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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., (© 2021 PMI. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

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., (© 2021. The Author(s).)

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.

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., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: Smoking is an important risk factor for the development of chronic obstructive pulmonary disease and cardiovascular diseases. This study aimed to further elucidate the role of ceramides, as a key lipid class dysregulated in disease states., Main Methods: In this article we developed and validated LC-MS/MS method for ceramides (Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1(15Z)) for the absolute quantification. We deployed it together with proteomics and transcriptomic analysis to assess the effects of cigarette smoke (CS) from the reference cigarette as well as aerosols from heat-not-burn (HnB) tobacco and e-vapor products in apolipoprotein E-deficient (ApoE -/- ) mice over several time points., Key Findings: In the lungs, CS exposure substantially elevated the ratios of Cer(d18:1/24:0) and Cer(d18:1/24:1) to Cer(d18:1/18:0) in two independent ApoE -/- mouse inhalation studies. Data from previous studies, in both ApoE -/- and wild-type mice, further confirmed the reproducibility of this finding. Elevation of these ceramide ratios was also observed in plasma/serum, the liver, and-for the Cer(d18:1/24:1(15Z)) to Cer(d18:1/18:0) ratio-the abdominal aorta. Also, the levels of acid ceramidase (Asah1) and glucocerebrosidase (Gba)-lysosomal enzymes involved in the hydrolysis of glucosylceramides-were consistently elevated in the lungs after CS exposure. In contrast, exposure to HnB tobacco product and e-vapor aerosols did not induce significant changes in the ceramide profiles or associated enzymes., Significance: Our work in mice contributes to the accumulating evidence on the importance of ceramide ratios as biologically relevant markers for respiratory disorders, adding to their already demonstrated role in cardiovascular disease risk assessment in humans., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Medical Informatics Association.)

Exposure to cigarette smoke (CS) causes detrimental health effects, increasing the risk of cardiovascular, pulmonary diseases and carcinogenesis in exposed individuals. The impact of CS on Inflammatory Bowel Disease (IBD) has been established by a number of epidemiological and clinical studies. In fact, CS is associated with a higher risk of developing Crohn's disease (CD) while inversely correlates with the development, disease risks, and relapse rate of ulcerative colitis (UC). To investigate the effect of CS exposure on experimental colitis, we performed a comprehensive and integrated comparative analysis of colon transcriptome and microbiome in mice exposed to dextran sodium sulfate (DSS) and CS. Colon transcriptome analysis revealed that CS downregulated specific pathways in a concentration-dependent manner, affecting both the inflammatory state and composition of the gut microbiome. Metagenomics analysis demonstrated that CS can modulate DSS-induced dysbiosis of specific bacterial genera, contributing to resolve the inflammation or accelerate recovery. The risks of smoking far outweigh any possible benefit, thus smoking cessation must always be encouraged because of its significant health benefits. However, the inverse association between active smoking and the development of UC cannot be ignored and the present study lays the foundation for investigating potential molecular mechanisms responsible for the attenuation of colitis by certain compounds of tobacco when decoupled from combustion.

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.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Justyna Szostak, Bjoern Titz, Emmanuel Guedj, Alain Sewer, Blaine Phillips, Patrice Leroy, Neau Laurent, Keyur Trivedi, Florian Martin, Nikolai V. Ivanov, Patrick Vanscheeuwijck, Manuel C. Peitsch, Julia Hoeng are employee of Philip Morris Products S.A., Neuchâtel, Switzerland. The work reported was paid by PMP S.A. Dr. Walter Schlage and Dr. Ansgar Buettner are consultants paid by Philip Morris Products S.A., Neuchâtel, Switzerland. Philip Morris Products S.A. is the sole source of funding and sponsor of this project., (Copyright © 2019. Published by Elsevier B.V.)

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., (Copyright © 2019. Published by Elsevier Ltd.)

