Assessment of inhalation toxicity of cigarette smoke and aerosols from flavor mixtures: 5-week study in A/J mice.

Most flavors used in e-liquids are generally recognized as safe for oral consumption, but their potential effects when inhaled are not well characterized. In vivo inhalation studies of flavor ingredients in e-liquids are scarce. A structure-based grouping approach was used to select 38 flavor group representatives (FGR) on the basis of known and in silico-predicted toxicological data. These FGRs were combined to create prototype e-liquid formulations and tested against cigarette smoke (CS) in a 5-week inhalation study. Female A/J mice were whole-body exposed for 6 h/day, 5 days/week, for 5 weeks to air, mainstream CS, or aerosols from (1) test formulations containing propylene glycol (PG), vegetable glycerol (VG), nicotine (N; 2% w/w), and flavor (F) mixtures at low (4.6% w/w), medium (9.3% w/w), or high (18.6% w/w) concentration or (2) base formulation (PG/VG/N). Male A/J mice were exposed to air, PG/VG/N, or PG/VG/N/F-high under the same exposure regimen. There were no significant mortality or in-life clinical findings in the treatment groups, with only transient weight loss during the early exposure adaptation period. While exposure to flavor aerosols did not cause notable lung inflammation, it caused only minimal adaptive changes in the larynx and nasal epithelia. In contrast, exposure to CS resulted in lung inflammation and moderate-to-severe changes in the epithelia of the nose, larynx, and trachea. In summary, the study evaluates an approach for assessing the inhalation toxicity potential of flavor mixtures, thereby informing the selection of flavor exposure concentrations (up to 18.6%) for a future chronic inhalation study.

A 6-month inhalation toxicology study in Apoe-/- mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe-/- mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations-(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)-or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure-volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.

Impact of whole-body versus nose-only inhalation exposure systems on systemic, respiratory, and cardiovascular endpoints in a 2-month cigarette smoke exposure study in the ApoE-/- mouse model.

Cigarette smoking is one major modifiable risk factor in the development and progression of chronic obstructive pulmonary disease and cardiovascular disease. To characterize and compare cigarette smoke (CS)-induced disease endpoints after exposure in either whole-body (WB) or nose-only (NO) exposure systems, we exposed apolipoprotein E-deficient mice to filtered air (Sham) or to the same total particulate matter (TPM) concentration of mainstream smoke from 3R4F reference cigarettes in NO or WB exposure chambers (EC) for 2 months. At matching TPM concentrations, we observed similar concentrations of carbon monoxide, acetaldehyde, and acrolein, but higher concentrations of nicotine and formaldehyde in NOEC than in WBEC. In both exposure systems, CS exposure led to the expected adaptive changes in nasal epithelia, altered lung function, lung inflammation, and pronounced changes in the nasal epithelial transcriptome and lung proteome. Exposure in the NOEC caused generally more severe histopathological changes in the nasal epithelia and a higher stress response as indicated by body weight decrease and lower blood lymphocyte counts compared with WB exposed mice. Erythropoiesis, and increases in total plasma triglyceride levels and atherosclerotic plaque area were observed only in CS-exposed mice in the WBEC group but not in the NOEC group. Although the composition of CS in the breathing zone is not completely comparable in the two exposure systems, the CS-induced respiratory disease endpoints were largely confirmed in both systems, with a higher magnitude of severity after NO exposure. CS-accelerated atherosclerosis and other pro-atherosclerotic factors were only significant in WBEC.

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.

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.

Reduced Chronic Toxicity and Carcinogenicity in A/J Mice in Response to Life-Time Exposure to Aerosol From a Heated Tobacco Product Compared With Cigarette Smoke.

