biostudies-arrayexpressYang XiangMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-9597Cigarette smoking causes serious diseases, including lung cancer, heart disease, and emphysema. While cessation remains the most effective approach to minimize smoking-related disease, alternative non-combustible tobacco-derived nicotine containing products may reduce disease risks among those unable or unwilling to quit. E-vapor aerosols typically contain significantly lower levels of smoke-related harmful and potentially harmful constituents; however, health risks of long-term inhalation exposures are unknown. We designed a 7-month inhalation study in C57BL/6 mice 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). There were no significant changes in in-life observations (body weights, clinical signs) in e-vapor groups compared to the Sham Control. The 3R4F CS group showed reduced respiratory function during exposure and had lower body weight and showed transient signs of distress post-exposure. Following 7 months of exposure, e-vapor aerosols resulted in no or minimal increase in pulmonary inflammation, while exposure to 3R4F CS led to impairment of lung function and caused marked lung inflammation and emphysematous changes. Biological changes observed in the Switching group were similar to the Cessation group. 3R4F CS exposure dysregulated lung and nasal tissue transcriptome, while these molecular effects were substantially lower in the e-vapor group. Results from this study demonstrate that in comparison with 3R4F CS, e-vapor aerosols induce substantially lower biological responses including pulmonary inflammation and emphysema, and that complete switching from CS to e-vapor products significantly reduces biological changes associated with cigarette smoke in C57BL/6 mice.biostudies-arrayexpressHybridization - Hybridization cocktails were prepared for each sample according to the manufacturer's instructions. The final cocktails were hybridized to GeneChip® Mouse Genome 430 2.0 arrays (Thermo Fisher, Santa Clara, CA, USA) at 45°C for 16h while rotating at 60 rpm.Nucleic Acid Extraction - Total RNA was isolated using a Qiagen miRNeasy Mini Kit with Qiazol lysis buffer. The quantity of the purified RNA was determined using a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), while the quality of the RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples with an RNA integrity number (RIN) ≥ 6 were processed further.Labeling - The target preparation workflow was performed using the Biomek FXP Target Prep Express liquid handling system (Beckman Coulter, Brea, CA, USA). In brief, 100 ng total RNA were reverse-transcribed to cDNA using the Gene Chip™ HT 3' IVT PLUS kit (Affymetrix, Santa Clara, CA, USA). The cDNA was then labeled, amplified to complementary RNA (cRNA), and fragmented. Successful amplification was evaluated with Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA) and fragmentation step was confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).Sample Treatment - A commercial e-vapor product (MarkTen® device [version 2.6.8]; “Test Red”) was supplied by Altria Client Services, LLC. The Test Red formulation was composed of aerosol formers (propylene glycol [PG] and vegetable glycerol [VG]), ~4% nicotine by weight, and flavors (non-menthol). 3R4F reference cigarettes (Lexington, KY) were purchased from the University of Kentucky. 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. During the first 2 weeks of exposure, exposure duration gradually increased from 0.5 h (Day 1, 2), to 1 h (Day 3, 4), to 2 h (Day 5, 6), to 3 h (Day 7, 8), and then to 4 h (starting on day 9 and continued up to month 7). 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.Scaning - Hybridized arrays were washed (Protocol FS450-0001) and stained on a FS450 DX GeneChip® Fluidics Station (Thermo Fisher, Santa Clara, CA, USA), and scanned using a 3000 7G GeneChip® Scanner (Thermo Fisher, Santa Clara, CA, USA) and the raw image data were saved in DAT files. Command Console Software (Thermo Fisher, Santa Clara, CA, USA) was used to automatically grid the DAT files and create the CEL files (probe intensity data).Growth Protocol - Female C57BL/6 mice were received from Charles River Kingston (Stone Ridge, NY). The light/dark cycle was maintained at 12 hours of light and dark cycles starting at 6:00 AM each day, and during the study the housing room conditions were maintained at a room temperature of 20.6℃ to 23.9℃ and a mean relative humidity of 50%. Mice had ad libitum access to water and certified feed (Purina Certified Rodent diet, LabDiet 5002), except when removed from the home cage for daily exposures or sample collection.Sample Collection - At the end of their respective exposure period, the animals were dissected and assigned to various endpoints for analysis.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsData Transformation - The raw data CEL files were preprocessed through a pipeline based on multiple Bioconductor packages developed for the R statistical software environment (https://www.r-project.org/; Huber et al., PMID 25633503). Data quality was first controlled by examining log-intensities plots, normalized unscaled standard error plots, relative log expression plots, polyA controls boxplots, RNA degradation plots, spike-in control boxplots, and pseudo- and raw images, all generated using the affyPLM and affy packages (Gautier et al., PMID 14960456; Brettschneider et al., arXiv:0710.0178, 2007). Arrays that fell below a set of thresholds on these quality control checks were excluded from further analysis. Admitted CEL files were then background-corrected, normalized, and summarized using frozen robust microarray analysis fRMA (v1.18.0) (McCall et al., PMID 20097884). The microarray expression values from all arrays were generated using the custom CDF environment mouse4302frmavecs (v1.3) (Dai et al., PMID 16284200).MetabolomicsUnknownTranscriptomicsGenomicsProteomicsAnimal - One-color microarrayMus musculusA 7-month inhalation toxicology study in C57BL/6 mice demonstrates reduced pulmonary inflammation and emphysema following smoking cessation or switching to e-vapor products.Ulrike KogelYang XiangAshutosh Kumar, Ulrike Kogel, Marja Talikka, Celine Merg, Emmanuel Guedj, Yang Xiang, Athanasios Kondylis, Bjoern Titz, Nikolai Ivanov, Julia Hoeng, Manuel Peitsch, Joshua Allen, Amit Gupta, Anthony Skowronek, Monica LeefalseA 7-month inhalation toxicology study in C57BL/6 mice demonstrates reduced pulmonary inflammation and emphysema following smoking cessation or switching to e-vapor products.Cigarette smoking causes serious diseases, including lung cancer, heart disease, and emphysema. While cessation remains the most effective approach to minimize smoking-related disease, alternative non-combustible tobacco-derived nicotine containing products may reduce disease risks among those unable or unwilling to quit. E-vapor aerosols typically contain significantly lower levels of smoke-related harmful and potentially harmful constituents; however, health risks of long-term inhalation exposures are unknown. We designed a 7-month inhalation study in C57BL/6 mice 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). There were no significant changes in in-life observations (body weights, clinical signs) in e-vapor groups compared to the Sham Control. The 3R4F CS group showed reduced respiratory function during exposure and had lower body weight and showed transient signs of distress post-exposure. Following 7 months of exposure, e-vapor aerosols resulted in no or minimal increase in pulmonary inflammation, while exposure to 3R4F CS led to impairment of lung function and caused marked lung inflammation and emphysematous changes. Biological changes observed in the Switching group were similar to the Cessation group. 3R4F CS exposure dysregulated lung and nasal tissue transcriptome, while these molecular effects were substantially lower in the e-vapor group. Results from this study demonstrate that in comparison with 3R4F CS, e-vapor aerosols induce substantially lower biological responses including pulmonary inflammation and emphysema, and that complete switching from CS to e-vapor products significantly reduces biological changes associated with cigarette smoke in C57BL/6 mice.2021-03-18T00:00:00Z2022-03-10T05:22:06.909Z2022-03-10T05:22:06.909ZE-MTAB-9597DOI: 10.1177/2397847321995875EFO_0003814EFO_0002944EFO_0003813EFO_0003789EFO_0005518EFO_0003816EFO_0003969EFO_0003815