Computational modeling method to estimate secondhand exposure potential from exhalations during e-vapor product use under various real-world scenarios.

Potential secondhand exposure of exhaled constituents from e-vapor product (EVP) use is a public health concern. We present a computational modeling method to predict air levels of exhaled constituents from EVP use. We measured select constituent levels in exhaled breath from adult e-vapor product users, then used a validated computational model to predict constituent levels under three scenarios (car, office, and restaurant) to estimate likely secondhand exposure to non-users. The model was based on physical/thermodynamic interactions between air, vapor, and particulate phase of the aerosol. Input variables included space setting, ventilation rate, total aerosol amount exhaled, and aerosol composition. Exhaled breath samples were analyzed after the use of four different e-liquids in a cartridge-based EVP. Nicotine, propylene glycol, glycerin, menthol, formaldehyde, acetaldehyde, and acrolein levels were measured and reported based on a linear mixed model for analysis of covariance. The ranges of nicotine, propylene glycol, glycerin, and formaldehyde in exhaled breath were 89.44-195.70 µg, 1199.7-3354.5 µg, 5366.8-6484.7 µg, and 0.25-0.34 µg, respectively. Acetaldehyde and acrolein were below detectable limits; thus, no estimated exposure to non-EVP users is reported. The model predicted that nicotine and formaldehyde exposure to non-users was substantially lower during EVPs use compared to cigarettes. The model also predicted that exposure to propylene glycol, glycerin, nicotine and formaldehyde among non-users was below permissible exposure limits.

Assessment of the Exposure to NNN in the Plasma of Smokeless Tobacco Users.

N-Nitrosonornicotine (NNN) is a human carcinogen present in cigarette smoke and smokeless tobacco. Urinary NNN is usually measured in order to assess the exposure to this toxicant for tobacco users. NNN excretion in urine can be highly biased due to the formation of NNN by nitrosation of nornicotine under acidic conditions, both endogenously and exogenously. Hence, urinary NNN levels may not necessarily correctly reflect the product-specific exposure. Measurement of plasma NNN may be less prone to endogenous formation due to the stable pH (7.4) of blood. We developed an LC-MS/MS method for the quantification of NNN using 1 mL of human plasma. Validation according to FDA guidelines proved that the method is selective and highly sensitive with an LLOQ of 0.3 pg/mL. Accuracy and precision averaged to 98.7 and 7.5% (CV), respectively. The assay was applied to plasma samples collected from 10 experienced moist smokeless tobacco users during and after a single use of 2 g of the product for 40 min under controlled use conditions. Blood was drawn at 15 time points over a 6 h time course. The maximum NNN concentration (Cmax) ranged from 3.5 to 10 pg/mL (mean: 7.1 pg/mL) at a tmax of 32 min. Plasma NNN and nicotine were found to have similar time courses. In conclusion, the determination of NNN in plasma may be fit-for-purpose to evaluate the product-use-specific exposure to this carcinogen.

Biomarkers of Exposure and Biomarkers of Potential Harm in Adult Smokers Who Switch to e-Vapor Products Relative to Cigarette Smoking in a 24-week, Randomized, Clinical Trial.

