Reference Cigarette Research Articles

79664 Complement Driven Auto-Reactive Antibodies in Lung Transplantation

ABSTRACT IMPACT: Our work unveils a novel mechanism of ischemia repurfusion injury driven by pre-existing autoimmunity following lung transplant and a potential therapeutic strategy for blocking complement-dependent injury thereby reducing risk of lung transplant rejection. OBJECTIVES/GOALS: Our goal was to determine if pre-existing autoimmune autoantibodies, such as those resulting from cigarette smoke (CS), contribute to graft rejection in lung transplantation (LTx) and if autoreactive-mediated graft injury is complement-dependent. METHODS/STUDY POPULATION: For in vivo experiments, we utilized our emphysema mouse model. Briefly, eight-week-old C57BL/6J mice are exposed to 3R4F reference cigarette smoke 5 hours per day, 5 days a week for 6 months. Upon completion, cigarette smoked (CS) mice and control (NS) mice received syngeneic orthotopic left-lung transplant from age-matched C57BL/6J donors. To determine if pre-existing autoreactivity mediated graft injury was complement-dependent we treated CS-LTx mice with a novel, bifunctional complement inhibitor. Autoantibody levels were measured by ELISA and lung injury was assessed by blinded histopathological analyses. Complement inhibition was verified by immunofluorescence. RESULTS/ANTICIPATED RESULTS: We found that CS-exposure leads to production of autoreactive antibodies towards extracellular matrix (ECM) components and contributes to graft injury. Interestingly, LTx into CS exposed mice further increased de-novo ECM autoantibody development. Lastly, treatment with our novel, bifunctional complement inhibitor blocked autoantibody spreading and significantly reduced graft rejection. DISCUSSION/SIGNIFICANCE OF FINDINGS: These data demonstrate that smoking induces pre-LTx autoreactivity to ECM proteins that promotes graft injury following LTx. Furthermore, complement inhibition reduces autoantibody production and protects the graft from injury.

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.

Profiling of volatile substances by direct thermal desorption gas chromatography high-resolution mass spectrometry for flagging a characterising flavour in cigarette tobacco

This paper describes an analytical method that supports the implementation of articles 9 and 10 of the WHO Framework Convention on Tobacco Control (FCTC) regarding the provisions on the reduction of the palatability and attractiveness of tobacco products regarding flavour ingredients. This study aimed to develop a screening method to identify cigarettes that may have a characterising flavour to support the implementation of the ban of characterising flavours of tobacco products, as laid down in the US and EU law. An analytical method combining direct thermal desorption and GC–QTOF MS was developed for acquiring the profile of volatile and semi-volatile substances in tobacco. A database of flavour additives was created comprising 133 compounds. A group of cigarettes without a declared characterising flavour was used to establish a reference profile of flavouring chemicals commonly present in tobacco products. A reference profile was modelled both by the means of principal component analysis (PCA) and based on the calculation of threshold values specified as 95th percentile of measured compounds’ relative responses. Cigarettes and roll-your-own tobacco labelled as flavoured were analysed to evaluate the discrimination power of the method. A constructed model of the reference cigarettes allowed the differentiation of the flavoured tobacco products from the reference group. The method allows drawing conclusions on the chemical profiles of flavour constituents of tobacco products at even sensorial subliminal concentration levels and is suitable for both the initial screening of products on the market for characterising flavours and for confirmatory purposes after sensory analysis.Graphical abstract

Qualitative and Quantitative Comparison of Flavor Chemicals in Tobacco Heating Products, Traditional Tobacco Products and Flavoring Capsules

A gas chromatography-mass spectrometric (GC-MS) method was developed for the qualitative and quantitative analysis of flavor chemicals in tobacco heating products (THPs), traditional tobacco products (TTPs) and their flavoring capsules. A total of 247 compounds were identified through nontargeted analysis, 151 compounds were selected as target analytes, and the final 302 compounds were selected to develop an analytical method. The lower limits of detection (LOD) of analytes were 0.00074 to 12 mg/kg and their LOD range was wide depending on the presence or absence in the reference cigarette. The precision of the 302 compounds was less than 24.5%, and the accuracy ranged from 80.0-120%. A total 190 flavors and 5 contaminants were determined in 21 THP, 10 TTP, 8 THP capsules and 11 TTP capsules. When comparing the total flavor content of flavors per cigarette, it was in the order of THP capsule > TTP capsule ≫ THP ≫ TTP. The correlations between the 53 cigarette products and 190 flavor compounds were analyzed using PCA. It has been demonstrated that PCA results can be a useful tool in differentiating brands and manufacturers of tobacco products.

