Kentucky Reference Cigarettes Research Articles

Flavored E-cigarette product aerosols induce transformation of human bronchial epithelial cells

ObjectivesE-cigarettes are the most commonly used nicotine containing products among youth. In vitro studies support the potential for e-cigarettes to cause cellular stress in vivo; however, there have been no studies to address whether exposure to e-liquid aerosols can induce cell transformation, a process strongly associated with pre-malignancy. We examined whether weekly exposure of human bronchial epithelial cell (HBEC) lines to e-cigarette aerosols would induce transformation and concomitant changes in gene expression and promoter hypermethylation. Materials and methodsAn aerosol delivery system exposed three HBEC lines to unflavored e-liquid with 1.2% nicotine, 3 flavored products with nicotine, or the Kentucky reference cigarette once a week for 12 weeks. Colony formation in soft agar, RNA-sequencing, and the EPIC Beadchip were used to evaluate transformation, genome-wide expression and methylation changes. ResultsJamestown e-liquid aerosol induced transformation of HBEC2 and HBEC26, while unflavored and Blue Pucker transformed HBEC26. Cigarette smoke aerosol transformed HBEC4 and HBEC26 at efficiencies up to 3-fold greater than e-liquids. Transformed clones exhibited extensive reprogramming of the transcriptome with common and distinct gene expression changes observed between the cigarette and e-liquids. Transformation by e-liquids induced alterations in canonical pathways implicated in lung cancer that included axonal guidance and NRF2. Gene methylation, while prominent in cigarette-induced transformed clones, also affected hundreds of genes in HBEC2 transformed by Jamestown. Many genes with altered expression or epigenetic-mediated silencing were also affected in lung tumors from smokers. ConclusionsThese studies show that exposure to e-liquid aerosols can induce a pre-malignant phenotype in lung epithelial cells. While the Food and Drug Administration banned the sale of flavored cartridge-based electric cigarettes, consumers switched to using flavored products through other devices. Our findings clearly support expanding studies to evaluate transformation potency for the major categories of e-liquid flavors to better inform risk from these complex mixtures.

Carbonyl Yields in Cigars under Three Smoking Regimens Using a Linear Smoking Machine.

This study used standard linear smoking machines and puffing protocols to generate data on carbonyl yields in mainstream smoke from 11 unfiltered sheet-wrapped cigars (SWC), seven leaf-wrapped cigars (LWC), and two Kentucky reference cigarettes (3R4F, 1R6F). Carbonyl yields in cigar and cigarette products were determined using three different smoking regimens: International Organization for Standardization (ISO), Canadian Intense (CI), and Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Recommended Method (CRM) No. 64 (CRM64, Routine Analytical Cigar-Smoking Machine─Specifications, Definitions and Standard Conditions). Mainstream tobacco smoke was collected using a smoking machine fitted with an impinger containing 2,4-dinitrophenylhydrazine (DNPH) and carbonyl compounds quantified using liquid chromatography with an ultraviolet detector. Commercial SWC and LWC generated comparable formaldehyde yields (SWC, 9.4-28 μg/cigar [ISO], 8.2-43 μg/cigar [CI], 8.6-13 μg/cigar [CRM64]; LWC, 11-13 μg/cigar [ISO], 11-22 μg/cigar [CI], 16-21 μg/cigar [CRM64]) and acrolein yields; however, LWC generated higher acetaldehyde yields compared to SWC, using CI and CRM64 regimens. Reference cigarettes using standard puffing regimens generated carbonyl yields within reported ranges and 5-10% RSDs, whereas the CRM64 regimen generated lower carbonyl yields and 12-14% RSDs. Reference cigarettes generated higher formaldehyde yields using cigarette smoking regimens (21-28 μg/cigarette under ISO, 76-96 μg/cigarette under CI) but comparable formaldehyde yields under CRM64 (12-14 μg/cigarette). In addition, this study evaluated physical parameters (e.g., tobacco weight, length, diameter, circumference, tobacco rod density) that show the correlation between tobacco weight, tobacco rod density, and acetaldehyde yields under the three smoking regimens. Carbonyl yields in the mainstream smoke of cigar products using the three smoking regimens were highly variable; however, the CI smoking regimen may provide meaningful analytical information regarding cigar smoke constituents, with lower likelihood of self-extinguishment due to the short puffing intervals.