We compared early biological changes in mice after inhalation exposures to cigarette smoke or e-vapor aerosols (MarkTen ® cartridge with Carrier, Test-1, or Test-2 formulations; 4% nicotine). Female C57BL/6 mice were exposed to 3R4F cigarette smoke or e-vapor aerosols by nose-only inhalation for up to 4 hours/day, 5 days/week, for 3 weeks. The 3R4F and e-vapor exposures were set to match the target nose port aerosol nicotine concentration (∼41 µg/L). Only the 3R4F group showed postexposure clinical signs such as tremors and lethargy. At necropsy, the 3R4F group had significant increases in lung weight and changes in bronchoalveolar lavage parameters, as well as microscopic findings in the respiratory tract. The e-vapor groups had minimal microscopic changes, including squamous metaplasia in laryngeal epiglottis, and histiocytic infiltrates in the lung (Test-2 group only). The 3R4F group had a higher incidence and severity of microscopic findings compared to any e-vapor group. Transcriptomic analysis also showed that the 3R4F group had the highest number of differentially expressed genes compared to Sham Control. Among e-vapor groups, Test-2 group had more differentially expressed genes but the magnitude of gene expression-based network perturbations in all e-vapor groups was ∼94% less than the 3R4F group. On proteome analysis in the lung, differentially regulated proteins were detected in the 3R4F group only. In conclusion, 3-weeks of 3R4F exposure induced molecular and microscopic changes associated with smoking-related diseases in the respiratory tract, while e-vapor exposures showed substantially reduced biological activities.

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., (Copyright © 2018 PMI R&D, Philip Morris Products, S.A. Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2018 PMI R&D, Philip Morris Products S.A. Published by Elsevier Ltd.. All rights reserved.)

Smoking is one of the major lifestyle-related risk factors for periodontal diseases. Modified risk tobacco products (MRTP) offer a promising alternative in the harm reduction strategy for adult smokers unable to quit. Using a systems toxicology approach, we investigated and compared the exposure effects of a reference cigarette (3R4F) and a heat-not-burn technology-based candidate MRTP, the Tobacco Heating System (THS) 2.2. Human gingival epithelial organotypic cultures were repeatedly exposed (3 days) for 28 min at two matching concentrations of cigarette smoke (CS) or THS2.2 aerosol. Results showed only minor histopathological alterations and minimal cytotoxicity upon THS2.2 aerosol exposure compared to CS (1% for THS2.2 aerosol vs. 30% for CS, at the high concentration). Among the 14 proinflammatory mediators analyzed, only 5 exhibited significant alterations with THS2.2 exposure compared with 11 upon CS exposure. Transcriptomic and metabolomic analysis indicated a general reduction of the impact in THS2.2 aerosol-exposed samples with respect to CS (∼79% lower biological impact for the high THS2.2 aerosol concentration compared to CS, and 13 metabolites significantly perturbed for THS2.2 vs. 181 for CS). This study indicates that exposure to THS2.2 aerosol had a lower impact on the pathophysiology of human gingival organotypic cultures than CS., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Experimental studies clearly demonstrate a causal effect of cigarette smoking on cardiovascular disease. To reduce the individual risk and population harm caused by smoking, alternative products to cigarettes are being developed. We recently reported on an apolipoprotein E-deficient (Apoe -/- ) mouse inhalation study that compared the effects of exposure to aerosol from a candidate modified risk tobacco product, Tobacco Heating System 2.2 (THS2.2), and smoke from the reference cigarette (3R4F) on pulmonary and vascular biology. Here, we applied a transcriptomics approach to evaluate the impact of the exposure to 3R4F smoke and THS2.2 aerosol on heart tissues from the same cohort of mice. The systems response profiles demonstrated that 3R4F smoke exposure led to time-dependent transcriptomics changes (False Discovery Rate (FDR) < 0.05; 44 differentially expressed genes at 3-months; 491 at 8-months). Analysis of differentially expressed genes in the heart tissue indicated that 3R4F exposure induced the downregulation of genes involved in cytoskeleton organization and the contractile function of the heart, notably genes that encode beta actin (Actb), actinin alpha 4 (Actn4), and filamin C (Flnc). This was accompanied by the downregulation of genes related to the inflammatory response. None of these effects were observed in the group exposed to THS2.2 aerosol., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study reports a comparative assessment of the biological impact of a heated tobacco aerosol from the tobacco heating system (THS) 2.2 and smoke from a combustible 3R4F cigarette. Human organotypic bronchial epithelial cultures were exposed to an aerosol from THS2.2 (a candidate modified-risk tobacco product) or 3R4F smoke at similar nicotine concentrations. A systems toxicology approach was applied to enable a comprehensive exposure impact assessment. Culture histology, cytotoxicity, secreted pro-inflammatory mediators, ciliary beating, and genome-wide mRNA/miRNA profiles were assessed at various time points post-exposure. Series of experimental repetitions were conducted to increase the robustness of the assessment. At similar nicotine concentrations, THS2.2 aerosol elicited lower cytotoxicity compared with 3R4F smoke. No morphological change was observed following exposure to THS2.2 aerosol, even at nicotine concentration three times that of 3R4F smoke. Lower levels of secreted mediators and fewer miRNA alterations were observed following exposure to THS2.2 aerosol than following 3R4F smoke. Based on the computational analysis of the gene expression changes, 3R4F (0.13 mg nicotine/L) elicited the highest biological impact (100%) in the context of Cell Fate, Cell Proliferation, Cell Stress, and Inflammatory Network Models at 4 h post-exposure. Whereas, the corresponding impact of THS2.2 (0.14 mg nicotine/L) was 7.6%., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In vitro toxicology approaches have evolved from a focus on molecular changes within a cell to understanding of toxicity-related mechanisms in systems that can mimic the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offers a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus, the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. Aligned with 3Rs strategy, this study aimed to explore the relevance of a human 3-D nasal culture model to assess the toxicological impact of aerosols generated from a candidate modified risk tobacco product (cMRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F. A series of experimental repetitions, where multiple concentrations of THS2.2 aerosol and 3R4F smoke were applied, were conducted to obtain reproducible measurements to understand the cellular/molecular changes that occur following exposure. In agreement with "Toxicity Testing in the 21st Century - a Vision and a Strategy", this study implemented a systems toxicology approach and found that for all tested concentrations the impact of 3R4F smoke was substantially greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles.

