An application of a systems toxicology approach using a human organotypic small airway cultures for a comparative assessment between a candidate modified-risk tobacco product aerosol and cigarette smoke.
ABSTRACT: Along the trachea-bronchial tree, including the small airway region, cigarette smoke exposure induces inflammation, which can exacerbate the development of chronic obstructive pulmonary disease (COPD). The small airway region is known as the primary location of airway blockage in COPD and asthma. Therefore, evaluating exposure impact on the small airway is relevant for risk assessment. Using an in vitro human small airway culture model and a Systems Toxicology approach, this present study reports an assessment of the biological impact of an aerosol from a candidate modified-risk tobacco product, tobacco heating system (THS) 2.2, as compared with 3R4F smoke, at similar nicotine concentratng other functional measures (e.g., cytotoxicity, ciliary beating function, secretion of pro-inflammatory mediators) and histological assessment. The NPA methodology provides not only a qualitative measure of the exposure impact, but also a quantification of the exposure effect: the highest biological impact was observed in cultures 4 h post-exposure to 3R4F smoke at 0.15 mg nicotine/L (100% impact). In contrast, THS2.2 aerosol at similar nicotine concentration, only elicited 15% relative biological impact at 4 h post-exposure in the context of various biological processes modeled in the networks: Cell Fate, Cell Proliferation, Cell Stress, and Inflammatory Process Networks. Consistently, ciliary beating function and culture morphology were not remarkably altered in samples exposed to THS2.2 aerosol, even at nicotine concentration three times that of 3R4F smoke.
Project description:Using an <i>in vitro</i> human small airway epithelium model, we assessed the biological impact of an aerosol from a candidate modified-risk tobacco product, the tobacco heating system (THS) 2.2, to investigate the potential reduced risk of THS2.2 aerosol exposure compared with cigarette smoke. Following the recommendations of the Institute of Medicine and the Tobacco Product Assessment Consortium, in which modified-risk tobacco products assessment should be performed in comparison with standard conventional products, the effects of the THS2.2 aerosol exposure on the small airway cultures were compared with those of 3R4F cigarette smoke. We used a <i>systems toxicology</i> approach whereby elucidation of toxic effects is derived not only from functional assay readouts but also from omics technologies. Cytotoxicity, ciliary beating function, secretion of pro-inflammatory mediators and histological assessment represented functional assays. The omics data included transcriptomic and miRNA profiles. Exposure-induced perturbations of causal biological networks were computed from the transcriptomic data. The results showed that THS2.2 aerosol exposure at the tested doses elicited lower cytotoxicity levels and lower changes in the secreted pro-inflammatory mediators than 3R4F smoke. Although THS2.2 exposure elicited alterations in the gene expression, a higher transcriptome-induced biological impact was observed following 3R4F smoke: The effects of THS2.2 aerosol exposure, if observed, were mostly transient and diminished more rapidly after exposure than those of 3R4F smoke. The study demonstrated that the systems toxicology approach can reveal changes at the cellular level that would be otherwise not detected from functional assays, thus increasing the sensitivity to detect potential toxicity of a treatment/exposure.
Project description:Along the trachea-bronchial tree, including the small airway region, cigarette smoke exposure induces inflammation, which can exacerbate the development of chronic obstructive pulmonary disease (COPD). The small airway region is known as the primary location of airway blockage in COPD and asthma. Therefore, evaluating exposure impact on the small airway is relevant for risk assessment. Using an in vitro human small airway culture model and a Systems Toxicology approach, this present study reports an assessment of the biological impact of an aerosol from a candidate modified-risk tobacco product, tobacco heating system (THS) 2.2, as compared with 3R4F smoke, at similar nicotine concentrating other functional measures (e.g., cytotoxicity, ciliary beating function, secretion of pro-inflammatory mediators) and histological assessment. The NPA methodology provides not only a qualitative measure of the exposure impact, but also a quantification of the exposure effect: the highest biological impact was observed in cultures 4 h post-exposure to 3R4F smoke at 0.15 mg nicotine/L (100% impact). In contrast, THS2.2 aerosol at similar nicotine concentration, only elicited 15% relative biological impact at 4 h post-exposure in the context of various biological processes modeled in the networks: Cell Fate, Cell Proliferation, Cell Stress, and Inflammatory Process Networks. Consistently, ciliary beating function and culture morphology were not remarkably altered in samples exposed to THS2.2 aerosol, even at nicotine concentration three times that of 3R4F smoke.
