3R4F Reference Cigarette Research Articles

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]

Impact of inhalation exposure to cigarette smoke on the pathogenesis of pulmonary hypertension primed by monocrotaline in rats.

Extensive, long-term exposure to cigarette smoke (CS) was recently suggested to be a risk factor for pulmonary hypertension, although further validation is required. The vascular effects of CS share similarities with the etiology of pulmonary hypertension, including vascular inflammation and remodeling. Thus, we examined the influence of CS exposure on the pathogenesis of monocrotaline (MCT)-induced pulmonary hypertension, hypothesizing that smoking might accelerate the development of primed pulmonary hypertension. CS was generated from 3R4F reference cigarettes, and rats were exposed to CS by inhalation at total particulate matter concentrations of 100-300 μg/L for 4h/day, 7days/week for 4weeks. Following 1 week of initial exposure, rats received 60 mg/kg MCT and were sacrificed and analyzed after an additional 3weeks of exposure. MCT induced hypertrophy in pulmonary arterioles and increased the Fulton index, a measure of right ventricular hypertrophy. Additional CS exposure exacerbated arteriolar hypertrophy but did not further elevate the Fulton index. No significant alterations were observed in levels of endothelin-1 and vascular endothelial growth factor, or in hematological and serum biochemical parameters. Short-term inhalation exposure to CS exacerbated arteriolar hypertrophy in the lung, although this effect did not directly aggravate the overworked heart under the current experimental conditions.

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.

Impact of different ventilation conditions on tobacco smoke-associated particulate matter emissions in a car cabin using the TAPaC platform

Despite antagonizing attempts from the tobacco industry, passive inhalation of tobacco smoke is known to be cancerogenic and toxic to human health for decades. Nonetheless, millions of non-smoking adults and children are still victims of second-hand smoke. Accumulation of particulate matter (PM) in confined spaces such as the car are particularly harmful due to high concentrations. We here aimed to analyze the specific effects of ventilation conditions in the setting of a car. By the use of the measuring platform TAPaC (tobacco-associated particulate matter emissions inside a car cabin), 3R4F reference cigarettes, Marlboro red, and Marlboro gold were smoked in a car interior with a volume of 3.709 m3. Seven different ventilation conditions (C1–C7) were analyzed. Under C1, all windows were closed. Under C2–C7, the car ventilation was turned on power level 2/4 with the air directed towards the windshield. Only the passenger side window was opened, where an outer placed fan could create an airstream speed of 15.9–17.4 km/h at one meter distance to simulate a driving car. C2: Window 10 cm opened. C3: Window 10 cm opened with the fan turned on. C4: Window half-opened. C5: Window half-opened with the fan turned on. C6: Window fully opened. C7: Window fully opened with the fan turned on. Cigarettes were remotely smoked by an automatic environmental tobacco smoke emitter and a cigarette smoking device. Depending on the ventilation condition the cigarettes emitted different mean PM concentrations after 10 min under condition C1 (PM10: 1272–1697 µg/m3, PM2.5: 1253–1659 µg/m3, PM1: 964–1263 µg/m3) under C2, C4, and C6 (PM10: 68.7–196.2 µg/m3, PM2.5: 68.2–194.7 µg/m3, PM1: 66.1–183.8 µg/m3) C3, C5, and C7 (PM10: 73.7–139 µg/m3, PM2.5: 72–137.9 µg/m3, PM1:68.9–131.9 µg/m3). Vehicle ventilation is insufficient to protect passengers from toxic second-hand smoke completely. Brand-specific variations of tobacco ingredients and mixtures markedly influence PM emissions under ventilation conditions. The most efficient ventilation mode to reduce PM exposure was achieved by opening the passenger´s window 10 cm and turning the onboard ventilation on power level 2/4. In-vehicle smoking should be banned to preserve innocent risk groups (e.g., children) from harm.

Non-nicotine constituents in cigarette smoke extract enhance nicotine addiction through monoamine oxidase A inhibition.

