Assessment of inhalation toxicity of cigarette smoke and aerosols from flavor mixtures: 5-week study in A/J mice.

Most flavors used in e-liquids are generally recognized as safe for oral consumption, but their potential effects when inhaled are not well characterized. In vivo inhalation studies of flavor ingredients in e-liquids are scarce. A structure-based grouping approach was used to select 38 flavor group representatives (FGR) on the basis of known and in silico-predicted toxicological data. These FGRs were combined to create prototype e-liquid formulations and tested against cigarette smoke (CS) in a 5-week inhalation study. Female A/J mice were whole-body exposed for 6 h/day, 5 days/week, for 5 weeks to air, mainstream CS, or aerosols from (1) test formulations containing propylene glycol (PG), vegetable glycerol (VG), nicotine (N; 2% w/w), and flavor (F) mixtures at low (4.6% w/w), medium (9.3% w/w), or high (18.6% w/w) concentration or (2) base formulation (PG/VG/N). Male A/J mice were exposed to air, PG/VG/N, or PG/VG/N/F-high under the same exposure regimen. There were no significant mortality or in-life clinical findings in the treatment groups, with only transient weight loss during the early exposure adaptation period. While exposure to flavor aerosols did not cause notable lung inflammation, it caused only minimal adaptive changes in the larynx and nasal epithelia. In contrast, exposure to CS resulted in lung inflammation and moderate-to-severe changes in the epithelia of the nose, larynx, and trachea. In summary, the study evaluates an approach for assessing the inhalation toxicity potential of flavor mixtures, thereby informing the selection of flavor exposure concentrations (up to 18.6%) for a future chronic inhalation study.

A 6-month inhalation toxicology study in Apoe-/- 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.

Impact of whole-body versus nose-only inhalation exposure systems on systemic, respiratory, and cardiovascular endpoints in a 2-month cigarette smoke exposure study in the ApoE-/- mouse model.

Cigarette smoking is one major modifiable risk factor in the development and progression of chronic obstructive pulmonary disease and cardiovascular disease. To characterize and compare cigarette smoke (CS)-induced disease endpoints after exposure in either whole-body (WB) or nose-only (NO) exposure systems, we exposed apolipoprotein E-deficient mice to filtered air (Sham) or to the same total particulate matter (TPM) concentration of mainstream smoke from 3R4F reference cigarettes in NO or WB exposure chambers (EC) for 2 months. At matching TPM concentrations, we observed similar concentrations of carbon monoxide, acetaldehyde, and acrolein, but higher concentrations of nicotine and formaldehyde in NOEC than in WBEC. In both exposure systems, CS exposure led to the expected adaptive changes in nasal epithelia, altered lung function, lung inflammation, and pronounced changes in the nasal epithelial transcriptome and lung proteome. Exposure in the NOEC caused generally more severe histopathological changes in the nasal epithelia and a higher stress response as indicated by body weight decrease and lower blood lymphocyte counts compared with WB exposed mice. Erythropoiesis, and increases in total plasma triglyceride levels and atherosclerotic plaque area were observed only in CS-exposed mice in the WBEC group but not in the NOEC group. Although the composition of CS in the breathing zone is not completely comparable in the two exposure systems, the CS-induced respiratory disease endpoints were largely confirmed in both systems, with a higher magnitude of severity after NO exposure. CS-accelerated atherosclerosis and other pro-atherosclerotic factors were only significant in WBEC.

A 6-month systems toxicology inhalation study in ApoE-/- mice demonstrates reduced cardiovascular effects of E-vapor aerosols compared with cigarette smoke.

