Toxicity of Waterpipe Tobacco Smoking: The Role of Flavors, Sweeteners, Humectants, and Charcoal.

Waterpipe tobacco (WPT) smoking is a public health concern, particularly among youth and young adults. The global spread of WPT use has surged since the introduction of pre-packaged flavored and sweetened WPT, which is widely marketed as a safer tobacco alternative. Besides flavorants and sugars, WPT additives include humectants, which enhance the moisture and sweetness of WPT, act as solvents for flavors, and impart smoothness to the smoke, thus increasing appeal to users. In the United States (U.S.), unlike cigarette tobacco flavoring (with the exception of menthol), there is no FDA product standard or policy in place prohibiting sales of flavored WPT. Research has shown that the numerous fruit, candy, and alcohol flavors added to WPT entice individuals to experience those flavors, putting them at an increased risk of exposure to WPT smoke-related toxicants. Additionally, burning charcoal briquettes-used as a heating source for WPT-contributes to the harmful health effects of WPT smoking. This review presents existing evidence on the potential toxicity resulting from humectants, sugars, and flavorants in WPT, and from the charcoal used to heat WPT. The review discusses relevant studies of inhalation toxicity in animal models and of biomarkers of exposure in humans. Current evidence suggests that more data are needed on toxicant emissions in WPT smoke to inform effective tobacco regulation to mitigate the adverse impact of WPT use on human health.

In vitro toxicity and chemical analysis of e-cigarette aerosol produced amid dry hitting.

Dry hitting, a phenomenon produced by e-cigarettes with refillable cartridges when the liquid in the coil is low, is a common occurrence among regular vapers despite being an unintended consequence of the device. This phenomenon's hazard to public health is still unknown and needs further investigation. Lung cells cultured at the air-liquid interface were exposed to vaped aerosol consisting of 3 % w/v ethyl maltol in propylene glycol for three-second puffs every 30 seconds for 80 total puffs with either dry hit or saturated conditions. Cytotoxicity was measured colorimetrically. The thermal degradation of the heating coils and wicks was visualized using scanning electron microscopy. The chemical byproducts in the aerosol were analyzed using proton nuclear magnetic resonance and inductively coupled plasma mass spectrometry. The results revealed a highly significant increase in cytotoxicity from dry hit treatments. Imaging showed thermal decomposition of the cotton wick after dry hitting, which was confirmed by energy dispersive x-ray spectroscopy with less oxygen in the dry hit cotton. Chemical byproducts were found via unique peaks in the dry hit condensate in the aromatic and alkene regions. Saturated condensate showed higher concentrations of detected metal species than dry-hit condensate. E-cigarette users should avoid dry hitting by refilling tanks or cartridges preemptively or by using disposable coils to avoid increased toxicity during vaping.

A review of the toxicity of ingredients in e-cigarettes, including those ingredients having the FDA's "Generally Recognized as Safe (GRAS)" regulatory status for use in food.

INTRODUCTION: Some firms and marketers of electronic cigarettes (e-cigarettes; a type of electronic nicotine delivery system (ENDS)) and refill liquids (e-liquids) have made claims about the safety of ingredients used in their products based on the term "GRAS or Generally Recognized As Safe" (GRAS). However, GRAS is a provision within the definition of a food additive under section 201(s) (21 U.S.C. 321(s)) of the U.S. Federal Food Drug and Cosmetic Act (FD&C Act). Food additives and GRAS substances are by the FD&C Act definition intended for use in food, thus safety is based on oral consumption; the term GRAS cannot serve as an indicator of the toxicity of e-cigarette ingredients when aerosolized and inhaled (i.e., vaped). There is no legal or scientific support for labeling e-cigarette product ingredients as "GRAS". This review discusses our concerns with the GRAS provision being applied to e-cigarette products and provides examples of chemical compounds that have been used as food ingredients but have been shown to lead to adverse health effects when inhaled. The review provides scientific insight into the toxicological evaluation of e-liquid ingredients and their aerosols to help determine the potential respiratory risks associated with their use in e-cigarettes. IMPLICATIONS: The rise in prevalence of e-cigarette use and emerging evidence of adverse effects, particularly on lung health, warrant assessing all aspects of e-cigarette toxicity. One development is manufacturers' stated or implied claims of the safety of using e-cigarette products containing ingredients determined to be "Generally Recognized As Safe" (GRAS) for use in food. Such claims, typically placed on e-cigarette product labels and used in marketing, are unfounded, as pointed out by the United States Food and Drug Administration (FDA)1 and the Flavor and Extract Manufacturers Association (FEMA)2. Assessment of inhalation health risks of all ingredients used in e-liquids, including those claimed to be GRAS, is warranted.