Tobacco addiction has been largely attributed to nicotine, a component in tobacco leaves and smoke. However, extensive evidence suggests that some non-nicotine components of smoke should not be overlooked when considering tobacco dependence. Yet, their individual effect and synergistic effect on nicotine reinforcement remain poorly understood. The study herein focused on the role of non-nicotine constituents in promoting the effects of nicotine and their independent reinforcing effects. Denicotinized cigarettes were prepared by chemical extracting of cut tobacco, and the cigarette smoke extracts (CSE, used as a proxy for non-nicotine ingredients) were obtained by machine-smoking the cigarettes and DMSO extraction. The compositions of harmful components, nicotine, and other minor alkaloids in both cut tobacco and the CSE of experimental denicotinized cigarettes were examined by GC-MS, and compared with 3R4F reference cigarettes. individually and in synergy with nicotine were determined by conditioned place preference (CPP), dopamine (DA) level detection, the open field test (OFT), and the elevated plus maze (EPM). Finally, the potential enhancement mechanism of non-nicotinic constituents was investigated by nicotine metabolism and monoamine oxidase A (MAOA) activity inhibition in the striatum of mice and human recombinant MAOA. Thenicotine content in smoke from the experimental denicotinized cigarettes (under ISO machine-smoking conditions) was reduced by 95.1% and retained most minor alkaloids, relative to the 3R4F reference cigarettes. It was found that non-nicotine constituents increased acute locomotor activities. This was especially pronounced for DA levels in NAc and CPP scores, decreased the time in center zone. There were no differences in these metrics with DNC group when compared to the NS group. Non-nicotine constituents alone did not show reinforcing effects in CPP or striatum DA levels in mice. However, in the presence of nicotine, non-nicotine constituents further increased the reinforcing effects. Furthermore, non-nicotine constituents may enhance nicotine's reinforcing effects by inhibiting striatum MAOA activity rather than affecting nicotine metabolism or total striatum DA content in mice. These findings expand our knowledge of the effect on smoking reinforcement of non-nicotine constituents found in tobacco products.

P07-38 Comparative cytotoxicity study of whole cigarette smoke condensates generated from heated tobacco products and 3R4F reference cigarette

Food-producing animals are infected by a number of ectoparasites that can result in production losses. Control of these parasites is largely based on the use of chemicals that are either applied topically to the skin or sometimes given parenterally. Most ectoparasiticides are neurotoxins, exerting their effect on the nervous system of the target parasite. As a result of frequent and inappropriate use, resistance of arthropods to the lethal effects of parasiticides has been reported in many parts of the world. Ectoparasiticides can also be toxic to mammals and restrictions are therefore applied to many of the compounds indicated for use in food-producing animals to ensure that unacceptable residues are not present in products intended for human consumption. Consequently many international organizations and countries have issued their own pesticide maximum residual limits in international trade.

TAPaC—tobacco-associated particulate matter emissions inside a car cabin: establishment of a new measuring platform

BackgroundParticulate matter (PM) emission caused by tobacco combustion leads to severe health burdens worldwide. Second-hand smoke exposure is extraordinarily high in enclosed spaces (e.g., indoor rooms, car cabins) and poses a particular threat to the health of vulnerable individuals (e.g., children, elderly, etc.). This study aimed to establish a new measuring platform and investigate PM emissions under four different ventilation conditions inside a car cabin without exposing any person to harmful tobacco smoke.MethodsPM concentrations were measured during the smoking of 3R4F reference cigarettes in a Mitsubishi Space Runner (interior volume 3.709 m3). The cigarettes were smoked with a machine, eliminating exposure of the researchers. Cigarettes were extinguished 4.5 min after ignition, and PM measurements continued until 10 min after ignition.ResultsHigh mean PM concentrations were measured for cigarettes without ventilation after 4.5 min (PM10: 1150 µg/m3, PM2.5: 1132 µg/m3, PM1: 861.6 µg/m3) and after 10 min (PM10: 1608 µg/m3, PM2.5: 1583 µg/m3, PM1: 1133 µg/m3). 3R4F smoked under conditions with turned on ventilation resulted in reduction of PM compared to those smoked without ventilation after 4.5 min (PM10:-47.5 to -58.4%, PM2.5:-47.2 to -58%, PM1:-39.6 to -50.2%) and after 10 min (PM10:-70.8 to -74.4%, PM2.5:-70.6 to -74.3%, PM1:-64.0 to -68.0%). Cigarettes smoked without ventilation generated high PM peaks at 4.5 min (PM10: 2207 µg/m3, PM2.5: 2166 µg/m3, PM1: 1421 µg/m3) and at 10 min (PM10: 1989 µg/m3, PM2.5: 1959 µg/m3, PM1: 1375 µg/m3). PM peaks of cigarettes smoked under different ventilation modes varied at 4.5 min (PM10: 630-845 µg/m3, PM2.5: 625-836 µg/m3, PM1: 543 - 693 µg/m3) and 10 min (PM10: 124 - 130 µg/m3, PM2.5: 124 - 129 µg/m3, PM1: 118 - 124 µg/m3).ConclusionThe new measuring platform provides a safer way for researchers to investigate PM emissions of cigarettes. These data are comparable to published research and show that smoking in a parked vehicle with the windows closed generates harmful PM emissions even when the vehicle ventilation is in operation. Future studies should be carried out using the new measuring platform investigating PM exposure and PM distribution of in-vehicle smoking under a wide range of conditions.

