Cigarette 3R4F Research Articles

Metabolomics Provides Novel Insights into the Potential Toxicity Associated with Heated Tobacco Products, Electronic Cigarettes, and Tobacco Cigarettes on Human Bronchial Epithelial BEAS-2B Cells.

Smoking is an established risk factor for various pathologies including lung cancer. Electronic cigarettes (e-cigs) and heated tobacco products (HTPs) have appeared on the market in recent years, but their safety or, conversely, their toxicity has not yet been demonstrated. This study aimed to compare the metabolome of human lung epithelial cells exposed to emissions of e-cigs, HTPs, or 3R4F cigarettes in order to highlight potential early markers of toxicity. BEAS-2B cells were cultured at the air-liquid interface and exposed to short-term emissions from e-cigs set up at low or medium power, HTPs, or 3R4F cigarettes. Untargeted metabolomic analyses were performed using liquid chromatography coupled with mass spectrometry. Compared to unexposed cells, both 3R4F cigarette and HTP emissions affected the profiles of exogenous compounds, one of which is carcinogenic, as well as those of endogenous metabolites from various pathways including oxidative stress, energy metabolism, and lipid metabolism. However, these effects were observed at lower doses for cigarettes (2 and 4 puffs) than for HTPs (60 and 120 puffs). No difference was observed after e-cig exposure, regardless of the power conditions. These results suggest a lower acute toxicity of e-cig emissions compared to cigarettes and HTPs in BEAS-2B cells. The pathways deregulated by HTP emissions are also described to be altered in respiratory diseases, emphasizing that the toxicity of HTPs should not be underestimated.

Comparison Between Smoked Tobacco and Medical Cannabis Cigarettes Concerning Particulate Matter.

Introduction: Cannabis is a widely used drug like tobacco and alcohol. In the meantime, it is also prescribed for medical treatment in some countries. Tobacco smoke contains chemical carcinogens and particulate matter (PM) that are both harmful to health. Method: In this study, we investigated PM levels in second-hand smoke (SHS) of hand-tamped cannabis cigarettes compared to cigarettes with tubing tobacco and the 3R4F reference cigarette. Results: It could be demonstrated that the largest proportion of the particle mass is attributable to particles with a diameter of less than 1μm and that every tested cigarette emitted more PM than the 3R4F reference cigarette. In addition, our data clearly revealed that cannabis smoke contains higher PM levels in SHS than tobacco cigarettes. Compared to the reference cigarette, the PM1 emissions of cannabis were 105% higher. Also, the cannabis mixed cigarettes had higher PM levels than the 3R4F cigarettes. For instance, the PM10 emissions were 93% higher. Also, the Gauloises Mélange tubing tobacco also reached higher PM concentrations than the 3R4F cigarette. Discussion: Regardless of negative health effects, cannabis is seen as a harmless drug in the public eye. We found strong indications for potential health risks by PM from cannabis products and, therefore, the public should be educated about a potential harm.

A Comparative In Vitro Toxicological Screening of a Closed-End Heated Tobacco Product *

SUMMARY Heated tobacco products (HTPs) are a recent category of tobacco products, with their relative safety compared to cigarette smoking and potential to help smokers to quit being two reasons why regulators may consider their market approval. Designed to heat tobacco rather than to burn in order to produce aerosol, different heating techniques are applied to commercial HTPs, which may result in differing aerosol formation. Therefore, each product requires separate assessment. This work focuses on a closed-end HTP (coded as HTP-A), which is electrically heated and designed to allow puffing air flow to bypass its tobacco section, resulting in reduced oxygen concentration within the tobacco section during heating and aerosol forming. To provide a preliminary aerosol chemistry and in vitro toxicological screening, this study assessed HTP-A against a commercial electrically heated HTP (IQOSTM, coded as HTP-B) and a 3R4F reference cigarette. Under Health Canada Intense (HCI) smoking regime, the levels of 9 regulatory priority toxicants in the aerosol of HTP-A were either reduced or comparable to those in HTP-B on a per-stick basis. Additionally, both HTPs showed significant reduction (greater than 90%) in comparison to those measured in mainstream smoke of 3R4F cigarette for these toxicants. Using a set of standard in vitro toxicological assays (Ames, Micronucleus and Neutral Red Uptake), the two HTPs showed no observable responses while significant toxicity responses were recorded for 3R4F’s total particulate matter. Based on these preliminary results, the novel closed-end HTP-A design may provide similar toxicological profiles to the comparator HTP-B. Further toxicological and clinical assessments are warranted to evaluate HTP-A’s potential for exposure or disease risk reduction. [Contrib. Tob. Nicotine Res. 32 (2023) 146–156]

