RNA sequencing analysis of early-stage atherosclerosis in vascular-on-a-chip and its application for comparing combustible cigarettes with heated tobacco products.

Our previous study showed promising results in replicating early-stage atherosclerosis when vascular endothelial cells (VECs) were exposed to cigarette smoke (CS) extract via M0 macrophages. We used an organ-on-a-chip system as an alternative to animal testing to model atherosclerosis, which is a complex disease involving endothelial and immune cell communications. By incorporating macrophages into the vascular-on-a-chip system, we aimed to mimic the indirect effects of inhalable substances, such as CS, on VECs. In the current study, we further examined the suitability of our in vitro system for mimicking early-stage atherosclerosis by transcriptomic analyses of VECs exposed to CS directly or indirectly via macrophages. We also incorporated M1 macrophages to replicate a preexisting inflammatory state. We found a greater number of differentially expressed genes (DEGs) in direct exposure methods than indirect exposure methods. However, a pathway analysis showed that the direct exposure of CS to VECs primarily caused cell death-related pathway alterations, and the "Atherosclerosis Signaling" pathway was predicted to be negatively regulated. Indirect exposure via M0 macrophages similarly showed that the identified DEGs were related to cell death, while the "Atherosclerosis Signaling" pathway was predicted to be activated. In contrast, cell death-related pathway alterations were not observed by indirect exposure of CS to VECs via M1 macrophages, but the pathway perturbations were similar to a pro-inflammatory positive control. In addition, the "Atherosclerosis Signaling" pathway was predicted to be activated in VECs that were indirectly exposed to CS via M1 macrophages. These results suggest that M0 or M1 macrophages contribute to atherogenic transcriptomic changes in VECs, although they affect cell death-related pathways differently. We also used indirect exposure methods to compare the effects of CS and heated tobacco product (HTP) aerosol. Notably, gene expression changes related to atherosclerosis were less pronounced in HTP aerosol-exposed VECs than CS. Our study highlights the utility of the vascular-on-a-chip system with indirect exposure of CS extract via macrophages for replicating atherogenesis and suggests a reduced risk potential of the HTP. This research contributes to advancing alternatives to animal testing for toxicological and disease modeling studies.

Proof of concept for quantitative adverse outcome pathway modeling of chronic toxicity in repeated exposure.

Adverse Outcome Pathway (AOP) is a useful tool to glean mode of action (MOE) of a chemical. However, in order to use it for the purpose of risk assessment, an AOP needs to be quantified using in vitro or in vivo data. Majority of quantitative AOPs developed so far, were for single exposure to progressively higher doses. Limited attempts were made to include time in the modeling. Here as a proof-of concept, we developed a hypothetical AOP, and quantified it using a virtual dataset for six repeated exposures using a Bayesian Network Analysis (BN) framework. The virtual data was generated using realistic assumptions. Effects of each exposure were analyzed separately using a static BN model and analyzed in combination using a dynamic BN (DBN) model. Our work shows that the DBN model can be used to calculate the probability of adverse outcome when other upstream KEs were observed earlier. These probabilities can help in identification of early indicators of AO. In addition, we also developed a data driven AOP pruning technique using a lasso-based subset selection, and show that the causal structure of AOP is itself dynamic and changes over time. This proof-of-concept study revealed the possibility for expanding the applicability of the AOP framework to incorporate biological dynamism in toxicity appearance by repeated insults.

A revision of the multiple-path particle dosimetry model focusing on tobacco product aerosol dynamics.

