Blood and urine multi-omics analysis of the impact of e-vaping, smoking, and cessation: from exposome to molecular responses.

Cigarette smoking is a major preventable cause of morbidity and mortality. While quitting smoking is the best option, switching from cigarettes to non-combustible alternatives (NCAs) such as e-vapor products is a viable harm reduction approach for smokers who would otherwise continue to smoke. A key challenge for the clinical assessment of NCAs is that self-reported product use can be unreliable, compromising the proper evaluation of their risk reduction potential. In this cross-sectional study of 205 healthy volunteers, we combined comprehensive exposure characterization with in-depth multi-omics profiling to compare effects across four study groups: cigarette smokers (CS), e-vapor users (EV), former smokers (FS), and never smokers (NS). Multi-omics analyses included metabolomics, transcriptomics, DNA methylomics, proteomics, and lipidomics. Comparison of the molecular effects between CS and NS recapitulated several previous observations, such as increased inflammatory markers in CS. Generally, FS and EV demonstrated intermediate molecular effects between the NS and CS groups. Stratification of the FS and EV by combustion exposure markers suggested that this position on the spectrum between CS and NS was partially driven by non-compliance/dual use. Overall, this study highlights the importance of in-depth exposure characterization before biological effect characterization for any NCA assessment study.

Tobacco Alkaloid Assessment in a DSS-Induced Colitis Mouse Model with a Fully Humanized Immune System.

Inflammatory bowel disease (IBD) refers to chronic intestinal immune-mediated diseases including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease (CD). Epidemiological, clinical, and preclinical evidence has highlighted the potential anti-inflammatory properties of naturally occurring alkaloids. In the present study, we investigated the potential anti-inflammatory activities of the tobacco alkaloids nicotine and anatabine in a dextran sulfate sodium (DSS)-induced UC mouse model with a fully humanized immune system. Our results show that nicotine significantly reduced all acute colitis symptoms and improved colitis-specific endpoints, including histopathologically assessed colon inflammation, tissue damage, and mononuclear cell infiltration. The tobacco alkaloid anatabine showed similar effectiveness trends, although they were generally weaker or not significant. Gene expression analysis in the context of biological network models of IBD further pinpointed a possible mechanism by which nicotine attenuated DSS-induced colitis in humanized mice. The current study enables further investigation of possible molecular mechanisms by which tobacco alkaloids attenuate UC symptoms.

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.

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

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

A meta-analysis of microRNAs expressed in human aerodigestive epithelial cultures and their role as potential biomarkers of exposure response to nicotine-containing products.

The expression of some microRNAs (miRNA) is modulated in response to cigarette smoke (CS), which is a leading cause of major preventable diseases. However, whether miRNA expression is also modulated by the aerosol/extract from potentially reduced-risk products is not well studied. The present work is a meta-analysis of 12 in vitro studies in human organotypic epithelial cultures of the aerodigestive tract (buccal, gingival, bronchial, nasal, and small airway epithelia). These studies compared the effects of exposure to aerosols from electronic vapor (e-vapor) products and heated tobacco products, and to extracts from Swedish snus products (in the present work, will be referred to as reduced-risk products [RRPs]) on miRNA expression with the effects of exposure to CS or its total particulate matter fraction. This meta-analysis evaluated 12 datasets of a total of 736 detected miRNAs and 2775 exposed culture inserts. The t-distributed stochastic neighbor embedding method was used to find similarities across the diversity of miRNA responses characterized by tissue type, exposure type, and product concentration. The CS-induced changes in miRNA expression in gingival cultures were close to those in buccal cultures; similarly, the alterations in miRNA expression in small airway, bronchial, and nasal tissues resembled each other. A supervised clustering was performed to identify miRNAs exhibiting particular response patterns. The analysis identified a set of miRNAs whose expression was altered in specific tissues upon exposure to CS (e.g., miR-125b-5p, miR-132-3p, miR-99a-5p, and 146a-5p). Finally, we investigated the impact of RRPs on miRNA expression in relation to that of CS by calculating the response ratio r between the RRP- and CS-induced alterations at an individual miRNA level, showing reduced alterations in miRNA expression following RRP exposure relative to CS exposure (94 % relative reduction). No specific miRNA response pattern indicating exposure to aerosols from heated tobacco products and e-vapor products, or extracts from Swedish snus was identifiable.