Characterization of Aerosol Deliveries from Combustible Cigarettes, Heated Tobacco Products, and Electronic Nicotine Delivery Systems Using the Vitrocell Ames 48

Introduction: The Vitrocell® Ames 48 whole-aerosol exposure module offers a high-throughput platform for air-agar-interface exposures that mimic human exposure conditions for assessing potential mutagenic activity of a test article. The aim of this study was to characterize the Vitrocell Ames 48 exposure module for assessment of aerosolized tobacco products utilizing multiple dosimetry techniques and nicotine deposition as determined by chemical analysis. Materials and Methods: Characterization was performed using the Vitrocell VC10® smoking machine and three nicotine containing tobacco product test articles: Kentucky Reference cigarettes (3R4F), a commercially available electrically heated tobacco product (eHTP), and an Electronic Nicotine Delivery System (ENDS). The following dosimetry tools were used: Aerosol photometers (using area under curve values, ∑AUC), free glycerol deposition in phosphate-buffered saline (PBS), fluorescence of anhydrous dimethyl sulfoxide (DMSO)-captured smoke particulate matter, and analytical determination of nicotine in PBS and DMSO. Characterization of 3R4F Kentucky Reference cigarettes was examined over a diluting airflow range 8 to 0.5 L/min; eHTP and ENDS were examined over a diluting airflow range of 4 to 0 L/min (undiluted). Results: Results from the dosimetry techniques showed that whole smoke from 3R4F cigarettes and whole aerosol from eHTP and ENDS demonstrated reproducible and consistent delivery, with results being consistent between experiments and within each airflow. Conclusion: These results show that this exposure module is fit for purpose using these three tobacco product types.

Nicotine Rather Than Non-Nicotine Substances in 3R4F WCSC Increases Behavioral Sensitization and Drug-Taking Behavior in Rats.

Nicotine increases reinforcing effects of cigarette smoking by upregulating glutamate and dopamine releases via stimulation of nicotinic acetylcholine receptors (nAChRs) in the dorsal striatum (CPu). The present study was conducted to evaluate whether non-nicotine substances in cigarette smoke potentiate nicotine-induced behaviors by increasing glutamate and dopamine concentrations in the CPu. Changes in the levels of glutamate and dopamine in the CPu were analyzed using a glutamate colorimetric assay and dopamine enzyme-linked immunosorbent assay, respectively, after repeated administration of nicotine or whole cigarette smoke condensate (WCSC) in male Sprague-Dawley rats. Changes in locomotion and drug-taking behavior were analyzed using the measurements of locomotor activity and self-administration under a fixed ratio 1 schedule in response to repeated administration of nicotine or WCSC. Repeated subcutaneous (s.c.) injections of nicotine (0.25mg/kg/day) for 7 consecutive days significantly increased the levels of glutamate and dopamine in the CPu. Similar results were obtained from repeated injections of WCSC (0.25mg/kg nicotine/day, s.c.) extracted from 3R4F Kentucky reference cigarettes. Parallel with the increases in the neurotransmitter levels in the CPu, both nicotine and WCSC increased locomotor activity and self-administration (0.03mg/kg nicotine/infusion). However, repeated injections of WCSC did not change the nicotine-induced increases in neurotransmitter levels, locomotor activity, and self-administration. Nicotine rather than non-nicotine substances in WCSC play a major role in potentiating behavioral sensitization and drug-taking behavior via elevation of glutamate and dopamine concentrations in the CPu of rats. WCSC does not augment the nicotine-induced increases in behavioral sensitization, drug-taking behavior, and glutamate and dopamine concentrations, suggesting that non-nicotine substances do not potentiate the nicotine-induced behaviors by increasing the concentrations of the neurotransmitters in the CPu. These findings imply that nicotine, but not non-nicotine substances in WCSC, may be a major contributor that induces tobacco dependence in rats.

