Project description:We previously put forward a multi-layer systems toxicology framework for in vitro assessment of e-liquids that is meant to complement the battery of classical assays for genotoxicity testing. The framework started with the first layer to screen e-liquids for their potential toxicity, followed by the second layer to investigate the toxicity-related mechanism of a selected e-liquid(s), and finally the third layer to evaluate the toxicity-related mechanism of the corresponding aerosol(s). In this work, we leveraged this framework to assess the biological impact of an e-liquid MESH™ “Classic Tobacco” and its aerosol in comparison with the impact of 3R4F reference cigarettes. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco liquid (containing humectants, nicotine, and flavors) and its Base liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F cigarette smoke (CS). In the second layer, we explored changes in specific markers using high content screening assays to identify potential toxicity-related mechanisms of the MESH Classic Tobacco and Base liquids beyond cell viability compared with the 3R4F TPM-induced effects. In the third layer, we compared the biological impact of exposure to the MESH Classic Tobacco aerosol with CS using human organotypic buccal and small airway epithelial cultures. The results showed that the cytotoxicity profile of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than the toxicity of 3R4F TPM at comparable nicotine concentrations. When compared with CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the global mRNA, global microRNA, and protein markers. The global mRNA changes following Classic Tobacco aerosol exposure indicated perturbations in processes related to cell fate, cell stress, and inflammatory response that were less than 20% of the perturbations following CS exposure. In the context of tobacco-harm reduction strategy, the framework is suitable to assess the potential reduced impact of EC aerosol relative to CS.

Project description:A 7-month inhalation study in C57BL/6 mice was conducted to evaluate long-term respiratory toxicity of e-vapor aerosols compared to cigarette smoke and to assess the impact of smoking cessation or switching to an e-vapor product after 3 months of exposure to 3R4F cigarette smoke (CS). In this study, we performed a chronic inhalation (4 h/day, 5 d/week, up to 7 months) study in C57BL/6 mice using a commercial (MarkTen®) e-vapor product and a combustible reference cigarette (3R4F) using a Switching and Cessation study design. A commercial e-vapor product (MarkTen® device [version 2.6.8]; “Test Red”) was supplied by Altria Client Services LLC (Richmond, VA, USA). The Test Red formulation was composed of aerosol formers (propylene glycol [PG] and vegetable glycerol [VG]), ~4% nicotine by weight, and flavors (non-menthol). The 3R4F commercial reference cigarettes were purchased from the University of Kentucky (Lexington, KY). HEPA filtered air at the testing facility (Battelle, West Jefferson, OH) was used as Sham Control. General procedures for animal care and housing met the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) recommendations and requirements stated in the “Guide for Care and Use of Laboratory Animals” [National Research Council (NRC)] and approved by the Institutional Animal Care and Use Committee (IACUC). Female C57BL/6 mice were received from Charles River Kingston (Stone Ridge, NY). Test atmosphere was generated from smoking machines and delivered to the mice through a nose-only exposure system. The modified Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Reference Method 81 regimen (55/30/5: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 5-second puff duration) was used to generate e-vapor aerosol for 130 puffs/cartridge. Mainstream smoke from 3R4F cigarette was generated using a modified Health Canada Intense regimen (55/30/2: a 55 ± 0.3 mL puff volume, a puff every 30 seconds, a 2-second puff duration, and a near-square puff profile) for 8 puffs/cigarette. Female C57BL/6 mice (~10 weeks old) were randomly assigned based on body weight to one of five exposure groups: Sham Control, 3R4F CS, Test Red, Switching, and Cessation. Mice were exposed to 3R4F CS (550 µg/L TPM) or e-vapor aerosols (Test Red; 1100 µg/L TPM) via nose-only inhalation up to 4 h/day, 5 d/week for up to 7 months. After the first 3 months of exposure, groups of 3R4F CS mice were subjected to exposures of: (1) Test Red aerosol (“Switching”) or (2) filtered air (“Cessation”), while a group of mice continued to be exposed to 3R4F CS. Here, the protein expression data for lung tissue assessed by iTRAQ®-based quantitative proteomics is reported.

Project description:In vitro toxicology approaches have evolved, from a focus on the molecular changes within a cell to understanding of toxicity-related mechanisms that simulate the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offer 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 relevancy and application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. The biological impact was assessed following exposure to aerosol generated from a candidate modified risk tobacco product (MRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F using an in vitro human 3-D nasal epithelial cultures. A series of experimental repetitions where multiple doses of the aerosol and smoke were applied, were conducted to obtain reproducible measurements and reliable observations to understand the cellular/molecular changes that occur following exposure. Aligned with the 3Rs Strategy and the Vision-and-Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was considerably greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles. In addition, to evaluate further the possible adverse effects of THS2.2 aerosol, a dose range assessment was conducted. A broader range of THS2.2 concentrations were exposed to the nasal cultures. Various dilutions of THS2.2 were applied to the cultures using the VitrocellÂ® 24/48 exposure system, corresponding to the concentrations of nicotine between 0.15 mg/L and 1.79 mg/L

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Exposure to aerosol of an e-vapor device with MESH™ technology had a lower biological impact than exposure to cigarette smoke on human bronchial and alveolar culture models: an in vitro systems toxicology assessment.

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