Mitochondria as a possible target for nicotine action.

Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.

Gastroparesis: Quality of Life and Health Care Utilization.

BACKGROUND AND AIMS: Gastroparesis (GP) patients suffer from recurrent symptoms of nausea, vomiting, early satiety, and abdominal pain. The impact of GP on quality of life (QoL), health care utilization and daily activities is not well understood. MATERIALS AND METHODS: Part 1: 398 adult patients (≥18 y) with documented GP (symptoms >6 mo) were surveyed to assess QoL and pain using the Short Form 36 and McGill pain questionnaires. Part 2: 491 adult GP patients were surveyed to evaluate employment status, work and daily activities, medication use, physician visits, diagnostic testing, emergency room visits, and hospitalizations related to their GP symptoms. Both protocols received Institutional Review Board approval. RESULTS: Part 1: 250 fully evaluable surveys were returned (63%). The mean age was 46.8 years (range, 18 to 84 y); 78% were women. Mean Short Form 36 scores for mental health (56.9) and social functioning (68.1) were analogous to scores for patients with serious chronic medical disorders and depression. Pain scores were lower in patients with idiopathic GP compared with diabetic GP. Part 2: 228 evaluable surveys were analyzed (58.3% response rate). The mean age was 49.6 (19 to 86 y); 77.2% were women. GP symptoms reduced daily activities in 67.5% and lowered annual income in 28.5%. In total, 11% were disabled due to GP symptoms. CONCLUSIONS: GP reduces patients' QoL and places a significant financial burden on the health care system. Treatment strategies for GP patients should be broad based and incorporate pain management, psychological evaluation and management, and strategies to improve overall well-being with a return to work and daily activities.

Understanding the limits of animal models as predictors of human biology: lessons learned from the sbv IMPROVER Species Translation Challenge.

MOTIVATION: Inferring how humans respond to external cues such as drugs, chemicals, viruses or hormones is an essential question in biomedicine. Very often, however, this question cannot be addressed because it is not possible to perform experiments in humans. A reasonable alternative consists of generating responses in animal models and 'translating' those results to humans. The limitations of such translation, however, are far from clear, and systematic assessments of its actual potential are urgently needed. sbv IMPROVER (systems biology verification for Industrial Methodology for PROcess VErification in Research) was designed as a series of challenges to address translatability between humans and rodents. This collaborative crowd-sourcing initiative invited scientists from around the world to apply their own computational methodologies on a multilayer systems biology dataset composed of phosphoproteomics, transcriptomics and cytokine data derived from normal human and rat bronchial epithelial cells exposed in parallel to 52 different stimuli under identical conditions. Our aim was to understand the limits of species-to-species translatability at different levels of biological organization: signaling, transcriptional and release of secreted factors (such as cytokines). Participating teams submitted 49 different solutions across the sub-challenges, two-thirds of which were statistically significantly better than random. Additionally, similar computational methods were found to range widely in their performance within the same challenge, and no single method emerged as a clear winner across all sub-challenges. Finally, computational methods were able to effectively translate some specific stimuli and biological processes in the lung epithelial system, such as DNA synthesis, cytoskeleton and extracellular matrix, translation, immune/inflammation and growth factor/proliferation pathways, better than the expected response similarity between species. CONTACT: pmeyerr@us.ibm.com or Julia.Hoeng@pmi.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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

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

In Vitro Systems Toxicology Assessment of a Candidate Modified Risk Tobacco Product Shows Reduced Toxicity Compared to That of a Conventional Cigarette.

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.

Assessment of in vitro COPD models for tobacco regulatory science: Workshop proceedings, conclusions and paths forward for in vitro model use.

The Family Smoking Prevention and Tobacco Control Act of 2009 established the Food and Drug Administration Center for Tobacco Products (FDA-CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 8-10 December 2014, IIVS organised a workshop conference, entitled Assessment of In Vitro COPD Models for Tobacco Regulatory Science, to bring together stakeholders representing regulatory agencies, academia, industry and animal protection, to address the research priorities articulated by the FDA-CTP. Specific topics were covered to assess the status of current in vitro technologies as they are applied to understanding the adverse pulmonary events resulting from tobacco product exposure, and in particular, the progression of chronic obstructive pulmonary disease (COPD). The four topics covered were: a) Inflammation and Oxidative Stress; b) Ciliary Dysfunction and Ion Transport; c) Goblet Cell Hyperplasia and Mucus Production; and d) Parenchymal/Bronchial Tissue Destruction and Remodelling. The 2.5 day workshop included 18 expert speakers, plus poster sessions, networking and breakout sessions, which identified key findings and provided recommendations to advance the in vitro technologies and assays used to evaluate tobacco-induced disease etiologies. The workshop summary was reported at the 2015 Society of Toxicology Annual Meeting, and the recommendations led to an IIVS-organised technical workshop in June 2015, entitled Goblet Cell Hyperplasia, Mucus Production, and Ciliary Beating Assays, to assess these assays and to conduct a proof-of-principle multi-laboratory exercise to determine their suitability for standardisation. Here, we report on the proceedings, recommendations and outcomes of the December 2014 workshop, including paths forward to continue the development of non-animal methods to evaluate tissue responses that model the disease processes that may lead to COPD, a major cause of mortality worldwide.