INTRODUCTION: Long-term health effects of e-vapor products (EVPs) are not well-established. We compared biomarkers of exposure (BoE) to select harmful and potentially harmful constituents and biomarkers of potential harm (BoPH) in adult smokers who switched to EVPs versus continued smoking for 24 weeks. METHODS: Adult smokers (n = 450, >10 cigarettes per day for ≥10 years) were randomly assigned to continue smoking (control) or switch to one of two cartridge-based EVPs (test 1: classic; test 2: menthol, 4% nicotine). BoE and BoPH were measured at baseline and 12 weeks. The results presented here are from a subset of 150 control and EVP subjects (switchers with exhaled carbon monoxide <8 ppm and <10% baseline cigarettes per day) followed for 24 total weeks. RESULTS: Total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and carboxyhemoglobin were significantly reduced (p < .0001) in tests 1 and 2 at 24 weeks. Urinary nicotine equivalents were not statistically significantly different between the control and EVP groups. At week 24, statistically significant reductions (p < .05) were observed for white blood cell counts, 11-dehydrothromboxane ß2, and sICAM in both test groups, and there were several significant changes in measures of pulmonary function. High-density lipoprotein cholesterol and 8-epi-prostaglandin-F2α were directionally favorable in both EVP groups versus control. CONCLUSIONS: We demonstrate that significant reductions of selected harmful and potentially harmful constituents in EVP aerosol results in significant reductions in BoEs and favorable changes in BoPHs after switching to EVPs for 24 weeks. These changes approached those reported for smoking cessation, suggesting that switching to exclusive use of the EVPs may be less harmful than continuing smoking. IMPLICATIONS: Cigarette smoking causes serious diseases. Switching from cigarettes to a noncombustible product is a potential harm reduction pathway for adult smokers unable or unwilling to quit. Long-term health effects of e-vapor products (EVPs) compared with continued smoking have not been extensively studied. We present biomarker of exposure evidence on select harmful and potentially harmful constituents and biomarkers of potential harm related to inflammation and oxidative stress in adult smokers switching to two EVPs. This study demonstrates significant reductions in biomarkers of exposure (except for nicotine) accompanied with favorable changes in various biomarkers of potential harm, including pulmonary function. The totality of evidence suggests that exclusive EVP use may present lower health risks compared with smoking cigarettes.

Assessment of the potential vaping-related exposure to carbonyls and epoxides using stable isotope-labeled precursors in the e-liquid.

The formation of carbonyls and epoxides in e-cigarette (EC) aerosol is possible due to heating of the liquid constituents. However, high background levels of these compounds have inhibited a clear assessment of exposure during use of ECs. An EC containing an e-liquid replaced with 10% of 13C-labeled propylene glycol and glycerol was used in a controlled use clinical study with 20 EC users. In addition, five smokers smoked cigarettes spiked with the described e-liquid. Seven carbonyls (formaldehyde, acetaldehyde, acrolein, acetone, crotonaldehyde, methacrolein, propionaldehyde) were measured in the aerosol and the mainstream smoke. Corresponding biomarkers of exposure were determined in the user's urine samples. 13C-labeled formaldehyde, acetaldehyde and acrolein were found in EC aerosol, while all seven labeled carbonyls were detected in smoke. The labeled biomarkers of exposure to formaldehyde (13C-thiazolidine carboxylic acid and 13C-N-(1,3-thiazolidine-4-carbonyl)glycine), acrolein (13C3-3-hydroxypropylmercapturic acid) and glycidol (13C3-dihydroxypropylmercapturic acid) were present in the urine of vapers indicating an EC use-specific exposure to these toxicants. However, other sources than vaping contribute to a much higher extent by several orders of magnitude to the overall exposure of these toxicants. Comparing data for the native (unlabeled) and the labeled (exposure-specific) biomarkers revealed vaping as a minor source of user's exposure to these toxicants while other carbonyls and epoxides were not detectable in the EC aerosol.

Characterization of puff topography of a prototype electronic cigarette in adult exclusive cigarette smokers and adult exclusive electronic cigarette users.

Puff topography is an important measure of how consumers use e-vapor products. The purpose of this study was to evaluate the feasibility of using SODIM Smoking Puff Analyzer Mobile Device (SPA/M) to measure puff topography during use of a prototype e-cigarette (e-cig) in exclusive cigarette smokers (CS) and e-cig users (EC) under ad lib conditions in a clinic. Adult CS (n = 13) and EC (EC; n = 10) completed a 7-hr use session with the e-cig (2% tobacco-derived nicotine by weight, cartridge based system approximately the size of a king size cigarette). E-liquid usage was determined from cartridge weight. CS also smoked a single cigarette with the SPA/M. The SPA/M reliably recorded puff parameters throughout the study period, with CS puffs averaging 47.9 ±â€¯18.2 ml volume, 2.3 ±â€¯0.8 s duration, and 21.5 ±â€¯4.6 ml/s flow rate. EC puffs averaged 53.4 ±â€¯19.2 ml volume, 3.0 ±â€¯1.3 s duration, and 19.6 ±â€¯5.0 flow rate. CS average e-liquid use was 292 ±â€¯214 mg and EC averaged 415 ±â€¯305 mg over 7 h. When compared to a single use of their own brand cigarettes, CS took longer (2.3 ±â€¯0.8 vs.1.7 ±â€¯0.4 s) puffs with similar puff volume (47.9 ±â€¯18.2 vs. 44.1 ±â€¯10.5 ml) from the e-cig prototype. The puff duration, flow rate and peak flow were significantly lower (p < 0.05) with the e-cigs compared to cigarettes. Experienced EC and CS appeared to use the e-cig prototype differently, which is consistent with the literature. The SPA/M could be a useful tool in assessing e-cig use behavior for regulatory purposes.