Modified risk tobacco products (MRTPs) are being developed with the aim of reducing smoking-related health risks. The Tobacco Heating System 2.2 (THS2.2) is a candidate MRTP that uses the heat-not-burn principle. Here, systems toxicology approaches were engaged to assess the respiratory effects of mentholated THS2.2 (THS2.2M) in a 90-day rat inhalation study (OECD test guideline 413). The standard endpoints were complemented by transcriptomics and quantitative proteomics analyses of respiratory nasal epithelium and lung tissue and by lipidomics analysis of lung tissue. The adaptive response of the respiratory nasal epithelium to conventional cigarette smoke (CS) included squamous cell metaplasia and an inflammatory response, with high correspondence between the molecular and histopathological results. In contrast to CS exposure, the adaptive tissue and molecular changes to THS2.2M aerosol exposure were much weaker and were limited mostly to the highest THS2.2M concentration in female rats. In the lung, CS exposure induced an inflammatory response, triggered cellular stress responses, and affected sphingolipid metabolism. These responses were not observed or were much lower after THS2.2M aerosol exposure. Overall, this system toxicology analysis complements and reconfirms the results from classical toxicological endpoints and further suggests potentially reduced health risks of THS2.2M., (Copyright © 2016. Published by Elsevier Inc.)

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.