HPHC Testing of Tobacco and Smoke to Examine Cigarette Temporal Variability

Summary Commercial cigarettes were analyzed for harmful and potentially harmful constituents (HPHCs) in tobacco and smoke to investigate temporal product variability independent of analytical variability over one week, one year, and three years. Cigarettes from the worldwide market with various design features were collected over a 3-year period, stored, and tested concurrently for HPHCs to minimize analytical variability; repeat testing of reference cigarette 3R4F was included as an analytical control for the study design. Physical parameters were found to be relatively consistent. No trends in variability were noted based on blend type, smoke analyte matrix, or magnitude of an HPHC's yield. Combustion-related HPHCs generally showed low variation. Long-term batch-to-batch variability was found to be higher than short-term variability for tobacco-related compounds that have the potential to vary over time due to weather and agronomic practices. “Tar”, nicotine, and carbon monoxide were tested in multiple labs and showed greater lab-to-lab variability than batch-to-batch variability across all phases. Based on the results of this study, commercial cigarette products appear to have relatively low product variability. The low analyte variability noted in this study with products tested under unconventionally controlled analytical conditions serves to indicate that analytical variability may be a significant contributor to overall variability for general product testing over time and in interlaboratory studies. Laboratory controls and using a matched reference product across studies and between laboratories are important to assess testing differences and variability.

A simple sampling method for quantification of hazardous volatile organic compounds in mainstream cigarette smoke: Method development and prestudy validation

Cigarette smoke can be fatal to human health, as it contains a plethora of hazardous compounds such as volatile organic compounds (VOCs). To assess the toxic effects of cigarette smoke, both qualitative and quantitative information about the VOCs in cigarette smoke are essential. However, sampling and analysis of cigarette smoke are not simple tasks and require complicated validation procedures. Here, we develop and verify a simple sampling method for the quantification of VOCs in mainstream cigarette smoke using three steps: direct collection of the mainstream cigarette smoke sample by gas-tight syringe, injection of the sample into a bag sampler for dilution, and extraction of VOCs into an adsorption-tube sampler for quantitation by thermal desorption-gas chromatography-mass spectrometry. This method has been employed successfully to quantify seven target VOCs (isoprene, methyl ethyl ketone, benzene, acrylonitrile, toluene, m-xylene, and styrene) in cigarette smoke from 3R4F reference cigarettes. The quantities of these major five VOCs (isoprene, methyl ethyl ketone, benzene, acrylonitrile, and toluene: average of 459 ± 646 ng mL−1), when measured from 3R4F samples based on our method, were comparable to or moderately higher than those measured based on the established standard methods (e.g., Health Canada Intense (462 ± 557 ng mL−1) and International Standardization Organization (356 ± 438 ng mL−1)). As such, we succeeded in reliably collecting and quantifying VOCs in cigarette smoke using a simple sampling method proposed in this research. The proposed method is thus recommended as an alternative to complicated and costly representative methods based on international guidelines.

A 3D in vitro comparison of two undiluted e-cigarette aerosol generating systems

In vitro studies play an important role in supporting the toxicological assessment of e-cigarettes, with many current methods reliant on sophisticated in vitro exposure systems designed for conventional cigarette testing. In this study, we have compared two distinct systems; the modified Vitrocell VC10 and Borgwaldt LM4E designed to deliver undiluted e-cigarette aerosol. We assessed the cytotoxicity response of 3D reconstituted lung tissue (MucilAir) exposed to undiluted aerosol from ePen3 (closed modular e-cigarette) using these two exposure systems. As the induced cytotoxicity profiles were comparable, we then compared these responses against historical eBox (open modular e-cigarette) and 3R4F reference cigarette data to show evolution of product technology. This latter approach was deemed possible by monitoring intrinsic donor-to-donor control variability over a three-year period, bridging between exposure systems and observed biological responses.Despite the differences in the technology, on a puff-by-puff basis these machines gave remarkably similar cytotoxicity profiles for ePen3, as determined by MTT, and consistency of pre-cytotoxicity markers: transepithelial electrical resistance (TEER), cilia beat frequency and cilia active area. When responses are compared as a function of exposed nicotine concentration, we see differences due to the dynamics of the exposure systems. The parity of responses between the systems in generated undiluted aerosol has allowed us to compare back to previously published eBox data, irrespective of aerosol generating system and MucilAir donor, showing how evolution from open systems to podmod e-cigarette design can make a step change in the cytotoxicity profile of the product.

