Quantitative modeling of in vitro data using an adverse outcome pathway for the risk assessment of decreased lung function in humans.

In the absence of epidemiological data, there is a need to develop computational models that convert in vitro findings to human disease risk predictions following toxicant exposure. In such efforts, in vitro data can be evaluated in the context of adverse outcome pathways (AOPs) that organize mechanistic knowledge based on empirical evidence into a sequence of molecular-, cellular-, tissue-, and organ-level key events that precede an adverse outcome (AO). Here we combined data from advanced in vitro organotypic airway models exposed to combustible cigarette (CC) smoke or Tobacco Heating System (THS) aerosol with an AOP for increased oxidative stress leads to decreased lung function. The mathematical modeling predicted reduced risk of decreased ciliary beating frequency (CBF) based on oxidative stress measurements and reduced risk of decreased mucociliary clearance (MCC) based on CBF measurements in THS aerosol- compared with CC smoke-exposed cultures. To extend the predictions to the AO of decreased lung function, we leveraged human MCC data from current smokers, nonsmokers, former smokers, and users of heated tobacco products. This approach provided a plausible prediction of diminished reduction in lung function in response to THS use compared with continued smoking. The current approach may also present a basis for an integrated approach to testing and assessment of tobacco products for future regulatory decision-making.

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.

Impact of aerosols on liver xenobiotic metabolism: A comparison of two methods of exposure.

Assessment of aerosols effects on liver CYP function generally involves aqueous fractions (AF). Although easy and efficient, this method has not been optimized recently or comparatively assessed against other aerosol exposure methods. Here, we comparatively evaluated the effects of the AFs of cigarette smoke (CS) and Tobacco Heating System (THS) aerosols on CYP activity in liver spheroids. We then used these data to develop a physiological aerosol exposure system combining a multi-organs-on-a-chip, 3D lung tissues, liver spheroids, and a direct aerosol exposure system. Liver spheroids incubated with CS AF showed a dose-dependent increase in CYP1A1/1B1, CYP1A2, and CYP2B6 activity and a dose-dependent decrease in CYP2C9, CYP2D6, and CYP3A4 activity relative to untreated tissues. In our physiological exposure system, repeated CS exposure of the bronchial tissues also caused CYP1A1/1B1 and CYP1A2 induction in the bronchial tissues and liver spheroids; but the spheroids showed an increase in CYP3A4 activity and no effect on CYP2C9 or CYP2D6 activity relative to air-exposed tissues, which resembles the results reported in smokers. THS aerosol did not affect CYP activity in bronchial or liver tissues, even at 4 times higher concentrations than CS. In conclusion, our system allows us to physiologically test the effects of CS or other aerosols on lung and liver tissues cultured in the same chip circuit, thus delivering more in vivo like data.

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.

Comparing the preclinical risk profile of inhalable candidate and potential candidate modified risk tobacco products: A bridging use case.

Cigarette smoking causes major preventable diseases, morbidity, and mortality worldwide. Smoking cessation and prevention of smoking initiation are the preferred means for reducing these risks. Less harmful tobacco products, termed modified-risk tobacco products (MRTP), are being developed as a potential alternative for current adult smokers who would otherwise continue smoking. According to a regulatory framework issued by the US Food and Drug Administration, a manufacturer must provide comprehensive scientific evidence that the product significantly reduces harm and the risk of tobacco-related diseases, in order to obtain marketing authorization for a new MRTP. For new tobacco products similar to an already approved predicate product, the FDA has foreseen a simplified procedure for assessing "substantial equivalence". In this article, we present a use case that bridges the nonclinical evidence from previous studies demonstrating the relatively reduced harm potential of two heat-not-burn products based on different tobacco heating principles. The nonclinical evidence was collected along a "causal chain of events leading to disease" (CELSD) to systematically follow the consequences of reduced exposure to toxicants (relative to cigarette smoke) through increasing levels of biological complexity up to disease manifestation in animal models of human disease. This approach leverages the principles of systems biology and toxicology as a basis for further extrapolation to human studies. The experimental results demonstrate a similarly reduced impact of both products on apical and molecular endpoints, no novel effects not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP that is comparable to that of complete smoking cessation. Ideally, a subset of representative assays from the presented sequence along the CELSD could be sufficient for predicting similarity or substantial equivalence in the nonclinical impact of novel products; this would require further validation, for which the present use case could serve as a starting point.