We conducted an inhalation study, in accordance with Organisation for Economic Co-operation and Development Test Guideline 453, exposing A/J mice to tobacco heating system (THS) 2.2 aerosol or 3R4F reference cigarette smoke (CS) for up to 18 months to evaluate chronic toxicity and carcinogenicity. All exposed mice showed lower thymus and spleen weight, blood lymphocyte counts, and serum lipid concentrations than sham mice, most likely because of stress and/or nicotine effects. Unlike THS 2.2 aerosol-exposed mice, CS-exposed mice showed increased heart weight, changes in red blood cell profiles and serum liver function parameters. Similarly, increased pulmonary inflammation, altered lung function, and emphysematous changes were observed only in CS-exposed mice. Histopathological changes in other respiratory tract organs were significantly lower in the THS 2.2 aerosol-exposed groups than in the CS-exposed group. Chronic exposure to THS 2.2 aerosol also did not increase the incidence or multiplicity of bronchioloalveolar adenomas or carcinomas relative to sham, whereas CS exposure did. Male THS 2.2 aerosol-exposed mice had a lower survival rate than sham mice, related to an increased incidence of urogenital issues that appears to be related to congenital factors rather than test item exposure. The lower impact of THS 2.2 aerosol exposure on tumor development and chronic toxicity is consistent with the significantly reduced levels of harmful and potentially harmful constituents in THS 2.2 aerosol relative to CS. The totality of the evidence from this study further supports the risk reduction potential of THS 2.2 for lung diseases in comparison with cigarettes.

Respiratory Effects of Exposure to Aerosol From the Candidate Modified-Risk Tobacco Product THS 2.2 in an 18-Month Systems Toxicology Study With A/J Mice.

Smoking cessation is the most effective measure for reducing the risk of smoking-related diseases. However, switching to less harmful products (modified-risk tobacco products [MRTP]) can be an alternative to help reduce the risk for adult smokers who would otherwise continue to smoke. In an 18-month chronic carcinogenicity/toxicity study in A/J mice (OECD Test Guideline 453), we assessed the aerosol of Tobacco Heating System 2.2 (THS 2.2), a candidate MRTP based on the heat-not-burn principle, compared with 3R4F cigarette smoke (CS). To capture toxicity- and disease-relevant mechanisms, we complemented standard toxicology endpoints with in-depth systems toxicology analyses. In this part of our publication series, we report on integrative assessment of the apical and molecular exposure effects on the respiratory tract (nose, larynx, and lungs). Across the respiratory tract, we found changes in inflammatory response following 3R4F CS exposure (eg, antimicrobial peptide response in the nose), with both shared and distinct oxidative and xenobiotic responses. Compared with 3R4F CS, THS 2.2 aerosol exerted far fewer effects on respiratory tract histology, including adaptive tissue changes in nasal and laryngeal epithelium and inflammation and emphysematous changes in the lungs. Integrative analysis of molecular changes confirmed the substantially lower impact of THS 2.2 aerosol than 3R4F CS on toxicologically and disease-relevant molecular processes such as inflammation, oxidative stress responses, and xenobiotic metabolism. In summary, this work exemplifies how apical and molecular endpoints can be combined effectively for toxicology assessment and further supports findings on the reduced respiratory health risks of THS 2.2 aerosol.

Evaluation of toxicity of aerosols from flavored e-liquids in Sprague-Dawley rats in a 90-day OECD inhalation study, complemented by transcriptomics analysis.

The use of flavoring substances is an important element in the development of reduced-risk products for adult smokers to increase product acceptance and encourage switching from cigarettes. In a first step towards characterizing the sub-chronic inhalation toxicity of neat flavoring substances, a study was conducted using a mixture of the substances in a base solution of e-liquid, where the standard toxicological endpoints of the nebulized aerosols were supplemented with transcriptomics analysis. The flavor mixture was produced by grouping 178 flavors into 26 distinct chemical groups based on structural similarities and potential metabolic and biological effects. Flavoring substances predicted to show the highest toxicological effect from each group were selected as the flavor group representatives (FGR). Following Organization for Economic Cooperation and Development Testing Guideline 413, rats were exposed to three concentrations of the FGR mixture in an e-liquid composed of nicotine (23 µg/L), propylene glycol (1520 µg/L), and vegetable glycerin (1890 µg/L), while non-flavored and no-nicotine mixtures were included as references to identify potential additive or synergistic effects between nicotine and the flavoring substances. The results indicated that the inhalation of an e-liquid containing the mixture of FGRs caused very minimal local and systemic toxic effects. In particular, there were no remarkable clinical (in-life) observations in flavored e-liquid-exposed rats. The biological effects related to exposure to the mixture of neat FGRs were limited and mainly nicotine-mediated, including changes in hematological and blood chemistry parameters and organ weight. These results indicate no significant additive biological changes following inhalation exposure to the nebulized FGR mixture above the nicotine effects measured in this sub-chronic inhalation study. In a subsequent study, e-liquids with FGR mixtures will be aerosolized by thermal treatment and assessed for toxicity.

