platforms, and cytokine elevation/secretion was
Conclusions
The three in vitro platforms for exposing respiratory epithelium to menthol each lead to similar conclusions. Concentrations of menthol within the range found in many EC fluids and aerosols produced rapid calcium influx followed by an increase in oxidative stress and inflammatory cytokines. These responses were inhibited by BCTC and siRNA knock-down of the TRPM8 receptor. Taken together, these data provide a strategy for evaluating the toxicity of inhaled chemicals by first screening in the MTT
Declaration of Competing Interest
The authors have no competing interests to declare.
Acknowledgements
Research reported in this publication was supported by NIDA, NIEHS, and the FDA Center for Tobacco Products (CTP) grant #s R01DA036493 and R01ES029741. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Food and Drug Administration. The Orbitrap Fusion mass spectrometer was purchased with funds from an NIH shared instrumentation grant (S10OD010669). We thank Lindsey Bustos for her help with the VITROCELL® exposures as
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The first two authors contributed equally to this work.