Cytotoxic impact of nicotine products on periodontal ligament cells.

OBJECTIVES: The primary objective of this in vitro experiment was an assessment of proliferative capacity, metabolic activity, and potential cellular detriment of human periodontal ligament cells (hPDL) exposed to cigarette smoke (CS), electronic cigarette vapor (eCV), and heated tobacco product aerosol (HTP), or air (control). MATERIALS AND METHODS: Using a CAD/CAM-designed exposition chamber, hPDL were exposed to CS, eCV, HTP, or air (control) based on the Health Canada Intense Smoking Regime. Cell proliferation, metabolic activity, and cellular detriment were assessed at various time points. RESULTS: Compared to the control, hPDL exposed to CS exhibited significantly decreased cell numbers at all time points. HTP exposure led to reduced cell numbers 48 h and 72 h post-exposure, while eCV-exposed cells showed no significant decrease. The metabolic activity of eCV-treated hPDL was slightly reduced at 7 h but recovered at 24 h and 48 h. In contrast, CS-treated cells exhibited significantly decreased metabolic activity at 24 h and 48 h, and HTP-exposed cells showed a significant decrease after 48 h. Flow cytometry indicated both apoptotic and necrotic cell death following CS exposure, with necrotic cell death being more pronounced. CONCLUSIONS: eCV and HTP demonstrated comparatively reduced detrimental effects on hPDL compared to CS. CLINICAL RELEVANCE: The findings suggest that conventional cigarette smoke poses a substantial risk to periodontal health by significantly impairing cell proliferation and metabolic activity. However, alternatives such as eCV and HTP may offer a comparatively reduced risk.

Toxicity of cigarette butts (CBs) leachate on Nile tilapia (Oreochromis niloticus): Blood biochemical parameters, oxidative stress biomarkers, and metabolic profile.

The aim of this study is to evaluate the toxic effects of different concentrations of cigarette butt leachate (CBL) (0.0, 0.5, 1, 1.5, and 2.0 µL L-1) on blood biochemistry, oxidative stress biomarkers, and the biochemical profile of the liver and muscle of Nile tilapia fish (Oreochromis niloticus) after 21 days. Increased activity of lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), and aspartate aminotransferase (AST) in plasma, and decreased activity of alkaline phosphatase (ALP) in fish exposed to CBL, indicated cytotoxicity. Elevated cholesterol, triglycerides, and glucose levels, coupled with reduced total protein, albumin, and globulin levels in the plasma, indicated impaired liver function in the fish. An increase in creatinine showed kidney damage. Increased superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, along with the decrease in liver glutathione (GSH) content and total antioxidant capacity in the hepatocytes of fish exposed to CBL, indicated the occurrence of oxidative stress. Malondialdehyde (MDA) elevation indicated heightened lipid peroxidation in CBL-exposed fish hepatocytes. Raman spectroscopy revealed altered biochemical profiles in fish liver and muscle post-CBL exposure. The results demonstrated that exposure to CBL led to a decrease in phospholipid levels, collagen destruction, changes in phenylalanine levels, and a decrease in the levels of lipids, proteins, and nucleic acids in fish liver and muscle tissue. Furthermore, the metabolites and compounds of cigarette butt juice were detectable in the liver and muscle tissue of fishes. In conclusion, this study showed that exposure to CBL can have adverse effects on fish health.

In vitro toxicological evaluation of glo menthol and non-menthol heated tobacco products.