To assess the health impact of inhaled aerosols, it is necessary to understand aerosol dynamics and the associated dosimetry in the human respiratory tract. Although several studies have measured or simulated the dosimetry of aerosol constituents, the respiratory tract focus areas have been limited. In particular, the aerosols generated from tobacco products are complex composites and simulating their dynamics in the respiratory tract is challenging. To assess the dosimetry of the aerosol constituents of tobacco products, we developed a revised version of the Multiple-Path Particle Dosimetry (MPPD) model, which employs (1) new geometry based on CT-scanned human respiratory tract data, (2) convective mixing in the oral cavity and deep lung, and (3) constituent partitioning between the tissue and air, and clearance. The sensitivity analysis was conducted using aerosols composed of four major constituents of electronic cigarette (EC) aerosols to investigate the parameters that have a significant impact on the results. In addition, the revised model was run with 4 and 10 constituents in ECs and conventional cigarettes (CCs), respectively. Sensitivity analysis revealed that the new modeling and the physicochemical properties of constituents had a considerable impact on the simulated aerosol concentration and dosimetry. The simulations could be carried out within 3 min even when 10 constituents of CC aerosols were analyzed simultaneously. The revised model based on MPPD is an efficient and easy-to-use tool for understanding the aerosol dynamics of CC and EC constituents and their effect on the human body.

Chemical and in vitro toxicological comparison of emissions from a heated tobacco product and the 1R6F reference cigarette.

It has previously been found that, compared with cigarette smoke, the aerosols generated by heated tobacco products contain fewer and lower harmful and potentially harmful constituents (HPHCs) and elicit lower biological activity in in vitro models and lower smoking-related exposure biomarker levels in clinical studies. It is important to accumulate such scientific evidences for heated tobacco products with a novel heating system, because different heating system may affect the quantitative aspect of the amount of HPHCs and the qualitative aspect of the biological activity of the aerosol generated. Here, the chemical properties of, and toxicological responses to aerosols emitted by DT3.0a, a new heated tobacco product with a novel heating system, and cigarette smoke (CS) were compared, using chemical analyses, in vitro battery (standardized genotoxicity and cytotoxicity) assays, and mechanistic (ToxTracker and two-dimensional cell culture) assays. Regular- and menthol-flavored DT3.0a and standard 1R6F reference cigarettes were tested. Selected HPHC yields were lower in DT3.0a aerosol than 1R6F CS. The genotoxicity-related assays indicated that DT3.0a aerosol was not genotoxic, regardless of metabolic activation. The other biological assays indicated that less cytotoxicity induction and oxidative stress response were elicited by DT3.0a aerosol compared with 1R6F CS. Similar results were found for both regular and menthol DT3.0a. Like previous reports for heated tobacco products with other heating systems, the results of this study indicated that DT3.0a aerosols have chemical and biological properties less likely to be harmful than 1R6F CS.

Binary classification of users of electronic cigarettes and smokeless tobacco through biomarkers to assess similarity with current and former smokers: machine learning applied to the population assessment of tobacco and health study.

BACKGROUND: Exposure to harmful and potentially harmful constituents in cigarette smoke is a risk factor for cardiovascular and respiratory diseases. Tobacco products that could reduce exposure to these constituents have been developed. However, the long-term effects of their use on health remain unclear. The Population Assessment of Tobacco and Health (PATH) study is a population-based study examining the health effects of smoking and cigarette smoking habits in the U.S. POPULATION: Participants include users of tobacco products, including electronic cigarettes and smokeless tobacco. In this study, we attempted to evaluate the population-wide effects of these products, using machine learning techniques and data from the PATH study. METHODS: Biomarkers of exposure (BoE) and potential harm (BoPH) in cigarette smokers and former smokers in wave 1 of PATH were used to create binary classification machine-learning models that classified participants as either current (BoE: N = 102, BoPH: N = 428) or former smokers (BoE: N = 102, BoPH: N = 428). Data on the BoE and BoPH of users of electronic cigarettes (BoE: N = 210, BoPH: N = 258) and smokeless tobacco (BoE: N = 206, BoPH: N = 242) were input into the models to investigate whether these product users were classified as current or former smokers. The disease status of individuals classified as either current or former smokers was investigated. RESULTS: The classification models for BoE and BoPH both had high model accuracy. More than 60% of participants who used either one of electronic cigarettes or smokeless tobacco were classified as former smokers in the classification model for BoE. Fewer than 15% of current smokers and dual users were classified as former smokers. A similar trend was found in the classification model for BoPH. Compared with those classified as former smokers, a higher percentage of those classified as current smokers had cardiovascular disease (9.9-10.9% vs. 6.3-6.4%) and respiratory diseases (19.4-22.2% vs. 14.2-16.7%). CONCLUSIONS: Users of electronic cigarettes or smokeless tobacco are likely to be similar to former smokers in their biomarkers of exposure and potential harm. This suggests that using these products helps to reduce exposure to the harmful constituents of cigarettes, and they are potentially less harmful than conventional cigarettes.