Anatabine ameliorates intestinal inflammation and reduces the production of pro-inflammatory factors in a dextran sulfate sodium mouse model of colitis.

BACKGROUND: Inflammatory bowel disease (IBD) is the collective term for chronic immune-mediated diseases of unknown, multifactorial etiology, arising from the interplay between genetic and environmental factors and including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease. In the last few decades, naturally occurring alkaloids have gained interest because of their substantial anti-inflammatory effects in several animal models of disease. Studies on mouse models of IBD have demonstrated the anti-inflammatory action of the main tobacco alkaloid, nicotine. In addition, anatabine, a minor tobacco alkaloid also present in peppers, tomato, and eggplant presents anti-inflammatory properties in vivo and in vitro. In this study, we aimed to evaluate the anti-inflammatory properties of nicotine and anatabine in a dextran sulfate sodium (DSS) mouse model of UC. RESULTS: Oral administration of anatabine, but not nicotine, reduced the clinical symptoms of DSS-induced colitis. The result of gene expression analysis suggested that anatabine had a restorative effect on global DSS-induced gene expression profiles, while nicotine only had limited effects. Accordingly, MAP findings revealed that anatabine reduced the colonic abundance of DSS-associated cytokines and increased IL-10 abundance. CONCLUSIONS: Our results support the amelioration of inflammatory effects by anatabine in the DSS mouse model of UC, and suggest that anatabine constitutes a promising therapeutic agent for IBD treatment.

The reduction of DSS-induced colitis severity in mice exposed to cigarette smoke is linked to immune modulation and microbial shifts.

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.

Systems toxicology assessment of a representative e-liquid formulation using human primary bronchial epithelial cells.

The development of reduced-risk products aims to provide alternatives to cigarettes that present less risk of harm for adult smokers. Responsible use of flavoring substances in these products may fulfill an important role in product acceptance. While most flavoring substances used in such products are also used by the food industry and are considered safe when ingested, their impact when inhaled may require further assessment. To aid in such an assessment, a three-step approach combining real-time cellular analysis, phenotypic high-content screening assays, and gene expression analysis was developed and tested in normal human bronchial epithelial cells with 28 flavoring substances commonly used in e-liquid formulations, dissolved individually or as a mixture in a base solution composed of propylene glycol, vegetable glycerin, and 0.6% nicotine. By employing this approach, we identified individual flavoring substances that potentially contribute greatly to the overall mixture effect (citronellol and alpha-pinene). By assessing modified mixtures, we showed that, although cytotoxic effects were found when assessed individually, alpha-pinene did not contribute to the overall mixture cytotoxicity. Most of the cytotoxic effect appeared to be attributable to citronellol, with the remaining substances contributing due to synergistic effects. We developed and used different scoring methods (Tox-Score, Phenotypic Score, and Biological Impact Factor/Network Perturbation Amplitude), ultimately enabling a ranking based on cytotoxicity, phenotypic outcome, and molecular network perturbations. This case study highlights the benefits of testing both individual flavoring substances and mixtures for e-liquid flavor assessment and emphasized the importance of data sharing for the benefit of consumer safety.

How complex should an in vitro model be? Evaluation of complex 3D alveolar model with transcriptomic data and computational biological network models.

