An in vitro evaluation of e-vapor products: The contributions of chemical adulteration, concentration, and device power.

Homemade e-liquids and power-adjustable vaping devices may carry higher risks than commercial formulations and fixed-power devices. This study used human macrophage-like and bronchial epithelial (NHBE) cell cultures to investigate toxicity of homemade e-liquids containing propylene glycol and vegetable glycerin (PG/VG), nicotine, vitamin E acetate (VEA), medium-chain fatty acids (MCFAs), phytol, and cannabidiol (CBD). SmallAir™ organotypic epithelial cultures were exposed to aerosols generated at different power settings (10-50 W). Carbonyl levels were measured, and endpoints reflecting epithelial function (ciliary beating frequency [CBF]), integrity (transepithelial electrical resistance [TEER]), and structure (histology) were investigated. Treatment with nicotine or VEA alone or with PG/VG did not impact cell viability. CBD, phytol, and lauric acid caused cytotoxicity in both culture systems and increased lipid-laden macrophages. Exposure of SmallAir™ organotypic cultures to CBD-containing aerosols resulted in tissue injury and loss of CBF and TEER, while PG/VG alone or with nicotine or VEA did not. Aerosols generated with higher power settings had higher carbonyl concentrations. In conclusion, the presence and concentration of certain chemicals and device power may induce cytotoxicity in vitro. These results raise concerns that power-adjustable devices may generate toxic compounds and suggest that toxicity assessments should be conducted for both e-liquid formulations and their aerosols.

Assessment of in vitro kinetics and biological impact of nebulized trehalose on human bronchial epithelium.

Trehalose is added in drug formulations to act as fillers or improve aerosolization performance. Its characteristics as a carrier molecule have been explored; however, the fate of trehalose in human airway tissues has not been thoroughly investigated. Here, we investigated the fate of nebulized trehalose using in vitro human air-liquid bronchial epithelial cultures. First, a tracing experiment was conducted using 13C12-trehalose; we measured trehalose distribution in different culture compartments (apical surface liquid, epithelial culture, and basal side medium) at various time points following acute exposure to 13C12-labeled trehalose. We found that 13C12-trehalose was metabolized into 13C6-glucose. The data was then used to model the kinetics of trehalose disappearance from the apical surface of bronchial cultures. Secondly, we evaluated the potential adverse effects of nebulized trehalose on the bronchial cultures after they were acutely exposed to nebulized trehalose up to a level just below its solubility limit (50 g/100 g water). We assessed the ciliary beating frequency and histological characteristics. We found that nebulized trehalose did not lead to marked alteration in ciliary beating frequency and morphology of the epithelial cultures. The in vitro testing approach used here may enable the early selection of excipients for future development of inhalation products.

Comparison of the biological impact of aerosol of e-vapor device with MESH® technology and cigarette smoke on human bronchial and alveolar cultures.

Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than cigarette smoke (CS) exposure. Although many studies on e-vapor products have tested the effects of liquid formulations on cell cultures, few have evaluated the effects of aerosolized formulations. We examined the effects of acute exposure to the aerosol of an e-vapor device that uses the MESH® technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette on human organotypic bronchial epithelial culture and alveolar triculture models. In contrast to 3R4F CS exposure, exposure to the IQOS MESH aerosol (Classic Tobacco flavor) did not cause cytotoxicity in bronchial epithelial cultures or alveolar tricultures despite its greater concentrations of deposited nicotine (3- and 4-fold, respectively). CS exposure caused a marked decrease in the frequency and active area of ciliary beating in bronchial cultures, whereas IQOS MESH aerosol exposure did not. Global mRNA expression and secreted protein profiles revealed a significantly lower impact of IQOS MESH aerosol exposure than 3R4F CS exposure. Overall, our whole aerosol exposure study shows a clearly reduced impact of IQOS MESH aerosol relative to CS in bronchial and alveolar cultures, even at greater nicotine doses.

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.

Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of Classic Tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESH™ technology.

We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air-liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.