Characterization of smoke and aerosol deliveries from combustible cigarettes, heated tobacco products and electronic nicotine delivery systems in the Vitrocell® Mammalian 6/48 exposure module

The rapid development associated with Next Generation Tobacco Products (NGTP) has necessitated the development of high throughput methodologies to test their genotoxic potential in vitro when compared to conventional cigarette smoke (CS). An assessment of two Vitrocell® Mammalian 6/48 exposure modules in three independent experiments was made by comparing results from multiple dosimetric techniques applied to aerosol generated from 3R4F Kentucky Reference cigarettes, commercially available electronically heated tobacco product (eHTP) and Electronic Nicotine Delivery System (ENDS) using the Vitrocell® VC10®. Real-time aerosol particle concentration was assessed by means of light scattering photometers and expressed as area under the curve (∑AUC). Nicotine concentrations were determined analytically by LC/MS. Humectant amount and distribution was assessed for eHTP and ENDS by the quantification of free glycerol in a phosphate buffered saline (PBS) trap, whereas total particulate matter (TPM) was assessed in the 3R4F cigarettes by the fluorescence of the particulate at 485 nm in anhydrous dimethyl sulfoxide (DMSO) trap within the exposure. Dose was adjusted by means of the addition of ambient air to dilute the whole smoke/aerosol in L/min and sampled into the system at a rate of 5 mL/min. Dilution of CS ranged from 8.0 to 0.5 L/min and for the eHTP and ENDS ranged from 4 to 0 L/min (undiluted). Dosimetric analysis of the system showed good concordance within replicates (p-values ranged from p = 0.3762 to p = 0.8926) and showed that the Vitrocell® Mammalian 6/48 is a viable means for genotoxic assessment of aerosol generated from both conventional cigarettes and NGTP. Results demonstrate the need to tailor dosimetry approaches to different aerosols due to variations in the physio-chemical composition, with a multi-dosimetry approach recommended.

Transcriptome analysis reveals lung-specific miRNAs associated with impaired mucociliary clearance induced by cigarette smoke in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is strongly associated with impaired mucociliary clearance (MCC), which has been implicated in the pathogenesis of CS-induced respiratory diseases, such as chronic obstructive pulmonary diseases (COPD). In this study, we aimed to identify microRNAs (miRNAs) that are associated with impaired MCC caused by CS in an in vitro human air-liquid-interface (ALI) airway tissue model. ALI cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min of clean air) from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 1week (a total of 3days, 40min/day). Transcriptome analyses of ALI cultures exposed to the high concentration of CS identified 5090 differentially expressed genes and 551 differentially expressed miRNAs after the third exposure. Genes involved in ciliary function and ciliogenesis were significantly perturbed by repeated CS exposures, leading to changes in cilia beating frequency and ciliary protein expression. In particular, a time-dependent decrease in the expression of miR-449a, a conserved miRNA highly enriched in ciliated airway epithelia and implicated in motile ciliogenesis, was observed in CS-exposed cultures. Similar alterations in miR-449a have been reported in smokers with COPD. Network analysis further indicates that downregulation of miR-449a by CS may derepress cell-cycle proteins, which, in turn, interferes with ciliogenesis. Investigating the effects of CS on transcriptome profile in human ALI cultures may provide not only mechanistic insights, but potential early biomarkers for CS exposure and harm.

Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model

Exposure to cigarette smoke (CS) is a known risk factor in the pathogenesis of smoking-caused diseases, such as chronic obstructive pulmonary diseases (COPD) and lung cancer. To assess the effects of CS on the function and phenotype of airway epithelial cells, we developed a novel repeated treatment protocol and comprehensively evaluated the progression of key molecular, functional, and structural abnormalities induced by CS in a human in vitro air–liquid-interface (ALI) airway tissue model. Cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min clean air) generated from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 4 weeks (3 days per week, 40 min/day). By integrating the transcriptomics-based approach with the in vitro pathophysiological measurements, we demonstrated CS-mediated effects on oxidative stress, pro-inflammatory cytokines and matrix metalloproteinases (MMPs), ciliary function, expression and secretion of mucins, and squamous cell differentiation that are highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the ALI cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses that were perturbed in response to CS exposure. These responses, in turn, may trigger aberrant tissue remodeling, eventually leading to the onset of respiratory diseases. Furthermore, changes of a panel of genes known to be disturbed in smokers with COPD were successfully reproduced in the ALI cultures exposed to CS. In summary, findings from this study suggest that such an integrative approach may be a useful tool for identifying genes and adverse cellular events caused by inhaled toxicants, like CS.

Impact of cigarette versus electronic cigarette aerosol conditioned media on aortic endothelial cells in a microfluidic cardiovascular model

Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0–1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.

Multi-endpoint analysis of human 3D airway epithelium following repeated exposure to whole electronic vapor product aerosol or cigarette smoke

Smoking is a cause of serious diseases in smokers including chronic respiratory diseases. This study aimed to evaluate the tobacco harm reduction (THR) potential of an electronic vapor product (EVP, myblu™) compared to a Kentucky Reference Cigarette (3R4F), and assessed endpoints related to chronic respiratory diseases. Endpoints included: cytotoxicity, barrier integrity (TEER), cilia function, immunohistochemistry, and pro-inflammatory markers. In order to more closely represent the user exposure scenario, we have employed the in vitro 3D organotypic model of human airway epithelium (MucilAir™, Epithelix) for respiratory assessment. The model was repeatedly exposed to either whole aerosol of the EVP, or whole 3R4F smoke, at the air liquid interface (ALI), for 4 weeks to either 30, 60 or 90 puffs on 3-exposure-per-week basis. 3R4F smoke generation used the ISO 20778:2018 regime and EVP aerosol used the ISO 20768:2018 vaping regime.Exposure to undiluted whole EVP aerosol did not trigger any significant changes in the level of pro-inflammatory mediators, cilia beating function, barrier integrity and cytotoxicity when compared with air controls. In contrast, exposure to diluted (1:17) whole cigarette smoke caused significant changes to all the endpoints mentioned above. To our knowledge, this is the first study evaluating the effects of repeated whole cigarette smoke and whole EVP aerosol exposure to a 3D lung model at the ALI. Our results add to the growing body of scientific literature supporting the THR potential of EVPs relative to combustible cigarettes and the applicability of the 3D lung models in human-relevant product risk assessments.

Genotoxicity evaluation of tobacco and nicotine delivery products: Part Two. In vitro micronucleus assay

In this study, a variety of test matrices from tobacco and nicotine delivery products were assessed against a 3R4F Kentucky reference cigarette using the in vitro micronucleus assay. Testing was conducted using two Chinese hamster cell lines (CHO and V79), and a human lymphoblastoid cell line (TK6), in accordance with established guidelines.Total particulate matter (TPM) from a 3R4F Reference cigarette was compared to an electronic cigarette e-liquid, electronic cigarette TPM and TPM from a commercial tobacco heating product using a standard and an extended treatment condition with recovery period.Cells were assessed with 3R4F TPM prior to assessment of the other tobacco and nicotine product test matrices. These cell lines gave varied responses to 3R4F TPM with the most robust response using V79 cells. The use of an extended exposure/recovery period was seen to increase assay sensitivity for CHO and V79 cell lines but was less clear for TK6 cells.Negative responses were observed for all products except 3R4F across all treatment conditions in V79 cells. The most potent response to cigarette smoke was following extended treatment with recovery, suggesting this may be a more appropriate treatment for the future assessment of tobacco and nicotine product test matrices.

Mainstream smoke constituents and in vitro toxicity comparative analysis of 3R4F and 1R6F reference cigarettes.

