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.

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.

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.

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.

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.

A six-month systems toxicology inhalation/cessation study in ApoE-/- mice to investigate cardiovascular and respiratory exposure effects of modified risk tobacco products, CHTP 1.2 and THS 2.2, compared with conventional cigarettes.

Smoking is one of the major modifiable risk factors in the development and progression of chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD). Modified-risk tobacco products (MRTP) are being developed to provide substitute products for smokers who are unable or unwilling to quit, to lessen the smoking-related health risks. In this study, the ApoE-/- mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F, or aerosol from two potential MRTPs based on the heat-not-burn principle, carbon heated tobacco product 1.2 (CHTP1.2) and tobacco heating system 2.2 (THS 2.2), on the cardiorespiratory system over a 6-month period. In addition, cessation or switching to CHTP1.2 after 3 months of CS exposure was assessed. A systems toxicology approach combining physiology, histology and molecular measurements was used to evaluate the impact of MRTP aerosols in comparison to CS. CHTP1.2 and THS2.2 aerosols, compared with CS, demonstrated lower impact on the cardiorespiratory system, including low to absent lung inflammation and emphysematous changes, and reduced atherosclerotic plaque formation. Molecular analyses confirmed the lower engagement of pathological mechanisms by MRTP aerosols than CS. Both cessation and switching to CHTP1.2 reduced the observed CS effects to almost sham exposure levels.

Delivery efficiencies of constituents of combustion-derived aerosols across the air-liquid interface during in vitro exposures.

In vitro aerosol exposure of epithelial cells grown at the air-liquid interface is an experimental methodology widely used in respiratory toxicology. The exposure depends to a large part on the physicochemical properties of individual aerosol constituents, as they determine the transfer kinetics from the aerosol into the cells. We characterized the transfer of 70 cigarette smoke constituents from the smoke into aqueous samples exposed in the Vitrocell® 24/48 aerosol exposure system. The amounts of these compounds in the applied smoke were determined by trapping whole smoke in N,N-dimethylformamide and then compared with their amounts in smoke-exposed, phosphate-buffered saline, yielding compound specific delivery efficiencies. Delivery efficiencies of different smoke constituents differed by up to five orders of magnitude, which indicates that the composition of the applied smoke is not necessarily representative for the delivered smoke. Therefore, dose metrics for in vitro exposure experiments should, if possible, be based on delivered and not applied doses. A comparison to literature on in vivo smoke retention in the respiratory tract indicated that the same applies for smoke retention in the respiratory tract.

A 90-day OECD TG 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of the aerosol from the carbon heated tobacco product version 1.2 (CHTP1.2) compared with cigarette smoke. I. Inhalation exposure, clinical pathology and histopathology.

Within the framework of a systems toxicology approach, the inhalation toxicity of aerosol from a novel tobacco-heating potentially modified risk tobacco product (MRTP), the carbon-heated tobacco product (CHTP) 1.2, was characterized and compared with that of mainstream smoke (CS) from the 3R4F reference cigarette in a 90-day nose-only rat inhalation study in general accordance with OECD TG 413. CHTP1.2 is a heat-not-burn product using a carbon heat source to produce an aerosol that contains nicotine and tobacco flavor. At equal or twice the nicotine concentration in the test atmospheres, inhalation of CHTP1.2 aerosol led to a significantly lower exposure to harmful constituents and induced less respiratory tract irritation, systemic, and pathological effects compared with CS. Nasal epithelial changes were less pronounced in the CHTP1.2- than in the CS-exposed groups and reverted in the nicotine concentration-matched group after a recovery period. Lung inflammation was minimal in the CHTP1.2-treated groups compared with the moderate extent seen in the 3R4F groups. Many other toxicological endpoints evaluated did not show CHTP1.2 aerosol exposure-related effects, and no effects not seen for 3R4F were observed. These observations were consistent with findings from previous studies in which rats were exposed to MRTP aerosols containing similar nicotine concentrations.