Project description: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%.
Project description:Systems biology combines comprehensive molecular analyses with quantitative modeling to understand the characteristics of a biological system as a whole. Leveraging a similar approach, systems toxicology aims to decipher complex biological responses following exposures. This work reports a systems toxicology meta-analysis in the context of in vitro assessment of a candidate modified-risk tobacco product (MRTP) using three human organotypic cultures of the aerodigestive tract (buccal, bronchial, and nasal epithelia). Complementing a series of functional measures, a causal network enrichment analysis of transcriptomic data was used to compare quantitatively the biological impact of aerosol from the Tobacco Heating System (THS) 2.2, a candidate MRTP, with 3R4F cigarette smoke (CS) at similar nicotine concentrations. Lower toxicity was observed in all cultures following exposure to THS2.2 aerosol compared with 3R4F CS. Because of their morphological differences, a smaller exposure impact was observed in the buccal (stratified epithelium) compared with the bronchial and nasal (pseudostratified epithelium). However, the causal network enrichment approach supported a similar mechanistic impact of CS across the three cultures, including the impact on xenobiotic, oxidative stress, and inflammatory responses. At comparable nicotine concentrations, THS2.2 aerosol elicited reduced and more transient effects on these processes. To demonstrate the benefits of additional data modalities, we employed a newly established targeted mass-spectrometry marker panel to further confirm the reduced cellular stress responses elicited by THS2.2 aerosol compared with 3R4F CS in the nasal culture. Overall, this work demonstrates the applicability and robustness of the systems toxicology approach for in vitro inhalation toxicity assessment.
Project description:Electronic cigarettes (e-cigarettes) use has increased globally and could potentially offer a lower risk alternative to cigarette smoking. Here, we assessed the transcriptional response of a primary 3D airway model acutely exposed to e-cigarette aerosol and cigarette (3R4F) smoke. Aerosols were generated with standard intense smoking regimens with careful consideration for dose by normalizing the exposures to nicotine. Two e-cigarette aerosol dilutions were tested for equivalent and higher nicotine delivery compared to 3R4F. RNA was extracted at 24?hrs and 48?hrs post exposure for RNA-seq. 873 and 205 RNAs were differentially expressed for 3R4F smoke at 24?hrs and 48?hrs using a pFDR?<?0.01 and a [fold change]?>?2 threshold. 113 RNAs were differentially expressed at the highest dose of e-cigarette aerosol using a looser threshold of pFDR?<?0.05, 3 RNAs exceeded a fold change of 2. Geneset enrichment analysis revealed a clear response from lung cancer, inflammation, and fibrosis associated genes after 3R4F smoke exposure. Metabolic/biosynthetic processes, extracellular membrane, apoptosis, and hypoxia were identified for e-cigarette exposures, albeit with a lower confidence score. Based on equivalent or higher nicotine delivery, an acute exposure to e-cigarette aerosol had a reduced impact on gene expression compared to 3R4F smoke exposure in vitro.
Project description:In the context of tobacco harm-reduction strategy, the potential reduced impact of electronic cigarette (EC) exposure should be evaluated relative to the impact of cigarette smoke exposure. We conducted a series of in vitro studies to compare the biological impact of an acute exposure to aerosols of "test mix" (flavors, nicotine, and humectants), "base" (nicotine and humectants), and "carrier" (humectants) formulations using MarkTen® EC devices with the impact of exposure to smoke of 3R4F reference cigarettes, at a matching puff number, using human organotypic air-liquid interface buccal and small airway cultures. We measured the concentrations of nicotine and carbonyls deposited in the exposure chamber after each exposure experiment. The deposited carbonyl concentrations were used as representative measures to assess the reduced exposure to potentially toxic volatile substances. We followed a systems toxicology approach whereby functional biological endpoints, such as histopathology and ciliary beating frequency, were complemented by multiplex and omics assays to measure secreted inflammatory proteins and whole-genome transcriptomes, respectively. Among the endpoints analyzed, the only parameters that showed a significant response to EC exposure were secretion of proteins and whole-genome transcriptomes. Based on the multiplex and omics analyzes, the cellular responses to EC aerosol exposure were tissue type-specific; however, those alterations were much smaller than those following cigarette smoke exposure, even when the EC aerosol exposure under the testing conditions resulted in a deposited nicotine concentration approximately 200 times that in saliva of EC users.