Cigarette smoke and tobacco heating aerosol on the aging of clear aligners.

The treatments with clear aligners (CA) showed an exponential higher percentage in the last years being almost invisible with major patient acceptability and a resulting improvement in quality of life. CA are composed of thermoplastic polyurethane not inert but subjected to changes due to heating and humidity, chewing forces, and prolonged exposure to enzymes in saliva in the oral cavity and external factors. Cigarette smoke (CS) and the aerosol produced by tobacco products with reduced health risks may be considered among the external factors affecting CA. The purpose of this study was the assessment of optical properties (absorbance and transmittance) and roughness of CA after in vitro aging due to exposition to CS and THS2.2, compared to controls. The secondary objective will be the investigation of CS and THS2.2 effects on resin composite inside aligners used for attachments during treatment with CA. A total number of 60 CA units will be used and equally divided in three different groups (20 in each): CS group exposed to reference cigarettes smoking, THS2.2 group exposed to aerosol from 20 heat-not-burn sticks, and control group, CG to pure air only. The aligners will present ten introflection for the attachments on the anterior part. In accordance with ISO standard 3402, 3R4F cigarettes and THS2.2 tobacco sticks must undergo conditioning for a minimum of 48 hours and a maximum of 21 days at a temperature of 22±1 °C and a relative humidity of 60±3%. Using spectrophotometry, the optical characteristics (absorbance and transmittance) of each aligner will be evaluated after aging (Jasco UV-vis V630PC, Tokyo, Japan). Each aligner will be cut with a rotating saw from canine to canine prior to the measurements in order to lop off the lingual region and displaying the labial barrier. The absorbance and transmittance measurements will be collected and statistically analyzed with a significance of P<0.05. Surface roughness of the aligners and attachments inside them will be assessed by 3D scanning microscopy (Infinite Focus G4h). Color of resin composite will be assessed using the (CIE Lab) Commission Internationale de l'Eclairage L*a*b* color space by means of Olympus CrystalEyef dental spectrophotometer. Clinicians should advise patients to refrain from smoking and drinking coffee or tea while wearing aligners for two reasons: first, the color change is unacceptably noticeable when wearing aligners, and second, the material's chemical composition may be slightly altered, which could affect the intended dental movements.

Comparison of the toxicological potential of two JUUL ENDS products to reference cigarette 3R4F and filtered air in a 90-day nose-only inhalation toxicity study.

Electronic nicotine delivery systems (ENDS) are generally recognized as less harmful alternatives for those who would otherwise continue to smoke cigarettes. The potential toxicity of aerosols generated from JUUL Device and Virginia Tobacco (VT3) or Menthol (ME3) JUULpods at 3.0% nicotine concentration was assessed in rats exposed at target aerosol concentrations of 1400μg/L for up to 6h/day on a 5 day/week basis for at least 90 days (general accordance with OECD 413). 3R4F reference cigarette smoke (250μg/L) and Filtered Air were used as comparators. JUUL ENDS product aerosol exposures at >5x the 3R4F cigarette smoke level resulted in greater plasma nicotine and cotinine levels (up to 2x). Notable cigarette smoke related effects included pronounced body weight reductions in male rats, pulmonary inflammation evidenced by elevated lactate dehydrogenase, pro-inflammatory cytokines and neutrophils in bronchoalveolar lavage fluid, increased heart and lung weights, and minimal to marked respiratory tract histopathology. In contrast, ENDS aerosol exposed animals had minimal body weight changes, no measurable inflammatory changes and minimal to mild laryngeal squamous metaplasia. Despite the higher exposure levels, VT3 and ME3 did not result in significant toxicity or appreciable respiratory histopathology relative to 3R4F cigarette smoke following 90 days administration.

