3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product

Abstract

ratory animals -to reduce the number of animals, to refine the design of procedures such that pain and distress are minimized, and to replace animal models with alternative methods and lower organisms when possible (Doke and Dhawale, 2015). In this regard, the capability to preserve human respiratory cells in culture provides a tool not only to generate mechanistic data, but also to improve the predicted effects of exposure hazard through inhalation in humans. An emphasis on assessment studies using human-derived in vitro culture systems will reduce the problems that are inherent to interspecies translatability (Manuppello and Sullivan, 2015).In vitro toxicology approaches have evolved from focusing on the molecular changes within a cell to understanding the toxicity-related mechanisms (cell-cell interactions) in models IntroductionMechanistic investigation of the impact of exposure on the lower airway remains challenging because access to lung tissues is limited. Although biopsy samples can be obtained from patients/donors for mechanistic studies of exposure-induced injury, a biopsy procedure is invasive and there are related ethical concerns (Peppercorn, 2013). Alternatively, animal studies allow the collection of biological samples from various animal models of human diseases. However, biological responses can vary among animal species, and findings obtained from animal studies may not apply to humans. Alternatives to animal testing have been proposed to overcome these drawbacks, including the 3Rs strategy -reduction, refinement, and replacement of labo- Research SummaryIn vitro toxicology approaches have evolved from a focus on molecular changes within a cell to understanding of toxicity-related mechanisms in systems that can mimic the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offers 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 application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. Aligned with 3Rs strategy, this study aimed to explore the relevance of a human 3-D nasal culture model to assess the toxicological impact of aerosols generated from a candidate modified risk tobacco product (cMRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F. A series of experimental repetitions, where multiple concentrations of THS2.2 aerosol and 3R4F smoke were applied, were conducted to obtain reproducible measurements to understand the cellular/molecular changes that occur following exposure. In agreement with "Toxicity Testing in the 21 st Century -a Vision and a Strategy", this study implemented a systems toxicology approach and found that for all tested concentrations the impa...

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

“…Targeted proteomics by PRM was conducted at the 24 h PE time for the ALI buccal epithelial cultures (N = 9 independent exposure experiments), and at the 48 h PE time for the ALI small airway epithelial cultures (N = 9 independent exposure experiments) following a previous published protocol (Iskandar et al 2017). The timepoint collection for each culture type was decided based on the previous studies in which we detected greater changes in the protein levels following exposure (data not shown).…”

“…Raw files from the PRM acquisition were analyzed with SpectroDive (version 8.0 Biognosys AG). Ion chromatograms for the endogenous peptides and the corresponding stable isotope-labeled reference peptides were extracted for all measured transitions [the list of transitions was reported in a previous publication (Iskandar et al 2017)] using the software vendor's default settings. For quantification, the area under the curve (AUC) intensities of all transitions were summed, and ratios of AUC sums of the endogenous and corresponding reference peptide signal were calculated.…”

Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics

“…Although current systems toxicology studies reported a lesser impact of e-cigarette and heated tobacco aerosols in vitro 5, 6 , other toxicological studies using targeted endpoints reported potential for DNA damage, oxidative stress and cytotoxicity 12, 13 . These discrepancies arise from the use of different cell models, products/devices, exposure matrices, times, and doses.…”

“…Examples of robust in vitro assessment studies of novel nicotine delivery devices (heated tobacco products and e-cigarettes) have used a relevant cell model, a well-considered exposure strategy combined with holistic untargeted omics screens to comprehensively assess the biological perturbations 5, 6 . Such in vitro- based systems toxicology approaches have been driven by the increasing demand to replace the use of low-throughput animal testing with high- throughput in vitro and computational technologies.…”

“…Following the Tox21 approache, a series of studies have used microarray-based transcriptomics analyses to demonstrate reduced impact of heated tobacco aerosol on 3D human in vitro airway cells compared to burned tobacco 5, 10 . More recently toxicogenomics RNA-seq-based analyses were applied to the testing of e-cigarette aerosol and also demonstrated reduced impact on the transcriptome 6 .…”