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.

Inter-Laboratory Reproducibility and Interchangeability of 3R4F and 1R6F Reference Cigarettes in Mainstream Smoke Chemical Analysis and In Vitro Toxicity Assays

Summary A new reference cigarette, 1R6F, produced by the Kentucky Tobacco Research and Development Center, has been manufactured as a substitute for the 3R4F reference cigarette because of a depletion of 3R4F stock. The purpose of the current study was to investigate the interchangeability of 1R6F and 3R4F by comparing the chemical and biological characteristics of the mainstream smoke and to assess the inter-laboratory reproducibility by comparing the results obtained in the current study with a previous report. We analyzed 45 priority chemicals required by Health Canada for regulatory reporting and assessed the toxicological effects of cigarette smoke using in vitro standard toxicological assays recommended by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) under the International Organization for Standardization (ISO) standard and intense smoking regimens. The results of the chemical analysis and standard toxicological assays showed a good inter-laboratory reproducibility for 1R6F as a reference cigarette, while there were some slight reproducible differences between 1R6F and 3R4F. In addition, we investigated the interchangeability of 1R6F with 3R4F in some additional toxicological assays that detect oxidative stress because oxidative stress is a principle endpoint used in tobacco research with next generation tobacco and nicotine delivery products (NGPs). Both 1R6F and 3R4F elicited comparable responses in the oxidative stress assays. Overall, our results showed inter-laboratory reproducibility in chemical and standard toxicological assessments of 1R6F; thus, suggesting the suitability of 1R6F as a reference cigarette. In addition, the results obtained in the oxidative stress assays provide insight into the interchangeability of 1R6F with 3R4F when used as a comparator for NGPs.

Comparison of the biological impact of aerosol of e-vapor device with MESH® technology and cigarette smoke on human bronchial and alveolar cultures

Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than cigarette smoke (CS) exposure. Although many studies on e-vapor products have tested the effects of liquid formulations on cell cultures, few have evaluated the effects of aerosolized formulations. We examined the effects of acute exposure to the aerosol of an e-vapor device that uses the MESH® technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette on human organotypic bronchial epithelial culture and alveolar triculture models. In contrast to 3R4F CS exposure, exposure to the IQOS MESH aerosol (Classic Tobacco flavor) did not cause cytotoxicity in bronchial epithelial cultures or alveolar tricultures despite its greater concentrations of deposited nicotine (3- and 4-fold, respectively). CS exposure caused a marked decrease in the frequency and active area of ciliary beating in bronchial cultures, whereas IQOS MESH aerosol exposure did not. Global mRNA expression and secreted protein profiles revealed a significantly lower impact of IQOS MESH aerosol exposure than 3R4F CS exposure. Overall, our whole aerosol exposure study shows a clearly reduced impact of IQOS MESH aerosol relative to CS in bronchial and alveolar cultures, even at greater nicotine doses.

The roles of standard cigarettes in assuring the ignition resistance of soft furnishings

SummaryRegulations for cigarette ignition resistance (CIR) of soft furnishings (beds and upholstered furniture) and less fire‐prone cigarettes have contributed substantially to the decrease in losses from cigarette‐initiated fires over time. Two standard reference cigarettes play key roles in mitigating these losses and in sustaining the effectiveness of the fire safety regulations as exogenous changes occur. SRM 1082 provides a uniform, durable supply of cigarettes for use in ASTM E2187 that assures manufacturers and regulators of compliance with regulations for reduced ignition propensity cigarettes; enables quality control of cigarette fire test performance; enables assurance of uniform interlaboratory test results; obviates effects on fire safety as tobacco crops and smokers change over time; and, when the original ASTM E2187 substrate material was no longer available, enabled adding a new, equivalent substrate. SRM 1196 provides a uniform, durable supply of cigarettes for assurance of consistent interlaboratory evaluation of the ignition resistance of soft furnishings using the mandated test methods; obviates possible unknown changes in soft furnishings' CIR due to the evolving ignition strength of the original test cigarette over time; and provided a test cigarette that was stronger than most cigarettes being smoked after manufacture of the original commercial test cigarette ceased.