Comparison of emissions across tobacco products: A slippery slope in tobacco control.

In this narrative review, we highlight the challenges of comparing emissions from different tobacco products under controlled laboratory settings (using smoking/vaping machines). We focus on tobacco products that generate inhalable smoke or aerosol, such as cigarettes, cigars, hookah, electronic cigarettes, and heated tobacco products. We discuss challenges associated with sample generation including variability of smoking/vaping machines, lack of standardized adaptors that connect smoking/vaping machines to different tobacco products, puffing protocols that are not representative of actual use, and sample generation session length (minutes or number of puffs) that depends on product characteristics. We also discuss the challenges of physically characterizing and trapping emissions from products with different aerosol characteristics. Challenges to analytical method development are also covered, highlighting matrix effects, order of magnitude differences in analyte levels, and the necessity of tailored quality control/quality assurance measures. The review highlights two approaches in selecting emissions to monitor across products, one focusing on toxicants that were detected and quantified with optimized methods for combustible cigarettes, and the other looking for product-specific toxicants using non-targeted analysis. The challenges of data reporting and statistical analysis that allow meaningful comparison across products are also discussed. We end the review by highlighting that even if the technical challenges are overcome, emission comparison may obscure the absolute exposure from novel products if we only focus on relative exposure compared to combustible products.

Chemical and physiological interactions between e-liquid constituents: cause for concern?

Studies of Electronic Nicotine Delivery Systems (ENDS) toxicity have largely focused on individual components such as flavour additives, base e-liquid ingredients (propylene glycol, glycerol), device characteristics (eg, model, components, wattage), use behaviour, etc. However, vaping involves inhalation of chemical mixtures and interactions between compounds can occur that can lead to different toxicities than toxicity of the individual components. Methods based on the additive toxicity of individual chemical components to estimate the health risks of complex mixtures can result in the overestimation or underestimation of exposure risks, since interactions between components are under-investigated. In the case of ENDS, the potential of elevated toxicity resulting from chemical reactions and interactions is enhanced due to high operating temperatures and the metallic surface of the heating element. With the recent availability of a wide range of e-liquid constituents and popularity of do-it-yourself creation of e-liquid mixtures, the need to understand chemical and physiological impacts of chemical combinations in ENDS e-liquids and aerosols is immediate. There is a significant current knowledge gap concerning how specific combinations of ENDS chemical ingredients result in synergistic or antagonistic interactions. This commentary aims to review the current understanding of chemical reactions between e-liquid components, interactions between additives, chemical reactions that occur during vaping and aerosol properties and biomolecular interactions, all of which may impact physiological health.

Chemical Interactions and Cytotoxicity of Terpene and Diluent Vaping Ingredients.