Multi-omics systems toxicology study of mouse lung assessing the effects of aerosols from two heat-not-burn tobacco products and cigarette smoke.

Cigarette smoke (CS) causes adverse health effects and, for smoker who do not quit, modified risk tobacco products (MRTPs) can be an alternative to reduce the risk of developing smoking-related diseases. Standard toxicological endpoints can lack sensitivity, with systems toxicology approaches yielding broader insights into toxicological mechanisms. In a 6-month systems toxicology study on ApoE-/- mice, we conducted an integrative multi-omics analysis to assess the effects of aerosols from the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2-a potential and a candidate MRTP based on the heat-not-burn (HnB) principle-compared with CS at matched nicotine concentrations. Molecular exposure effects in the lungs were measured by mRNA/microRNA transcriptomics, proteomics, metabolomics, and lipidomics. Integrative data analysis included Multi-Omics Factor Analysis and multi-modality functional network interpretation. Across all five data modalities, CS exposure was associated with an increased inflammatory and oxidative stress response, and lipid/surfactant alterations. Upon HnB aerosol exposure these effects were much more limited or absent, with reversal of CS-induced effects upon cessation and switching to CHTP 1.2. Functional network analysis revealed CS-induced complex immunoregulatory interactions across the investigated molecular layers (e.g., itaconate, quinolinate, and miR-146) and highlighted the engagement of the heme-Hmox-bilirubin oxidative stress axis by CS. This work exemplifies how multi-omics approaches can be leveraged within systems toxicology studies and the generated multi-omics data set can facilitate the development of analysis methods and can yield further insights into the effects of toxicological exposures on the lung of mice.

E-vapor aerosols do not compromise bone integrity relative to cigarette smoke after 6-month inhalation in an ApoE-/- mouse model.

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE-/- mice were exposed to aerosols from three different e-vapor formulations-CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)-to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.

A 6-month systems toxicology inhalation study in ApoE-/- mice demonstrates reduced cardiovascular effects of E-vapor aerosols compared with cigarette smoke.

Smoking cigarettes is harmful to the cardiovascular system. Considerable attention has been paid to the reduced harm potential of alternative nicotine-containing inhalable products such as e-cigarettes. We investigated the effects of E-vapor aerosols or cigarette smoke (CS) on atherosclerosis progression, cardiovascular function, and molecular changes in the heart and aorta of female apolipoprotein E-deficient (ApoE-/-) mice. The mice were exposed to aerosols from three different E-vapor formulations: 1) carrier (propylene glycol and vegetable glycerol), 2) base (carrier and nicotine), or 3) test (base and flavor) or to CS from 3R4F reference cigarettes for up to 6 mo. Concentrations of CS and base or test aerosols were matched at 35 µg nicotine/L. Exposure to CS, compared with sham-exposed fresh air controls, accelerated atherosclerotic plaque formation, whereas no such effect was seen for any of the three E-vapor aerosols. Molecular changes indicated disease mechanisms related to oxidative stress and inflammation in general, plus changes in calcium regulation, and altered cytoskeletal organization and microtubule dynamics in the left ventricle. While ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following E-vapor aerosols exposure, the nicotine-containing base and test aerosols caused an increase in isovolumic relaxation time similar to CS. A nicotine-related increase in pulse wave velocity and arterial stiffness was also observed, but it was significantly lower for base and test aerosols than for CS. These results demonstrate that in comparison with CS, E-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular diseases.NEW & NOTEWORTHY Analysis of key urinary oxidative stress markers and proinflammatory cytokines showed an absence of oxidative stress and inflammation in the animals exposed to E-vapor aerosols. Conversely, animals exposed to conventional cigarette smoke had high urinary levels of these markers. When compared with conventional cigarette smoke, E-vapor aerosols induced smaller atherosclerotic plaque surface area and volume. Systolic and diastolic cardiac function, as well as endothelial function, were further significantly less affected by electronic cigarette aerosols than conventional cigarette smoke. Molecular analysis demonstrated that E-vapor aerosols induce significantly smaller transcriptomic dysregulation in the heart and aorta compared with conventional cigarette smoke.