Project description:The harmful effects of cigarette smoke exposure on the respiratory tract are widely known. Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than CS exposure. Many studies have assessed the potential toxicity of the aerosol from e-vapor products in vitro. However, most studies have only tested the effects of liquid formulations applied directly to cell cultures but not those of the aerosolized formulations. In this study, we examined the effects of acute exposure on human organotypic bronchial epithelial culture and alveolar tri-culture models to an aerosol generated by an e-vapor device that uses the MESH™ technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette. In contrast to 3R4F CS exposure, exposure to the IQOS MESH Classic Tobacco aerosol did not cause cytotoxicity in either of the two lung culture models, despite resulting in greater concentrations of deposited nicotine. Ciliary beating frequency in bronchial cultures was not impacted in response to IQOS MESH aerosol exposure, whereas CS exposure caused a marked decrease in the frequency and the cilia beating active area. We complemented the histological and functional findings with quantitative analysis of the molecular changes in the exposed cultures. Global mRNA expression profiles and secreted protein profiles revealed a significantly lower impact of exposure to IQOS MESH aerosol than to 3R4F CS exposure. Overall, our study using whole aerosols for the exposure shows a much reduced impact of IQOS MESH aerosol in comparison to CS exposure in both bronchial and alveolar cultures, even at greater nicotine doses.
Project description:Cigarette smoking is a major risk factor for the development and progression of diseases such as cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD). Modified risk tobacco products (MRTP) are designed to reduce smoking-related health risks. Suitable animal models are important for understanding smoke-induced pathogenesis. Over an 8-month period, hallmarks of CVD were investigated in ApoE?/? mice exposed to conventional cigarette smoke (CS) or to an aerosol from a candidate RRP, the tobacco heating system (THS2.2). In addition to chronic exposure, cessation or switching to THS2.2 after 2 months of CS were investigated.? The aim of this study was to evaluate the transcriptomics response of mouse heart tissue after exposure to 3R4F and a potential reduced risk tobacco product, Tobacco Heating System 2.2 (THS2.2). The transcriptomic analysis of heart tissue, showed that cigarette smoke strongly impacted the differential expression of genes at long term of smoke exposure at 6 and 8 month, whereas the new THS2.2 not burning product don?t present any significant impact on the genes expression on cardiovascular tissue. In a similar manner, cessation and switching exposure don?t present any significant regulation of genes. Functional analysis of differentially expressed genes shown that 3R4F exposure strongly regulate the cytoskeleton organization and contraction and shown a down regulation of actin, actinin alpha 4, the main component of sarcomere structure. These preliminary results suggest that the conventional smoking product may favours the development of cardiomyopathy via a direct impact of the cardiomyocytes structure and that the inflammatory process could also be affected by the smoke exposure and could be responsible for the non-adaptive response of heart to a stress.
Project description:Modified-risk tobacco products (MRTP) are designed to reduce the individual risk of tobacco-related disease as well as population harm compared to smoking cigarettes. Experimental proof of their benefit needs to be provided at multiple levels in research fields. Here, we examined microRNA (miRNA) levels in the lungs of rats exposed to a candidate modified-risk tobacco products, the Tobacco Heating System 2.2 (THS2.2) in a 90-day OECD TG-413 inhalation study. Our aim was to assess the miRNA response to THS2.2 aerosol compared with the response to combustible cigarettes (CC) smoke from the reference cigarette 3R4F. CC smoke exposure, but not THS2.2 aerosol exposure, caused global miRNA downregulation, which may be explained by the interference of CC smoke constituents with the miRNA processing machinery. Upregulation of specific miRNA species, such as miR-146a/b and miR-182, indicated that they are causal elements in the inflammatory response in CC-exposed lung, but they were reduced after THS2.2 aerosol exposure. Transforming transcriptomic data into protein activity based on corresponding downstream gene expression, we identified potential mechanisms for miR-146a/b and miR-182 that were activated by CC smoke but not by THS2.2 aerosol and possibly involved in the regulation of those miRNAs. The inclusion of miRNA profiling in systems toxicology approaches increases the mechanistic understanding of the complex exposure responses.
Project description: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.