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Inflammatory and Oxidative Responses Induced by Exposure to Commonly Used e-Cigarette Flavoring Chemicals and Flavored e-Liquids without Nicotine.

ABSTRACT: Background: The respiratory health effects of inhalation exposure to e-cigarette flavoring chemicals are not well understood. We focused our study on the immuno-toxicological and the oxidative stress effects by these e-cigarette flavoring chemicals on two types of human monocytic cell lines, Mono Mac 6 (MM6) and U937. The potential to cause oxidative stress by these flavoring chemicals was assessed by measuring the production of reactive oxygen species (ROS). We hypothesized that the flavoring chemicals used in e-juices/e-liquids induce an inflammatory response, cellular toxicity, and ROS production. Methods: Two monocytic cell types, MM6 and U937 were exposed to commonly used e-cigarette flavoring chemicals; diacetyl, cinnamaldehyde, acetoin, pentanedione, o-vanillin, maltol and coumarin at different doses between 10 and 1,000 ?M. Cell viability and the concentrations of the secreted inflammatory cytokine interleukin 8 (IL-8) were measured in the conditioned media. Cell-free ROS produced by these commonly used flavoring chemicals were also measured using a 2',7'dichlorofluorescein diacetate probe. These DCF fluorescence data were expressed as hydrogen peroxide (H2O2) equivalents. Cytotoxicity due to the exposure to selected e-liquids was assessed by cell viability and the IL-8 inflammatory cytokine response in the conditioned media. Results: Treatment of the cells with flavoring chemicals and flavored e-liquid without nicotine caused cytotoxicity dose-dependently. The exposed monocytic cells secreted interleukin 8 (IL-8) chemokine in a dose-dependent manner compared to the unexposed cell groups depicting a biologically significant inflammatory response. The measurement of cell-free ROS by the flavoring chemicals and e-liquids showed significantly increased levels of H2O2 equivalents in a dose-dependent manner compared to the control reagents. Mixing a variety of flavors resulted in greater cytotoxicity and cell-free ROS levels compared to the treatments with individual flavors, suggesting that mixing of multiple flavors of e-liquids are more harmful to the users. Conclusions: Our data suggest that the flavorings used in e-juices can trigger an inflammatory response in monocytes, mediated by ROS production, providing insights into potential pulmonary toxicity and tissue damage in e-cigarette users.

SUBMITTER: Muthumalage T

PROVIDER: S-EPMC5768608 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Project description:Numerous flavoring chemicals are added to e-cigarette liquids to create various flavors. Flavorings provide sensory experience to users and increase product appeal; however, concerns have been raised about their potential inhalation toxicity. Estimating potential health risk of inhaling these chemicals has been challenging since little is known about their actual concentrations in e-cigarette products. To date, a limited number of analytical methods exist to measure the concentrations of flavoring chemicals in e-cigarette products. We have developed an analytical method that accurately and precisely measures the concentrations of 20 flavoring chemicals of potential inhalation risk concerns: 2,3,5-trimethylpyrazine, acetoin, benzaldehyde, benzyl alcohol, butanoic acid, dl-limonene, ethyl maltol, ethyl salicylate, ethyl vanillin, eucalyptol, eugenol, furaneol, isovanillin, l-menthol, maltol, methyl salicylate, pulegone, trans-cinnamaldehyde, triacetin, and vanillin. Calibration and QC solutions were prepared in 50:50 propylene glycol (PG):vegetable glycerin (VG) and 5% H2O and flavoring concentrations ranging from 0.02 to 10.00 mg/ml. Samples of commercial e-cigarette liquids, calibration and QC solutions were combined with 30 µL of an internal standard mix (benzene-d6, pyridine-d5, chlorobenzene-d5, naphthalene-d8 and acenaphthene-d10; 1 mg/ml each) and were diluted 100-fold into methanol. Analysis was performed on an Agilent 7890B/7250 GC/Q-TOF using a DB-624UI column (30 m x 0.25 mmID x 1.4 μm film thickness), with a total runtime of 13.5 min. Calibration curves were fit using a weighted quadratic model and correlations of determination (r 2) values exceeded 0.990 for all chemicals. Bias and precision tests yielded values less than 20% and lower limits of quantitation (LLOQ) ranged from 0.02 to 0.63 mg/ml. Over 200 commercially available products, purchased or collected from adult e-cigarette users and spanning a range of flavor categories, were evaluated with this method. Concentrations of pulegone, a suspected carcinogen, varied from below limit of quantitation (BLOQ) to 0.32 mg/ml, while acetoin and vanillin, known precursors to more cytotoxic byproducts, ranged from BLOQ to 1.52 mg/ml and from BLOQ to 16.22 mg/ml, respectively. This method features a wide dynamic working range and allows for a rapid routine analysis of flavoring additives in commercial e-cigarette liquids.