Human vasculature-on-a-chip with macrophage-mediated endothelial activation: The biological effect of aerosol from heated tobacco products on monocyte adhesion.

Heated tobacco products (HTPs) are expected to have the potential to reduce risks of smoking-associated cardiovascular disease (CVD). However, mechanism-based investigations of the effect of HTPs on atherosclerosis remain insufficient and further studies under human-relevant situations are desired for deeper understanding of the reduced risk potential of HTPs. In this study, we first developed an in vitro model of monocyte adhesion by considering macrophage-derived proinflammatory cytokine-mediated endothelial activation using an organ-on-a-chip (OoC), which provided great opportunities to mimic major aspects of human physiology. Then biological activities of aerosol from three different types of HTPs in terms of monocyte adhesion were compared with that of cigarette smoke (CS). Our model showed that the effective concentration ranges of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were close to the actual condition in CVD pathogenesis. The model also showed that monocyte adhesion was less induced by each HTP aerosol than CS, which may be caused by less proinflammatory cytokine secretion. In summary, our vasculature-on-a-chip model assessed the difference in biological effects between cigarettes and HTPs, and suggested a reduced risk potential of HTPs for atherosclerosis.

Oxidative stress-mediated epidermal growth factor receptor activation by cigarette smoke or heated tobacco aerosol in human primary bronchial epithelial cells from multiple donors.

The epidermal growth factor receptor (EGFR) signaling pathway has essential roles in maintaining homeostasis of various tissues by regulating cell proliferation and differentiation. Deregulation of the EGFR signaling pathway is associated with various chronic diseases including chronic obstructive pulmonary disease. Cigarette smoke (CS) is known to activate EGFR, which is linked to chronic obstructive pulmonary disease. The biological sequence from CS exposure to EGFR activation is initiated by oxidative stress caused by intracellular reactive oxygen species (ROS) and the depletion of glutathione, which led to EGFR ligand secretion and EGFR activation. We hypothesized that reducing exposure to CS constituents contributes to preventing CS-inducible EGFR activation. Therefore, we examined the aerosol from heated tobacco products (HTPs) because the aerosol contains fewer chemical constituents at lower levels than CS. We exposed primary human bronchial epithelial cells from four donors to the extracts of CS from a 1R6F reference cigarette or HTP aerosol from three in-market products, including our DT3.0a. The biological sequence from ROS to EGFR activation was assessed. CS induced all the tested endpoints although inter-donor differences were observed, whereas HTPs elicited most of the biological events at higher concentrations; however, EGFR phosphorylation was not observed even at fivefold higher concentration than CS. Overall, our results indicate that HTPs are less effective than CS to elicit ROS-induced EGFR activation. The reduced-risk potential of HTPs on EGFR-related diseases should be investigated further. In addition, testing with multiple donors is warranted when considering the individual differences in responses of primary cells to stimuli.

Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance.

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used - Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

Donor-to-donor variability of a human three-dimensional bronchial epithelial model: A case study of cigarette smoke exposure.