Cigarette Smoke Extract Produces Superoxide in Aqueous Media by Reacting with Bicarbonate.

The toxicity of cigarette smoke (CS) is largely attributed to its ability to generate reactive oxygen species (ROS). Reportedly, CS generates superoxide in cell culture systems by stimulating the cells to produce superoxide and through direct chemical reactions with components of the culture media. In this study, we investigated CS-induced superoxide formation in biocompatible aqueous media and its characteristics. Cigarette smoke extract (CSE) and total particulate matter (TPM) were prepared from the mainstream smoke of 3R4F reference cigarettes. CSE and TPM generated superoxide in Hank’s balanced salt solution (HBSS), Dulbecco’s modified Eagle media (DMEM), and blood plasma, but not in distilled water and phosphate-buffered saline. Each constituent of HBSS in solution was tested, and bicarbonate was found to be responsible for the superoxide generation. More than half of the superoxide formation was abolished by pretreating CSE or TPM with peroxidase, indicating that the substrates of peroxidase, presumably peroxides and peroxy acids, mainly contributed to the superoxide production. In conclusion, the presence of bicarbonate in experimental conditions should be considered carefully in studies of the biological activity of CS. Furthermore, the local amount of bicarbonate in exposed tissues may be a determinant of tissue sensitivity to oxidative damage by CS.

Effects of antioxidant capacity on micronucleus induction by cigarette smoke in mammalian cells

We have compared micronucleus (MN) induction by cigarette smoke in the L5178Y, TK6, and CHL/IU cell lines. The test sample was total particulate matter of 3R4F reference cigarette smoke, suspended in DMSO. After 3-h treatment, with or without a rat liver S9 metabolic activation system, followed by 24-h recovery, dose-dependent MN increases were seen in all cell lines. However, CHL/IU and TK6 cells were more resistant than L5178Y cells (comparison by Benchmark Doses with PROAST software). 3R4F smoke generates reactive oxygen species (ROS). Therefore, we explored the relationship between the sensitivities to 3R4F smoke and the antioxidant capacities of the cell lines. While the total antioxidant capacities were not significantly different among the cell lines, cellular glutathione (GSH) was higher in CHL/IU cells than in L5178Y cells. Pretreatment of CHL/IU cells with a GSH precursor, N-acetylcysteine (NAC), reduced the genotoxicity/cytotoxicity of 3R4F, whereas an inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO), enhanced it. The effects of NAC and BSO were also seen after treatment with allyl isothiocyanate, a ROS-generating chemical, but not with mitomycin C, a ROS-independent genotoxicant. Pretreatment with NAC increased cellular thiol levels. From the present results, the genotoxicity and cytotoxicity of cigarette smoke differs among these cell lines in a manner that may be related to their antioxidant thiol levels.

Targeted Characterization of the Chemical Composition of JUUL Systems Aerosol and Comparison with 3R4F Reference Cigarettes and IQOS Heat Sticks

Aerosol constituent yields have been reported from a wide range of electronic nicotine delivery systems. No comprehensive study has been published on the aerosol constituents generated from the JUUL system. Targeted analyses of 53 aerosol constituents from the four JUUL products currently on the US market (Virginia Tobacco and Menthol flavored e-liquids in both 5.0% and 3.0% nicotine concentration by weight) was performed using non-intense and intense puffing regimens. All measurements were conducted by an ISO 17025 accredited contract research organization. JUUL product aerosol constituents were compared to published values for the 3R4F research cigarette and IQOS Regular and Menthol heated tobacco products. Across the four JUUL products and two puffing regimes, only 10/53 analytes were quantifiable, including only two carbonyls (known propylene glycol or glycerol degradants). The remaining analytes were primary ingredients, nicotine degradants and water. Average analyte reductions (excluding primary ingredients and water) for all four JUUL system aerosols tested were greater than 98% lower than 3R4F mainstream smoke, and greater than 88% lower than IQOS aerosol. In summary, chemical characterization and evaluation of JUUL product aerosols demonstrates a significant reduction in toxicants when compared to mainstream cigarette smoke from 3R4F reference cigarettes or aerosols from IQOS-heated tobacco products.