Project description:IntroductionSugars are major constituents and additives in traditional tobacco products, but little is known about their content or related toxins (formaldehyde, acetaldehyde, and acrolein) in electronic cigarette (e-cigarette) liquids. This study quantified levels of sugars and aldehydes in e-cigarette liquids across brands, flavors, and nicotine concentrations (n = 66).MethodsUnheated e-cigarette liquids were analyzed using liquid chromatography mass spectrometry and enzymatic test kits. Generalized linear models, Fisher's exact test, and Pearson's correlation coefficient assessed sugar, aldehyde, and nicotine concentration associations.ResultsGlucose, fructose and sucrose levels exceeded the limits of quantification in 22%, 53% and 53% of the samples. Sucrose levels were significantly higher than glucose [χ2(1) = 85.9, p < .0001] and fructose [χ2(1) = 10.6, p = .001] levels. Formaldehyde, acetaldehyde, and acrolein levels exceeded the limits of quantification in 72%, 84%, and 75% of the samples. Acetaldehyde levels were significantly higher than formaldehyde [χ2(1) = 11.7, p = .0006] and acrolein [χ2(1) = 119.5, p < .0001] levels. Differences between nicotine-based and zero-nicotine labeled e-cigarette liquids were not statistically significant for sugars or aldehydes. We found significant correlations between formaldehyde and fructose (-0.22, p = .004) and sucrose (-0.25, p = .002) and acrolein and fructose (-0.26, p = .0006) and sucrose (-0.21, p = .0006). There were no significant correlations between acetaldehyde and any of the sugars or any of the aldehydes and glucose.ConclusionsSugars and related aldehydes were identified in unheated e-cigarette liquids and their composition may influence experimentation in naïve users and their potential toxicity.ImplicationsThe data can inform the regulation of specific flavor constituents in tobacco products as a strategy to protect young people from using e-cigarettes, while balancing FDA's interest in how these emerging products could potentially benefit adult smokers who are seeking to safely quit cigarette smoking. The data can also be used to educate consumers about ingredients in products that may contain nicotine and inform future FDA regulatory policies related to product standards and accurate and comprehensible labeling of e-cigarette liquids.

Project description:IntroductionTo examine the interaction between an added flavoring (cherry) and nicotine on the perception of electronic cigarette (e-cigarette) aerosol and how this impacts the appeal of flavored liquids for e-cigarette (e-liquids).MethodsA total of 19 subjects (13 male, 6 female) vaped six commercially available e-liquids with varying contents of nicotine (0, 6, 12 mg/mL) and cherry flavor (4.7% or 9.3% vol/vol). For each e-liquid, subjects first rated overall liking/disliking of the aerosol using the Labeled Hedonic Scale, followed by perceived intensities of sweetness, bitterness, harshness (irritation), and cherry flavor of the aerosol using the general version of Labeled Magnitude Scale.ResultsThe main findings were that (1) added nicotine increased perceived irritation and bitterness, and decreased the perceived sweetness of the e-cigarette aerosol; (2) cherry flavoring added a characteristic "cherry flavor" and an increase in the flavoring concentration from 4.7% to 9.3% tended to increase perceived intensities of sweetness, harshness, and bitterness; and (3) hedonic ratings of the e-cigarette aerosol decreased as nicotine level increased, but were not affected by flavor level.ConclusionsOur findings indicate that the appeal of the e-cigarette aerosol decreases as nicotine concentration increases. Conversely, perceived sweetness improved liking. An increase in the concentration of cherry flavoring did not appear to impact any of the measured attributes to a significant degree.ImplicationsThis work demonstrates that the perception of specific sensory attributes of e-cigarettes and their overall appeal are affected by the e-liquid constituents. Most significantly, the results suggest that nicotine decreases the sensory appeal of e-cigarettes by contributing to the perceived irritation and bitterness of the aerosol. These data have implications for the role that nicotine plays in the sensory perception and appeal of e-cigarettes aerosol and further how these sensory factors can be modulated by sweet flavoring.

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Inflammatory and Oxidative Responses Induced by Exposure to Commonly Used e-Cigarette Flavoring Chemicals and Flavored e-Liquids without Nicotine.

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