Three-dimensional (3D) cultured primary cells are used to predict the toxicity of substances towards humans because these 3D cultures closely mimic the physiological architecture of tissues. Nonetheless, it is important to consider primary-cell-specific variability for endpoint selection and appropriate evaluation of toxicity because donor-dependent characteristics may be retained even in in vitro cell cultures. In this report, 3D differentiated bronchial epithelial cells from three donors were used to investigate donor-to-donor variability, with an aqueous extract of cigarette smoke (CS) used as the test substance. Ciliary function, cytokine secretion, and histopathology, which are affected by CS, were examined, and transcriptomic analysis was also performed. The results revealed that interleukin-8 secretion and oxidative stress-related gene expression were consistently altered for all donors; however, their amplitudes varied. Moreover, one of the donors showed unique responses to CS, suggesting that this donor was an outlier. This donor showed intrinsic differences in histology, cytokine secretion, and gene expression profile. Such donors may help evaluate potential toxicological concerns and aid our understanding of disease pathogenesis. Conversely, these donors may confound toxicological assessment and endpoint selection. Fit-for-purpose handling of inter-donor variability is warranted.

Human TRPV1 and TRPA1 are receptors for bacterial quorum sensing molecules.

In this study, we investigated the activation of TRPV1 and TRPA1 by N-acyl homoserine lactones, quorum sensing molecules produced by Gram-negative bacteria, and the inhibitory effect of TRPV1 and TRPA1 by autoinducing peptides (AIPs), quorum sensing molecules produced by Gram-positive bacteria, using human embryonic kidney 293T cell lines stably expressing human TRPV1 and TRPA1, respectively. As a result, we found that some N-acyl homoserine lactones, such as N-octanoyl-L-homoserine lactone (C8-HSL), N-nonanoyl-L-homoserine lactone (C9-HSL) and N-decanoyl-L-homoserine lactone (C10-HSL), activated both TRPV1 and TRPA1. In addition, we clarified that some N-acyl homoserine lactones, such as N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), only activated TRPV1 and N-acyl homoserine lactones having saturated short acyl chain, such as N-acetyl-L-homoserine lactone (C2-HSL) and N-butyryl-L-homoserine lactone (C4-HSL), only activated TRPA1. Furthermore, we found that an AIP, simple linear peptide CHWPR, inhibited both TRPV1 and TRPA1 and peptide having thiolactone ring DICNAYF, the thiolactone ring were formed between C3 to F7, strongly inhibited only the TRPV1. Although the specificity of TRPV1 and TRPA1 for quorum sensing molecules was different, these data suggest that both TRPV1 and TRPA1 would function as receptors for quorum sensing molecule produced by bacteria. Graphical Abstract.

LC/MS analysis of three-dimensional model cells exposed to cigarette smoke or aerosol from a novel tobacco vapor product.

A novel tobacco vapor product (NTV) contains tobacco leaves and generates nicotine-containing aerosols using heating elements. Subchronic biological effects have been evaluated previously using three-dimensional bronchial epithelial model cells by repeated exposure to cigarette smoke (CS) and the NTV aerosols; however, the intracellular exposure characteristics have not been studied in detail. In this study, cells were initially exposed to an aqueous extract (AqE) of cigarette smoke (CS) at two concentration levels, and the cell lysate underwent untargeted analysis by LC-high resolution mass spectrometry to determine the exogenous compounds present in the cells. Among the thousands of peaks detected, four peaks showed a CS-dependency, which were reproducibly detected. Two of the peaks were nicotine and nicotine N-oxide, and the other two putative compounds were myosmine and norharman. The cells were then exposed to an AqE of CS in various combinations of exposure and post-exposure culture durations. Post-exposure culturing of cells with fresh medium markedly decreased the peak areas of the four compounds. The in-vitro switching study of CS to NTV aerosols was investigated by intermittently exposing cells to an AqE of CS four times, followed by exposure to either an AqE of CS, NTV aerosol or medium another four times. Switching to NTV reduced myosmine and norharman levels, which are known CS constituents. The results indicate that extracellular compounds inside cells reflect the exposure state outside cells. Thus, monitoring functional changes to cells in these exposure experiments is feasible.

