The Family Smoking Prevention and Tobacco Control Act of 2009 banned the use of all natural and artificial compounds characterized as flavors in combustible cigarettes and any of their component parts to eliminate flavored tobacco products that held special appeal in the youth market. In 2020, the US FDA finalized their enforcement policy on flavored cartridge based e-cigarette products, including fruit and mint flavors, but excluded menthol and tobacco flavors.However, since this enforcement policy only applies to prefilled cartridges, flavored eliquids in bottles and refillable empty cartridges are still commercially available. Flavored products can be legally marketed in the USA if they receive a tobacco product marketing order from the FDA, and this may become common in the coming years. There is sufficient evidence to suggest that flavorings influence the perception, use, and safety of e-cigarettes.Flavor compounds,particularly sweet ones, have the potential to mask and/or ameliorate irritant and bitter sensations, such as the taste of nicotine.The route of delivery has an impact on the toxicology of a compound. Indeed, many flavor compounds have only been tested for safety following oral delivery , and to date have not been tested for inhalation safety. The potential risk of flavor compounds when inhaled rather than ingested has been demonstrated by occupational and consumer inhalation of high doses of the buttery flavor diacetyl , which leads to irreversible lung disease, namely bronchiolitis obliterans or “popcorn lung” in microwave popcorn factory workers.At one time, diacetyl was found in 70% of 159 sweet flavorings used in e-cigarettes,while a more recent study found diacetyl in only a few flavorings. Flavored e-liquids and individual flavors induce toxicity and/ or exert biological effects: Sassano et al. found vanillin is the most common flavor.The number of flavors used varies considerably across e-liquid products. Notably, in 20 e-liquids, Hua et al. found 99 different flavors.Sassano et al. studied 148 eliquids and found 100 flavor constituents.
They also found the more flavors contained in an e-liquid, the more likely it was to be cytotoxic,cannabis indoor greenhouse while concentrations of vanillin and cinnamaldehyde in e-liquids significantly correlated with toxicity.Cinnamaldehyde, was found to be >7.6 mM in multiple e-liquids, and in some cases, levels exceeded 1 M.These concentrations were sufficient to induce cytotoxicity and ciliary dysfunction. However, flavor concentrations in most e-liquids have not yet been determined. Menthol is biologically active and can activate the transient receptor potential channel in pulmonary neurons to suppress cough and irritation,making it easier to tolerate cigarette smoking. Menthol flavors are significantly more popular among African Americans and tobacco companies have marketed them accordingly.Flavorings including vanillin and cinnamaldehyde are aldehydes that have the potential to form adducts with proteins and DNA. Vanillin can also activate TRP channels to exert biological effects .They can also react with base constituents in e-liquids, leading to the formation of acetals, which can stimulate irritant receptors.Sweet and bitter taste receptors are G-protein-coupled receptors expressed in airway epithelia where they regulate innate immunity. The sweet and bitter taste receptors are expressed in the nasal passages/upper airways, while only bitter/T2R taste receptors are expressed in the lower airways.This raises the possibility that inhalation of flavor compounds may stimulate airway taste receptors and affect immune function. Their activation may disrupt innate airway defense by suppressing the release of antimicrobial peptides that are capable of killing a variety of respiratory pathogens. In the lower airways, T2R activation leads to an increase in ciliary beating and may have other physiological functions via its effects on cytoplasmic Ca2þ, a universal second messenger. There is some evidence that toxicity is cell type-dependent, suggesting that mechanistic investigations must be cell-specific.In summary, the adverse impact of flavor compounds in e-cigarettes include the potential for increased appeal of these products, particularly to the youth market, influence on patterns of use and smoking topography, changes in cell signaling, and increased cellular toxicity .PG and VG are commercially available in different mixture ratios, and the ratio of PG to VG in the e-liquid can affect taste sensation, the amount of aerosol generated, the amount of nicotine delivered, and the overall user experience.Predominantly PG-based e-liquids deliver more nicotine systemically, but taste less pleasant than VG-rich e-liquids.PG is used to deliver pharmaceuticals intravenously and while it is generally considered safe, higher PG doses can lead to metabolic acidosis, acute renal injury, and sepsis-like syndrome.