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Alternatives to Animal Use in Risk Assessment of Mixtures.

Risk assessment of chemical mixtures has emerged as a focus of research efforts, but traditional toxicology testing in mammals is costly, time-consuming, and subject to ethical scrutiny in the context of recent trends to reduce reliance on animal testing. In this review, which is a summary of presentations given at a workshop in Havana, Cuba, in April 2019, we survey the utility of zebra fish as an alternative laboratory model in whole-mixture and component-based testing, as well as in vitro modeling in 3-dimensional organotypic cultures from primary human cells cultured at the air-liquid interface and organ-on-a-chip platforms. Finally, we discuss the complexities of assessing the dynamics and delivery of multispecies liquid aerosol mixtures along the human respiratory tract, with examples of alternative and computational approaches to aerosol dosimetry. The workshop contributed to the professional development of Cuban toxicologists, an underserved segment of the global scientific community, delivering a set of tools and recommendations that could potentially provide cost-effective solutions for scientists with limited research resources.

Comparison of the basic morphology and function of 3D lung epithelial cultures derived from several donors.

In vitro models of the human lung play an essential role in evaluating the toxicity of inhaled compounds and understanding the development of respiratory diseases. Three-dimensional (3D) organotypic models derived from lung basal epithelial cells and grown at the air-liquid interface resemble human airway epithelium in multiple aspects, including morphology, cell composition, transcriptional profile, and xenobiotic metabolism. Whether the different characteristics of basal cell donors have an impact on model characteristics and responses remains unknown. In addition, studies are often conducted with 3D cultures from one donor, assuming a representative response on the population level. Whether this assumption is correct requires further investigation. In this study, we compared the morphology and functionality of 3D organotypic bronchial and small airway cultures from different donors at different weeks after air-lift to assess the interdonor variability in these parameters. The thickness, cell type composition, and transepithelial electrical resistance varied among the donors and over time after air-lift. Cilia beating frequency increased in response to isoproterenol treatment in both culture types, independent of the donor. The cultures presented low basal cytochrome P450 (CYP) 1A1/1B1 activity, but 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment induced CYP1A1/1B1 activity regardless of the donor. In conclusion, lung epithelial cultures prepared from different donors present diverse morphology but similar functionality and metabolic activity, with certain variability in their response to stimulation.

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.

A lung/liver-on-a-chip platform for acute and chronic toxicity studies.

The merging of three-dimensional in vitro models with multi-organ-on-a-chip (MOC) technology has taken in vitro assessment of chemicals to an unprecedented level. By connecting multiple organotypic models, MOC allows for the crosstalk between different organs to be studied to evaluate a compound's safety and efficacy better than with single cultures. The technology could also improve the toxicological assessment of aerosols that have been implicated in the development of chronic obstructive pulmonary disease, asthma, or lung cancer. Here we report the development of a lung/liver-on-a-chip, connecting in a single circuit, normal human bronchial epithelial (NHBE) cells cultured at the air-liquid interface (ALI), and HepaRG™ liver spheroids. Maintenance of the individual tissues in the chip increased NHBE ALI tissue transepithelial electrical resistance and decreased HepaRG™ spheroid adenosine triphosphate content as well as cytochrome P450 (CYP) 1A1/1B1 inducibility. CYP inducibility was partly restored when HepaRG™ spheroids were cocultured with NHBE ALI tissues. Both tissues remained viable and functional for 28 days when cocultured in the chip. The capacity of the HepaRG™ spheroids to metabolize compounds present in the medium and to modulate their toxicity was proven using aflatoxin B1 (AFB1). AFB1 toxicity in NHBE ALI tissues decreased when HepaRG™ spheroids were present in the same chip circuit, proving that the HepaRG™-mediated detoxification is protecting/decreasing from AFB1-mediated cytotoxicity. The lung/liver-on-a-chip platform presented here offers new opportunities to study the toxicity of inhaled aerosols or to demonstrate the safety and efficacy of new drug candidates targeting the human lung.

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.

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%.