Nicotine and Microvascular Responses in Skeletal Muscle from Acute Exposure to Cigarettes and Vaping.

Despite claims of safety or harm reduction for electronic cigarettes (E-cig) use (also known as vaping), emerging evidence indicates that E-cigs are not likely safe, or necessarily safer than traditional cigarettes, when considering the user's risk of developing vascular dysfunction/disease. E-cigs are different from regular cigarettes in that E-cig devices are highly customizable, and users can change the e-liquid composition (such as the base solution, flavors, and nicotine level). Since the effects of E-cigs on the microvascular responses in skeletal muscle are poorly understood, we used intravital microscopy with an acute (one-time 10 puff) exposure paradigm to evaluate the individual components of e-liquid on vascular tone and endothelial function in the arterioles of the gluteus maximus muscle of anesthetized C57Bl/6 mice. Consistent with the molecular responses seen with endothelial cells, we found that the peripheral vasoconstriction response was similar between mice exposed to E-cig aerosol or cigarette smoke (i.e., 3R4F reference cigarette); this response was not nicotine dependent, and endothelial cell-mediated vasodilation was not altered within this acute exposure paradigm. We also report that, regardless of the base solution component [i.e., vegetable glycerin (VG)-only or propylene glycol (PG)-only], the vasoconstriction responses were the same in mice with inhalation exposure to 3R4F cigarette smoke or E-cig aerosol. Key findings from this work reveal that some component other than nicotine, in inhaled smoke or aerosol, is responsible for triggering peripheral vasoconstriction in skeletal muscle, and that regardless of one's preference for an E-cig base solution composition (i.e., ratio of VG-to-PG), the acute physiological response to blood vessels appears to be the same. The data suggest that vaping is not likely to be 'safer' than smoking towards blood vessels and can be expected to produce and/or result in the same adverse vascular health outcomes associated with smoking cigarettes.

Should IQOS Emissions Be Considered as Smoke and Harmful to Health? A Review of the Chemical Evidence.

The chemical evidence that IQOS emissions fit the definition of both an aerosol and smoke, and that IQOS and potentially other heated tobacco products (HTPs) pose some harmful health threats from the range of compounds released even at somewhat lower concentrations is reviewed. Further, we address the yields of harmful and potentially harmful compounds (HPHCs), including polycyclic aromatic hydrocarbons (PAHs), and the constituents of IQOS emission that are diagnostic of pyrolysis to provide information on the temperatures reached in IQOS tobacco sticks. The HPHCs present in IQOS emissions are the same as in conventional cigarette smoke (CCs), analogous to emissions from earlier generation of HTPs classed as smoke. However, Philip Morris International (PMI) studies have to some degree underestimated IQOS aerosol HPHC yields, which are a factor of between 3.2 and 3.6 higher when expressed on a tobacco rather than an IQOS stick basis compared to the reference 3R4F cigarette. Further, IQOS emissions contain carbon particles, which fit definition of both aerosol and smoke. Continual reheating of deposited tar in the IQOS device will occur with real-life use, likely leading to generation of even higher concentrations of HPHCs and particulate matter. Despite IQOS not exceeding 350 °C, local hot spots could exist, causing formation of species (phenol/cresols, PAHs). It is recommended that the impact of repeated use to determine the levels of black carbon (insoluble organic matter) in the particulate matter, and the extent to which compounds in IQOS emissions are formed by pyrolysis need to be assessed rigorously. To address whether uneven temperature profiles in heat sticks can lead to potential hot spots that could, for example, lead to PAH formation, it is recommended that pyrolysis studies on tobacco and other constituents of HTPs are required in conjunction with more effort on heating tobacco blends under controlled temperature/time conditions.