Vaping devices have risen in popularity since their inception in 2007. The practice involves using a variety of commercially available devices. Internal heating systems in devices aerosolize e-liquid formulations of complex mixtures including an active ingredient (e.g., THC, CBD, and nicotine), diluents (or cutting agents), solvents, and flavoring agents (e.g., terpenes and aldehydes). The vaping toxicology literature consists of cytotoxicity studies of individual chemicals and commercial formulas. Because of the variation of e-liquid composition, there is a limited understanding of the toxicity of ingredient combinations. This study analyzed the cytotoxic effects after exposure to individual and binary mixtures of a representative terpene (+-R-limonene) and diluent (triethyl citrate) on human lung cell models. Data were analyzed to determine the effects of 97:3 and 80:20% v/v (triethyl citrate/limonene) binary mixtures. BEAS-2B cells, a bronchial epithelial cell, and A549 cells, a type II alveolar epithelial cell, served as models for comparison. LC50 values were calculated and isobolograms were used to assess chemical interactions. Results show that limonene was more cytotoxic than triethyl citrate. Isobolographic analyses confirmed that the 97:3% v/v mixture resulted in an antagonistic chemical interaction. The 80:20% v/v mixture resulted in a similar result. Further testing of different ratios of binary mixtures is needed for chemical interaction screening to inform safety assessments.

Vaping Cannabinoid Acetates Leads to Ketene Formation.

Δ8-THC acetate is a relatively new psychoactive cannabis product that is available online and in vape shops across the United States since it is currently largely unregulated. Because it contains a similar substructure to vitamin E acetate, which has been shown to form the poison gas ketene during vaping, we investigated potential ketene formation from Δ8-THC acetate, as well as two other cannabinoids acetates, CBN acetate and CBD acetate, under vaping conditions. Ketene was consistently observed in vaped condensates from all three cannabinoid acetates as well as from a commercial Δ8-THC acetate product purchased online.

Emerging ENDS products and challenges in tobacco control toxicity research.

Electronic nicotine delivery systems (ENDS) continue to rapidly evolve. Current products pose unique challenges and opportunities for researchers and regulators. This commentary aims to highlight research gaps, particularly in toxicity research, and provide guidance on priority research questions for the tobacco regulatory community. Disposable flavoured ENDS have become the most popular device class among youth and may contain higher nicotine levels than JUUL devices. They also exhibit enhanced harmful and potentially harmful constituents production, contain elevated levels of synthetic coolants and pose environmental concerns. Synthetic nicotine and flavour capsules are innovations that have recently enabled the circumvention of Food and Drug Administration oversight. Coil-less ENDS offer the promise of delivering fewer toxicants due to the absence of heating coils, but initial studies show that these products exhibit similar toxicological profiles compared with JUULs. Each of these topic areas requires further research to understand and mitigate their impact on human health, especially their risks to young users.

Effects of Common e-Liquid Flavorants and Added Nicotine on Toxicant Formation during Vaping Analyzed by 1H NMR Spectroscopy.

A broad variety of e-liquids are used by e-cigarette consumers. Additives to the e-liquid carrier solvents, propylene glycol and glycerol, often include flavorants and nicotine at various concentrations. Flavorants in general have been reported to increase toxicant formation in e-cigarette aerosols, yet there is still much that remains unknown about the effects of flavorants, nicotine, and flavorants + nicotine on harmful and potentially harmful constituents (HPHCs) when aerosolizing e-liquids. Common flavorants benzaldehyde, vanillin, benzyl alcohol, and trans-cinnamaldehyde have been identified as some of the most concentrated flavorants in some commercial e-liquids, yet there is limited information on their effects on HPHC formation. E-liquids containing flavorants + nicotine are also common, but the specific effects of flavorants + nicotine on toxicant formation remain understudied. We used 1H NMR spectroscopy to evaluate HPHCs and herein report that benzaldehyde, vanillin, benzyl alcohol, trans-cinnamaldehyde, and mixtures of these flavorants significantly increased toxicant formation produced during e-liquid aerosolization compared to unflavored e-liquids. However, e-liquids aerosolized with flavorants + nicotine decreased the HPHCs for benzaldehyde, vanillin, benzyl alcohol, and a "flavorant mixture" but increased the HPHCs for e-liquids containing trans-cinnamaldehyde compared to e-liquids with flavorants and no nicotine. We determined how nicotine affects the production of HPHCs from e-liquids with flavorant + nicotine versus flavorant, herein referred to as the "nicotine degradation factor". Benzaldehyde, vanillin, benzyl alcohol, and a "flavorant mixture" with nicotine showed lower HPHC levels, having nicotine degradation factors <1 for acetaldehyde, acrolein, and total formaldehyde. HPHC formation was most inhibited in e-liquids containing vanillin + nicotine, with a degradation factor of ∼0.5, while trans-cinnamaldehyde gave more HPHC formation when nicotine was present, with a degradation factor of ∼2.5 under the conditions studied. Thus, the effects of flavorant molecules and nicotine are complex and warrant further studies on their impacts in other e-liquid formulations as well as with more devices and heating element types.