Ceramide ratios are affected by cigarette smoke but not heat-not-burn or e-vapor aerosols across four independent mouse studies

AimsSmoking is an important risk factor for the development of chronic obstructive pulmonary disease and cardiovascular diseases. This study aimed to further elucidate the role of ceramides, as a key lipid class dysregulated in disease states. Main methodsIn this article we developed and validated LC–MS/MS method for ceramides (Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1(15Z)) for the absolute quantification. We deployed it together with proteomics and transcriptomic analysis to assess the effects of cigarette smoke (CS) from the reference cigarette as well as aerosols from heat-not-burn (HnB) tobacco and e-vapor products in apolipoprotein E-deficient (ApoE−/−) mice over several time points. Key findingsIn the lungs, CS exposure substantially elevated the ratios of Cer(d18:1/24:0) and Cer(d18:1/24:1) to Cer(d18:1/18:0) in two independent ApoE−/− mouse inhalation studies. Data from previous studies, in both ApoE−/− and wild-type mice, further confirmed the reproducibility of this finding. Elevation of these ceramide ratios was also observed in plasma/serum, the liver, and—for the Cer(d18:1/24:1(15Z)) to Cer(d18:1/18:0) ratio—the abdominal aorta. Also, the levels of acid ceramidase (Asah1) and glucocerebrosidase (Gba)—lysosomal enzymes involved in the hydrolysis of glucosylceramides—were consistently elevated in the lungs after CS exposure. In contrast, exposure to HnB tobacco product and e-vapor aerosols did not induce significant changes in the ceramide profiles or associated enzymes. SignificanceOur work in mice contributes to the accumulating evidence on the importance of ceramide ratios as biologically relevant markers for respiratory disorders, adding to their already demonstrated role in cardiovascular disease risk assessment in humans.

Development of a novel method to measure material surface staining by cigarette, e-cigarette or tobacco heating product aerosols

Tobacco smoke (CS) may visually stain indoor surfaces including ceilings, walls and soft furnishings over time. Potentially reduced risk products (PRRPs) such as e-cigarettes (EC) and tobacco heating products (THP) produce chemically less complex aerosols with significantly reduced levels of toxicants, particles and odour. However, the potential effects of EC and THP aerosols on the staining of indoor surfaces are currently unknown. In this study, an exposure chamber was developed as a model system to enable the accelerated staining of wallpaper and cotton samples by a scientific reference cigarette (3R4F), three THP (glo™, glo™ pro, glo™ sens) and an e-cigarette (iSwitch Maxx).Exposure to 3R4F reference cigarettes caused the greatest level of staining, which was significantly higher than glo™, glo™ pro, glo™ sens or iSwitch Maxx aerosols, all of which showed relatively little colour change. Exposure to 200–1000 puffs of 3R4F cigarette smoke resulted in a visible dose response effect to wallpaper and cotton samples which was not observed following exposure to glo™, glo™ pro, glo™ sens or iSwitch Maxx aerosols. Aging of the samples for 4 weeks post-exposure resulted in changes to the staining levels, however PRRP staining levels were minimal and significantly lower than 3R4F exposed samples.For the first time, diverse PRRPs across the tobacco and nicotine products risk continuum have been assessed in vitro for their impact on surface staining. CS exposure significantly increased the level of wallpaper and cotton staining, whereas exposure to glo™, glo™ pro, glo™ sens or iSwitch Maxx aerosols resulted in significantly reduced levels of staining, staining levels were also comparable to untreated control samples.

Reduced Chronic Toxicity and Carcinogenicity in A/J Mice in Response to Life-Time Exposure to Aerosol From a Heated Tobacco Product Compared With Cigarette Smoke.