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.

Volatile organic compounds released in the mainstream smoke of flavor capsule cigarettes

In this study, the composition of mainstream smoke was investigated with an emphasis on a list of volatile organic compounds (VOCs: e.g., isoprene, acrylonitrile, methyl ethyl ketone, benzene, toluene, m-xylene and styrene) using the two types of flavor capsule cigarettes (FCCs, here coded as F1 and F2) in reference to one commercial, non-flavored (NF) and 3R4F cigarette. The concentrations of all the target compounds from FCCs were quantified under two contrasting conditions (i.e., with and without breaking the capsules). The effect of breaking the capsule was apparent in the FCC products with the enhancement of VOC levels, specifically between after and before breaking the capsules (e.g., 1.10–1.58 folds (benzene) and 1.30–1.53 folds (acetonitrile)). Such increases were apparent in both FCC samples if assessed in terms of the total amount of VOCs (TVOC): (1) F1 (from 2159 to 2530 μg cig−1 (p = 9.42 × 10−6)) and (2) F2 (from 1470 to 2014 μg cig−1 (p = 0.05)). In addition, these TVOC levels determined from the FCCs were 1.62- to 1.83- and 1.29- to 1.46-fold higher than those of the NF cigarette and the 3R4F cigarette, respectively. Thus, these FCC products are suspected to play a role as stronger sources of VOCs than the general cigarette products.

In Utero Exposure to Environmental Tobacco Smoke Increases Neuroinflammation in Offspring.

The embryonic stage is the most vulnerable period for congenital abnormalities. Due to its prolonged developmental course, the central nervous system (CNS) is susceptible to numerous genetic, epigenetic, and environmental influences. During embryo implantation, the CNS is more vulnerable to external influences such as environmental tobacco smoke (ETS), increasing the risk for delayed fetal growth, sudden infant death syndrome, and immune system abnormalities. This study aimed to evaluate the effects of in utero exposure to ETS on neuroinflammation in the offspring of pregnant mice challenged or not with lipopolysaccharide (LPS). After the confirmation of mating by the presence of the vaginal plug until offspring birth, pregnant C57BL/6 mice were exposed to either 3R4F cigarettes smoke (Kentucky University) or compressed air, twice a day (1h each), for 21 days. Enhanced glial cell and mixed cell cultures were prepared from 3-day-old mouse pups. After cell maturation, both cells were stimulated with LPS or saline. To inhibit microglia activation, minocycline was added to the mixed cell culture media 24 h before LPS challenge. To verify the influence of in utero exposure to ETS on the development of neuroinflammatory events in adulthood, a different set of 8-week-old animals was submitted to the Autoimmune Experimental Encephalomyelitis (EAE) model. The results indicate that cells from LPS-challenged pups exposed to ETS in utero presented high levels of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNFα) and decreased cell viability. Such a proinflammatory environment could modulate fetal programming by an increase in microglia and astrocytes miRNA155. This scenario may lead to the more severe EAE observed in pups exposed to ETS in utero.

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.

Comparison of 3R4F cigarette smoke and IQOS heated tobacco product aerosol emissions

In this study, the smoke from a 3R4F research cigarette and the aerosol generated by the Heated Tobacco Product IQOS, also referred to as the Tobacco Heating System (THS) 2.2 in the literature, were compared. The objective was to characterize the gas and suspended particulate matter compositions in the mainstream smoke from a combusted 3R4F cigarette and in the aerosol generated by IQOS during use. The results indicated that the determined aerosol emissions from IQOS were notably lower than in the cigarette smoke under a Health Canada Intense puffing regimen. As an interesting detail in this study, the maximum nicotine concentrations within a puff were practically the same in both the 3R4F smoke and the IQOS aerosol, but the average concentration was lower for the IQOS aerosol. For both products, water constituted a significant proportion of the particulate matter, although it was substantially higher in the IQOS aerosol. Furthermore, combustion-related solid particles observed in the 3R4F smoke contained elements such as carbon, oxygen, potassium, calcium, and silicon. In contrast, IQOS aerosol particulate matter was composed of semi-volatile organic constituents with some minor traces of oxygen and silicon. The particulate matter found in the IQOS aerosol was volatile, which was especially noticeable when exposed to the electron beam of the scanning electron microscope (SEM) and Transmission Electron Microscope (TEM).