Structural, functional, and molecular impact on the cardiovascular system in ApoE-/- mice exposed to aerosol from candidate modified risk tobacco products, Carbon Heated Tobacco Product 1.2 and Tobacco Heating System 2.2, compared with cigarette smoke.

AIM: To investigate the molecular, structural, and functional impact of aerosols from candidate modified risk tobacco products (cMRTP), the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2, compared with that of mainstream cigarette smoke (CS) on the cardiovascular system of ApoE-/- mice. METHODS: Female ApoE-/- mice were exposed to aerosols from THS 2.2 and CHTP 1.2 or to CS from the 3R4F reference cigarette for up to 6 months at matching nicotine concentrations. A Cessation and a Switching group (3 months exposure to 3R4F CS followed by filtered air or CHTP 1.2 for 3 months) were included. Cardiovascular effects were investigated by echocardiographic, histopathological, immunohistochemical, and transcriptomics analyses. RESULTS: Continuous exposure to cMRTP aerosols did not affect atherosclerosis progression, heart function, left ventricular (LV) structure, or the cardiovascular transcriptome. Exposure to 3R4F CS triggered atherosclerosis progression, reduced systolic ejection fraction and fractional shortening, caused heart LV hypertrophy, and initiated significant dysregulation in the transcriptomes of the heart ventricle and thoracic aorta. Importantly, the structural, functional, and molecular changes caused by 3R4F CS were improved in the smoking cessation and switching groups. CONCLUSION: Exposure to cMRTP aerosols lacked most of the CS exposure-related functional, structural, and molecular effects. Smoking cessation or switching to CHTP 1.2 aerosol caused similar recovery from the 3R4F CS effects in the ApoE-/- model, with no further acceleration of plaque progression beyond the aging-related rate.

Systems toxicology assessment of a representative e-liquid formulation using human primary bronchial epithelial cells.

The development of reduced-risk products aims to provide alternatives to cigarettes that present less risk of harm for adult smokers. Responsible use of flavoring substances in these products may fulfill an important role in product acceptance. While most flavoring substances used in such products are also used by the food industry and are considered safe when ingested, their impact when inhaled may require further assessment. To aid in such an assessment, a three-step approach combining real-time cellular analysis, phenotypic high-content screening assays, and gene expression analysis was developed and tested in normal human bronchial epithelial cells with 28 flavoring substances commonly used in e-liquid formulations, dissolved individually or as a mixture in a base solution composed of propylene glycol, vegetable glycerin, and 0.6% nicotine. By employing this approach, we identified individual flavoring substances that potentially contribute greatly to the overall mixture effect (citronellol and alpha-pinene). By assessing modified mixtures, we showed that, although cytotoxic effects were found when assessed individually, alpha-pinene did not contribute to the overall mixture cytotoxicity. Most of the cytotoxic effect appeared to be attributable to citronellol, with the remaining substances contributing due to synergistic effects. We developed and used different scoring methods (Tox-Score, Phenotypic Score, and Biological Impact Factor/Network Perturbation Amplitude), ultimately enabling a ranking based on cytotoxicity, phenotypic outcome, and molecular network perturbations. This case study highlights the benefits of testing both individual flavoring substances and mixtures for e-liquid flavor assessment and emphasized the importance of data sharing for the benefit of consumer safety.

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.

Antiparasitic properties of leaf extracts derived from selected Nicotiana species and Nicotiana tabacum varieties.

