biostudies-arrayexpressVincenzo BelcastroHomo sapiensR/Bioconductor gcrma packageAffymetrix AGCChttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-5179In 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 Vitrocell® 24/48 exposure system, corresponding to the concentrations of nicotine between 0.15 mg/L and 1.79 mg/Lbiostudies-arrayexpressSample Collection - The tissues were collected for RNA isolation at each post-exposure time points (4h, 24h, 48h, and 72h). The tissues were first washed 3 times with 1X PBS in both apical and basal side. PBS was removed. Tissue inserts were then detached gently and transfered into labeled MagnaLyzer tubes (Roche) containing 700uL of QIAZOL Lysis Reagent (miRNeasy mini kit, QIAGEN). Samples were then vortexed 10 seconds and stored at -80C.Nucleic Acid Extraction - miRNeasy Mini Kit (Qiagen) was used to extract and purify both mRNA and microRNA. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile.Scaning - Command Console Software (Affymetrix) was used to automatically grid the DAT files and create the CEL files (probe cell intensity data).Scaning - The arrays washed (using Fluidics station FS450-0001 protocol) and scanned using the GeneChip Scanner 3000 7G and the raw image data was saved in DAT files.Sample Collection - A series of batches of 3D organotypic human bronchial epithelial tissue cultures MucilAir™ was obtained for each of the experimental repetitions and dose range assessment. Two organotypic bronchial epithelial culture models were used in this study: - EpiAirway™ (MatTek Corporation, Ashland, MA, USA) reconstituted from primary bronchial epithelial cells of a 23-year old male, non-smoker, no pathology reported (used in experimental repetition 1 and 2; also known as study phase 2 and 3, respectively) and - MucilAir™ Bronchial (Epithelix Sàrl, Geneva, Switzerland) reconstituted from primary bronchial epithelial cells of a 28-year old male, non-smoker, no pathology reported (used for experimental repetition 3, 4, 5, and 6; also known as study phase 4, 6, 7, and 8, respectively). The cultures were grown in 6.5 mm Transwell® inserts; the dimension of the insert was chosen based on its fitting for the Cultivation Base Module of the Vitrocell® 24/48 exposure system. Upon arrival, the EpiAirway™ bronchial cultures were placed in their fresh culture media (5.5 mL/well) provided by the supplier, pre-warmed to 37°C, in 12-well culture plates. Similarly, upon arrival, the MucilAir™ bronchial cultures were placed in their fresh culture media (0.7 mL/well) provided by the supplier, pre-warmed to 37°C, in 24-well culture plates. All cultures were maintained at the air-liquid interface at 37°C (5% CO2, 90% humidity) with medium change every 2–3 days according to the supplier’s instructions. The cultures were regularly inspected microscopically to check for potential morphological changes, as well as for bacterial or fungal contamination. Three days before exposure (referred to as an exposure run), the apical sides of the cultures were washed gently by a dropwise addition of a total of 200 µL culture media (pre-warmed to 37°C) that was subsequently aspirated slowly. After exposure, the cultures were maintained without medium change until sample collections (according to the experimental design).Hybridization - The biotin-labeled complementary RNA was Hybridized overnight on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array.Nucleic Acid Extraction - Standard Qiazol lysis reagent (Qiagen) and its protocol were used for cell lysisSample Treatment - 3R4F reference cigarettes were purchased from the University of Kentucky (Kentucky Tobacco Research & Development Center). The candidate modified-risk tobacco product, termed tobacco heating system (THS) 2.2, was obtained from Philip Morris International R&D, Neuchâtel, Switzerland. THS2.2 uses a “heat-not-burn” based technology that heats tobacco instead of burning it. The 3R4F cigarettes and THS2.2 sticks were conditioned for at least 48 h and up to 21 d at 22 ± 1°C with a relative humidity of 60 ± 3%, according to ISO standard 3402. 3R4F smoke and THS2.2 aerosol were generated according to the Health Canada smoking protocol (55 mL puff over two sec, twice per min with an 8 sec pump exhaust time). Each 3R4F cigarette was smoked to a standard butt length (approximately 35 mm), and each THS2.2 was aerosolized for a total of 12 puffs per stick. For an exposure run, 3R4F reference cigarettes were smoked using a 30-port carousel smoking machine (SM) (SM2000; Philip Morris, International) connected to a Vitrocell® 24/48 exposure system (Vitrocell Systems GmbH, Waldkirch, Germany). Another dedicated 30-port carousel smoking machine was used to generate the aerosol from THS2.2 that was connected to another Vitrocell® 24/48 exposure system. For a 28-min exposure run, 10 3R4F cigarettes were smoked and 10 THS2.2 sticks were aerosolized. The duration of