Cytotoxicity, oxidative stress, and inflammatory response of smokeless tobacco extracts and cytotoxicity of combustible cigarette whole smoke in a 3D oral organotypic buccal cell model

Oral disease is frequently associated with viral and environmental exposures and oral hygiene. The use of tobacco is a risk factor in the development of oral disease. Cytotoxicity, inflammatory response, and oxidative stress have been reported to have a role in the development of oral disease. These three endpoints were evaluated in a 3D human oral buccal model, EpiOral™, following exposure to CORESTA reference smokeless tobacco products (CRPs) and cigarette whole smoke. CRPs for Swedish style snus (CRP1), moist snuff (CRP2), and dry snuff (CRP3) were each extracted in complete artificial saliva (CAS) with a ratio of 300 mg CRP to 1 mL of CAS. Each of the CRP extracts (15–300 mg/ml) were applied to the apical side of a 3D organotypic buccal cell model for 24 or 48 h continuously, then cytotoxicity (LDH), oxidative stress (8-isoprostane), and inflammatory response (IP10, IL-1α, and IL-8) were measured. Experiments with 3R4F cigarettes were conducted by exposing the buccal tissues to whole smoke for a maximum of 2.5 h. Cytotoxicity (MTT) was measured 24 h post-exposure. Exposure of buccal tissues to whole smoke from a cigarette induced a dose-dependent cytotoxic response. In contrast, the CRP extracts elicited minimal cytotoxicity (<15%) when compared to CAS (vehicle control), but time- and dose-dependent effects on oxidative stress and inflammatory response were observed. Collectively, these data demonstrate that a 3D organotypic buccal human model may be used to assess biological mechanisms (MOAs) involved in the development of oral disease following exposure to smokeless tobacco products and may be applicable for differentiation between tobacco product categories.

Comparison of the in vivo genotoxicity of electronic and conventional cigarettes aerosols after subacute, subchronic and chronic exposures.

Tobacco smoking is classified as a human carcinogen. A wide variety of new products, in particular electronic cigarettes (e-cigs), have recently appeared on the market as an alternative to smoking. Although the in vitro toxicity of e-cigs is relatively well known, there is currently a lack of data on their long-term health effects. In this context, the aim of our study was to compare, on a mouse model and using a nose-only exposure system, the in vivo genotoxic and mutagenic potential of e-cig aerosols tested at two power settings (18 W and 30 W) and conventional cigarette (3R4F) smoke. The standard comet assay, micronucleus test and Pig-a gene mutation assay were performed after subacute (4 days), subchronic (3 months) and chronic (6 months) exposure. The generation of oxidative stress was also assessed by measuring the 8-hydroxy-2'-deoxyguanosine and by using the hOGG1-modified comet assay. Our results show that only the high-power e-cig and the 3R4F cigarette induced oxidative DNA damage in the lung and the liver of exposed mice. In return, no significant increase in chromosomal aberrations or gene mutations were noted whatever the type of product. This study demonstrates that e-cigs, at high-power setting, should be considered, contrary to popular belief, as hazardous products in terms of genotoxicity in mouse model.