A new Kentucky reference cigarette, 1R6F, has been manufactured to replace the depleting 3R4F reference cigarette. The 3R4F Kentucky reference cigarettes have been widely used as monitor or comparator cigarettes for mainstream smoke analysis and in vitro and in vivo toxicological data of cigarettes and novel tobacco products. Both reference cigarettes were analyzed in the same laboratory during the same period of time with the goal of performing a comparison of 3R4F and 1R6F. On the basis of the results obtained from aerosol chemistry and in vitro assays, we consider that the 1R6F reference cigarette is a suitable replacement for the 3R4F reference cigarette as a comparator/monitor cigarette. Its specific use as a comparator for novel tobacco products was checked on the basis of a comparative test with the Tobacco Heating System 2.2 as an example.

Determination of Hydrogen Cyanide in Cigarette Smoke by Continuous Flow Analysis Method Using Safer Chemistry

Summary A new safe and sensitive method to determine hydrogen cyanide (HCN) in cigarette smoke using continuous flow analyser (CFA) has been developed and validated. The use of highly toxic potassium cyanide (KCN) as a standard necessitates the development of a safer method for the determination of HCN in cigarette smoke. In this described method KCN is replaced by less toxic potassium tetracyanozincate (Lethal Dose LD50 oral is 7.49 mg/kg for KCN and 2000 mg/kg for potassium tetracyanozincate). Furthermore, the new method uses isonicotinic acid-barbituric acid (coupling reagent) instead of pyridine-pyrazolone as a reagent for the determination of HCN, and hence eliminates the use of pyridine. In this method HCN is trapped on both the Cambridge Filter Pad, then extracted with aqueous sodium hydroxide solution, and in an impinger containing the same solution. The solution thus extracted is oxidised to cyanogen chloride by Chloramine-T and treated with coupling reagent, the resulting stable chromophore was measured colorimetrically at 600 nm. The regression equation was linear in the range of 1 to 25 μg/mL for cyanide with a correlation coefficient (R2) > 0.9998. The limit of detection (LOD) was 0.76 μg/cig and the overall relative standard deviation (RSD) of the method was less than 10%. Excellent recoveries of cyanide were obtained in the range from 92% to 112% and the HCN yields from the Kentucky Reference Cigarette 3R4F obtained from the newly developed method are in good agreement with those from the conventional KCN method. The proposed method is robust, reliable, selective and safer than any of the existing methods for determination of hydrogen cyanide in mainstream as well as in sidestream cigarette smoke.

Aspirin abrogates impairment of mammary gland differentiation induced by early in life second-hand smoke in mice.

Epidemiological studies show that there is limited evidence that tobacco smoking causes breast cancer in humans. In rodents, many tobacco smoke chemicals cause mammary gland tumors. This study evaluated the mammary gland differentiation in mice exposed to environmental cigarette smoke (ECS), using 3R4F Kentucky reference cigarettes, starting after birth and continuing daily for 10 weeks (total particulate exposure 95 mg/m3; CO 610 ppm). We also analyzed the effects of oral administration of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin (1600 mg/kg) or naproxen (320 mg/kg), on mammary gland differentiation, either in unexposed or ECS-exposed mice. The ECS exposure caused delay of mammary glands development. We speculate that this delay may result from aryl hydrocarbon receptor (AHR) signaling activation, which has an antiestrogenic effect and crosstalk to the estrogen metabolism pathway. Similarly, naproxen impaired gland differentiation in unexposed and ECS-exposed mice, while aspirin hindered its development only in unexposed mice. The lack of differentiation induced by the NSAIDs could be explained by their antiestrogenic effect through inhibition of aldo-keto reductases. In ECS-exposed animals, aspirin induced intense lobular formation, which could indicate that aspirin is counteracting the AHR signaling induced by ECS. Based on the differentiation induced by aspirin in ECS-exposed animals, we postulate that these mice would be less susceptible to mammary carcinogenesis. Our results suggest that exposure to smoke at an early age impairs the development of the mammary gland, thus resulting in a longer period of susceptibility and increased risk of breast cancer. However, addition of aspirin can abrogate this effect.