Smoking cigarettes is harmful to the cardiovascular system. Considerable attention has been paid to the reduced harm potential of alternative nicotine-containing inhalable products such as e-cigarettes. We investigated the effects of E-vapor aerosols or cigarette smoke (CS) on atherosclerosis progression, cardiovascular function, and molecular changes in the heart and aorta of female apolipoprotein E-deficient (ApoE-/-) mice. The mice were exposed 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 for up to 6 mo. Concentrations of CS and base or test aerosols were matched at 35 µg nicotine/L. Exposure to CS, compared with sham-exposed fresh air controls, accelerated atherosclerotic plaque formation, whereas no such effect was seen for any of the three E-vapor aerosols. Molecular changes indicated disease mechanisms related to oxidative stress and inflammation in general, plus changes in calcium regulation, and altered cytoskeletal organization and microtubule dynamics in the left ventricle. While ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following E-vapor aerosols exposure, the nicotine-containing base and test aerosols caused an increase in isovolumic relaxation time similar to CS. A nicotine-related increase in pulse wave velocity and arterial stiffness was also observed, but it was significantly lower for base and test aerosols than for CS. These results demonstrate that in comparison with CS, E-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular diseases.NEW & NOTEWORTHY Analysis of key urinary oxidative stress markers and proinflammatory cytokines showed an absence of oxidative stress and inflammation in the animals exposed to E-vapor aerosols. Conversely, animals exposed to conventional cigarette smoke had high urinary levels of these markers. When compared with conventional cigarette smoke, E-vapor aerosols induced smaller atherosclerotic plaque surface area and volume. Systolic and diastolic cardiac function, as well as endothelial function, were further significantly less affected by electronic cigarette aerosols than conventional cigarette smoke. Molecular analysis demonstrated that E-vapor aerosols induce significantly smaller transcriptomic dysregulation in the heart and aorta compared with conventional cigarette smoke.

Evaluation of toxicity of aerosols from flavored e-liquids in Sprague-Dawley rats in a 90-day OECD inhalation study, complemented by transcriptomics analysis.

The use of flavoring substances is an important element in the development of reduced-risk products for adult smokers to increase product acceptance and encourage switching from cigarettes. In a first step towards characterizing the sub-chronic inhalation toxicity of neat flavoring substances, a study was conducted using a mixture of the substances in a base solution of e-liquid, where the standard toxicological endpoints of the nebulized aerosols were supplemented with transcriptomics analysis. The flavor mixture was produced by grouping 178 flavors into 26 distinct chemical groups based on structural similarities and potential metabolic and biological effects. Flavoring substances predicted to show the highest toxicological effect from each group were selected as the flavor group representatives (FGR). Following Organization for Economic Cooperation and Development Testing Guideline 413, rats were exposed to three concentrations of the FGR mixture in an e-liquid composed of nicotine (23 µg/L), propylene glycol (1520 µg/L), and vegetable glycerin (1890 µg/L), while non-flavored and no-nicotine mixtures were included as references to identify potential additive or synergistic effects between nicotine and the flavoring substances. The results indicated that the inhalation of an e-liquid containing the mixture of FGRs caused very minimal local and systemic toxic effects. In particular, there were no remarkable clinical (in-life) observations in flavored e-liquid-exposed rats. The biological effects related to exposure to the mixture of neat FGRs were limited and mainly nicotine-mediated, including changes in hematological and blood chemistry parameters and organ weight. These results indicate no significant additive biological changes following inhalation exposure to the nebulized FGR mixture above the nicotine effects measured in this sub-chronic inhalation study. In a subsequent study, e-liquids with FGR mixtures will be aerosolized by thermal treatment and assessed for toxicity.

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

Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of Classic Tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESH™ technology.

We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air-liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.

Systems Toxicology Assessment of the Biological Impact of a Candidate Modified Risk Tobacco Product on Human Organotypic Oral Epithelial Cultures.

