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.

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.

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.

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.

Efficacy of RyR2 inhibitor EL20 in induced pluripotent stem cell-derived cardiomyocytes from a patient with catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal-dominant variants in the cardiac ryanodine receptor type-2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca2+ ) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca2+ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca2+ handling and arrhythmic events in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg-176-Glu (R176Q) and a mutation-negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC-CMs were derived using the StemdiffTM kit. Confocal Ca2+ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC-CMs harbouring the R176Q variant demonstrated spontaneous SR Ca2+ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC50  = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca2+ handling in control iPSC-CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca2+ handling and suppresses arrhythmogenic activity in iPSC-CMs derived from a CPVT patient. Hence, this study confirms that this RyR2-inhibitor represents a promising therapeutic candidate for treatment of CPVT.

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.

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.

Correction: Functional synthetic probes for selective targeting and multi-analyte detection and imaging.

Correction for 'Functional synthetic probes for selective targeting and multi-analyte detection and imaging' by Yongkang Yue et al., Chem. Soc. Rev., 2019, DOI: 10.1039/c8cs01006d.

Functional synthetic probes for selective targeting and multi-analyte detection and imaging.

In contrast to the classical design of a probe with one binding site to target one specific analyte, probes with multiple interaction sites or, alternatively, with single sites promoting tandem reactions to target one or multiple analytes, have been developed. They have been used in addressing the inherent challenges of selective targeting in the presence of structurally similar compounds and in complex matrices, as well as the visualization of the in vivo interaction or crosstalk between the analytes. Examples of analytes include reactive sulfur species, reactive oxygen species, nucleotides and enzymes. This review focuses on recent innovations in probe design, detection mechanisms and the investigation of biological processes. The vision is to promote the ongoing development of fluorescent probes to enable deeper insight into the physiology of bioactive analytes.

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.

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.

Free-Base Nicotine Is Nearly Absent in Aerosol from IQOS Heat-Not-Burn Devices, As Determined by 1H NMR Spectroscopy.

Heat-not-burn products, eg, I quit ordinary smoking (IQOS), are becoming popular alternative tobacco products. The nicotine aerosol protonation state has addiction implications due to differences in absorption kinetics and harshness. Nicotine free-base fraction (αfb) ranges from 0 to 1. Herein, we report αfb for IQOS aerosols by exchange-averaged 1H NMR chemical shifts of the nicotine methyl protons in bulk aerosol and verified by headspace-solid phase microextraction-gas chromatography-mass spectrometry. The αfb ≈ 0 for products tested; likely a result of proton transfer from acetic acid and/or other additives in the largely aqueous aerosol. Others reported higher αfb for these products, however, their methods were subject to error due to solvent perturbation.

Altering Fundamental Trends in the Emission of Xanthene Dyes.

Fluorescent small molecules enable researchers and clinicians to visualize biological events in living cells, tissues, and organs in real time. Herein, the focus is on the structure and properties of the relatively rare benzo[ a]xanthenes that exhibit enhanced steric and electronic interactions due to their annulated structures. Three types of fluorophores were synthesized: (i) pH- and solvent-dependent seminaphthorhodafluors, (ii) pH- and solvent-independent seminaphthorhodafluors, and (iii) pH-independent but solvent-sensitive seminaphthorhodamines. The probes exhibited promising far-red to near-infrared (NIR) emission, large Stoke shifts, broad full width at half-maximum (fwhm), relatively high quantum yields, and utility in immunofluorescence staining. Deviation of the π-system from planarity due to changes in the fluorophore ionization state resulted in fluorescence properties that are atypical of common xanthene dyes.

In Situ Lysosomal Cysteine-Specific Targeting and Imaging during Dexamethasone-Induced Apoptosis.

Herein we utilize the similar though divergent nucleophilic properties of cysteine, homocysteine, and glutathione to achieve the selective detection of cysteine under mildly acidic conditions. This enables the specific in situ detection of lysosomal cysteine. Employing time-dependent fluorescent imaging of probe-labeled A549 cells, we demonstrate that dexamethasone-induced apoptosis is not dependent on lysosomal cysteine. This methodology can thus produce useful information about pathogenesis associated with cysteine and lysosomes.

Fluorescent Probes with Multiple Binding Sites for the Discrimination of Cys, Hcy, and GSH.

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play crucial roles in maintaining redox homeostasis in biological systems. This Minireview summarizes the most significant current challenges in the field of thiol-reactive probes for biomedical research and diagnostics, emphasizing the needs and opportunities that have been under-investigated by chemists in the selective probe and sensor field. Progress on multiple binding site probes to distinguish Cys, Hcy, and GSH is highlighted as a creative new direction in the field that can enable simultaneous, accurate ratiometric monitoring. New probe design strategies and researcher priorities can better help address current challenges, including the monitoring of disease states such as autism and chronic diseases involving oxidative stress that are characterized by divergent levels of GSH, Cys, and Hcy.

Systemic Delivery and Biodistribution of Cisplatin in Vivo.

Cisplatin is widely used to treat a variety of cancers. However, ototoxicity and nephrotoxicity remain serious side effects of cisplatin-based chemotherapy. In order to inform the study of cisplatin's off-target effects, a new drug-fluorophore conjugate was synthesized that exhibited utility as a tracer to determine the cellular uptake and in vivo distribution of cisplatin. This probe will serve as a useful tool to facilitate investigations into the kinetics and biodistribution of cisplatin and its associated side effects in preclinical models after systemic administration.