In vitro long-term repeated exposure and exposure switching of a novel tobacco vapor product in a human organotypic culture of bronchial epithelial cells.

Next-generation tobacco products and nicotine delivery systems such as heat-not-burn tobacco products and electronic cigarettes, the usage of which is expected to have a beneficial impact on public health, have gained popularity over the past decade. However, the risks associated with the long-term use of such products are still incompletely understood. Although the risks of these products should be clarified through epidemiological studies, such studies are normally performed based on each product category, not product-by-product. Therefore, investigation of the risk on a product-by-product basis is important to provide specific scientific evidence. In the current study, we performed the 40-day repeated exposure of in vitro human bronchial epithelial tissues to cigarette smoke (CS) or vapor from our proprietary novel tobacco vapor product (NTV). In addition, tissue samples exposed to CS were switched to NTV or CS exposure was stopped at 20 days to reflect a situation where smokers switched to NTV or ceased to smoke. All tissue samples were assessed in terms of toxicity, inflammation and transcriptomic alterations. Tissue samples switched to NTV and the cessation of exposure samples showed recovery from CS-induced damage although there was a time-course difference. Moreover, repeated exposure to NTV produced negligible effects on the tissue samples while CS produced cumulative effects. Our results suggest that the use of NTV, including switching to NTV from cigarette smoking, has fewer effects on bronchial epithelial tissues than continuing smoking.

Novel biomarker genes which distinguish between smokers and chronic obstructive pulmonary disease patients with machine learning approach.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is combination of progressive lung diseases. The diagnosis of COPD is generally based on the pulmonary function testing, however, difficulties underlie in prognosis of smokers or early stage of COPD patients due to the complexity and heterogeneity of the pathogenesis. Computational analyses of omics technologies are expected as one of the solutions to resolve such complexities. METHODS: We obtained transcriptomic data by in vitro testing with exposures of human bronchial epithelial cells to the inducers for early events of COPD to identify the potential descriptive marker genes. With the identified genes, the machine learning technique was employed with the publicly available transcriptome data obtained from the lung specimens of COPD and non-COPD patients to develop the model that can reflect the risk continuum across smoking and COPD. RESULTS: The expression levels of 15 genes were commonly altered among in vitro tissues exposed to known inducible factors for earlier events of COPD (exposure to cigarette smoke, DNA damage, oxidative stress, and inflammation), and 10 of these genes and their corresponding proteins have not previously reported as COPD biomarkers. Although these genes were able to predict each group with 65% accuracy, the accuracy with which they were able to discriminate COPD subjects from smokers was only 29%. Furthermore, logistic regression enabled the conversion of gene expression levels to a numerical index, which we named the "potential risk factor (PRF)" index. The highest significant index value was recorded in COPD subjects (0.56 at the median), followed by smokers (0.30) and non-smokers (0.02). In vitro tissues exposed to cigarette smoke displayed dose-dependent increases of PRF, suggesting its utility for prospective risk estimation of tobacco products. CONCLUSIONS: Our experimental-based transcriptomic analysis identified novel genes associated with COPD, and the 15 genes could distinguish smokers and COPD subjects from non-smokers via machine-learning classification with remarkable accuracy. We also suggested a PRF index that can quantitatively reflect the risk continuum across smoking and COPD pathogenesis, and we believe it will provide an improved understanding of smoking effects and new insights into COPD.

An inter-laboratory in vitro assessment of cigarettes and next generation nicotine delivery products.