Smoking cigarettes is a major risk factor in the development and progression of cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD). Modified risk tobacco products (MRTPs) are being developed to reduce smoking-related health risks. The goal of this study was to investigate hallmarks of COPD and CVD over an 8-month period in apolipoprotein E-deficient mice exposed to conventional cigarette smoke (CS) or to the aerosol of a candidate MRTP, tobacco heating system (THS) 2.2. In addition to chronic exposure, cessation or switching to THS2.2 after 2 months of CS exposure was assessed. Engaging a systems toxicology approach, exposure effects were investigated using physiology and histology combined with transcriptomics, lipidomics, and proteomics. CS induced nasal epithelial hyperplasia and metaplasia, lung inflammation, and emphysematous changes (impaired pulmonary function and alveolar damage). Atherogenic effects of CS exposure included altered lipid profiles and aortic plaque formation. Exposure to THS2.2 aerosol (nicotine concentration matched to CS, 29.9 mg/m(3)) neither induced lung inflammation or emphysema nor did it consistently change the lipid profile or enhance the plaque area. Cessation or switching to THS2.2 reversed the inflammatory responses and halted progression of initial emphysematous changes and the aortic plaque area. Biological processes, including senescence, inflammation, and proliferation, were significantly impacted by CS but not by THS2.2 aerosol. Both, cessation and switching to THS2.2 reduced these perturbations to almost sham exposure levels. In conclusion, in this mouse model cessation or switching to THS2.2 retarded the progression of CS-induced atherosclerotic and emphysematous changes, while THS2.2 aerosol alone had minimal adverse effects., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.)

The impact of cigarette smoke (CS), a major cause of lung diseases, on the composition and metabolism of lung lipids is incompletely understood. Here, we integrated quantitative lipidomics and proteomics to investigate exposure effects on lung lipid metabolism in a C57BL/6 and an Apolipoprotein E-deficient (Apoe(-/-)) mouse study. In these studies, mice were exposed to high concentrations of 3R4F reference CS, aerosol from potential modified risk tobacco products (MRTPs) or filtered air (Sham) for up to 8 months. The 2 assessed MRTPs, the prototypical MRTP for C57BL/6 mice and the Tobacco Heating System 2.2 for Apoe(-/-) mice, utilize "heat-not-burn" technologies and were each matched in nicotine concentrations to the 3R4F CS. After 2 months of CS exposure, some groups were either switched to the MRTP or underwent cessation. In both mouse strains, CS strongly affected several categories of lung lipids and lipid-related proteins. Candidate surfactant lipids, surfactant proteins, and surfactant metabolizing proteins were increased. Inflammatory eicosanoids, their metabolic enzymes, and several ceramide classes were elevated. Overall, CS induced a coordinated lipid response controlled by transcription regulators such as SREBP proteins and supported by other metabolic adaptations. In contrast, most of these changes were absent in the mice exposed to the potential MRTPs, in the cessation group, and the switching group. Our findings demonstrate the complex biological response of the lungs to CS exposure and support the benefits of cessation or switching to a heat-not-burn product using a design such as those employed in this study., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.)

Cigarette smoke increases the risk for respiratory and other diseases. Although smoking prevalence has declined over the years, millions of adults choose to continue to smoke. Modified risk tobacco products (MRTPs) are potentially valuable tools for adult smokers that are unwilling to quit their habit. Here, we investigated the biological impact of a candidate MRTP, the tobacco-heating system (THS) 2.2, compared to that of the 3R4F reference cigarette in normal primary human bronchial epithelial cells. Chemical characterization of the THS 2.2 aerosol showed reduced levels of harmful constituents compared to those of a combustible cigarette. Multiparametric indicators of cellular toxicity were measured via real-time cellular analysis and high-content screening. The study was complemented by a whole transcriptome analysis, followed by computational approaches to identify and quantify perturbed molecular pathways. Exposure of cells to 3R4F cigarette smoke resulted in a dose-dependent response in most toxicity end points. Moreover, we found a significant level of perturbation in multiple biological pathways, particularly in those related to cellular stress. By contrast, exposure to THS 2.2 resulted in an overall lower biological impact. At 3R4F doses, no toxic effects were observed. A toxic response was observed for THS 2.2 in some functional end points, but the responses occurred at doses between 3 and 15 times higher than those of 3R4F. The level of biological network perturbation was also significantly reduced following THS 2.2 aerosol exposure compared to that of 3R4F cigarette smoke. Taken together, the data suggest that THS 2.2 aerosol is less toxic than combustible cigarette smoke and thus may have the potential to reduce the risk for smoke-related diseases.