We conducted an inhalation study, in accordance with Organisation for Economic Co-operation and Development Test Guideline 453, exposing A/J mice to tobacco heating system (THS) 2.2 aerosol or 3R4F reference cigarette smoke (CS) for up to 18 months to evaluate chronic toxicity and carcinogenicity. All exposed mice showed lower thymus and spleen weight, blood lymphocyte counts, and serum lipid concentrations than sham mice, most likely because of stress and/or nicotine effects. Unlike THS 2.2 aerosol-exposed mice, CS-exposed mice showed increased heart weight, changes in red blood cell profiles and serum liver function parameters. Similarly, increased pulmonary inflammation, altered lung function, and emphysematous changes were observed only in CS-exposed mice. Histopathological changes in other respiratory tract organs were significantly lower in the THS 2.2 aerosol-exposed groups than in the CS-exposed group. Chronic exposure to THS 2.2 aerosol also did not increase the incidence or multiplicity of bronchioloalveolar adenomas or carcinomas relative to sham, whereas CS exposure did. Male THS 2.2 aerosol-exposed mice had a lower survival rate than sham mice, related to an increased incidence of urogenital issues that appears to be related to congenital factors rather than test item exposure. The lower impact of THS 2.2 aerosol exposure on tumor development and chronic toxicity is consistent with the significantly reduced levels of harmful and potentially harmful constituents in THS 2.2 aerosol relative to CS. The totality of the evidence from this study further supports the risk reduction potential of THS 2.2 for lung diseases in comparison with cigarettes.

Robustness of HPHC Reduction for THS 2.2 Aerosol Compared with 3R4F Reference Cigarette Smoke Under High Intensity Puffing Conditions

Summary In the absence of standards specific for testing the reduction robustness of the levels of harmful and potentially harmful constituents (HPHCs), the aerosol from the THS 2.2, a heated tobacco product, was compared with the mainstream smoke of the 3R4F reference cigarette over a broad range of machine-smoking regimes. The average reduction and the introduced concept of threshold limits of robust reduction were derived from HPHC concentrations, in mass per tobacco-stick normalized per total puff volume, to propose an alternative for the assessment of products where nicotine-adjusted yields would be inappropriate. In addition, this study explores the influence of 3R4F reference cigarette filter ventilation, and discusses the roles of temperature and precursors in the present context of robustness of HPHC reduction. Fifty-four HPHCs were analyzed under multiple regimes in THS 2.2 aerosol and 3R4F cigarette smoke. The average reduction of HPHC concentrations compared across all regimes characterized the robustness. Threshold limits of reduction of individual HPHCs were statistically determined across all regimes. The results observed under Health Canada Intense (HCI) and more intense regimes indicated that on average the reductions in HPHCs levels investigated in THS 2.2 aerosol were more than 90% and that the majority of the 54 HPHCs investigated in THS 2.2 aerosol showed more than 90% reduction. The robustness of THS 2.2 in maintaining the levels of reduction of representative HPHCs, whatever the puffing regime, can be quantified. The mass of HPHC per tobacco-stick normalized per total puff volume is a valuable approach to compare the robustness of the performance of a product over a large range of puffing conditions. Our findings will greatly complement the assessment for robustness of current and future similar products where classical approaches would present limitations.

The use of human induced pluripotent stem cells to screen for developmental toxicity potential indicates reduced potential for non-combusted products, when compared to cigarettes

devTOX quickPredict (devTOXqP) is a metabolomics biomarker-based assay that utilises human induced pluripotent stem (iPS) cells to screen for potential early stage embryonic developmental toxicity in vitro. Developmental toxicity potential is assessed based on the assay endpoint of the alteration in the ratio of key unrelated biomarkers, ornithine and cystine (o/c).This work aimed to compare the developmental toxicity potential of tobacco-containing and tobacco-free non-combustible nicotine products to cigarette smoke. Smoke and aerosol from test articles were produced using a Vitrocell VC10 smoke/aerosol exposure system and bubbled into phosphate buffered saline (bPBS). iPS cells were exposed to concentrations of up to 10% bPBS. Assay sensitivity was assessed through a spiking study with a known developmental toxicant, all-trans-retinoic acid (ATRA), in combination with cigarette smoke extract.The bPBS extracts of reference cigarettes (1R6F and 3R4F) and a heated tobacco product (HTP) were predicted to have the potential to induce developmental toxicity, in this screening assay. The bPBS concentration at which these extracts exceeded the developmental toxicity threshold was 0.6% (1R6F), 1.3% (3R4F), and 4.3% (HTP) added to the cell media. Effects from cigarette smoke and HTP aerosol were driven largely by cytotoxicity, with the cell viability and o/c ratio dose–response curves crossing the developmental toxicity thresholds at very similar concentrations of added bPBS. The hybrid product and all the electronic cigarette (e-cigarette) aerosols were not predicted to be potential early developmental toxicants, under the conditions of this screening assay.