exposure (28 min) was chosen according to a previous report, in which a 28-min exposure compared with 7, 14, and 21 min exposure of 3R4F exposure induced the highest concentration of secreted matrix metalloproteinase (MMP)-1 in bronchial organotypic cultures. The secretion of MMP-1 was considered as a control to demonstrate the responsiveness of organotypic airway cultures following CS exposure. The Vitrocell® 24/48 exposure system is equipped with a Dilution/Distribution Module where the smoke/aerosol stream can be mixed with fresh air. The set-up allows adjustment of the smoke/aerosol concentrations applied to the organotypic bronchial cultures, which are located in the Cultivation Base Module of the Vitrocell® 24/48 exposure system. In this study, for the exposure run, two different concentrations of smoke from 3R4F cigarettes were applied: 0.13 mg nicotine/L and 0.25 mg nicotine/L, corresponding to smoke concentrations in the Dilution/Distribution Module of approximately 8% and 15% (v/v) 3R4F smoke in filtered air, respectively. These concentrations were chosen based on previous observations from which measurable effects were observed without overt tissue destruction. For the assessment of THS2.2, three different concentrations of THS2.2 aerosol were applied: 0.14 mg nicotine/L, 0.25 mg nicotine /L, and 0.42 mg nicotine/L, corresponding to aerosol concentrations in the Dilution/Distribution Module of 13%, 24%, and 31% aerosol in air (v/v), respectively. Two of the concentrations were matched to the nicotine concentrations in the diluted 3R4F smoke. Nicotine was used as the internal reference compound to compare the impact of 3R4F smoke and THS2.2 aerosol (the nicotine yield from one 3R4F cigarette is roughly 1.4-fold greater than from one THS2.2 stick. A similar exposure set-up was applied for the dose range assessment of THS2.2, in which seven different concentrations of THS2.2 aerosol were tested: 0.14 mg; 0.22 mg; 0.37 mg; 0.55 mg; 0.77 mg; 1.02 mg; and 1.79 mg nicotine/L aerosol. For all exposure runs, a paired design was implemented in which air-exposed samples for each of the endpoints and post-exposure collection time points were included (in every exposure run).Nucleic Acid Extraction - The inserts were disrupted in 700 microL QIAzol lysis buffer (Qiagen, Hilden, Germany) followed by RNA extraction using a Qiagen miRNeasy Mini Kit and a QIAcube robot (Qiagen, Hilden, Germany). The quantity of the purified RNA was determined using the NanoDrop ND8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), while the quality of the RNA was analyzed using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).Labeling - Labeling of 100ng using the HT 3’IVT Express Kit (Affymetrix) according to the supplier’s manual for experiment 1 and 2 (study phase 2 and 3, respectively). Then, 100ng using HT 3'IVT Plus (Affymetrix) according to the manufacture's instructions for experiment 3, 4, 5, 6 (study phase 4, 6, 7, and 8, respectively).MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsData Transformation - Raw CEL files are background-corrected, normalized, and summarized using frozen-Robust Microarray Analysis (fRMA). Background correction and quantile normalization is used to generate microarray expression values from all arrays passing quality control checks, which is performed using the custom CDF (Chip Description File) environment HGU133Plus2_Hs_ENTREZG v16.0. Quality controls, including log-intensities, normalized-unscaled standard error (NUSE), relative log expression (RLE), median absolute value RLE (MARLE) and pseudo-images as well as raw image plots, are performed with the affyPLM package (Bioconductor). CEL files that fulfil at least one of the criteria described below dropped for further analysis. a) Pseudo-image displaying a spatial pattern covering approximately 10% of the pseudo-image. b) Median NUSE >1.05 c) |Median RLE| >0.1 d) |(MARLE-median(MARLE))|/ (1.4826 *mad(MARLE)) >1/?0.01 ; (where mad is the median absolute deviation)MetabolomicsUnknownTranscriptomicsGenomicsProteomicsAffymetrix GeneChip Scanner 3000 7Gtranscription profiling by arrayHomo sapiensA 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.Vincenzo BelcastroSam AnsariIskandar AnitaCarole MathisAnita R Iskandar, Carole Mathis, Walter K Schlage, Stefan Frentzel, Patrice Leroy, Yang Xiang, Alain Sewer, Shoaib Majeed, Laura Ortega-Torres, Stephanie Johne, Emmanuel Guedj, Keyur Trivedi, Gilles Kratzer, Celine Merg, Ashraf Elamin, Florian Martin, Nikolai V Ivanov, Manuel C Peitsch, Julia Hoeng.falseIN VITRO ASSESSMENT OF ACUTE EXPOSURE OF THS2.2 AEROSOL ON ORGANOTYPIC ACUTE HUMAN BRONCHIAL TISSUE CULTURESIn 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 Vitrocell® 24/48 exposure system, corresponding to the concentrations of nicotine between 0.15 mg/L and 1.79 mg/L2016-12-01T00:00:00Z2022-03-03T15:35:44.601Z2022-03-03T15:35:44.601ZE-MTAB-5179EFO_0002768