Heated tobacco products (HTPs) are non-combustible, inhaled tobacco products that generate an aerosol with fewer and lower levels of toxicants, with a potential to reduce risk relative to cigarette smoking. Here, we assessed in vitro toxicological effects of three menthol (glo neo neoCLICK, neo Smooth Menthol and Fresh Menthol) and one non-menthol (neo Smooth Tobacco) variants of glo HTP, along with market comparators for cigarettes and HTPs. Limited chemical characterization of the study products revealed significantly lower levels of acetaldehyde, acrolein, crotanaldehyde and formaldehyde in test samples from HTPs than those from cigarettes. The glo HTPs were non-mutagenic in the bacterial reverse mutagenesis assay. Although, the whole aerosol exposures of glo HTPs were classified as genotoxic in the in vitro micronucleus assay, and cytotoxic in the NRU (monolayer) and MTT (3 dimensional EpiAirway™ tissues) assays, the cigarette comparators were the most toxic study products in each of these assessments. Further, glo HTPs elicited oxidative stress responses only at the highest dose tested, whereas the cigarette comparators were potent inducers of oxidative stress at substantially lower doses in the EpiAirway tissues. The comparator (non-glo) HTP results were similar to the glo HTPs in these assays. Thus, the glo HTPs exhibit substantially lower toxicity compared to cigarettes.

Reduction of the environmental and health consequences of cigarette butt recycling by removal of toxic and carcinogenic compounds from its leachate.

Cigarette butt is a hazardous waste, and its management faces many challenges. The generation of leachate containing many pollutants including heavy metals is one of the limitations of recycling cigarette butts. The aim of this study was to reduce organic compounds and heavy metals in leachate resulting from cigarette butt recycling in an electrocoagulation reactor. For this purpose, two samples, including freshly smoked cigarette butts and littered cigarette butts, were processed and the treatment of leachate resulting from them was studied in an electrocoagulation reactor. The efficiency of leachate treatment was investigated in the treatment time of 10-40 min and current intensity of 20-100 mA. The results showed that the minimum reduction of chemical oxygen demand and turbidity was 25.3% and 33.4%, respectively. Increasing the current intensity and treatment time increased the efficiency of removing turbidity and chemical oxygen demand up to 47.1% and 41.2%, respectively, in optimum conditions. The reduction of nickel, chromium, cadmium, and lead in the lowest current density and minimum treatment time was more than 90%, which increased to more than 99% with the increase of the current density and treatment time. The use of electrocoagulation is a suitable solution to reduce heavy metals in leachate resulting from cigarette butt recycling, while the proper reduction of organic compounds in this type of leachate requires further treatment processes.

An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

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.

Key Challenges for In Vitro Testing of Tobacco Products for Regulatory Applications: Recommendations for the In Vitro Mouse Lymphoma Assay.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across traditional tobacco and various tobacco and nicotine next-generation products (NGPs), including Heated Tobacco Products (HTPs) and Electronic Nicotine Delivery Systems (ENDS). This report was developed by a working group composed of attendees of the seventh IIVS workshop, 'Approaches and recommendations for conducting the mouse lymphoma gene mutation assay (MLA) and introduction to in vitro disease models', which was held virtually on 21-23 June 2022. This publication provides a background overview of the MLA, and includes the description of assay conduct and data interpretation, key challenges and recommended best practices for evaluating tobacco and nicotine products, with a focus on the evaluation of NGPs, and a summary of how the assay has been used to evaluate and compare tobacco and nicotine products.

Waterborne Toxicity to Neotropical Invertebrates and Hazard of Cigarette Butt Leachates in Marine Environments.

Cigarette butts (CBs) are the most common type of beach litter worldwide and contain a complex mixture of chemicals. Given the recent interest in this emerging problem, it is important to assess the toxicity of CB leachates to a range of species from different regions, sensitivities, and ecological traits. We evaluated the waterborne toxicity of smoked CB to tropical invertebrates. Leachates were prepared in the laboratory and characterized for trace elements (Mn, Fe, Co, Ni, Cu, Zn, As, Cd, and Pb), ammonia nitrogen, and polycyclic aromatic hydrocarbons. Then a set of toxicity tests with marine invertebrates was performed as follows: the brine shrimp Artemia sp. (nontoxic); the amphipod Tiburonella viscana (median lethal concentration [LC50] of 0.038 CB/L); the tanaid Monokalliapseudes schubarti (LC50 of 0.126 CB/L); the copepods Tisbe biminiensis (median effect concentration [EC50] of 0.038 CB/L) and Nitokra sp. (EC50 of 0.009 CB/L); pluteus stage larvae of the sea urchin Echinometra lucunter (EC50 of 0.152 CB/L); the sand dollar Mellita quinquiesperforata (EC50 of 0.054 CB/L); and D-stage larvae of the mussel Perna perna (EC50 of 0.005 CB/L). The predicted no-effect concentration was estimated using species sensitivity distribution, producing a 5th percentile hazard concentration of 0.015 CB/L. This preliminary threshold allowed us to estimate the potential impact of a single CB to 67 L of seawater via leaching, contributing to the advancement of knowledge regarding the contamination, toxicity, and ecological risks of cigarette waste. Environ Toxicol Chem 2024;43:374-384. © 2023 SETAC.