Cigarette smoke (CS) has been reported to increase predisposition to oral cancer and is also recognized as a risk factor for many conditions including periodontal diseases, gingivitis, and other benign mucosal disorders. Smoking cessation remains the most effective approach for minimizing the risk of smoking-related diseases. However, reduction of harmful constituents by heating rather than combusting tobacco, without modifying the amount of nicotine, is a promising new paradigm in harm reduction. In this study, we compared effects of exposure to aerosol derived from a candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, with those of CS generated from the 3R4F reference cigarette. Human organotypic oral epithelial tissue cultures (EpiOral, MatTek Corporation) were exposed for 28 min to 3R4F CS or THS2.2 aerosol, both diluted with air to comparable nicotine concentrations (0.32 or 0.51 mg nicotine/L aerosol/CS for 3R4F and 0.31 or 0.46 mg/L for THS2.2). We also tested one higher concentration (1.09 mg/L) of THS2.2. A systems toxicology approach was employed combining cellular assays (i.e., cytotoxicity and cytochrome P450 activity assays), comprehensive molecular investigations of the buccal epithelial transcriptome (mRNA and miRNA) by means of computational network biology, measurements of secreted proinflammatory markers, and histopathological analysis. We observed that the impact of 3R4F CS was greater than THS2.2 aerosol in terms of cytotoxicity, morphological tissue alterations, and secretion of inflammatory mediators. Analysis of the transcriptomic changes in the exposed oral cultures revealed significant perturbations in various network models such as apoptosis, necroptosis, senescence, xenobiotic metabolism, oxidative stress, and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) signaling. The stress responses following THS2.2 aerosol exposure were markedly decreased, and the exposed cultures recovered more completely compared with those exposed to 3R4F CS.

Effects of cigarette smoke, cessation and switching to a candidate modified risk tobacco product on the liver in Apoe -/- mice--a systems toxicology analysis.

The liver is one of the most important organs involved in elimination of xenobiotic and potentially toxic substances. Cigarette smoke (CS) contains more than 7000 chemicals, including those that exert biological effects and cause smoking-related diseases. Though CS is not directly hepatotoxic, a growing body of evidence suggests that it may exacerbate pre-existing chronic liver disease. In this study, we integrated toxicological endpoints with molecular measurements and computational analyses to investigate effects of exposures on the livers of Apoe(-/- )mice. Mice were exposed to 3R4F reference CS, to an aerosol from the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product (MRTP) or to filtered air (Sham) for up to 8 months. THS2.2 takes advantage of a "heat-not-burn" technology that, by heating tobacco, avoids pyrogenesis and pyrosynthesis. After CS exposure for 2 months, some groups were either switched to the MRTP or filtered air. While no group showed clear signs of hepatotoxicity, integrative analysis of proteomics and transcriptomics data showed a CS-dependent impairment of specific biological networks. These networks included lipid and xenobiotic metabolism and iron homeostasis that likely contributed synergistically to exacerbating oxidative stress. In contrast, most proteomic and transcriptomic changes were lower in mice exposed to THS2.2 and in the cessation and switching groups compared to the CS group. Our findings elucidate the complex biological responses of the liver to CS exposure. Furthermore, they provide evidence that THS2.2 aerosol has reduced biological effects, as compared with CS, on the livers of Apoe(-/- )mice.

Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarette smoke.

The objective of the study was to characterize the toxicity from sub-chronic inhalation of test atmospheres from the candidate modified risk tobacco product (MRTP), Tobacco Heating System version 2.2 (THS2.2), and to compare it with that of the 3R4F reference cigarette. A 90-day nose-only inhalation study on Sprague-Dawley rats was performed, combining classical and systems toxicology approaches. Reduction in respiratory minute volume, degree of lung inflammation, and histopathological findings in the respiratory tract organs were significantly less pronounced in THS2.2-exposed groups compared with 3R4F-exposed groups. Transcriptomics data obtained from nasal epithelium and lung parenchyma showed concentration-dependent differential gene expression following 3R4F exposure that was less pronounced in the THS2.2-exposed groups. Molecular network analysis showed that inflammatory processes were the most affected by 3R4F, while the extent of THS2.2 impact was much lower. Most other toxicological endpoints evaluated did not show exposure-related effects. Where findings were observed, the effects were similar in 3R4F- and THS2.2-exposed animals. In summary, toxicological changes observed in the respiratory tract organs of THS2.2 aerosol-exposed rats were much less pronounced than in 3R4F-exposed rats while other toxicological endpoints either showed no exposure-related effects or were comparable to what was observed in the 3R4F-exposed rats.