Identifying a role for the interaction of homocysteine and copper in promoting cardiovascular-related damage.

Observations that copper and homocysteine levels are simultaneously elevated in patients with cardiovascular disease has generated interest in investigating the interactions between copper and homocysteine. Several prior studies have shown that complexes of copper and homocysteine are toxic, leading to cardiovascular damage in vitro. It is not clear, however, why related effects do not occur with other structurally similar, more abundant cellular thiols such as glutathione and cysteine. Herein, a mechanism for a selective redox interaction between copper and homocysteine is demonstrated. It involves a kinetically favored intramolecular hydrogen atom transfer that results in an alpha-amino carbon-centered radical known to promote biomolecular damage.

The influence of terpenes on the release of volatile organic compounds and active ingredients to cannabis vaping aerosols.

Dabbing and vaping cannabis extracts have gained large popularity in the United States as alternatives to cannabis smoking, but diversity in both available products and consumption habits make it difficult to assess consumer exposure to psychoactive ingredients and potentially harmful components. This work studies the how relative ratios of the two primary components of cannabis extracts, Δ9-tetrahydrocannabinol (THC) and terpenes, affect dosage of these and exposure to harmful or potentially harmful components (HPHCs). THC contains a monoterpene moiety and has been previously shown to emit similar volatile degradation products to terpenes when vaporized. Herein, the major thermal degradation mechanisms for THC and ß-myrcene are elucidated via analysis of their aerosol gas phase products using automated thermal desorption-gas chromatography-mass spectrometry with the aid of isotopic labelling and chemical mechanism modelling. Four abundant products - isoprene, 2-methyl-2-butene, 3-methylcrotonaldehyde, and 3-methyl-1-butene - are shown to derive from a common radical intermediate for both THC and ß-myrcene and these products comprise 18-30% of the aerosol gas phase. The relative levels of these four products are highly correlated with applied power to the e-cigarette, which indicates formation of these products is temperature dependent. Vaping THC-ß-myrcene mixtures with increasing % mass of ß-myrcene is correlated with less degradation of the starting material and a product distribution suggestive of a lower aerosolization temperature. By contrast, dabbing THC-ß-myrcene mixtures with increasing % mass of ß-myrcene is associated with higher levels of HPHCs, and isotopic labelling showed this is due to increased reactivity of ß-myrcene relative to THC.

A simple predictive model for estimating relative e-cigarette toxic carbonyl levels.

E-cigarette devices are wide ranging, leading to significant differences in levels of toxic carbonyls in their respective aerosols. Power can be a useful method in predicting relative toxin concentrations within the same device, but does not correlate well to inter-device levels. Herein, we have developed a simple mathematical model utilizing parameters of an e-cigarette's coil and wick in order to predict relative levels of e-liquid solvent degradation. Model 1, which is coil length/(wick surface area*wraps), performed in the moderate-to-substantial range as a predictive tool (R2 = 0.69). Twelve devices, spanning a range of coil and wick styles, were analyzed. Model 1 was evaluated against twelve alternative models and displayed the best predictability. Relationships that included power settings displayed weak predictability, validating that power levels cannot be reliably compared between devices due to differing wicking and coil components and heat transfer efficiencies.

Comparison of Chemotherapeutic Activities of Rhodamine-Based GUMBOS and NanoGUMBOS.