Evaluation of toxicity of heated tobacco products aerosol and cigarette smoke to BEAS-2B cells based on 3D biomimetic chip model.

It is still a controversial topic about evaluating whether heated tobacco products (HTP) really reduce harm, which involves the choice of an experimental model. Here, a three-dimensional (3D) biomimetic chip model was used to evaluate the toxicity of aerosols came from HTP and smoke produced by cigarettes (Cig). Based on cell-related experiments, we found that the toxicity of Cig smoke extract diluted four times was also much higher than that of undiluted HTP, showing higher oxidative stress response and cause mitochondrial dysfunction. Meanwhile, both tobacco products all affect the tricarboxylic acid cycle (TCA), which is manifested by a significant decrease in the mRNA expression of TCA key rate-limiting enzymes. Summarily, 3D Biomimetic chip technology can be used as an ideal model to evaluate HTP. It can provide important data for tobacco risk assessment when 3D chip model was used. Our experimental results showed that HTP may be less harmful than tobacco cigarettes, but it does show significant cytotoxicity with the increase of dose. Therefore, the potential clinical effects of HTP on targeted organs such as lung should be further studied.

A multi-organ, lung-derived inflammatory response following in vitro airway exposure to cigarette smoke and next-generation nicotine delivery products.

Despite increasing use of in vitro models that closely resemble in vivo human biology, their application in understanding downstream effects of airway toxicity, such as inflammation, are at an early stage. In this study, we used various assays to examine the inflammatory response induced in MucilAir™ tissues and A549 cells exposed to three products known to induce toxicity. Reduced barrier integrity was observed in tissues following exposure to each product, with reduced viability and increased cytotoxicity also shown. Similar changes in viability were also observed in A549 cells. Furthermore, whole cigarette smoke (CS) induced downstream phenotypic THP-1 changes and endothelial cell adhesion, an early marker of atherosclerosis. In contrast, exposure to next-generation delivery product (NGP) aerosol did not induce this response. Cytokine, histological and RNA analysis highlighted increased biomarkers linked to inflammatory pathways and immune cell differentiation following exposure to whole cigarette smoke, including GM-CSF, IL-1ß, cleaved caspase-3 and cytochrome P450 enzymes. As a result of similar observations in human airway inflammation, we propose that our exposure platform could act as a representative model for studying such events in vitro. Furthermore, this model could be used to test the inflammatory or anti-inflammatory impact posed by inhaled compounds delivered to the lung.

Oral nicotine pouches with an aftertaste? Part 2: in vitro toxicity in human gingival fibroblasts.