Analysis of furans and pyridines from new generation heated tobacco product in Japan

BackgroundIn recent years, heated tobacco products (HTPs), which are widely used in Japan, have been sold by various brands using additives such as flavors. It has been reported that the components of mainstream smoke are different from those of conventional cigarettes. In this study, we established an analytical method for furans and pyridines in the mainstream smoke, which are characteristic of HTPs and particularly harmful among the generated components, and investigated the amount of component to which the smokers are exposed.MethodsWe established a simple analytical method for simultaneous analysis of gaseous and particulate compounds in the mainstream smoke of HTPs (IQOS, glo, ploom S) in Japan by combining a sorbent cartridge and glass fiber filter (Cambridge filter pad (CFP)). Both the sorbent cartridge and CFP were extracted using 2-propanol and analyzed via GC-MS/MS to determine the concentration of furans and pyridines generated from each HTP.ResultsThe results showed that the levels of target furans such as furfural, 2-furanmethanol, 2(5H)-furanone, and 5-methylfurfural tended to be higher in the mainstream smoke of glo than in standard cigarettes (3R4F). Pyridine, which is generated at a high level in 3R4F as a combustion component, and 4-ethenylpyridine (EP), which is a known marker of environmental tobacco smoke, were detected. Among these components, 2-furanmethanol and pyridine are classified as Group 2B (possibly carcinogenic to humans) by the International Agency for Research on Cancer (IARC). Therefore, it is possible that they will contribute to the health effects caused by use of HTPs.ConclusionsUsing the new collection and analytical method for furans and pyridines in the mainstream smoke of HTPs, the level of each compound to which smokers are exposed could be clarified. By comprehensively combining information on the amount of ingredients and toxicity, it will be possible to perform a more detailed calculation of the health risks of using HTPs. In addition, the components detected in this study may be the causative substances of indoor pollution through exhaled smoke and sidestream smoke; therefore, environmental research on the chemicals generated from HTPs would be warranted in future studies.

Robustness of HPHC Reduction in THS 2.2 Aerosol Relative to 3R4F Reference Cigarette Smoke under Extreme Climatic Conditions

Summary In order to assess robustness for the reduction of harmful and potentially harmful constituent (HPHC) levels generated by the Tobacco Heating System 2.2 (THS 2.2), a heated tobacco product, we compared the aerosol of this product with mainstream smoke from the 3R4F reference cigarette under different conditions of temperature and humidity. The desired climatic conditions were achieved by using an air-conditioning system coupled with the smoking-machine housing. Two extreme climatic conditions were selected, representing a “Hot and Dry” climate (30 °C and 35% relative humidity RH) and a “Hot and Very Humid” climate (30 °C and 75% RH). In addition, aerosol and smoke were generated using the standard conditions recognized for smoking-machine analyses of tobacco products (22 °C and 60% RH), which were close to the climatic conditions defined for “Subtropical and Mediterranean” environments (25 °C and 60% RH). The experimental conditions were chosen to simulate the use of THS 2.2 and cigarettes under extreme conditions of temperature and humidity. HeatSticks and cigarettes taken from freshly opened packs were subjected to short-term conditioning from two to a few more days under the same experimental conditions. We analyzed 54 HPHCs in THS 2.2 aerosol and 3R4F cigarette smoke, generated in accordance with the Health Canada Intense (HCI) standard, using modified temperature and humidity conditions for sample conditioning and machine-smoking experiments. We used a volume-adjusted approach for comparing HPHC reductions across the different climatic conditions investigated. Although a single puffing regimen was used, the total puff volume recorded for the 3R4F cigarette smoke varied due to the influence of temperature and humidity on combustion rate, which justified the use of a volume-adjusted approach. Volume-adjusted yields were derived from HPHC yields expressed in mass-per-tobacco stick normalized per total puff volume. The results indicated that, regardless of the considered climatic conditions, the HPHC levels investigated in THS 2.2 aerosol were reduced by at least 90%, on average, when compared with the concentrations in 3R4F cigarette mainstream smoke. This confirmed the robustness in performance for THS 2.2 to deliver reduced levels of HPHCs under the extreme climatic conditions investigated in this study. In order to further characterize the robustness of these reductions, the lowest reduction performance achieved for individual HPHCs across all climatic conditions was used to define the threshold for a robust reduction. The majority of the 54 HPHCs investigated in THS 2.2 aerosol showed more than 90% reduction. Calculations derived from nicotine-adjusted yields also confirmed robust reductions for all investigated HPHCs. The small differences in absolute reduction between the volume- and nicotine-adjusted approaches were predominantly attributed to a combination of the differences in both nominal nicotine deliveries and total puff volumes between THS 2.2 and 3R4F cigarettes; however, this did not influence the determination of robustness. Our findings confirm the value of this approach for assessing the robustness of a product’s performance under different climatic conditions.