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

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

A vascular biology network model focused on inflammatory processes to investigate atherogenesis and plaque instability.

BACKGROUND: Numerous inflammation-related pathways have been shown to play important roles in atherogenesis. Rapid and efficient assessment of the relative influence of each of those pathways is a challenge in the era of "omics" data generation. The aim of the present work was to develop a network model of inflammation-related molecular pathways underlying vascular disease to assess the degree of translatability of preclinical molecular data to the human clinical setting. METHODS: We constructed and evaluated the Vascular Inflammatory Processes Network (V-IPN), a model representing a collection of vascular processes modulated by inflammatory stimuli that lead to the development of atherosclerosis. RESULTS: Utilizing the V-IPN as a platform for biological discovery, we have identified key vascular processes and mechanisms captured by gene expression profiling data from four independent datasets from human endothelial cells (ECs) and human and murine intact vessels. Primary ECs in culture from multiple donors revealed a richer mapping of mechanisms identified by the V-IPN compared to an immortalized EC line. Furthermore, an evaluation of gene expression datasets from aortas of old ApoE-/- mice (78 weeks) and human coronary arteries with advanced atherosclerotic lesions identified significant commonalities in the two species, as well as several mechanisms specific to human arteries that are consistent with the development of unstable atherosclerotic plaques. CONCLUSIONS: We have generated a new biological network model of atherogenic processes that demonstrates the power of network analysis to advance integrative, systems biology-based knowledge of cross-species translatability, plaque development and potential mechanisms leading to plaque instability.

Systems biology approach for evaluating the biological impact of environmental toxicants in vitro.

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.

The response of human nasal and bronchial organotypic tissue cultures to repeated whole cigarette smoke exposure.

Exposure to cigarette smoke (CS) is linked to the development of respiratory diseases, and there is a need to understand the mechanisms whereby CS causes damage. Although animal models have provided valuable insights into smoking-related respiratory tract damage, modern toxicity testing calls for reliable in vitro models as alternatives for animal experimentation. We report on a repeated whole mainstream CS exposure of nasal and bronchial organotypic tissue cultures that mimic the morphological, physiological, and molecular attributes of the human respiratory tract. Despite the similar cellular staining and cytokine secretion in both tissue types, the transcriptomic analyses in the context of biological network models identified similar and diverse biological processes that were impacted by CS-exposed nasal and bronchial cultures. Our results demonstrate that nasal and bronchial tissue cultures are appropriate in vitro models for the assessment of CS-induced adverse effects in the respiratory system and promising alternative to animal experimentation.

In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers.

Organotypic culture of human primary bronchial epithelial cells is a useful in vitro system to study normal biological processes and lung disease mechanisms, to develop new therapies, and to assess the biological perturbations induced by environmental pollutants. Herein, we investigate whether the perturbations induced by cigarette smoke (CS) and observed in the epithelium of smokers' airways are reproducible in this in vitro system (AIR-100 tissue), which has been shown to recapitulate most of the characteristics of the human bronchial epithelium. Human AIR-100 tissues were exposed to mainstream CS for 7, 14, 21, or 28 min at the air-liquid interface, and we investigated various biological endpoints [e.g., gene expression and microRNA profiles, matrix metalloproteinase 1 (MMP-1) release] at multiple postexposure time points (0.5, 2, 4, 24, 48 h). By performing a Gene Set Enrichment Analysis, we observed a significant enrichment of human smokers' bronchial epithelium gene signatures derived from different public transcriptomics datasets in CS-exposed AIR-100 tissue. Comparison of in vitro microRNA profiles with microRNA data from healthy smokers highlighted various highly translatable microRNAs associated with inflammation or with cell cycle processes that are known to be perturbed by CS in lung tissue. We also found a dose-dependent increase of MMP-1 release by AIR-100 tissue 48 h after CS exposure in agreement with the known effect of CS on this collagenase expression in smokers' tissues. In conclusion, a similar biological perturbation than the one observed in vivo in smokers' airway epithelium could be induced after a single CS exposure of a human organotypic bronchial epithelium-like tissue culture.

Nicotine-mediated effects in neuronal and mouse models of synucleinopathy.