A lower impact of an acute exposure to electronic cigarette aerosols than to cigarette smoke in human organotypic buccal and small airway cultures was demonstrated using systems toxicology assessment.

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.

Assessment of a 72-hour repeated exposure to Swedish snus extract and total particulate matter from 3R4F cigarette smoke on gingival organotypic cultures.

Swedish snus is a smokeless tobacco product that contains reduced levels of harmful compounds compared with cigarette smoke. In Sweden, where snus use exceeds smoking among men, relatively low rates of major smoking-related diseases have been recorded. To better understand how snus use could align with current tobacco harm reduction strategies, its potential mechanisms of toxicity must be investigated. This study aimed to determine, via a systems toxicology approach, the biological impact of repeated 72-hour exposure of human gingival epithelial organotypic cultures to extracts from both a commercial and a reference snus and the total particulate matter (TPM) from cigarette smoke. At concentrations relevant for human use, cultures treated with snus extracts induced mild, generally reversible biological changes, while TPM treatment induced substantial morphological and inflammatory alterations. Network enrichment analysis and integrative analysis of the global mRNA and miRNA expression profiles indicated a limited and mostly transient impact of the snus extracts, in particular on xenobiotic metabolism, while the effects of TPM were marked and sustained over time. High-confidence miRNAs that might be related to pathological conditions in vivo were identified. This study highlights the limited biological impact of Swedish snus extract on human organotypic gingival cultures.

Assessment of the impact of aerosol from a potential modified risk tobacco product compared with cigarette smoke on human organotypic oral epithelial cultures under different exposure regimens.

Cigarette smoke (CS) is affecting considerably the oral mucosa. Heating, instead of burning, tobacco reduces consistently the amount of toxic compounds and may exert a lower impact on oral health than combusted cigarettes. The carbon-heated tobacco product 1.2 (CHTP1.2) is a potential modified risk tobacco product (MRTP) based on heat-not-burn technology. Using a systems toxicology assessment framework, we compared the effects of exposure to CHTP1.2 aerosol with those of CS from a reference cigarette (3R4F). Human organotypic cultures derived from buccal and gingival epithelia were exposed acutely (28-min) or repeatedly (28 min/day for 3 days), respectively, to two matching concentrations of CHTP1.2 aerosol or 3R4F CS, and a non-diluted (100%) CHTP1.2 aerosol. The results showed an absence of cytotoxicity, reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP1.2 aerosol compared with 3R4F CS. Changes in mRNA and miRNA expression were linked by an integrative analysis approach, suggesting a regulatory role of miRNAs in several smoke/disease-relevant biological processes induced by 3R4F CS. The identification of mechanisms by which potential MRTPs can reduce the impact of tobacco use on biological systems is of great importance in understanding the molecular basis of the smoking harm reduction paradigm.

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.

3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product.

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

Comparative effects of a candidate modified-risk tobacco product Aerosol and cigarette smoke on human organotypic small airway cultures: a systems toxicology approach.

Using an in vitro 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 systems toxicology 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.

A systems toxicology approach for comparative assessment: Biological impact of an aerosol from a candidate modified-risk tobacco product and cigarette smoke on human organotypic bronchial epithelial cultures.

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%.

ß-Cryptoxanthin Reduced Lung Tumor Multiplicity and Inhibited Lung Cancer Cell Motility by Downregulating Nicotinic Acetylcholine Receptor α7 Signaling.