E-vapor aerosols do not compromise bone integrity relative to cigarette smoke after 6-month inhalation in an ApoE\u2013/\u2013 mouse model

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE–/– mice were exposed to aerosols from three different e-vapor formulations—CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)—to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.

Free Radical and Nicotine Yields in Mainstream Smoke of Chinese Marketed Cigarettes: Variation with Smoking Regimens and Cigarette Brands.

Free radicals and nicotine are components of cigarette smoke that are thought to contribute to the development of smoking-induced diseases. China has the largest number of smokers in the world, yet little is known about the yields of tobacco smoke constituents in different Chinese brands of cigarettes. In this study, gas-phase and particulate-phase free radicals as well as nicotine yields were quantified in mainstream cigarette smoke from five popular Chinese brands and two research cigarettes (3R4F and 1R6F). Mainstream smoke was generated under International Organization of Standardization (ISO) and Canadian Intense (CI) smoking regimens using a linear smoking machine. Levels of free radicals and nicotine were measured by electron paramagnetic resonance spectroscopy (EPR) and gas chromatography with flame-ionization detection, respectively. Under the ISO puffing regimen, Chinese brand cigarettes produced an average of 3.0 ± 1.2 nmol/cig gas-phase radicals, 118 ± 44.7 pmol/cig particulate-phase radicals, and 0.6 ± 0.2 mg/cig nicotine. Under the CI puffing regimen, Chinese brand cigarettes produced an average of 5.6 ± 1.2 nmol/cig gas-phase radicals, 282 ± 92.1 pmol/cig particulate-phase radicals, and 2.1 ± 0.4 mg/cig nicotine. Overall, both gas- and particulate-phase free radicals were substantially lower compared to the research cigarettes under both regimens, whereas no significant differences were observed for nicotine levels. When Chinese brands were compared, the highest free radical and nicotine yields were found in "LL" and "BS" brands, while lowest levels were found in "YY". These results suggested that the lower radical delivery by Chinese cigarettes compared to United States reference cigarettes may be associated with reductions in oxidant-related harm.

A comparative in vitro toxicity assessment of electronic vaping product e-liquids and aerosols with tobacco cigarette smoke

The use of electronic vaping products (EVPs) continues to increase worldwide among adult smokers in parallel with accumulating information on their potential toxicity and relative safety compared to tobacco smoke. At this time, in vitro assessments of many widely available EVPs are limited. In this study, an in vitro battery of established assays was used to examine the cytotoxic (Neutral red uptake), genotoxic (In vitro micronucleus) and mutagenic (Bacterial reverse mutation) responses of two commercial EVPs (blu GO™ disposable and blu PLUS+™ rechargeable) when compared to smoke from a reference cigarette (3R4F). In total, 12 commercial products were tested as e-liquids and as aerosols. In addition, two experimental base liquids containing 1.2% and 2.4% nicotine were also assessed to determine the effect of flavour and nicotine on all three assays.In the bacterial reverse mutation (Ames) and in vitro micronucleus (IVM) assays, exposures to e-liquids and EVP aerosols, with and without nicotine and in a range of flavourings, showed no mutagenic or genotoxic effects compared to tobacco smoke. The neutral red uptake (NRU) assay showed significantly reduced cytotoxicity (P < .05) for whole undiluted EVP aerosols compared to tobacco smoke, which by contrast was markedly cytotoxic even when diluted.The reduced in vitro toxicological responses of the EVPs add to the increasing body of scientific weight-of-evidence supporting the role of high-quality EVPs as a harm reduction tool for adult smokers.

Mitochondrial Network and Biogenesis in Response to Short and Long-Term Exposure of Human BEAS-2B Cells to Aerosol Extracts from the Tobacco Heating System 2.2.

Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.