A comprehensive evaluation of the toxicology of cigarette ingredients: essential oils and resins.

CONTEXT: A total of 32 essential oils and resins were added individually to experimental cigarettes. OBJECTIVE: A battery of tests was used to compare the toxicity of mainstream smoke from these experimental cigarettes. The lowest target inclusion level was 100 ppm and the highest was 100,000 ppm. MATERIALS AND METHODS: Smoke from each of the experimental cigarette was evaluated using analytical chemistry and in vitro bacterial (Salmonella, five strains) mutagenicity and cytotoxicity (neutral red uptake) assays. For seven of the ingredients (carob bean, carob bean extract, carrageenan, chamomile flower Hungarian oil, guar gum, peppermint oil, and spearmint oil), 90-day smoke inhalation studies with rats were also performed. RESULTS: In general, inclusion levels resulted in minimal changes in smoke chemistry; the exceptions were PO and SO, where reductions to 40-60% of control values were noted, possibly indicating a tobacco displacement effect. Cytotoxicity and mutagenicity were unaffected by any of the test ingredients, except for a dose-related reduction in cytotoxicity for SO. There were very few statistically significant differences within any of the seven inhalation studies; when present, the differences were sporadic and inconsistent between sexes. The addition of SO appeared to depress body weight gain and increase the atrophy of olfactory epithelia, but only in males. CONCLUSION: The essential oils and resins tested here as ingredients in experimental cigarettes show minimal toxicological sequelae, even at high inclusion levels. The highest inclusion level for SO showed some equivocal responses.

A comprehensive evaluation of the toxicology of cigarette ingredients: aliphatic carbonyl compounds.

CONTEXT: Aliphatic carbonyl compounds are used as ingredients in cigarette tobacco or cigarette filters. OBJECTIVE: A battery of tests was used to compare toxicity of mainstream smoke from experimental cigarettes containing 15 aliphatic carbonyl compounds that were added individually to experimental cigarettes at three different levels. MATERIALS AND METHODS: Smoke from experimental and control cigarettes were evaluated using analytical chemistry, in vitro cytotoxicity (neutral red uptake), and mutagenicity (five bacterial strains) studies. For one compound, glycerol triacetate (GTA), two 90-day inhalation studies were also performed, using different inclusion levels into either tobacco or cigarette filter. RESULTS: Several smoke constituent concentrations were reduced with the highest inclusion level of GTA in tobacco; incorporation of GTA into the filter, and the other compounds into tobacco, produced effectively no changes. Cytotoxicity was reduced by the highest inclusion of GTA into tobacco for both gas-vapor and particulate phases of smoke; incorporation of GTA into the filter, and the other compounds into tobacco, showed no changes. Mutagenicity was reduced by the middle and high inclusion levels of GTA into tobacco (TA1537 strain with S9); incorporation of GTA into the filter, and the other compounds into tobacco, showed no changes. CONCLUSION: Inclusion of GTA in tobacco at 100,000 ppm reduced the biological effects of the smoke in the various test systems reported in this study, although inclusion into the filter did not appear to have any major effect on the endpoints studied. The other 14 aliphatic carbonyl compounds that were tested lacked a toxicological response.

Gene expression profiling of peripheral blood leukocytes identifies potential novel biomarkers of chronic obstructive pulmonary disease in current and former smokers.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease with associated systemic effects. OBJECTIVE: To use gene expression microarrays in peripheral blood leukocytes of current and former cigarette smokers to identify differences associated with COPD. MATERIALS AND METHODS: Random forest modelling and a split-sample case-control approach were used to identify candidate predictors. RESULTS: We identified 1013 genes and one smoking exposure variable that differentiated current and former smokers with or without COPD. This predictor set was reduced to a nine-gene classifier (IL6R, CCR2, PPP2CB, RASSF2, WTAP, DNTTIP2, GDAP1, LIPE and RPL14). CONCLUSION: These gene expression profiles represent potential biomarkers for COPD and may help increase mechanistic understanding of the disease.