Despite the consistent association between a higher intake of the provitamin A carotenoid ß-cryptoxanthin (BCX) and a lower risk of lung cancer among smokers, potential mechanisms supporting BCX as a chemopreventive agent are needed. We first examined the effects of BCX on 4-[methyl nitrosamino]-1-[3-pyridyl]-1-butanone (NNK)-induced lung tumorigenesis in A/J mice. BCX supplementation was given daily to the mice starting 2 weeks prior to the injection of NNK and continued 16 weeks after NNK injection. BCX supplementation resulted in a dose-dependent increase of BCX concentration in both serum and lungs of the mice without a significant alteration of vitamin A (retinol and retinyl palmitate) concentration. BCX significantly reduced the multiplicity of the NNK-induced lung tumor by 52% to 63% compared with the NNK-treated mice without BCX supplementation. The protective effect of BCX in the lungs was associated with reductions of both mRNA and protein of the homopentameric neuronal nicotinic acetylcholine receptor α7 (α7-nAChR), which has been implicated in lung tumorigenesis. We then conducted an in vitro cell culture study and found that BCX treatment suppressed α7-nAChR expression and inhibited the migration and invasion of α7-nAChR-positive lung cancer cells but not in cells lacking α7-nAChR. The activities of BCX were significantly attenuated by activators of α7-nAChR/PI3K signaling or by overexpression of constitutively active PI3K. Collectively, the results suggest that BCX inhibits lung tumorigenesis and cancer cell motility through the downregulation of α7-nAChR/PI3K signaling, independent of its provitamin A activity. Therefore, BCX can be used as a chemopreventive agent or a chemotherapeutic compound against lung cancer. Cancer Prev Res; 9(11); 875-86. ©2016 AACR.

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.

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.

Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts.

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

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.

Systems approaches evaluating the perturbation of xenobiotic metabolism in response to cigarette smoke exposure in nasal and bronchial tissues.

Capturing the effects of exposure in a specific target organ is a major challenge in risk assessment. Exposure to cigarette smoke (CS) implicates the field of tissue injury in the lung as well as nasal and airway epithelia. Xenobiotic metabolism in particular becomes an attractive tool for chemical risk assessment because of its responsiveness against toxic compounds, including those present in CS. This study describes an efficient integration from transcriptomic data to quantitative measures, which reflect the responses against xenobiotics that are captured in a biological network model. We show here that our novel systems approach can quantify the perturbation in the network model of xenobiotic metabolism. We further show that this approach efficiently compares the perturbation upon CS exposure in bronchial and nasal epithelial cells in vivo samples obtained from smokers. Our observation suggests the xenobiotic responses in the bronchial and nasal epithelial cells of smokers were similar to those observed in their respective organotypic models exposed to CS. Furthermore, the results suggest that nasal tissue is a reliable surrogate to measure xenobiotic responses in bronchial tissue.

ß-cryptoxanthin restores nicotine-reduced lung SIRT1 to normal levels and inhibits nicotine-promoted lung tumorigenesis and emphysema in A/J mice.

Nicotine, a large constituent of cigarette smoke, is associated with an increased risk of lung cancer, but the data supporting this relationship are inconsistent. Here, we found that nicotine treatment not only induced emphysema but also increased both lung tumor multiplicity and volume in 4-nitrosamino-1-(3-pyridyl)-1-butanone (NNK)-initiated lung cancer in A/J mice. This tumor-promoting effect of nicotine was accompanied by significant reductions in survival probability and lung Sirtuin 1 (SIRT1) expression, which has been proposed as a tumor suppressor. The decreased level of SIRT1 was associated with increased levels of AKT phosphorylation and interleukin (il)-6 mRNA but decreased tumor suppressor p53 and retinoic acid receptor (RAR)-ß mRNA levels in the lungs. Using this mouse model, we then determined whether ß-cryptoxanthin (BCX), a xanthophyll that is strongly associated with a reduced risk of lung cancer in several cohort studies, can inhibit nicotine-induced emphysema and lung tumorigenesis. We found that BCX supplementation at two different doses was associated with reductions of the nicotine-promoted lung tumor multiplicity and volume, as well as emphysema in mice treated with both NNK and nicotine. Moreover, BCX supplementation restored the nicotine-suppressed expression of lung SIRT1, p53, and RAR-ß to that of the control group, increased survival probability, and decreased the levels of lung il-6 mRNA and phosphorylation of AKT. The present study indicates that BCX is a preventive agent against emphysema and lung cancer with SIRT1 as a potential target. In addition, our study establishes a relevant animal lung cancer model for studying tumor growth within emphysematous microenvironments.