In Sprague-Dawley rats, nasally inhaled PG of up to 2.2 mg/L for 90 days led to nasal hemorrhaging. Similar short-term exposures of mice to PG/VG resulted in changes to tissue elasticity, static compliance, and airway resistance, although these effects waned after 1 month of exposure, suggesting that there may be a long-term adaptive response.In contrast, a recent study funded by Philip Morris International found that nasal exposure to PG/VG mixtures of up to 1.5 mg/L PG and 1.9 mg/L VG for 90 days had minimal effects on respiratory organs, gene transcription, proteomics, and lipid profiles in Sprague-Dawley rats.Lipid-laden macrophages were recently observed in mice that were chronically-exposed to PG/VG.While this exposure was not fatal, these mice had decreased macrophage function and were more vulnerable to influenza A infection.Mucin abnormalities correlate with a decline in forced expiratory volume in 1 s in chronic obstructive pulmonary disease patients,indicating that mucins are important biomarkers of harm. Importantly, increased MUC5AC mucin levels were also detected in human e-cigarette users’ bronchial epithelia obtained by bronchoscopy and in sputum,and these increases in MUC5AC levels could be replicated in the laboratory by exposing both primary bronchial epithelial cultures and mice airways to PG/VG.While the underlying mechanism whereby PG/VG can exert their effects are unknown, Ghosh et al. found that PG/VG rapidly alters membrane rheology.Altered membrane properties could affect a number of aspects of fundamental cell biology including endocytosis, exocytosis, and cell division. In vitro,cannabis growing equipment higher levels of both PG and VG can prevent cell growth and/or induce cell death. However, more work will be required to fully appreciate the effects of PG/ VG. The potential effects of PG/VG at the cellular level are summarized in Figure 2. The concentrations of PG and VG saw in the lung and systemically after vaping are poorly understood, and additional studies to determine PG/VG pharmacokinetics and pharmacodynamics after inhalation are required.This may subject them to chemical reactions that result in the formation of new compounds including reactive oxygen species . For example, the hydroxyl radical was produced from PG/VG at higher power settings.
Carbonyl compounds were formed in e-liquid aerosols as a result of dehydration and oxidation, and was dependent on the PG/VG ratio, the wattage used to heat the e-liquid, as well as other factors including brand, and type of e-liquid used.Exposing biological tissues to carbonyl compounds can deplete glutathione, induce DNA damage, alter ion channel function, and elicit cell death.Thus, both reactive aldehyde production and subsequent reactive aldehyde metabolism in biological tissues need to be considered. This may be particularly important since nearly 8% of the world’s population has an impaired capability to metabolize reactive aldehydes, and they may show altered responses to e-cigarette/reactive aldehyde exposure.Importantly, aldehydes can form adducts with both proteinsand DNA.Adduct binding can impair protein function, as recently noted for the short palate and nasal epithelial clone 1 , which is an innate defense protein expressed in the lung. Here, crotonaldehyde bound to SPLUNC1, which prevented it from regulating lung hydration.Similarly, acrolein can form adducts with surfactant protein A, which leads to impaired innate defense by decreasing antimicrobial activity and reducing phagocytosis by macrophages.Aldehyde adducts can also bind to DNA, leading to frame shift and base-pair substitution mutations, which may contribute to cytotoxic and genotoxic effects.These results indicate that as a result of reactive aldehyde inhalation from heat-coil aerosolization of PG/VG, the lung may be especially vulnerable to adduct formation and the associated macromolecule damage. In addition to decomposition products resulting from heating e-liquids, emissions may also contain contaminants from components of the e-cigarette device itself. These can include toxic metals such as chromium, nickel, and lead.Therefore, understanding the potential health effects of thermal decomposition products remains key to delineating the overall e-cigarette health effects.There is a common perception e-cigarettes may be safer than combustible cigarettes, since they deliver much lower levels of oxidants, volatile organic chemicals, and other noxious chemicals associated with tobacco cigarette smoke and cardiovascular risk.However, both combustible tobacco products and e-cigarettes deliver oxidants, toxic metals, and potentially toxic carbonyls, which have been associated with cardiovascular disease.Moreover, e-cigarette-derived particles are spread among a wider size range than those generated by standard cigarettes. Known toxicants in e-cigarettes may also contribute to cardiovascular damage in a different manner than toxicant-induced cardiovascular damage from combustible cigarettes. There is an urgent need to determine both the acute and the long-term effects of e-cigarettes on the hearts and blood vessels of healthy adults and children, as well as those with either risk factors for cardiovascular disease or outright cardiovascular disease and to determine the comparative safety of e-cigarettes relative to combustible cigarettes.