Introduction: Alpha-synuclein (α-Syn) aggregation, transmission, and contribution to neurotoxicity represent central mechanisms underlying Parkinson's disease. The plant alkaloid "nicotine" was reported to attenuate α-Syn aggregation in different models, but its precise mode of action remains unclear. Methods: In this study, we investigated the effect of 2-week chronic nicotine treatment on α-Syn aggregation, neuroinflammation, neurodegeneration, and motor deficits in D-line α-Syn transgenic mice. We also established a novel humanized neuronal model of α-Syn aggregation and toxicity based on treatment of dopaminergic neurons derived from human induced pluripotent stem cells (iPSC) with α-Syn preformed fibrils (PFF) and applied this model to investigate the effects of nicotine and other compounds and their modes of action. Results and discussion: Overall, our results showed that nicotine attenuated α-Syn-provoked neuropathology in both models. Moreover, when investigating the role of nicotinic acetylcholine receptor (nAChR) signaling in nicotine's neuroprotective effects in iPSC-derived dopaminergic neurons, we observed that while α4-specific antagonists reduced the nicotine-induced calcium response, α4 agonists (e.g., AZD1446 and anatabine) mediated similar neuroprotective responses against α-Syn PFF-provoked neurodegeneration. Our results show that nicotine attenuates α-Syn-provoked neuropathology in vivo and in a humanized neuronal model of synucleinopathy and that activation of α4ß2 nicotinic receptors might mediate these neuroprotective effects.

Causal biological network models for reactive astrogliosis: a systems approach to neuroinflammation.

Astrocytes play a central role in the neuroimmune response by responding to CNS pathologies with diverse molecular and morphological changes during the process of reactive astrogliosis. Here, we used a computational biological network model and mathematical algorithms that allow the interpretation of high-throughput transcriptomic datasets in the context of known biology to study reactive astrogliosis. We gathered available mechanistic information from the literature into a comprehensive causal biological network (CBN) model of astrocyte reactivity. The CBN model was built in the Biological Expression Language, which is both human-readable and computable. We characterized the CBN with a network analysis of highly connected nodes and demonstrated that the CBN captures relevant astrocyte biology. Subsequently, we used the CBN and transcriptomic data to identify key molecular pathways driving the astrocyte phenotype in four CNS pathologies: samples from mouse models of lipopolysaccharide-induced endotoxemia, Alzheimer's disease, and amyotrophic lateral sclerosis; and samples from multiple sclerosis patients. The astrocyte CBN provides a new tool to identify causal mechanisms and quantify astrogliosis based on transcriptomic data.

Toxicological Assessment of Flavor Ingredients in E-Vapor Products.

Many flavor ingredients are often used in potentially reduced-risk tobacco products (such as e-vapor products). Although most are "generally recognized as safe (GRAS)" when used in food, there is limited information available on their long-term health effects when delivered by inhalation. While obtaining route-of-exposure-specific toxicological data on flavor ingredients is critical to product evaluation, the large number of individual flavor ingredients available and their potential combinations render classical toxicological assessment approaches impractical, as they may require years of preclinical investigations and thousands of laboratory animals. Therefore, we propose a pragmatic approach in which flavor ingredients are initially assigned to groups of structurally related compounds (Flavor Groups), from which flavor group representatives (FGR) are then selected and tested individually and as a mixture in vitro and in vivo. The premise is that structurally related compounds would have comparable metabolic and biological activity and that the data generated using FGRs could support the toxicological assessment of other structurally related flavor ingredients of their respective Flavor Groups. This approach is explained in a step-wise manner and exemplified by a case study, along with its strengths, limitations as well as recommendations for further confirmatory testing. Once completed, this FGR approach could significantly reduce the time and resources required for filling the data gap in understanding the health risks of many flavor ingredients while also minimizing the need for laboratory animals.

Nogo-a regulates neural precursor migration in the embryonic mouse cortex.

Although Nogo-A has been intensively studied for its inhibitory effect on axonal regeneration in the adult central nervous system, little is known about its function during brain development. In the embryonic mouse cortex, Nogo-A is expressed by radial precursor/glial cells and by tangentially migrating as well as postmigratory neurons. We studied radially migrating neuroblasts in wild-type and Nogo-A knockout (KO) mouse embryos. In vitro analysis showed that Nogo-A and its receptor components NgR, Lingo-1, TROY, and p75 are expressed in cells emigrating from embryonic forebrain-derived neurospheres. Live imaging revealed an increased cell motility when Nogo-A was knocked out or blocked with antibodies. Antibodies blocking NgR or Lingo-1 showed the same motility-enhancing effect supporting a direct role of surface Nogo-A on migration. Bromodeoxyuridine (BrdU) labeling of embryonic day (E)15.5 embryos demonstrated that Nogo-A influences the radial migration of neuronal precursors. At E17.5, the normal transient accumulation of radially migrating precursors within the subventricular zone was not detectable in the Nogo-A KO mouse cortex. At E19, migration to the upper cortical layers was disturbed. These findings suggest that Nogo-A and its receptor complex play a role in the interplay of adhesive and repulsive cell interactions in radial migration during cortical development.

Effects of plant alkaloids on mitochondrial bioenergetic parameters.

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 µM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 µM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.

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.

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.

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.