Proteomic biomarkers in plasma that differentiate rapid and slow decline in lung function in adult cigarette smokers with chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of morbidity and mortality in the United States and cigarette smoking is a primary determinant of the disease. COPD is characterized by chronic airflow limitation as measured by the forced expiratory volume in one second (FEV(1)). In this study, the plasma proteomes of 38 middle-aged or older adult smokers with mild to moderate COPD, with FEV(1) decline characterized as either rapid (RPD, n = 20) or slow or absent (SLW, n = 18), were interrogated using a comprehensive high-throughput proteomic approach, the accurate mass and time (AMT) tag technology. This technology is based upon a putative mass and time tag database (PMT), high-resolution LC separations and high mass accuracy measurements using FT-ICR MS with a 9.4-T magnetic field. The peptide and protein data were analyzed using three statistical approaches to address ambiguities related to the high proportion of missing data inherent to proteomic analysis. The RPD and SLW groups were differentiated by 55 peptides which mapped to 33 unique proteins. Twelve of the proteins have known roles in the complement or coagulation cascade and, despite an inability to adjust for some factors known to affect lung function decline, suggest potential mechanistic biomarkers associated with the rate of lung function decline in COPD. Whether these proteins are the cause or result of accelerated decline will require further research.

High-resolution mass spectrometry proteomics for the identification of candidate plasma protein biomarkers for chronic obstructive pulmonary disease.

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n = 18) or slow (n = 18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.

Chemical analysis of cigarette smoke particulate generated in the MSB-01 in vitro whole smoke exposure system.

Cigarette mainstream smoke (MS) is a dynamic aerosol consisting of a gas-vapor phase and a particulate phase. In recent years, novel in vitro whole smoke exposure systems have been developed to expose cells directly to whole MS. One such system is the Burghart Mimic Smoker-01 (MSB-01). Our previous data using the MSB-01 indicated that a 50 +/- 10% loss of particulate matter occurred prior to MS delivery into the exposure chamber. Additionally, a change in aerosol particle diameter was also measured, suggesting that the chemical composition of MS might be changing within the system. In this study, we have expanded on our previous work and compared the particulate phase chemical composition of undiluted and diluted MS generated by the instrument and that of the MS delivered into the exposure chamber. The average percent delivery of cigarette smoke condensate (CSC) detected for all the measured chemical constituents was 35 +/- 13% for undiluted MS and 23 +/- 8% for 1:1 diluted MS. The data also indicate that under our experimental conditions, incomplete mixing of the freshly generated MS occurs during its dilution by the system. Taken together, the data presented here show that significant chemical changes occur between the generation of MS by the system and its delivery into the exposure chamber. This indicates that due to the dynamic nature of cigarette smoke, it is important to characterize the exposure conditions in order to gain the best insight and accurately correlate exposure with biological endpoints.

Cigarette smoke extract induced protein phosphorylation changes in human microvascular endothelial cells in vitro.

Phosphorylation is the most widely studied posttranslational modification (PTM) and is an important regulatory mechanism used during cellular responses to external stimuli. The kinases and phosphatases that regulate protein phosphorylation are known to be affected in many human diseases. Cigarette smoking causes cardiovascular disease (CVD). Endothelial cells play a pivotal role in CVD initiation and development; however, there have been limited investigations of the specific signaling cascades and protein phosphorylations activated by cigarette smoke in endothelial cells. The purpose of this research was to better understand the differential protein phosphorylation in endothelial cells stimulated with extracts of cigarette smoke total particulate matter (CS-TPM) in vitro. Human microvascular endothelial cells were exposed in vitro to CS-TPM at concentrations that were shown to cause endothelial cell dysfunction. The phosphorylated proteins were isolated using phosphoprotein-specific chromatography, followed by enzymatic digestion and nano-flow capillary liquid chromatography (ncap-LC) coupled to high resolution mass spectrometry. This study putatively identified 94 proteins in human microvascular endothelial cells that were differentially bound to a phosphoprotein-specific chromatography column following exposure to CS-TPM suggesting differential phosphorylation. Pathway analysis has also been conducted and confirmations of several observations have been made using immunoaffinity-based techniques (e.g., Western blotting).