Optimized method for determination of 16 FDA polycyclic aromatic hydrocarbons (PAHs) in mainstream cigarette smoke by gas chromatography\u2013mass spectrometry

A gas chromatography–mass spectrometry (GC–MS) method was validated for the determination of 16 polycyclic aromatic hydrocarbons (PAHs) from the FDA list of 93 harmful or potentially harmful constituents of mainstream cigarette smoke (MCS). Target analytes were extracted from total particulate matter using accelerated solvent extraction with a toluene/ethanol solvent mixture. Matrix artefacts were removed by two-step solid-phase extraction process. Three different GC–MS systems [GC–MS (single quadrupole), GC–MS/MS (triple quadrupole) and GC–HRMS (high resolution, magnetic sector)] using the same separation conditions were compared for the analysis of MCS of 3R4F Kentucky reference cigarettes generated under ISO and intense smoking regimes. The high mass resolution (m/∆m ≥ 10,000) and associated selectivity of detection by GC–HRMS provided the highest quality data for the target PAHs in MCS. Owing to the HR data acquisition mode enabling measurement of accurate mass, limits of quantification for PAHs were 5 to 15-fold lower for GC–HRMS than for GC–MS/MS and GC–MS. The presented study illustrates that the optimised sample preparation strategy followed by GC–HRMS analysis provides a fit-for-purpose and robust analytical approach allowing measurement of PAHs at (ultra)low concentrations in MCS. Furthermore, the study illustrates the importance and benefits of robust sample preparation and clean-up to compensate for limited selectivity when low-resolution MS is used.

Development, qualification, validation and application of the Ames test using a VITROCELL® VC10® smoke exposure system.

The Ames test has established use in the assessment of potential mutagenicity of tobacco products but has generally been performed using partitioned exposures (e.g. total particulate matter [TPM], gas vapor phase [GVP]) rather than whole smoke (WS). The VITROCELL®VC10® smoke exposure system offers multiple platforms for air liquid interface (ALI), or air agar interface (AAI) in the case of the Ames test exposure to mimic in vivo-like conditions for assessing the toxicological impact of fresh WS in in vitro assays. The goals of this study were to 1) qualify the VITROCELL®VC10® to demonstrate functionality of the system, 2) develop and validate the Ames test following WS exposure with the VITROCELL®VC10® and 3) assess the ability of the Ames test to differentiate between a reference combustible product (3R4F Kentucky reference cigarette) and a primarily tobacco heating product (Eclipse). Based on critical function assessments, the VITROCELL®VC10® was demonstrated to be fit for the purpose of consistent generation of WS. Assay validation was conducted for 5 bacterial strains (TA97, TA98, TA100, TA1535 and TA102) and reproducible exposure-related changes in revertants were observed for TA98 and TA100 in the presence of rat liver S-9 following exposure to 3R4F WS. In the comparative studies, exposure-related changes in in vitro mutagenicity following exposure of TA98 and TA100 in the presence of S9 to both 3R4F and Eclipse WS were observed, with the response for Eclipse being significantly less than that for 3R4F (p < 0.001) which is consistent with the fewer chemical constituents liberated by primarily-heating the product.

Determination of Mercury in Aerosol by Inductively Coupled Plasma Mass Spectrometry