To more accurately model inhalation toxicity in vitro, we developed a tetra-culture system that combines lung alveolar epithelial cells, endothelial cells, macrophages, and mast cells in a three-dimensional orientation. We characterized the influence of the added complexity using network perturbation analysis and gene expression data. This will allow us to gain insight into the steady-state profile of the assembled, complete three-dimensional model using all four cell types and of simpler models of one, two, or three cell types. Gene expression data were analyzed using cause-and-effect biological network models, together with a quantitative network-scoring algorithm, to determine the biological impact of co-culturing the various cell types. In the assembled tetra-culture, macrophages appeared to be the largest contributors to overall network perturbations, promoting high basal levels of oxidative stress and inflammation. This finding led to further optimization of the model using rested macrophages; the addition of rested macrophages decreased the basal inflammatory and cell stress status of the co-culture. Finally, we compared transcriptional profiles from publicly available datasets of conventional in vitro models representative of the airways and of healthy human lung tissues to assess similarities between our model and other in vitro models and the human lung. On the transcriptional level, we found an increasing correlation between airway models and normal human lung tissue, particularly as cell types became more physiologically relevant and the complexity of the system increased. This indicates that the combination of multiple lung-relevant cell types in vitro does indeed increase similarity to the physiological counterpart.

In vitro systems toxicology-based assessment of the potential modified risk tobacco product CHTP 1.2 for vascular inflammation- and cytotoxicity-associated mechanisms promoting adhesion of monocytic cells to human coronary arterial endothelial cells.

Cigarette smoking causes cardiovascular diseases. Heating tobacco instead of burning it reduces the amount of toxic compounds in the aerosol and may exert a reduced impact on health compared with cigarette smoke. Aqueous extract from the aerosol of a potential modified risk tobacco product, the Carbon Heated Tobacco Product (CHTP) 1.2, was compared in vitro with aqueous extract from the smoke of a 3R4F reference cigarette for its impact on the adhesion of monocytic cells to artery endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated for 4 h with conditioned media from human monocytic Mono Mac 6 (MM6) cells exposed to CHTP1.2 or 3R4F extracts for 2 h or directly with those extracts freshly generated. In vitro monocyte-endothelial cell adhesion was measured concomitantly with inflammatory, oxidative stress, cytotoxicity, and death markers. Furthermore, transcriptomics analyses enabled to quantify the level of perturbation in HCAECs, and provide biological interpretation for the underlying molecular changes following exposure to 3R4F or CHTP1.2 extract. Our systems toxicology study demonstrated that approximately 10-15-fold higher concentrations of the CHTP 1.2 aerosol extract were needed to elicit similar effects as the 3R4F smoke extract on cardiovascular disease-relevant inflammation and cytotoxicity-related mechanisms and markers investigated in vitro.

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.

The biological effects of long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product.

Cigarette smoking is the leading cause of preventable lung cancer (LC). Reduction of harmful constituents by heating rather than combusting tobacco may have the potential to reduce the risk of LC. We evaluated functional and molecular changes in human bronchial epithelial BEAS-2B cells following a 12-week exposure to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product (cMRTP) in comparison with those following exposure to TPM from the 3R4F reference cigarette. Endpoints linked to lung carcinogenesis were assessed. Four-week 3R4F TPM exposure resulted in crisis and epithelial to mesenchymal transition (EMT) accompanied by decreased barrier function and disrupted cell-to-cell contacts. By week eight, cells regained E-cadherin expression, suggesting that EMT was reversible. Increased levels of inflammatory mediators were noted in cells treated to 3R4F TPM but not in cells treated to the same or a five-fold higher concentration of cMRTP TPM. A 20-fold higher concentration of cMRTP TPM increased oxidative stress and DNA damage and caused reversible EMT. Anchorage-independent growth was observed in cells treated to 3R4F or a high concentration of cMRTP TPM. 3R4F TPM-derived clones were invasive, while cMRTP TPM-derived clones were not. Long-term exposure to TPM from the cMRTP had a lower biological impact on BEAS-2B cells compared with that of exposure to TPM from 3R4F.