In vitro testing can facilitate the rapid assessment of next generation nicotine delivery products (NGPs) with comparisons to combustible tobacco products. In vitro assays for cytotoxicity and oxidative stress were employed at BAT (UK) and JT (Japan) to test total particulate matter (TPM) of a scientific reference cigarette and aerosol collected mass (ACM) of a commercially available E-cigarette and two tobacco heating products (THP). 3R4F TPMs were generated using the Health Canada intense (HCI) regimen, a modified regime (mHCI) for the THP ACMs and the CORESTA recommended method no. 81 for the E-cigarette ACM. Human lung cells were exposed to the test product TPM/ACMs at concentrations between 0-200 µg/ml followed by the employment of commercially available assays for endpoint analysis that included reactive oxygen species (ROS) generation, the glutathione ratio (GSH:GSSG), activation of the antioxidant response elements (ARE) and cellular viability. TPM/ACM nicotine concentrations were quantified using a UPLC-PDA technique. At both laboratories the 3R4F TPM induced significant and dose-dependent responses in all in vitro assays, whereas no significant responses could be measured for the NGP ACMs. In conclusion, both laboratories obtained comparable results across all endpoints therefore demonstrating the utility of the in vitro techniques combined with standardised test products to support the assessment of NGPs.

Multi-omics analysis: Repeated exposure of a 3D bronchial tissue culture to whole-cigarette smoke.

Cigarette smoke (CS) is a major risk factor in the development of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease. A comprehensive investigation of the biological impacts of chronic CS exposure on lung tissue is therefore important for understanding the pathogenesis of lung disease. We used three-dimensional (3D) organotypic human bronchial tissue cultures and metabolomics, transcriptomics, and proteomics to investigate changes in biological processes affected by repeated whole-CS exposure. We found that CS perturbed central carbon metabolism in relation with oxidative stress responses. Epidermal growth factor receptor, which is involved in the early-stage pathogenesis of airway diseases, was identified as a key regulator of the perturbed processes. Proteomic analysis of proteins in the apical surface liquid of the 3D bronchial tissue cultures indicated that repeated whole-CS exposure induced alterations in the secretion of several known biomarkers of airway diseases, including mucins and matrix metalloproteinases. These findings are consistent with observations from lung disease patients. Overall, our results suggest that 3D bronchial tissue cultures can provide valuable information on tissue-specific alterations in biological processes induced by chronic exposure to CS.

Regulation of NRF2, AP-1 and NF-κB by cigarette smoke exposure in three-dimensional human bronchial epithelial cells.

Cigarette smoke (CS) is a complex mixture of chemicals and interacts with various physiological processes. We previously reported that nuclear factor erythroid 2-related factor 2 (NRF2) was the most sensitive transcription factor to aqueous CS extract (AqCSE) exposure in monolayer cultured human bronchial epithelial cell lines. Recently, in vitro three-dimensional (3D) culture models have been used to supplement pharmacological and toxicological assessments. Bronchial epithelium models in particular are useful for the evaluation of substances that directly contact the respiratory tract, such as CS. In the present study, we used 3D-cultured human bronchial epithelial cells (HBECs) to assess activation of transcription factors and relevant gene expression in response to AqCSE, primarily focusing on NRF2 and nuclear factor-kappa B (NF-κB) pathways. The 3D-cultured HBECs exposed to AqCSE showed expression of NRF2 and its nuclear translocation in addition to upregulation of genes related to oxidative stress. Our results suggest that the NRF2 pathway was the dominant pathway when 3D-cultured HBECs were exposed to AqCSE at a low dose, supporting our previous findings that NRF2 was the most sensitive transcription factor in response to AqCSE. Expression and nuclear translocation of NF-κB were not increased, although proinflammatory genes were upregulated. However, another inflammation-related transcription factor, activation protein 1, was induced by AqCSE. Gene classification analysis suggested that induction of the inflammatory response by AqCSE was dependent on NRF2 and activation protein 1 rather than NF-κB.

Effects of repeated cigarette smoke extract exposure over one month on human bronchial epithelial organotypic culture.