Summary Based on the knowledge gained from published studies, a new analytical method has been developed for the quantification of mercury (Hg) in the gas-vapor phase of mainstream cigarette smoke and in heated tobacco aerosol generated by a tobacco heating system (THS) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). From a preliminary test, the mercury concentration in the particulate matter of mainstream smoke from Kentucky reference cigarettes 3R4F generated under the International Organization for Standardization (ISO) smoking regimen was compared with the mercury concentration in the gasvapor phase to assure that mercury is only measurable in the gas-vapor phase, as reported in an earlier published study. The particulate matter was collected using an electrostatic precipitation trap and was analyzed by ICP-MS after a mineralization step. The gas-vapor phase was trapped in the same smoking run as for the particulate matter using two impingers containing a nitric acid-hydrochloric acid-gold solution. The impingers were connected in series behind the electrostatic precipitation trap and the combined impinger solution was analyzed by ICP-MS after sample dilution without further sample treatment. The addition of gold has shown to be efficient for maintaining mercury in an ionized form in the impinger solution and to minimize the mercury memory effect in the sample introduction system of the ICP-MS. Only mercury in the gas-vapor phase could be quantified whereas the signal for mercury in the particulate matter was found close to those of blank solutions and was not measurable, as already mentioned in an earlier study. Following this preliminary test, the electrostatic precipitation trap was replaced by a Cambridge filter pad for the separation of the gas-vapor phase from the particulate matter where only mercury in the gas-vapor phase was quantified. The method for the quantification of mercury in the gas-vapor phase of aerosols obtained under Health Canada (HC) and ISO smoking regimens was validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and Association of Official Analytical Chemists (AOAC) guidelines. Accuracy profiles were evaluated as described in Association Française de Normalisation (AFNOR). The regression curve was shown to be linear within the evaluated concentration range from 25 pg/mL to 1000 pg/mL with a weighting factor 1/x. The coefficients of variation for repeatability (r) were 3.6% for 3R4F and 4.8% for THS under HC smoking regimen and 3.6% for 3R4F and 4.6% for THS under ISO smoking regimen. The coefficients of variation for intermediate precision (IP) were 7.7% for 3R4F and 7.7% for THS under HC smoking regimen and 4.7% for 3R4F and 4.6% for THS under ISO smoking regimen. The nominal mercury concentrations for 3R4F obtained during the validation under both HC and ISO smoking regimens were found to be in line with results reported in a previously published CORESTA study.

The Determination of Diacetyl and Acetylpropionyl in Aerosols From Electronic Smoking Devices Using Gas Chromatography Triple Quad Mass Spectrometry

Summary A reliable and sensitive method for the measurement of the level of diacetyl (2,3-butanedione) and acetylpropionyl (2,3-pentanedione) in the aerosol (both the particles and the suspending gas) of electronic smoking devices (e-cigarettes) has been developed. The method uses a gas chromatographic separation on a Carbowax type column with the measurement of the analytes on a triplequadrupole mass spectrometer working in positive MRM mode. The method has been validated using standard requirements regarding selectivity, sensitivity, recovery, accuracy, and repeatability. The limit of quantitation (LOQ) for the method was determined to be 0.41 ng/mL for diacetyl and 0.21 ng/mL for acetylpropionyl as measured for standards. These values translate to an LOQ of 0.082 ng/puff for diacetyl and 0.042 ng/puff for acetylpropionyl as measured for an e-cigarette with 50 puffs placed in 10 mL acetone. The samples analyzed included collected aerosols from several e-cigarettes, and a number of liquids used in electronic cigarettes (e-liquids). 3R4F Kentucky reference cigarette was also analyzed for evaluating the accuracy of the procedure, with good agreement with data from the literature. Diacetyl and acetylpropionyl were distributed in both particulate phase and also in vapor phase. The levels of diacetyl and acetylpropionyl in particulate phase collected from 3R4F cigarettes were found to represent only about 22% for diacetyl and only 31% for acetylpropionyl, while the vapor phase for diacetyl represented 78% and for acetylpropionyl 69% of the total analyte. The levels of diacetyl and acetylpropionyl in the aerosols of most electronic smoking devices were found to be very low, with a few exceptions. The analysis of the two analytes in several e-liquids available on the market showed a very large range of levels. Some of the e-liquids from the market are likely to have diacetyl and/or acetylpropionyl intentionally added.

Tobacco Smoke Constituents Trigger Cytoplasmic Calcium Release.