Nicotine pouches contain fewer characteristic toxicants than conventional tobacco products. However, the associated risks in terms of toxicity and addiction potential are still unclear. Therefore, endpoints of toxicity and contents of flavoring substances were investigated in this study. The in vitro toxicity of five different nicotine pouches and the reference snus CRP1.1 were studied in human gingival fibroblasts (HGF-1). Cells were exposed to product extracts (nicotine contents: 0.03-1.34 mg/mL) and sampled at different time points. Cytotoxicity, total cellular reactive oxygen species (ROS) levels, and changes in the expression levels of inflammatory and oxidative stress genes were assessed. Flavor compounds used in the nicotine pouches were identified by GC-MS. Cytotoxicity was observed in two nicotine pouches. Gene expression of interleukin 6 (IL6) and heme oxygenase 1 (HMOX1) was upregulated by one and three pouches, respectively. ROS production was either increased or decreased, by one pouch each. CRP1.1 caused an upregulation of IL6 and elevated ROS production. Toxicity was not directly dependent on nicotine concentration and osmolarity. A total of 56 flavorings were detected in the five nicotine pouches. Seven flavorings were classified according to the harmonized hazard classification system as laid down in the European Classification, Labelling and Packaging regulation. Nine flavorings are known cytotoxins. Cytotoxicity, inflammation, and oxidative stress responses indicate that adverse effects such as local lesions in the buccal mucosa may occur after chronic product use. In conclusion, flavorings used in nicotine pouches likely contribute to the toxicity of nicotine pouches.

Comparison of the toxicological potential of two JUUL ENDS products to reference cigarette 3R4F and filtered air in a 90-day nose-only inhalation toxicity study.

Electronic nicotine delivery systems (ENDS) are generally recognized as less harmful alternatives for those who would otherwise continue to smoke cigarettes. The potential toxicity of aerosols generated from JUUL Device and Virginia Tobacco (VT3) or Menthol (ME3) JUULpods at 3.0% nicotine concentration was assessed in rats exposed at target aerosol concentrations of 1400 µg/L for up to 6 h/day on a 5 day/week basis for at least 90 days (general accordance with OECD 413). 3R4F reference cigarette smoke (250 µg/L) and Filtered Air were used as comparators. JUUL ENDS product aerosol exposures at >5x the 3R4F cigarette smoke level resulted in greater plasma nicotine and cotinine levels (up to 2x). Notable cigarette smoke related effects included pronounced body weight reductions in male rats, pulmonary inflammation evidenced by elevated lactate dehydrogenase, pro-inflammatory cytokines and neutrophils in bronchoalveolar lavage fluid, increased heart and lung weights, and minimal to marked respiratory tract histopathology. In contrast, ENDS aerosol exposed animals had minimal body weight changes, no measurable inflammatory changes and minimal to mild laryngeal squamous metaplasia. Despite the higher exposure levels, VT3 and ME3 did not result in significant toxicity or appreciable respiratory histopathology relative to 3R4F cigarette smoke following 90 days administration.

Effects of heated tobacco product aerosol extracts on DNA methylation and gene transcription in lung epithelial cells.

AIMS: Smoking causes DNA methylation (DNAm) alterations that lead to lung cancer development. Although the use of heated tobacco products (HTPs) has recently increased, their impact on health remains unclear. This study aimed to evaluate the effects of HTPs on DNAm and gene transcription in human lung epithelial cells in vitro. MAIN METHODS: Human lung adenocarcinoma (A549) cells with type II alveolar epithelial characteristics were treated with aerosol extracts of two HTPs or a smoke extract of combustible reference cigarette (RC). Global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels were quantified using dot blot analysis. Furthermore, reduced representation of bisulfite sequencing, DNA microarray, and quantitative PCR analyses were performed to determine CpG methylation and gene transcription changes induced by HTP and RC. KEY FINDINGS: Global 5-mC and 5-hmC levels were decreased by the RC extract but not the HTP extracts. However, an HTP extract altered the CpG methylation pattern, and Gene Ontology enrichment analysis of the differentially methylated regions of the RC and HTP groups showed a similar pattern. The HTP extract affected gene expression, albeit to a lesser extent than the RC extract. In particular, the HTP extract markedly affected the mRNA expression and promoter methylation of cytochrome P450 family 1 subfamily A member 1 (CYP1A1), which is associated with carcinogenic risk. SIGNIFICANCE: The study results suggest that HTPs as well as conventional combustible cigarettes can alter CpG methylation and gene transcription in lung epithelial cells.

Pulmonary immune response regulation, genotoxicity, and metabolic reprogramming by menthol- and tobacco-flavored e-cigarette exposures in mice.