A cross-category puffing topography, mouth level exposure and consumption study among Italian users of tobacco and nicotine products

Actual use studies play a key part in evaluating the reduced risk potential of tobacco and nicotine products. This study was undertaken to determine the puffing topography, mouth level exposure (MLE) and average daily consumption (ADC) relating to two commercially available tobacco heating products (THPs) and a prototype electronic cigarette (or e-cigarette) among Italian non-mentholated 7 mg ISO tar cigarette smokers. The study was conducted in Milan, Italy, with three groups of approximately 50 participants. Groups 1 and 3 included adult smokers of 7 mg ISO tar tobacco cigarettes, and Group 2 consisted of both solus vapers and dual users of vapour and tobacco products. Amongst smokers, e-cigarette mean puff volumes (41.6 mL vs 41.3 mL) and mean puff durations (1.4 s vs 1.5 s) were similar to that of the cigarette, although the average usage session was significantly longer (1064.8 s vs 289.5 s) with a higher total number of puffs (58.6 vs 17.3), however this may be attributable to differences in product operation. There were no significant differences across puffing topography measurements observed between smokers (Group 1) and regular vapers/dual users (Group 2) when using the e-cigarette. As consistent with previous research, users took, on average, larger mean puff volumes when using a THP compared to the reference cigarette (C651), although puff numbers and puff durations remained similar. The average interval between puffs was considerably shorter for THP1.0(T) compared to THS2.4(T) (11.0 s vs 17.1 s). MLE to nicotine-free dry particulate matter and nicotine was significantly reduced for THP1.0(T) and THS2.4(T) compared to the tobacco cigarette (C651). MLE to nicotine was also significantly reduced for the e-cigarette (IS1.0(T)) compared to C651. The average daily consumption (ADC) of cigarettes by groups 1 and 3 were higher than the respective ADCs of both THP consumables. There were no significant differences in ADC when comparing the same product between different groups. Differences seen between sensory scores for each of the product categories may be attributed to fundamental differences in design and mode of operation resulting in very different characteristics of the aerosol generated.

Comparative and cumulative quantitative risk assessments on a novel heated tobacco product versus the 3R4F reference cigarette

Novel tobacco products that heat rather than burn tobacco (heated tobacco products or HTPs) have been shown to produce lower levels of harmful and potentially harmful constituents than conventional combusted cigarettes. The present study uses a quantitative risk assessment approach to compare non-cancer and cancer risk estimates for emissions generated by an HTP with smoke from a reference cigarette (3R4F). Fifty-four analytes were evaluated from the HTP aerosol and the 3R4F cigarette smoke. Emissions were generated using the ISO and the Health Canada Intense smoking regimes. The measured values were extrapolated to define a conservative exposure assumption for per day use and lifetime use based on an estimated maximum usage level of 400 puffs per day i.e., approximately 8 HTP tobacco capsules or 40 combustible cigarettes. Non-cancer and cancer risk estimates were calculated using these exposure assumptions for individual and per health outcome domains based on toxicological reference values derived by regulatory and/or public health agencies. The results of this assessment showed a reduction of non-cancer and cancer risk estimates by more than 90 % for the HTP versus the 3R4F cigarette, regardless of the smoking regime.

The genotoxicological assessment of a tobacco heating product relative to cigarette smoke using the in vitro micronucleus assay

In vitro studies have supported the toxicological evaluation of chemicals and complex mixtures including cigarette smoke and novel tobacco and nicotine products which include tobacco heating products (THP). This new environment requires faster testing, higher throughput and appropriate in vitro studies, to support product innovation and development.In this study, total particulate matter (TPM) from a commercially available THP and a reference cigarette (3R4F) were assessed up to 500 μg/mL using two in vitro micronucleus techniques. V79 and TK6 cells were assessed using conventional OECD 487 manual scoring techniques, whereas, CHO cells were assessed using contemporary, automated high content screening approaches (Cellomics ArrayScan® VTI).V79 cells gave the most consistent response with all three treatment conditions producing a clear positive genotoxic response. Human TK6 cells only produced dose-dependent response, indicative of a weak-positive response. CHO cells demonstrated a positive response with TPM using long (24 h) -S9 conditions. All three cell lines equally demonstrated a negative response with THP TPM up to 500 μg/mL.In conclusion, THP TPM did not increase micronuclei formation above control levels even at doses far exceeding that tested with reference cigarette smoke, in most cases up to 10x the dose delivered compared to that of cigarette smoke. This study supports the growing belief that THPs are less risky than conventional cigarettes and that 21st century screening techniques can be employed to support product design and decision making, as a potential 1st screen prior to more traditional assessments.