Characterization of a whole smoke in vitro exposure system (Burghart Mimic Smoker-01).

In vitro systems are frequently used to study mechanisms of mainstream cigarette smoke (MS)-induced lung injury. Traditional methods of exposure involve the capture of MS particulate phase with filter pads or bubbling MS through phosphate buffered saline (PBS) or cell culture medium. Although useful for in vitro experiments, these exposure methods may fail to capture potential interactions between the gas and particulate phases. To better understand the effect of MS on the human airway, in vitro whole smoke exposure systems that utilize freshly generated whole smoke are needed. Here we report the characterization of a new in vitro whole smoke exposure system (Burghart Mimic Smoker-01 (MSB-01)). This system uses a smoke distribution manifold to simultaneously deliver MS to each well of a 96-well plate. Intraday and interday variations for particulate matter deposition were less than 5% and 13% respectively. Cytotoxicity measurements using lung epithelial BEAS-2B cells indicate variations in calculated EC(50) (half maximal effective concentration) values of 13% intraday and 20% interday. Smoke particulate losses and changes in particle size distribution were also analyzed. The data indicate that 45-50% of the MS generated at the smoking ports is lost within the system prior to delivery into the exposure chamber; however, no changes in particle size distribution were detected throughout the system. Overall, the MSB-01 reproducibly delivered mainstream cigarette smoke in a dose dependent manner across the multiwell plate. The MSB-01 is a high throughput system capable of exposing cells to both the MS particulate and gas/vapor phases simultaneously.

Inability of transforming growth factor-beta to cause SnoN degradation leads to resistance to transforming growth factor-beta-induced growth arrest in esophageal cancer cells.

It is well established that loss of a growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers including esophageal cancer. However, the molecular basis for the abrogation of this key homeostatic mechanism is poorly understood. In esophageal cancer cell lines that are resistant to TGF-beta-induced growth inhibition, TGF-beta also fails to decrease transcription of c-myc despite the presence of functional signaling components. Consequently, to gain a better understanding of the mechanisms leading to resistance to TGF-beta-induced growth arrest, the basis for the inability to decrease c-myc transcription was investigated. Regardless of sensitivity to TGF-beta-induced growth arrest, TGF-beta enhanced the ability of Smad3-protein complexes to bind c-myc regulatory elements. However, in a growth inhibition-resistant esophageal cancer cell line, the Smad3-protein complexes contained the SnoN oncoprotein. Furthermore, in esophageal cancer cell lines that are resistant to TGF-beta-induced growth arrest, TGF-beta does not cause degradation of SnoN. Analyses of the effect of modulating SnoN expression in both growth inhibition-sensitive and growth inhibition-resistant cell lines showed that degradation of SnoN is a prerequisite for both TGF-beta-induced repression of c-myc transcription and growth arrest. The data indicate that SnoN-Smad3 complexes do not cause repression of c-myc transcription but rather prevent functionality of active repressor complexes. Thus, these studies reveal a novel mechanism for resistance to TGF-beta-induced growth inhibition in esophageal cancer, namely the failure to degrade SnoN. In addition, they show that SnoN can block TGF-beta repression of gene transcription.

Heterogeneity in the transforming growth factor beta response of esophageal cancer cells.

The role of the transforming growth factor beta (TGFbeta) pathway in the development and progression of esophageal cancers is poorly understood. As an initial step in clarification of this issue, the functional status of the TGFbeta pathway was evaluated in a panel of esophageal cancer derived cell lines. Both adenocarcinoma and squamous cell carcinoma derived lines were represented. Although the TGFbeta pathway was intact and functional in four of the five cell lines, only one of them was growth inhibited by TGFbeta. In one cell line, the loss of a growth inhibitory response to TGFbeta could be explained by decreased expression of Smad 4 and a general inability to activate TGFbeta responsive promoters. In the other three cell lines, TGFbeta was able to activate transcription of TGFbeta responsive promoters, but unable to downregulate transcription of c-myc. Taken together these findings indicate that a selective loss in the ability of TGFbeta to regulate expression of a key component of the growth inhibitory pathway may contribute to the poor prognosis of esophageal cancers.