Menthol and tobacco flavors are available for almost all tobacco products, including electronic cigarettes (e-cigs). These flavors are a mixture of chemicals with overlapping constituents. There are no comparative toxicity studies of these flavors produced by different manufacturers. We hypothesized that acute exposure to menthol and tobacco-flavored e-cig aerosols induces inflammatory, genotoxicity, and metabolic responses in mouse lungs. We compared two brands, A and B, of e-cig flavors (PG/VG, menthol, and tobacco) with and without nicotine for their inflammatory response, genotoxic markers, and altered genes and proteins in the context of metabolism by exposing mouse strains, C57BL/6J (Th1-mediated) and BALB/cJ (Th2-mediated). Brand A nicotine-free menthol exposure caused increased neutrophils and differential T-lymphocyte influx in bronchoalveolar lavage fluid and induced significant immunosuppression, while brand A tobacco with nicotine elicited an allergic inflammatory response with increased Eotaxin, IL-6, and RANTES levels. Brand B elicited a similar inflammatory response in menthol flavor exposure. Upon e-cig exposure, genotoxicity markers significantly increased in lung tissue. These inflammatory and genotoxicity responses were associated with altered NLRP3 inflammasome and TRPA1 induction by menthol flavor. Nicotine decreased surfactant protein D and increased PAI-1 by menthol and tobacco flavors, respectively. Integration of inflammatory and metabolic pathway gene expression analysis showed immunometabolic regulation in T cells via PI3K/Akt/p70S6k-mTOR axis associated with suppressed immunity/allergic immune response. Overall, this study showed the comparative toxicity of flavored e-cig aerosols, unraveling potential signaling pathways of nicotine and flavor-mediated pulmonary toxicological responses, and emphasized the need for standardized toxicity testing for appropriate premarket authorization of e-cigarette products.

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

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

A contextualised e-cigarette testing strategy shows flavourings do not impact lung toxicity in vitro.

Vaping has the potential to reduce the individual health risks associated with smoking and e-cigarette flavours have been reported to help smokers' transition from cigarettes. In this manuscript, we provide evidence to support the reduced risk potential of e-cigarette aerosols and flavours by assessing commercially available e-liquids (Vuse ePod - Manufactured by British American Tobacco) in a 2D in vitro screening approach. We also analysed selected flavours using a more physiologically relevant 3D (MucilAir) whole aerosol exposure model, measuring toxicity and functional endpoints such as Trans Epithelial Electrical Resistance, Cilia Beat Frequency and Active Area. To contextualise responses, we have compared e-cigarette aerosol to cigarette smoke (1R6F research cigarette) and calculated the percentage reduction using a point of departure approach. We show that aerosolised flavoured e-liquids, (appropriately stewarded) do not increase the overall measured aerosol toxicity when compared to cigarette smoke. In fact, we demonstrate that the measured in vitro cellular toxicity of flavoured e-cigarette products remains > 95% reduced when compared to cigarette smoke toxicity, using point of departure (IC80) approach. These data indicate that the overall product toxicity is not increased in a flavour dependent manner and that flavoured e-cigarette products can potentially play a role in tobacco harm reduction.

Characterisation of a smoke/ aerosol exposure in vitro system (SAEIVS) for delivery of complex mixtures directly to cells at the air-liquid interface.

In vitro testing is important to characterise biological effects of consumer products, including nicotine delivery products such as cigarettes, e-cigarettes and heated tobacco products. Users' cells are exposed to these products' aerosols, of variant chemical compositions, as they move along the respiratory tract. In vitro exposure systems are available to model such exposures, including delivery of whole aerosols to cells, and at the air-liquid interface. Whilst there are clear advantages of such systems, factors including time to aerosol delivery, aerosol losses and number of cell cultures that can be exposed at one time could be improved. This study aimed to characterise a custom-built smoke/ aerosol exposure in vitro system (SAEIVS) using 1R6F reference cigarette smoke. This system contains five parallel smoking chambers and delivers different dilutions of smoke/ aerosol to two separate cell culture exposure chambers in <10 s. Using two dosimetry measures (optical density 400 nm [OD400 ]; mass spectrometric nicotine quantification), the SAEIVS demonstrated excellent linearity of smoke dilution prior to exposure (R2  = 0.9951 for mass spectrometric quantification; R2  = 0.9965 for OD400 ) and consistent puff-wise exposures across 24 and 96 well plates in cell culture relevant formats (e.g., within inserts). Smoke loss was lower than previously reported for other systems (OD400 : 16%; nicotine measurement: 20%). There was good correlation of OD400 and nicotine measurements, indicating that OD was a useful surrogate for exposure dosimetry for the product tested. The findings demonstrated that the SAEIVS is a fit-for-purpose exposure system for the reproducible dose-wise exposure assessment of nicotine delivery product aerosols.

Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Combined biological effects and lung proteomics analysis in mice reveal different toxic impacts of electronic cigarette aerosol and combustible cigarette smoke on the respiratory system.

Combustible cigarettes produce many toxic substances that have been linked to diseases, such as lung cancer and chronic obstructive pulmonary disease. For those smokers unable or unwilling to quit, electronic cigarettes (e-cigarettes) could be used as an alternative to cigarettes. However, the effects and mechanisms of e-cigarette aerosol (ECA) on respiratory function have not been fully elucidated, and in vivo studies of its safety are limited compared to cigarette smoke (CS). In this article, we chose nicotine levels as dosing references and C57BL/6 mice for a 10-week subchronic inhalation toxicity study. A comprehensive set of toxicological endpoints was used to study the effect of exposure. Both CS (6 mg/kg) and ECA (6 or 12 mg/kg) inhalation had decreased the animal's lung function and increased levels of inflammation markers, along with pathological changes in the airways and lungs, with ECA displaying a relatively small effect at the same dose. Proteomic analysis of lung tissue showed greater overall protein changes by CS than that of ECA, with more severe inflammatory network perturbations. Compared with ECA, KEGG analysis of CS revealed upregulation of more inflammatory and virus-related pathways. Protein-protein interactions (PPI) showed that both ECA and CS significantly changed ribosome and complement system-related proteins in mouse lung tissue. The results support that e-cigarette aerosol is less harmful to the respiratory system than cigarette smoke at the same dose using this animal model, thus providing additional evidence for the relative safety of e-cigarettes.

Effect of heated tobacco products and traditional cigarettes on pulmonary toxicity and SARS-CoV-2-induced lung injury.

Cigarette smoke (CS) significantly contributes to the development of chronic obstructive pulmonary disease (COPD). Heated tobacco products (HTPs), newly developed cigarette products, have been proposed as an alternative for safe cigarette smoking. Although it is plausible to think that replacing traditional cigarettes with HTPs would lower the risks of COPD, this notion requires confirmation by further investigations from sources independent of the tobacco industry. COPD is characterized by an ongoing inflammatory process in the lungs, and the renin-angiotensin system (RAS) has been implicated in the pathogenesis of COPD. Angiotensin-converting enzyme-2 (ACE2) functions as a negative regulator of RAS and has been suggested as a cellular receptor for the causative agent of SARS-CoV-2. It has been shown that smoking is most likely associated with the negative progression and adverse outcomes of SARS-CoV-2. In this study, we found that cigarette smoke extracts from traditional cigarettes (CSE) caused higher cytotoxicity and higher oxidative stress levels than extracts from HTPs (HTPE) in two lung cell lines (Calu-3 and Beas-2B). CSE and HTPE induced RAS activation, MAPK activation, and NF-kB inflammatory pathway activation, resulting in the production of inflammatory cytokines. Furthermore, CSE and a high dose of HTPE reduced tight junction proteins, including claudin 1, E-cadherin, and ZO-1, and disrupted lung epidermal tight junctions at the air-liquid interface (ALI). Finally, CSE and HTPE enhanced the spike protein S1-induced lung injury response. Together, these results suggest that HTPE induced similar lung pathogenesis relevant to COPD and SARS-CoV-2-induced lung injury caused by CSE.