Evaluation of the Tobacco Heating System 2.2. Part 6: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of a mentholated version compared with mentholated and non-mentholated cigarette smoke.

The toxicity of a mentholated version of the Tobacco Heating System (THS2.2M), a candidate modified risk tobacco product (MRTP), was characterized in a 90-day OECD inhalation study. Differential gene and protein expression analysis of nasal epithelium and lung tissue was also performed to record exposure effects at the molecular level. Rats were exposed to filtered air (sham), to THS2.2M (at 15, 23 and 50 µg nicotine/l), to two mentholated reference cigarettes (MRC) (at 23 µg nicotine/l), or to the 3R4F reference cigarette (at 23 µg nicotine/l). MRCs were designed to meet 3R4F specifications. Test atmosphere analyses demonstrated that aldehydes were reduced by 75%-90% and carbon monoxide by 98% in THS2.2M aerosol compared with MRC smoke; aerosol uptake was confirmed by carboxyhemoglobin and menthol concentrations in blood, and by the quantities of urinary nicotine metabolites. Systemic toxicity and alterations in the respiratory tract were significantly lower in THS2.2M-exposed rats compared with MRC and 3R4F. Pulmonary inflammation and the magnitude of the changes in gene and protein expression were also dramatically lower after THS2.2M exposure compared with MRCs and 3R4F. No menthol-related effects were observed after MRC mainstream smoke-exposure compared with 3R4F.

Alterations in Serum Polyunsaturated Fatty Acids and Eicosanoids in Patients with Mild to Moderate Chronic Obstructive Pulmonary Disease (COPD).

Smoking is a major risk factor for several diseases including chronic obstructive pulmonary disease (COPD). To better understand the systemic effects of cigarette smoke exposure and mild to moderate COPD-and to support future biomarker development-we profiled the serum lipidomes of healthy smokers, smokers with mild to moderate COPD (GOLD stages 1 and 2), former smokers, and never-smokers (n = 40 per group) (ClinicalTrials.gov registration: NCT01780298). Serum lipidome profiling was conducted with untargeted and targeted mass spectrometry-based lipidomics. Guided by weighted lipid co-expression network analysis, we identified three main trends comparing smokers, especially those with COPD, with non-smokers: a general increase in glycero(phospho)lipids, including triglycerols; changes in fatty acid desaturation (decrease in ω-3 polyunsaturated fatty acids, and an increase in monounsaturated fatty acids); and an imbalance in eicosanoids (increase in 11,12- and 14,15-DHETs (dihydroxyeicosatrienoic acids), and a decrease in 9- and 13-HODEs (hydroxyoctadecadienoic acids)). The lipidome profiles supported classification of study subjects as smokers or non-smokers, but were not sufficient to distinguish between smokers with and without COPD. Overall, our study yielded further insights into the complex interplay between smoke exposure, lung disease, and systemic alterations in serum lipid profiles.

A framework for in vitro systems toxicology assessment of e-liquids.

Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an in vitro systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air-liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust in vitro assessment of e-liquids and their aerosols.

Toxicity of aerosols of nicotine and pyruvic acid (separate and combined) in Sprague-Dawley rats in a 28-day OECD 412 inhalation study and assessment of systems toxicology.