A 90-day OECD TG 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of the aerosol from the carbon heated tobacco product version 1.2 (CHTP1.2) compared with cigarette smoke. II. Systems toxicology assessment.

Modified risk tobacco products (MRTPs) have the potential to reduce smoking-related health risks. The Carbon Heated Tobacco Product 1.2 (CHTP1.2) is a potential MRTP that uses a pressed carbon heat source to generate an aerosol by heating tobacco. Here, we report the results from the systems toxicology arm of a 90-day rat inhalation study (OECD test guideline 413) to assess the effects of CHTP1.2 aerosol compared with cigarette smoke (CS). Transcriptomics, proteomics, and lipidomics analyses complemented the standard endpoints. In the respiratory nasal epithelium, CS induced an adaptive tissue and inflammatory response, which was much weaker after CHTP1.2 aerosol exposure, mostly limited to the highest CHTP1.2 concentration (at twice the 3R4F CS concentration: 50 vs. 23 µg nicotine/L), in female rats. In the lungs, the effects of CS exposure included inflammatory and cellular stress responses, which were absent or much lower after CHTP1.2 aerosol exposure. Outside of the respiratory tract, CS and CHTP1.2 aerosol induced effects that were previously associated with exposure to any nicotine-containing aerosol, e.g., lower lipid concentrations in serum. Overall, this systems toxicology analysis complements and confirms the results from classical toxicological endpoints and further suggests potentially reduced respiratory health risks of CHTP1.2.

Assessment of the impact of aerosol from a potential modified risk tobacco product compared with cigarette smoke on human organotypic oral epithelial cultures under different exposure regimens.

Cigarette smoke (CS) is affecting considerably the oral mucosa. Heating, instead of burning, tobacco reduces consistently the amount of toxic compounds and may exert a lower impact on oral health than combusted cigarettes. The carbon-heated tobacco product 1.2 (CHTP1.2) is a potential modified risk tobacco product (MRTP) based on heat-not-burn technology. Using a systems toxicology assessment framework, we compared the effects of exposure to CHTP1.2 aerosol with those of CS from a reference cigarette (3R4F). Human organotypic cultures derived from buccal and gingival epithelia were exposed acutely (28-min) or repeatedly (28 min/day for 3 days), respectively, to two matching concentrations of CHTP1.2 aerosol or 3R4F CS, and a non-diluted (100%) CHTP1.2 aerosol. The results showed an absence of cytotoxicity, reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP1.2 aerosol compared with 3R4F CS. Changes in mRNA and miRNA expression were linked by an integrative analysis approach, suggesting a regulatory role of miRNAs in several smoke/disease-relevant biological processes induced by 3R4F CS. The identification of mechanisms by which potential MRTPs can reduce the impact of tobacco use on biological systems is of great importance in understanding the molecular basis of the smoking harm reduction paradigm.

Comparative biological impacts of an aerosol from carbon-heated tobacco and smoke from cigarettes on human respiratory epithelial cultures: A systems toxicology assessment.

The biological impact of an aerosol of a potential modified-risk tobacco product, carbon heated tobacco product 1.2 (CHTP1.2), was comprehensively assessed for the first time in vitro using human small airway and nasal epithelial models following a systems toxicology approach. The potentially reduced effects of CHTP1.2 aerosol exposure were benchmarked against those of 3R4F cigarette smoke at similar nicotine concentrations. Experimental repetitions were conducted for which new batches of small airway and nasal cultures were exposed to CHTP1.2 aerosol or 3R4F smoke for 28 minutes. The biological impacts were determined based on a collection of endpoints including morphology, cytotoxicity, proinflammatory mediator profiles, cytochrome P450 1A1/1B1 activity, global mRNA and microRNA changes and proteome profiles. Alterations in mRNA expression were detected in cultures exposed to CHTP1.2 aerosol, without noticeable morphological changes and cytotoxicity, and minimal impact on proinflammatory mediator and proteome profiles. The changes linked to CHTP1.2 aerosol exposure, when observed, were transient. However, the impact of 3R4F smoke exposure persisted long post-exposure and greater than CHTP1.2 aerosol. Morphological changes were observed only in cultures exposed to 3R4F smoke. The lower biological effects of CHTP1.2 aerosol than 3R4F smoke exposure were observed similarly in both small airway and nasal epithelial cultures.