Cigarette smoke is a known risk factor for inflammatory diseases in the respiratory tract, and inflammatory exacerbation is considered pivotal to the pathogenesis of these diseases. Here, we performed two repeated exposure studies in which we exposed human bronchial epithelial tissues in an organotypic culture model to cigarette smoke extract (CSE); the first study was conducted over a four-day period to determine the suitable dose range for the extended exposure period, and the second was a one-month exposure study to elucidate the exposure-by-exposure effects in bronchial tissues. We focused on matrix metalloproteinase (MMP)-9 and -1/3 and the inflammatory cytokines interleukin (IL)-8 and growth factor related oncogene to evaluate the transition into an inflammatory state. Even at CSE doses with no or low toxicity for a single exposure, the repetition of exposure induced cumulative effects on both the inflammatory responses, specifically the IL-8 and MMPs levels, and tissue morphology. Interestingly, untreated controls initially had relatively high baseline levels of these secreted proteins; these levels gradually declined, after which they showed periodic level changes, suggesting an acclimation period may be needed for this system. These results demonstrate the usability of this system for the elucidation of sub-chronic effects in vitro.

Oxidative stress responses in human bronchial epithelial cells exposed to cigarette smoke and vapor from tobacco- and nicotine-containing products.

The use of novel tobacco- and nicotine-containing vapor products that do not combust tobacco leaves is on the rise worldwide. The emissions of these products typically contain lower numbers and levels of potentially harmful chemicals compared with conventional cigarette smoke. These vapor products may therefore elicit fewer adverse biological effects. We compared the effects of emissions from different types of such products, i.e., our proprietary novel tobacco vapor product (NTV), a commercially available heat-not-burn tobacco product (HnB), and e-cigarette (E-CIG), and a combustible cigarette in a human bronchial epithelial cell line. The aqueous extract (AqE) of the test product was prepared by bubbling the produced aerosol into medium. Cells were exposed to the AqEs of test products, and then glutathione oxidation, Nrf2 activation, and secretion of IL-8 and GM-CSF were examined. We found that all endpoints were similarly perturbed by exposure to each AqE, but the effective dose ranges were different between cigarette smoke and the tobacco- and nicotine-containing vapors. These results demonstrate that the employed assays detect differences between product exposures, and thus may be useful to understand the relative potential biological effects of tobacco- and nicotine-containing products.

Application of a direct aerosol exposure system for the assessment of biological effects of cigarette smoke and novel tobacco product vapor on human bronchial epithelial cultures.

Recent advancements in in vitro exposure systems and cell culture technology enable direct exposure to cigarette smoke (CS) of human organotypic bronchial epithelial cultures. MucilAir organotypic bronchial epithelial cultures were exposed, using a Vitrocell exposure system, to mainstream aerosols from the 3R4F cigarette or from a recently developed novel tobacco vapor product (NTV). The exposure aerosol dose was controlled by dilution flow and the number of products smoked; there were five exposure conditions for 3R4F smoke and three for NTV vapor. The amount of nicotine delivered to the tissues under each condition was analyzed and that of the total particulate matter (TPM) was estimated using nicotine data. The nicotine dose was similar for the two products at the highest dose, but the estimated TPM levels from the NTV were 3.7 times the levels from the 3R4F. Following 3R4F smoke exposure, a dose dependent increase was observed in cytotoxicity, cytokine secretion, and differential gene expression. However, no changes were detected in these endpoints following NTV vapor exposure, suggesting the biological effects of NTV vapor are lower than those of conventional combustible CS. Our study design, which includes collection of biological data and dosimetry data, is applicable to assessing novel tobacco products.

A 3D epithelial-mesenchymal co-culture model of human bronchial tissue recapitulates multiple features of airway tissue remodeling by TGF-ß1 treatment.

BACKGROUND: The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated. METHODS: We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) ß1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis. RESULTS: TGF-ß1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-ß1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model. CONCLUSION: Our findings indicate that TGF-ß1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process.