Rhodamine derivatives have been widely investigated for their mitochondrial targeting and chemotherapeutic properties that result from their lipophilic cationic structures. In previous research, we have found that conversion of Rhodamine 6G into nanoGUMBOS, i.e., nanomaterials derived from a group of uniform materials based on organic salts (GUMBOS), led to selective chemotherapeutic toxicity for cancer cells over normal cells. Herein, we investigate the chemotherapeutic activity of GUMBOS derived from four different rhodamine derivatives, two bearing an ester group, i.e., Rhodamine 123 (R123) and SNAFR-5, and two bearing a carboxylic acid group, i.e., rhodamine 110 (R110) and rhodamine B (RB). In this study, we evaluate (1) relative hydrophobicity via octanol-water partition coefficients, (2) cytotoxicity, and (3) cellular uptake in order to evaluate possible structure-activity relationships between these different compounds. Intriguingly, we found that while GUMBOS derived from R123 and SNAFR-5 formed nanoGUMBOS in aqueous medium, no distinct nanoparticles are observed for RB and R110 GUMBOS. Further investigation revealed that the relatively high water solubility of R110 and RB GUMBOS hinders nanoparticle formation. Subsequently, while R123 and SNAFR-5 displayed selective chemotherapeutic toxicity similar to that of previously investigated R6G nanoGUMBOS, the R110 and RB GUMBOS were lacking in this property. Additionally, the chemotherapeutic toxicities of R123 and SNAFR-5 nanoGUMBOS were also significantly greater than R110 and RB GUMBOS. Observed results were consistent with decreased cellular uptake of R110 and RB as compared to R123 and SNAFR-5 compounds. Moreover, these results are also consistent with previous observations that suggest that nanoparticle formation is critical to the observed selective chemotherapeutic properties as well as the chemotherapeutic efficacy of rhodamine nanoGUMBOS.

Pine rosin identified as a toxic cannabis extract adulterant.

Pine rosin (colophony) has been identified as a potentially new adulterant in cannabis oil. Its inhalation toxicity poses a significant health concern to users. For example, pine rosin fumes are released during soldering, and have been cited as a causative agent of occupational asthma. Symptoms also include desquamation of bronchial epithelium, which has also been observed in e-cigarette or vaping product used-associated lung injury (EVALI) patients. The sample analyzed herein was acquired from a cannabis industry source, also contains medium chain triglycerides and oleamide, the latter of which is a hypnotic that is commonly found in the synthetic marijuana product Spice, or K2. A combination of proton nuclear magnetic resonance (1H NMR) and high pressure liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) was used to unambiguously identify major pine rosin ingredients such as abietic and other resin acids. Comparison to commercial samples of pure pine rosin confirmed the assignment.

A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase.

We report the first diselenide-based probe for the selective detection of thioredoxin reductase (TrxR), an enzyme commonly overexpressed in melanomas. The probe design involves conjugation of a seminaphthorhodafluor dye with a diselenide moiety. TrxR reduces the diselenide bond, triggering a fluorescence turn-on response of the probe. Kinetic studies reveal favorable binding of the probe with TrxR with a Michaelis-Menten constant (Km ) of 15.89 µm. Computational docking simulations predict a greater binding affinity to the TrxR active site in comparison to its disulfide analogue. In vitro imaging studies further confirmed the diselenide probe exhibited improved signaling of TrxR activity compared to the disulfide analogue.

E-Cigarette or Vaping Product Use-associated Lung Injury: Developing a Research Agenda. An NIH Workshop Report.

The NHLBI convened a working group on October 23, 2019, to identify the most relevant and urgent research priorities and prevailing challenges in e-cigarette or vaping product use-associated lung injury (EVALI). Experts across multiple disciplines discussed the complexities of the EVALI outbreak, identified research priorities, and recommended strategies to address most effectively its causal factors and improve diagnosis, treatment, and prevention of this disease. Many research priorities were identified, including the need to create national and international registries of patients with EVALI, to track accurately those affected and assess outcomes. The group concluded that biospecimens from subjects with EVALI are urgently needed to help define EVALI pathogenesis and that vaping has disease risks that are disparate from smoking, with the occurrence of EVALI highlighting the importance of broadening e-cigarette research beyond comparators to smoking-related diseases.