Cytosolic Ca2+ is a universal second messenger that is involved in many processes throughout the body, including the regulation of cell growth/cell division, apoptosis, and the secretion of both ions, and macromolecules. Tobacco smoke exerts multiple effects on airway epithelia and we have previously shown that Kentucky reference cigarette smoke exposure elevated the second messenger Ca2+, leading to dysfunctional ion secretion. In this study, we tested whether little cigar and commercial cigarette smoke exposure exerts similar effects on intracellular Ca2+ levels. Indeed, Swisher Sweets, Captain Black, and Cheyenne little cigars, as well as Camel, Marlboro, and Newport cigarettes, triggered a comparable increase in intracellular Ca2+ as seen with Kentucky reference cigarettes in human bronchial epithelia. We also found that Kentucky reference cigarette smoke exposure caused increases in Ca2+ in HEK293T cells and that similar increases in Ca2+ were seen with the tobacco smoke metabolites 1-NH2-naphthalene, formaldehyde, nicotine, and nicotine-derived nitrosamine ketone. Given the large number of physiological processes governed by changes in cytosolic Ca2+, our data suggest that Ca2+ signaling is a useful and reproducible assay that can be used to probe the propensity of tobacco products and their constituents to cause toxicity.

Rapid determination of 9 aromatic amines in mainstream cigarette smoke by modified dispersive liquid liquid microextraction and ultraperformance convergence chromatography tandem mass spectrometry

Aromatic amines in mainstream cigarette smoke have long been monitored due to their carcinogenic toxicity. In this work, a reliable and rapid method was developed for the simultaneous determination of 9 aromatic amines in mainstream cigarette smoke by modified dispersive liquid liquid microextraction (DLLME) and ultraperformance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Briefly, the particulate phase of the cigarette smoke was captured by a Cambridge filter pad, and diluted hydrogen chloride aqueous solution is employed to extract the aromatic amines under mechanical shaking. After alkalization with sodium hydroxide solution, small amount of toluene was introduced to further extract and enrich aromatic amines by modified DLLME under vortexing. After centrifugation, toluene phase was purified by a universal QuEChERS cleanup kit and was finally analyzed by UPC2-MS/MS. Attributing to the superior performance of UPC2-MS/MS, this novel approach allowed the separation and determination of 9 aromatic amines within 5.0min with satisfactory resolution and sensitivity. The proposed method was finally validated using Kentucky reference cigarette 3R4F, and emission levels of targeted aromatic amines determined were comparable to previously reported methods At three different spiked levels, the recoveries of most analytes were ranged from 74.01% to 120.50% with relative standard deviation (RSD) less than 12%, except that the recovery of p-toluidine at low spiked level and 3-aminobiphenyl at medium spiked level was 62.77% and 69.37% respectively. Thus, this work provides a novel alternative method for the simultaneous analysis of 9 aromatic amines in mainstream cigarette smoke.

Studies on the contributions of smoke constituents, individually and in mixtures, in a range of in vitro bioactivity assays

Tobacco smoke is a complex mixture with over 8700 identified constituents. Smoking causes many diseases including lung cancer, cardiovascular disease, and chronic obstructive pulmonary disease. However, the mechanisms of how cigarette smoke impacts disease initiation or progression are not well understood and individual smoke constituents causing these effects are not generally agreed upon. The studies reported here were part of a series of investigations into the contributions of selected smoke constituents to the biological activity of cigarette smoke. In vitro cytotoxicity measured by the neutral red uptake (NRU) assay and in vitro mutagenicity determined in the Ames bacterial mutagenicity assay (BMA) were selected because these assays are known to produce reproducible, quantitative results for cigarette smoke under standardized exposure conditions. In order to determine the contribution of individual cigarette smoke constituents, a fingerprinting method was developed to semi-quantify the mainstream smoke yields. For cytotoxicity, 90% of gas vapor phase (GVP) cytotoxicity of the Kentucky Reference cigarette 1R4F was explained by 3 aldehydes and 40% of the 1R4F particulate phase cytotoxicity by 10 smoke constituents, e.g., hydroquinone. In the microsuspension version of the BMA, 4 aldehydes accounted for approximately 70% of the GVP mutagenicity. Finally, the benefits of performing such studies along with the difficulties in interpretation in the context of smoking are discussed.