Organotypic 3D cultures of epithelial cells are grown at the air-liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in coculture models in which additional cell types such as fibroblasts were incorporated, the presence of another cell type could mask the response of the other. This study reports the impact of whole cigarette smoke (CS) exposure on organotypic mono- and coculture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: monoculture of bronchial epithelial cells without fibroblasts (BR) and coculture with fibroblasts (BRF) models. Adenylate kinase (AK)-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators into the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators was identified in the basolateral media of the monoculture than in the coculture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the monoculture epithelium model without fibroblasts. Finally, our results indicated that an in vivo smoking-induced xenobiotic metabolism response of bronchial epithelial cells was better reflected from the in vitro CS-exposed coculture model., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.)

Background: Mouse models are useful for studying cigarette smoke (CS)-induced chronic pulmonary pathologies such as lung emphysema. To enhance translation of large-scale omics data from mechanistic studies into pathophysiological changes, we have developed computational tools based on reverse causal reasoning (RCR)., Objective: In the present study we applied a systems biology approach leveraging RCR to identify molecular mechanistic explanations of pathophysiological changes associated with CS-induced lung emphysema in susceptible mice., Methods: The lung transcriptomes of five mouse models (C57BL/6, ApoE (-/-) , A/J, CD1, and Nrf2 (-/-) ) were analyzed following 5-7 months of CS exposure., Results: We predicted 39 molecular changes mostly related to inflammatory processes including known key emphysema drivers such as NF-κB and TLR4 signaling, and increased levels of TNF-α, CSF2, and several interleukins. More importantly, RCR predicted potential molecular mechanisms that are less well-established, including increased transcriptional activity of PU.1, STAT1, C/EBP, FOXM1, YY1, and N-COR, and reduced protein abundance of ITGB6 and CFTR. We corroborated several predictions using targeted proteomic approaches, demonstrating increased abundance of CSF2, C/EBPα, C/EBPβ, PU.1, BRCA1, and STAT1., Conclusion: These systems biology-derived candidate mechanisms common to susceptible mouse models may enhance understanding of CS-induced molecular processes underlying emphysema development in mice and their relevancy for human chronic obstructive pulmonary disease.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

Exposure to cigarette smoke is a leading cause of lung diseases including chronic obstructive pulmonary disease and cancer. Cigarette smoke is a complex aerosol containing over 6000 chemicals and thus it is difficult to determine individual contributions to overall toxicity as well as the molecular mechanisms by which smoke constituents exert their effects. We selected three well-known harmful and potentially harmful constituents (HPHCs) in tobacco smoke, acrolein, formaldehyde and catechol, and established a high-content screening method using normal human bronchial epithelial cells, which are the first bronchial cells in contact with cigarette smoke. The impact of each HPHC was investigated using 13 indicators of cellular toxicity complemented with a microarray-based whole-transcriptome analysis followed by a computational approach leveraging mechanistic network models to identify and quantify perturbed molecular pathways. HPHCs were evaluated over a wide range of concentrations and at different exposure time points (4, 8, and 24 h). By high-content screening, the toxic effects of the three HPHCs could be observed only at the highest doses. Whole-genome transcriptomics unraveled toxicity mechanisms at lower doses and earlier time points. The most prevalent toxicity mechanisms observed were DNA damage/growth arrest, oxidative stress, mitochondrial stress, and apoptosis/necrosis. A combination of multiple toxicological end points with a systems-based impact assessment allows for a more robust scientific basis for the toxicological assessment of HPHCs, allowing insight into time- and dose-dependent molecular perturbations of specific biological pathways. This approach allowed us to establish an in vitro systems toxicology platform that can be applied to a broader selection of HPHCs and their mixtures and can serve more generally as the basis for testing the impact of other environmental toxicants on normal bronchial epithelial cells.