Within the traditional pharmacopeia, tobacco (Nicotiana spp.) is often cited as an efficient pesticide. This activity is generally attributed to nicotine, but tobacco plants contain other alkaloids that could potentially contribute to this effect. In this study, we tested methanolic extracts of N. glutinosa, N. glauca, N. debneyi, and N. tabacum (putrescine N-methyltransferase line, burley TN90 and Stella, Virginia ITB 683 and K326), selected according to alkaloid content. Their antiparasitic activity was evaluated in bioassays against adult fleas (Ctenocephalides felis), blowfly (Lucilia cuprina) larvae, nematodes (Caenorhabditis elegans), and ticks (Rhipicephalus sanguineus larvae and adults, Ixodes ricinus nymphs). None of the extracts killed fleas and blowfly larvae effectively at the concentrations tested. Only N. tabacum K326 and N. glutinosa exhibited moderate anthelmintic activity. All extracts significantly repelled R. sanguineus ticks, but not I. ricinus, and the nicotine-rich extracts rapidly knocked down all tick species and stages at high concentrations. The link between nicotine and tick knockdown was confirmed by successfully testing the pure alkaloid at concentrations found in the tobacco extracts. In contrast, repellent activity could not be correlated to the individually tested alkaloids (nicotine, nornicotine, anabasine, anatabine), although anatabine and nornicotine were active in the tick bioassay at high concentrations.

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.

A six-month systems toxicology inhalation/cessation study in ApoE-/- mice to investigate cardiovascular and respiratory exposure effects of modified risk tobacco products, CHTP 1.2 and THS 2.2, compared with conventional cigarettes.

Smoking is one of the major modifiable risk factors in the development and progression of chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD). Modified-risk tobacco products (MRTP) are being developed to provide substitute products for smokers who are unable or unwilling to quit, to lessen the smoking-related health risks. In this study, the ApoE-/- mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F, or aerosol from two potential MRTPs based on the heat-not-burn principle, carbon heated tobacco product 1.2 (CHTP1.2) and tobacco heating system 2.2 (THS 2.2), on the cardiorespiratory system over a 6-month period. In addition, cessation or switching to CHTP1.2 after 3 months of CS exposure was assessed. A systems toxicology approach combining physiology, histology and molecular measurements was used to evaluate the impact of MRTP aerosols in comparison to CS. CHTP1.2 and THS2.2 aerosols, compared with CS, demonstrated lower impact on the cardiorespiratory system, including low to absent lung inflammation and emphysematous changes, and reduced atherosclerotic plaque formation. Molecular analyses confirmed the lower engagement of pathological mechanisms by MRTP aerosols than CS. Both cessation and switching to CHTP1.2 reduced the observed CS effects to almost sham exposure levels.

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 90-day OECD TG 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of the aerosol from the carbon heated tobacco product version 1.2 (CHTP1.2) compared with cigarette smoke. I. Inhalation exposure, clinical pathology and histopathology.

Within the framework of a systems toxicology approach, the inhalation toxicity of aerosol from a novel tobacco-heating potentially modified risk tobacco product (MRTP), the carbon-heated tobacco product (CHTP) 1.2, was characterized and compared with that of mainstream smoke (CS) from the 3R4F reference cigarette in a 90-day nose-only rat inhalation study in general accordance with OECD TG 413. CHTP1.2 is a heat-not-burn product using a carbon heat source to produce an aerosol that contains nicotine and tobacco flavor. At equal or twice the nicotine concentration in the test atmospheres, inhalation of CHTP1.2 aerosol led to a significantly lower exposure to harmful constituents and induced less respiratory tract irritation, systemic, and pathological effects compared with CS. Nasal epithelial changes were less pronounced in the CHTP1.2- than in the CS-exposed groups and reverted in the nicotine concentration-matched group after a recovery period. Lung inflammation was minimal in the CHTP1.2-treated groups compared with the moderate extent seen in the 3R4F groups. Many other toxicological endpoints evaluated did not show CHTP1.2 aerosol exposure-related effects, and no effects not seen for 3R4F were observed. These observations were consistent with findings from previous studies in which rats were exposed to MRTP aerosols containing similar nicotine concentrations.