Quantifying the risk-reduction potential of new Modified Risk Tobacco Products.

Quantitative risk assessment of novel Modified Risk Tobacco Products (MRTP) must rest on indirect measurements that are indicative of disease development prior to epidemiological data becoming available. For this purpose, a Population Health Impact Model (PHIM) has been developed to estimate the reduction in the number of deaths from smoking-related diseases following the introduction of an MRTP. One key parameter of the model, the F-factor, describes the effective dose upon switching from cigarette smoking to using an MRTP. Biomarker data, collected in clinical studies, can be analyzed to estimate the effects of switching to an MRTP as compared to quitting smoking. Based on transparent assumptions, a link function is formulated that translates these effects into the F-factor. The concepts of 'lack of sufficiency' and 'necessity' are introduced, allowing for a parametrization of a family of link functions. These can be uniformly sampled, thus providing different 'scenarios' on how biomarker-based evidence can be translated into the F-factor to inform the PHIM.

Toxicity of the main electronic cigarette components, propylene glycol, glycerin, and nicotine, in Sprague-Dawley rats in a 90-day OECD inhalation study complemented by molecular endpoints.

While the toxicity of the main constituents of electronic cigarette (ECIG) liquids, nicotine, propylene glycol (PG), and vegetable glycerin (VG), has been assessed individually in separate studies, limited data on the inhalation toxicity of them is available when in mixtures. In this 90-day subchronic inhalation study, Sprague-Dawley rats were nose-only exposed to filtered air, nebulized vehicle (saline), or three concentrations of PG/VG mixtures, with and without nicotine. Standard toxicological endpoints were complemented by molecular analyses using transcriptomics, proteomics, and lipidomics. Compared with vehicle exposure, the PG/VG aerosols showed only very limited biological effects with no signs of toxicity. Addition of nicotine to the PG/VG aerosols resulted in effects in line with nicotine effects observed in previous studies, including up-regulation of xenobiotic enzymes (Cyp1a1/Fmo3) in the lung and metabolic effects, such as reduced serum lipid concentrations and expression changes of hepatic metabolic enzymes. No toxicologically relevant effects of PG/VG aerosols (up to 1.520  mg PG/L + 1.890 mg VG/L) were observed, and no adverse effects for PG/VG/nicotine were observed up to 438/544/6.6 mg/kg/day. This study demonstrates how complementary systems toxicology analyses can reveal, even in the absence of observable adverse effects, subtoxic and adaptive responses to pharmacologically active compounds such as nicotine.

Mechanistic Evaluation of the Impact of Smoking and Chronic Obstructive Pulmonary Disease on the Nasal Epithelium.

Chronic obstructive pulmonary disease (COPD) is one of the major causes of chronic morbidity and mortality worldwide. The development of markers of COPD onset is hampered by the lack of accessibility to the primary target tissue, and there is a need to consider other sample sources as surrogates for biomarker research. Airborne toxicants pass through the nasal epithelium before reaching the lower airways, and the similarity with bronchial histology makes it an attractive surrogate for lower airways. In this work, we describe the transcriptomics findings from the nasal epithelia of subjects enrolled in a clinical study focusing on the identification of COPD biomarkers. Transcriptomic data were analyzed using the biological network approach that enabled us to pinpoint the biological processes affected in the upper respiratory tract in response to smoking and mild-to-moderate COPD. Our results indicated that nasal and lower airway immune responses were considerably different in COPD subjects and caution should be exercised when using upper airway samples as a surrogate for the lower airway. Nevertheless, the network approach described here could present a sensitive means of identifying smokers at risk of developing COPD.

Multicomponent aerosol particle deposition in a realistic cast of the human upper respiratory tract.