Toxicity of nebulized nicotine (Nic) and nicotine/pyruvic acid mixtures (Nic/Pyr) was characterized in a 28-day Organization for Economic Co-operation and Development 412 inhalation study with additional transcriptomic and lipidomic analyses. Sprague-Dawley rats were nose-only exposed, 6 h/day, 5 days/week to filtered air, saline, nicotine (50 µg/l), sodium pyruvate (NaPyr, 33.9 µg/l) or equimolar Nic/Pyr mixtures (18, 25 and 50 µg nicotine/l). Saline and NaPyr caused no health effects, but rats exposed to nicotine-containing aerosols had decreased body weight gains and concentration-dependent increases in liver weight. Blood neutrophil counts were increased and lymphocyte counts decreased in rats exposed to nicotine; activities of alkaline phosphatase and alanine aminotransferase were increased, and levels of cholesterol and glucose decreased. The only histopathologic finding in non-respiratory tract organs was increased liver vacuolation and glycogen content. Respiratory tract findings upon nicotine exposure (but also some phosphate-buffered saline aerosol effects) were observed only in the larynx and were limited to adaptive changes. Gene expression changes in the lung and liver were very weak. Nic and Nic/Pyr caused few significant changes (including Cyp1a1 gene upregulation). Changes were predominantly related to energy metabolism and fatty acid metabolism but did not indicate an obvious toxicity-related response. Nicotine exposure lowered plasma lipids, including cholesteryl ester (CE) and free cholesterol and, in the liver, phospholipids and sphingolipids. Nic, NaPyr and Nic/Pyr decreased hepatic triacylglycerol and CE. In the lung, Nic and Nic/Pyr increased CE levels. These data suggest that only minor biologic effects related to inhalation of Nic or Nic/Pyr aerosols were observed in this 28-day study.

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.

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.

Structural, functional, and molecular impact on the cardiovascular system in ApoE-/- mice exposed to aerosol from candidate modified risk tobacco products, Carbon Heated Tobacco Product 1.2 and Tobacco Heating System 2.2, compared with cigarette smoke.

AIM: To investigate the molecular, structural, and functional impact of aerosols from candidate modified risk tobacco products (cMRTP), the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2, compared with that of mainstream cigarette smoke (CS) on the cardiovascular system of ApoE-/- mice. METHODS: Female ApoE-/- mice were exposed to aerosols from THS 2.2 and CHTP 1.2 or to CS from the 3R4F reference cigarette for up to 6 months at matching nicotine concentrations. A Cessation and a Switching group (3 months exposure to 3R4F CS followed by filtered air or CHTP 1.2 for 3 months) were included. Cardiovascular effects were investigated by echocardiographic, histopathological, immunohistochemical, and transcriptomics analyses. RESULTS: Continuous exposure to cMRTP aerosols did not affect atherosclerosis progression, heart function, left ventricular (LV) structure, or the cardiovascular transcriptome. Exposure to 3R4F CS triggered atherosclerosis progression, reduced systolic ejection fraction and fractional shortening, caused heart LV hypertrophy, and initiated significant dysregulation in the transcriptomes of the heart ventricle and thoracic aorta. Importantly, the structural, functional, and molecular changes caused by 3R4F CS were improved in the smoking cessation and switching groups. CONCLUSION: Exposure to cMRTP aerosols lacked most of the CS exposure-related functional, structural, and molecular effects. Smoking cessation or switching to CHTP 1.2 aerosol caused similar recovery from the 3R4F CS effects in the ApoE-/- model, with no further acceleration of plaque progression beyond the aging-related rate.

Comparing the preclinical risk profile of inhalable candidate and potential candidate modified risk tobacco products: A bridging use case.

Cigarette smoking causes major preventable diseases, morbidity, and mortality worldwide. Smoking cessation and prevention of smoking initiation are the preferred means for reducing these risks. Less harmful tobacco products, termed modified-risk tobacco products (MRTP), are being developed as a potential alternative for current adult smokers who would otherwise continue smoking. According to a regulatory framework issued by the US Food and Drug Administration, a manufacturer must provide comprehensive scientific evidence that the product significantly reduces harm and the risk of tobacco-related diseases, in order to obtain marketing authorization for a new MRTP. For new tobacco products similar to an already approved predicate product, the FDA has foreseen a simplified procedure for assessing "substantial equivalence". In this article, we present a use case that bridges the nonclinical evidence from previous studies demonstrating the relatively reduced harm potential of two heat-not-burn products based on different tobacco heating principles. The nonclinical evidence was collected along a "causal chain of events leading to disease" (CELSD) to systematically follow the consequences of reduced exposure to toxicants (relative to cigarette smoke) through increasing levels of biological complexity up to disease manifestation in animal models of human disease. This approach leverages the principles of systems biology and toxicology as a basis for further extrapolation to human studies. The experimental results demonstrate a similarly reduced impact of both products on apical and molecular endpoints, no novel effects not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP that is comparable to that of complete smoking cessation. Ideally, a subset of representative assays from the presented sequence along the CELSD could be sufficient for predicting similarity or substantial equivalence in the nonclinical impact of novel products; this would require further validation, for which the present use case could serve as a starting point.