E-cigarettes induce toxicity comparable to tobacco cigarettes in airway epithelium from patients with COPD

BackgroundThe health effects of e-cigarettes in patients with pre-existing lung disease are unknown. The aim of this study was to investigate whether aerosols from a fourth-generation e-cigarette produces similar in-vitro cytotoxic, DNA damage and inflammatory effects on bronchial epithelial cells (BECs) from patients with COPD, as cigarette smoke. MethodsBECs from patients with COPD who underwent surgery for lung cancer and comparator (immortalised 16HBE) cells were grown at air liquid interface (ALI). BECs were exposed to aerosols from a JUUL® e-cigarette (Virginia Tobacco and Menthol pods at 5% nicotine strength) or reference 3R4F cigarette for 30 min at ALI. Cell cytotoxicity, DNA damage and inflammation were measured. ResultsIn response to the Virginia Tobacco and Menthol flavoured e-cigarette aerosols, COPD BECs showed comparable LDH release (cell cytotoxicity, p = 0.59, p = 0.67 respectively), DNA damage (p = 0.41, p = 0.51) and inflammation (IL-8, p = 0.20, p = 0.89 and IL-6, p = 0.24, p = 0.93), to cigarette smoke. 16HBE cells also showed comparable cellular responses to cigarette smoke. ConclusionIn airway cells from patients with COPD, aerosols from a fourth-generation e-cigarette were associated with similar toxicity to cigarette smoke. These results have potential implications for the safety of e-cigarette use in patients with lung disease.

B-Cell Activating Factor secreted by neutrophils is a critical player in lung inflammation to cigarette smoke exposure

B cell activating factor (BAFF) is a TNF family cytokine mainly produced by adaptive immune cells in particular B and T lymphocytes. Depending on the context, innate immune cells can also produce BAFF. First of all, BAFF was known to play an important role in the maturation and survival of B lymphocytes in humoral immune response. Recent data indicate that BAFF may also participate in the regulation of innate immune responses and in the pathophysiology of pulmonary diseases. In models of mice chronically exposed to cigarette smoke (CS), BAFF plays a major role in the generation of pulmonary antinuclear antibodies and tertiary lymphoid follicles. However, the implication of BAFF in the development of innate immunity to cigarette smoking remains unknown. Wild-type or BAFF deficient mice were exposed to 3R4F cigarette smoke 3 times a day for 4 days. Mice were treated or not with mouse anti-GR1 or recombinant mouse BAFF during CS exposure. Broncho-alveolar lavage fluids (BALF) were done and cell infiltration was determined. Different cytokines and chemokines were measured in BALF and lung homogenates. Immunostaining on cytospin were done. Bone marrow derived neutrophils (BMDN) and Murine tracheal epithelial cells (MTEC) were stimulated in vitro by CSE (Cigarette Smoke Extract). Cell viability and BAFF levels were determined. Acute CS exposure promotes BAFF expression in airway recruited neutrophils. Immunostaining analysis revealed that neutrophils are the major source of BAFF. We confirmed in vitro that neutrophils secrete BAFF in response to CSE stimulation. Neutrophil depletion partially decreases BAFF expression in lung tissue and bronchoalveolar space suggesting additional sources of BAFF. Importantly, BAFF deficient mice displayed decreased airway neutrophil recruiting chemokines and neutrophil influx while the addition of exogenous BAFF significantly enhanced this CS-induced neutrophilic inflammation. These results demonstrate that BAFF is a key pro-inflammatory cytokine and that innate immune cells in particular neutrophils, are an unconsidered source of BAFF in early stages of CS-induced innate immunity.