Investigation of the content of carbonyl compounds and specific nitrosamines of tobacco in cigarette smoke and in the aerosol of the tobacco heating system

Investigation of some toxic compounds concentrations in aerosol of new class of tobacco products - tobacco heating systems and comparing their levels with regular cigarettes aerosol is an important step in understanding whether these products are low-risk, as some manufacturers claim. Three popular in Russia cigarette brands, reference cigarette 3R4F and new heated tobacco products (HTP) were tested for levels of emissions of some harmful and potentially harmful constituents (HPHC). Aerosols of these products were collected under the ISO intense smoking regime and quantified for levels of N'-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, acrolein, acetaldehyde, formaldehyde. Levels of above mentioned HPHCs were measured by the high-performance liquid chromatography / mass spectrometry (HPLC/MS-MS). The results demonstrate that highest levels of measured HPHCs are obtained for reference cigarette 3R4F. As for popular in Russia cigarette brands their levels vary greatly and for some compounds can be very close to levels of 3R4F aerosol. Levels of all measured HPHCs in aerosol of HTP were significantly lower compared to all cigarettes. This can be explained by specific mechanism of HTPs aerosol formation without pyrolytic and pyrosynthetic reactions which are typical to all combustive tobacco products due to their high temperature of the process.

Systems toxicology study reveals reduced impact of heated tobacco product aerosol extract relative to cigarette smoke on premature aging and exacerbation effects in aged aortic cells in vitro

Aging and smoking are major risk factors for cardiovascular diseases (CVD). Our in vitro study compared, in the context of aging, the effects of the aerosol of Tobacco Heating System 2.2 (THS; an electrically heated tobacco product) and 3R4F reference cigarette smoke (CS) on processes that contribute to vascular pathomechanisms leading to CVD. Young and old human aortic smooth muscle cells (HAoSMC) were exposed to various concentrations of aqueous extracts (AE) from 3R4F CS [0.014–0.22 puffs/mL] or THS aerosol [0.11–1.76 puffs/mL] for 24 h. Key markers were measured by high-content imaging, transcriptomics profiling and multianalyte profiling. In our study, in vitro aging increased senescence, DNA damage, and inflammation and decreased proliferation in the HAoSMCs. At higher concentrations of 3R4F AE, young HAoSMCs behaved similarly to aged cells, while old HAoSMCs showed additional DNA damage and apoptosis effects. At 3R4F AE concentrations with the maximum effect, the THS AE showed no significant effect in young or old HAoSMCs. It required an approximately ten-fold higher concentration of THS AE to induce effects similar to those observed with 3R4F. These effects were independent of nicotine, which did not show a significant effect on HAoSMCs at any tested concentration. Our results show that 3R4F AE accelerates aging in young HAoSMCs and exacerbates the aging effect in old HAoSMCs in vitro, consistent with CS-related contributions to the risk of CVD. Relative to 3R4F AE, the THS AE showed a significantly reduced impact on HAoSMCs, suggesting its lower risk for vascular SMC-associated pathomechanisms leading to CVD.

Impact of 6-Month Exposure to Aerosols From Potential Modified Risk Tobacco Products Relative to Cigarette Smoke on the Rodent Gastrointestinal Tract.

Cigarette smoking causes adverse health effects that might occur shortly after smoking initiation and lead to the development of inflammation and cardiorespiratory disease. Emerging studies have demonstrated the role of the intestinal microbiome in disease pathogenesis. The intestinal microbiome is susceptible to the influence of environmental factors such as smoking, and recent studies have indicated microbiome changes in smokers. Candidate modified risk tobacco products (CMRTP) are being developed to provide substitute products to lower smoking-related health risks in smokers who are unable or unwilling to quit. In this study, the ApoE–/– mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F and aerosols from two CMRTPs based on the heat-not-burn principle [carbon-heated tobacco product 1.2 (CHTP 1.2) and tobacco heating system 2.2 (THS 2.2)] on the intestinal microbiome over a 6-month period. The effect of cessation or switching to CHTP 1.2 after 3 months of CS exposure was also assessed. Next-generation sequencing was used to evaluate the impact of CMRTP aerosols in comparison to CS on microbiome composition and gene expression in the digestive tract of mice. Our analyses highlighted significant gene dysregulation in response to 3R4F exposure at 4 and 6 months. The findings showed an increase in the abundance of Akkermansiaceae upon CS exposure, which was reversed upon cessation. Cessation resulted in a significant decrease in Akkemansiaceae abundance, whereas switching to CHTP 1.2 resulted in an increase in Lactobacillaceae abundance. These microbial changes could be important for understanding the effect of CS on gut function and its relevance to disease pathogenesis via the microbiome.

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.

A 6-month inhalation toxicology study in Apoe\u2212/\u2212 mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe−/− mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations—(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)—or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure–volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.