Aerosol Gas-Phase Components from Cannabis E-Cigarettes and Dabbing: Mechanistic Insight and Quantitative Risk Analysis.

Consumption of cannabis by nontraditional methods has surged since the advent of legalization in North America and worldwide. Inhaling cannabis extracts using vaporizers and via dabbing has risen in popularity, while concerns over product safety have not hindered their proliferation. The work herein is the first step toward assessing the safety of vaporizing and dabbing concentrated cannabis extracts as a function of gas-phase reaction products. The gas-phase thermal degradants of Δ9-tetrahydrocannabinol (THC) have not been previously investigated. It was found that users may be exposed to concerning degradants such as methacrolein, benzene, and methyl vinyl ketone when using cartridge vaporizers and dabbing. It was shown that THC alone and mixed with terpenes generated similar degradation products and, most notably, elevated levels of isoprene. Importantly, it was shown that added terpenes led to higher levels of gas-phase products compared to THC alone. To estimate cancer and noncancer risks associated with exposure to these and other degradants, quantitative risk assessment was applied to experimentally determined values for dabbing and vaping and literature-sourced levels of hazardous components in cannabis smoke. Overall, gas-phase aerosol products had significantly lower values in dabbing and vaporizing compared to cannabis smoking, although these results should be interpreted in light of potential variations in degradant levels due to disparate usage patterns and the dangers of the higher aerosol concentration of THC.

Assessment of human pancreas cancer tissue and precursor lesions via a fluorophore with inherent PDAC selectivity.

The current five-year survival rate of <5% for pancreatic ductal adenocarcinoma (PDAC) is compounded by late diagnosis, a lack of PDAC-specific intraoperative guidance to ensure complete resection, and the ineffectiveness of current therapies. Previously, utilizing compound 1, a fluorophore with inherent PDAC selectivity, PDAC was visualized both in vivo and ex vivo in a murine model. In the current study, human PDAC tissue is targeted. Compound 1 selectively stains ducts of the adenocarcinoma versus the surrounding stroma, enabling the imaging of PDAC in frozen tissue sections with high contrast. To enhance the potential of 1 for intraoperative applications, the ex vivo staining protocol was optimized for rapid margin assessment, with a final staining time of ~15 min. To measure diagnostic performance, the area under a receiver operating characteristic (ROC) curve was measured for the identification of ductal adenocarcinoma vs. stroma. The bright fluorescence contrast enabled quantitative determination of PDAC (or precancerous PanIN lesions) versus healthy pancreas tissue in human tissue array samples.

Sucralose-Enhanced Degradation of Electronic Cigarette Liquids during Vaping.

Electronic cigarette liquids (e-liquids) with sweetener additives such as sucralose, a synthetic chlorinated disaccharide, are popular among some e-cigarette consumers; sucralose can be added either by the manufacturer or by the consumer. The prevalence of sucralose in commercial e-liquids is not known, nor is the typical concentration of sucralose when present; labels are not required to disclose ingredient information. Here, we report the effects of sucralose on e-liquid degradation upon e-cigarette vaping as studied using 1H NMR spectroscopy, ion chromatography, and gas chromatography coupled with detection by mass spectrometry or flame ionization detector. Sucralose was found to be subject to degradation when included in propylene glycol + glycerol based e-liquids and vaped; the presence of sucralose in the e-liquids also resulted in altered and enhanced solvent degradation. In particular, production of aldehydes (carbonyls) and hemiacetals (which have implications for health) was enhanced, as demonstrated by 1H NMR. The presence of sucralose at 0.03 mol % (0.14 wt %) in an e-liquid also resulted in production of potentially harmful organochlorine compounds and catalyzed the cyclization of aldehydes with solvents to acetals upon vaping; the presence of chloride in e-liquid aerosols was confirmed by ion chromatography. Quantities of sucralose as low as 0.05 mol % (0.24 wt %) in e-liquids lead to significant production of solvent degradation products.