Inhalation of aerosols generated by electronic cigarettes leads to deposition of multiple chemical compounds in the human airways. In this work, an experimental method to determine regional deposition of multicomponent aerosols in an in vitro segmented, realistic human lung geometry was developed and applied to two aerosols, i.e. a monodisperse glycerol aerosol and a multicomponent aerosol. The method comprised the following steps: (1) lung cast model preparation, (2) aerosol generation and exposure, (3) extraction of deposited mass, (4) chemical quantification and (5) data processing. The method showed good agreement with literature data for the deposition efficiency when using a monodisperse glycerol aerosol, with a mass median aerodynamic diameter (MMAD) of 2.3 µm and a constant flow rate of 15 L/min. The highest deposition surface density rate was observed in the bifurcation segments, indicating inertial impaction deposition. The experimental method was also applied to the deposition of a nebulized multicomponent aerosol with a MMAD of 0.50 µm and a constant flow rate of 15 L/min. The deposited amounts of glycerol, propylene glycol and nicotine were quantified. The three analyzed compounds showed similar deposition patterns and fractions as for the monodisperse glycerol aerosol, indicating that the compounds most likely deposited as parts of the same droplets. The developed method can be used to determine regional deposition for multicomponent aerosols, provided that the compounds are of low volatility. The generated data can be used to validate aerosol deposition simulations and to gain insight in deposition of electronic cigarette aerosols in human airways.

Characterization of the Vitrocell® 24/48 aerosol exposure system for its use in exposures to liquid aerosols.

BACKGROUND: The Vitrocell® 24/48 is an advanced aerosol exposure system that has been widely used and characterized for exposure studies of cigarette smoke, but not for exposure to liquid aerosols with a low gas-vapor phase content such as the ones generated by electronic cigarettes. An experimental system characterization for this specific application was therefore performed. METHODS: Glycerol model aerosols of different particle size distributions, produced by a condensation monodisperse aerosol generator, were used for exposing small volumes of phosphate-buffered saline in the Vitrocell® 24/48. Disodium fluorescein, added as a tracer in the aerosol, allowed the exact aerosol mass deposition to be quantified fluorometrically. RESULTS: The aerosol mass delivery efficiency within the system showed variations in the range of ±25%. Aerosol dilution was not fully reflected in aerosol delivery, the achieved aerosol delivery should therefore be determined experimentally. Quartz crystal microbalances underestimated the deposition of liquid aerosols. Unequal delivery of particles of different sizes was detectable, although this effect is unlikely to be relevant under applied experimental conditions. CONCLUSIONS: The Vitrocell® 24/48 aerosol exposure system can be used for exposures to liquid aerosols, such as those generated by electronic cigarettes. However, our results indicate that, compared with aerosol studies of cigarettes, a higher variability is to be expected.

Physiological and biological characterization of smokers with and without COPD.

Chronic obstructive pulmonary disease (COPD) is a common inflammatory airway disease predominantly associated with cigarette smoking, and its incidence is increasing worldwide. According to the Global Initiative for Obstructive Lung Disease (GOLD) guidelines, spirometry is used to diagnose the disease. However, owing to its complexity, spirometry alone may not account for the multitude of COPD phenotypes or the early, asymptomatic lung damage seen in younger smokers. In addition, suitable biomarkers enabling early diagnosis, guiding treatment and estimating prognosis are still scarce, although large scale 'omics analyses have added to the spectrum of potential biomarkers that could be used for these purposes. The aim of the current study was to comprehensively profile patients with mild-to-moderate COPD and compare the profiles to i) a group of currently smoking asymptomatic subjects, ii) a group of healthy former smokers, and iii) a group of healthy subjects that had never smoked. The assessment was conducted at the molecular level using proteomics, transcriptomics, and lipidomics and complemented by a series of measurements of traditional and emerging indicators of lung health (ClinicalTrials.gov identifier: NCT01780298). In this data note, we provide a comprehensive description of the study population's physiological characteristics including full lung function, lung appearance on chest computed tomography, impulse oscillometry, and exercise tolerance and quality of life (QoL) measures.

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.