Respiratory Effects of Exposure to Aerosol From the Candidate Modified-Risk Tobacco Product THS 2.2 in an 18-Month Systems Toxicology Study With A/J Mice.

Smoking cessation is the most effective measure for reducing the risk of smoking-related diseases. However, switching to less harmful products (modified-risk tobacco products [MRTP]) can be an alternative to help reduce the risk for adult smokers who would otherwise continue to smoke. In an 18-month chronic carcinogenicity/toxicity study in A/J mice (OECD Test Guideline 453), we assessed the aerosol of Tobacco Heating System 2.2 (THS 2.2), a candidate MRTP based on the heat-not-burn principle, compared with 3R4F cigarette smoke (CS). To capture toxicity- and disease-relevant mechanisms, we complemented standard toxicology endpoints with in-depth systems toxicology analyses. In this part of our publication series, we report on integrative assessment of the apical and molecular exposure effects on the respiratory tract (nose, larynx, and lungs). Across the respiratory tract, we found changes in inflammatory response following 3R4F CS exposure (eg, antimicrobial peptide response in the nose), with both shared and distinct oxidative and xenobiotic responses. Compared with 3R4F CS, THS 2.2 aerosol exerted far fewer effects on respiratory tract histology, including adaptive tissue changes in nasal and laryngeal epithelium and inflammation and emphysematous changes in the lungs. Integrative analysis of molecular changes confirmed the substantially lower impact of THS 2.2 aerosol than 3R4F CS on toxicologically and disease-relevant molecular processes such as inflammation, oxidative stress responses, and xenobiotic metabolism. In summary, this work exemplifies how apical and molecular endpoints can be combined effectively for toxicology assessment and further supports findings on the reduced respiratory health risks of THS 2.2 aerosol.

Multi-omics systems toxicology study of mouse lung assessing the effects of aerosols from two heat-not-burn tobacco products and cigarette smoke.

Cigarette smoke (CS) causes adverse health effects and, for smoker who do not quit, modified risk tobacco products (MRTPs) can be an alternative to reduce the risk of developing smoking-related diseases. Standard toxicological endpoints can lack sensitivity, with systems toxicology approaches yielding broader insights into toxicological mechanisms. In a 6-month systems toxicology study on ApoE-/- mice, we conducted an integrative multi-omics analysis to assess the effects of aerosols from the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2-a potential and a candidate MRTP based on the heat-not-burn (HnB) principle-compared with CS at matched nicotine concentrations. Molecular exposure effects in the lungs were measured by mRNA/microRNA transcriptomics, proteomics, metabolomics, and lipidomics. Integrative data analysis included Multi-Omics Factor Analysis and multi-modality functional network interpretation. Across all five data modalities, CS exposure was associated with an increased inflammatory and oxidative stress response, and lipid/surfactant alterations. Upon HnB aerosol exposure these effects were much more limited or absent, with reversal of CS-induced effects upon cessation and switching to CHTP 1.2. Functional network analysis revealed CS-induced complex immunoregulatory interactions across the investigated molecular layers (e.g., itaconate, quinolinate, and miR-146) and highlighted the engagement of the heme-Hmox-bilirubin oxidative stress axis by CS. This work exemplifies how multi-omics approaches can be leveraged within systems toxicology studies and the generated multi-omics data set can facilitate the development of analysis methods and can yield further insights into the effects of toxicological exposures on the lung of mice.