A summary of the potential cardiovascular biomarkers of exposure/harm following vaping are shown in Table 1 and are discussed in more detail below.E-cigarette use has been consistently connected to reductions in vascular function and damage to ECs. For example, several studies have established that e-cigarette inhalation leads to increased arterial stiffness in humans and rodents, as evidenced by increases in augmentation index and pulse wave velocity.One crossover design study compared e-cigarette vaping 6 nicotine and found that pulse wave velocity, aortic pulse pressure, augmentation index corrected for heart rate, and sub-endocardial viability ratio were all significantly increased, but only when subjects vaped with nicotine.However, another study found that inhalation of nicotine free e-cigarette vapor caused an increase in aortic pulse wave velocity and resistivity index.These results have made it unclear as to whether nicotine is required to elicit these adverse effects: Additional factors such as e-cigarette wattage, which affects toxicant production, and/or the presence of flavors may also affect arterial stiffness. In addition to arterial stiffness, e-cigarette use impaired endothelial nitric oxide synthase signaling,which could be considered a biomarker of endothelial dysfunction in several vascular diseases including hypertension and atherosclerosis. Participants exposed to e-cigarettes showed significantly reduced flow-mediated dilation of the brachial artery, demonstrating endothelial dysfunction compared to non-users.However, in a separate study, smokers who switched to e-cigarettes for one month showed an improvement in FMD, suggesting that there may be a difference between acute and chronic effects of vaping on FMD.The mechanisms by which electronic cigarettes lead to these adverse vascular effects remain incompletely defined. However, a number of studies suggested that e-cigarettes might cause ROSmediated damage, including damage to ECs.Carnevale et al. performed a crossover study of 40 healthy subjects, half of whom were smokers. The subjects were asked to smoke combustible cigarettes for 1 week and were then crossed over to e-cigarettes.Both combustible cigarettes and e-cigarettes increased markers of oxidative stress and worsened FMD after a single use. Lee et al. exposed human-induced pluri potent stem cell-derived ECs to various flavored e-cigarette liquids and assessed endothelial integrity. A cinnamon-flavored e-cigarette product was most potent in reducing cell viability and increasing ROS levels.In multiple studies, subjects who used e-cigarettes either acutely or chronically were found to have altered blood and plasma biomarkers linked to oxidative stress and cardiovascular disease, including increased myeloperoxidase,increased isoprostanes such as 8-iso-PGF2a, reduced NO bio-availability, increased levels of the oxidantgenerating enzyme nicotinamide adenine dinucleotide phosphate oxidase ,and reduced levels of the non-enzymatic antioxidant vitamin E.Anderson et al.showed that e-cigarette aerosol exposure induced ROS in vitro, which caused DNA damage and cell death. Of note, the antioxidants alpha-tocopherol and n-acetyl cysteine were effective in alleviating the damage. After e-cigarette exposure, activated ECs may have been the source of vascular ROS: Chatterjee et al. exposed serum to e-liquids and observed a NOX2-dependent increase in ROS, coupled with inhibition of NADPH oxidase 2 reduced ROS production by 75%.Kuntic et al. extended these observations and demonstrated that e-cigarette-induced ROS burden and endothelial dysfunction could be rescued by NOX2 inhibition or NOX2 gene knockout in mice.These findings were nicotine-independent, and acrolein treatment alone was capable of causing NOX2- dependent ROS production in primary murine ECs. Together, these studies link e-cigarette use with NOX2 activation, endothelial oxidative stress, and subsequent endothelial damage/dysfunction, which may contribute to adverse vascular outcomes.