Discriminating Spontaneous From Cigarette Smoke and THS 2.2 Aerosol Exposure-Related Proliferative Lung Lesions in A/J Mice by Using Gene Expression and Mutation Spectrum Data.

Mice, especially A/J mice, have been widely employed to elucidate the underlying mechanisms of lung tumor formation and progression and to derive human-relevant modes of action. Cigarette smoke (CS) exposure induces tumors in the lungs; but, non-exposed A/J mice will also develop lung tumors spontaneously with age, which raises the question of discriminating CS-related lung tumors from spontaneous ones. However, the challenge is that spontaneous tumors are histologically indistinguishable from the tumors occurring in CS-exposed mice. We conducted an 18-month inhalation study in A/J mice to assess the impact of lifetime exposure to Tobacco Heating System (THS) 2.2 aerosol relative to exposure to 3R4F cigarette smoke (CS) on toxicity and carcinogenicity endpoints. To tackle the above challenge, a 13-gene gene signature was developed based on an independent A/J mouse CS exposure study, following by a one-class classifier development based on the current study. Identifying gene signature in one data set and building classifier in another data set addresses the feature/gene selection bias which is a well-known problem in literature. Applied to data from this study, this gene signature classifier distinguished tumors in CS-exposed animals from spontaneous tumors. Lung tumors from THS 2.2 aerosol-exposed mice were significantly different from those of CS-exposed mice but not from spontaneous tumors. The signature was also applied to human lung adenocarcinoma gene expression data (from The Cancer Genome Atlas) and discriminated cancers in never-smokers from those in ever-smokers, suggesting translatability of our signature genes from mice to humans. A possible application of this gene signature is to discriminate lung cancer patients who may benefit from specific treatments (i.e., EGFR tyrosine kinase inhibitors). Mutational spectra from a subset of samples were also utilized for tumor classification, yielding similar results. “Landscaping” the molecular features of A/J mouse lung tumors highlighted, for the first time, a number of events that are also known to play a role in human lung tumorigenesis, such as Lrp1b mutation and Ros1 overexpression. This study shows that omics and computational tools provide useful means of tumor classification where histopathological evaluation alone may be unsatisfactory to distinguish between age- and exposure-related lung tumors.

Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx.

The larynx is an essential organ in the respiratory tract and necessary for airway protection, respiration, and phonation. Cigarette smoking is a significant risk factor associated with benign and malignant laryngeal diseases. Despite this association, the underlying mechanisms by which cigarette smoke (CS) drives disease development are not well elucidated. In the current study, we developed a short-term murine whole body inhalation model to evaluate the first CS-induced cellular responses in the glottic [i.e. vocal fold (VF)] and subglottic regions of the larynx. Specifically, we investigated epithelial cell proliferation, cell death, surface topography, and mucus production, at various time points (1 day, 5 days, 10 days) after ∼ 2 h exposure to 3R4F cigarettes (Delivered dose: 5.6968 mg/kg per cigarette) and following cessation for 5 days after a 5 day CS exposure (CSE). CSE elevated levels of BrdU labeled proliferative cells and p63 labeled epithelial basal cells on day 1 in the VF. CSE increased proliferative cells in the subglottis at days 5, 10 and following cessation in the subglottis. Cleaved caspase-3 apoptotic activity was absent in VF at all time points and increased at day 1 in the subglottis. Evaluation of the VF surface by scanning electron microscopy (SEM) revealed significant epithelial microprojection damage at day 10 and early signs of necrosis at days 5 and 10 post-CSE. SEM visualizations additionally indicated the presence of deformed cilia at days 5 and 10 after CSE and post-cessation in the respiratory epithelium lined subglottis. In terms of mucin content, the impact of short-term CSE was observed only at day 10, with decreasing acidic mucin levels and increasing neutral mucin levels. Overall, these findings reveal regional differences in murine laryngeal cellular responses following short-term CSE and provide insight into potential mechanisms underlying CS-induced laryngeal disease development.