<i>In Vitro</i> Systems Toxicology Assessment of a Candidate Modified Risk Tobacco Product Shows Reduced Toxicity Compared to That of a Conventional Cigarette

Cigarette smoke increases the risk for respiratory and other diseases. Although smoking prevalence has declined over the years, millions of adults choose to continue to smoke. Modified risk tobacco products (MRTPs) are potentially valuable tools for adult smokers that are unwilling to quit their habit. Here, we investigated the biological impact of a candidate MRTP, the tobacco-heating system (THS) 2.2, compared to that of the 3R4F reference cigarette in normal primary human bronchial epithelial cells. Chemical characterization of the THS 2.2 aerosol showed reduced levels of harmful constituents compared to those of a combustible cigarette. Multiparametric indicators of cellular toxicity were measured via real-time cellular analysis and high-content screening. The study was complemented by a whole transcriptome analysis, followed by computational approaches to identify and quantify perturbed molecular pathways. Exposure of cells to 3R4F cigarette smoke resulted in a dose-dependent response in most toxicity end points. Moreover, we found a significant level of perturbation in multiple biological pathways, particularly in those related to cellular stress. By contrast, exposure to THS 2.2 resulted in an overall lower biological impact. At 3R4F doses, no toxic effects were observed. A toxic response was observed for THS 2.2 in some functional end points, but the responses occurred at doses between 3 and 15 times higher than those of 3R4F. The level of biological network perturbation was also significantly reduced following THS 2.2 aerosol exposure compared to that of 3R4F cigarette smoke. Taken together, the data suggest that THS 2.2 aerosol is less toxic than combustible cigarette smoke and thus may have the potential to reduce the risk for smoke-related diseases

A framework for <i>in vitro</i> systems toxicology assessment of e-liquids

<p>Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an <i>in vitro</i> systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air–liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust <i>in vitro</i> assessment of e-liquids and their aerosols.</p

Supplementary Table 2. Results of phosphoprotein measurements in primary normal human bronchial epithelial cells (NHBE) and normal rat bronchial epithelial cells (NRBE) using antibody-bead based assays for the experimental screening of 270 stimuli.

<p>Supplementary Table 2. Results of phosphoprotein measurements in primary normal human bronchial epithelial cells (NHBE) and normal rat bronchial epithelial cells (NRBE) using antibody-bead based assays for the experimental screening of 270 stimuli.</p> <p>After an exposure of 5 minutes to each stimulus, cells were lysed and used to measure phosphoproteins. Data were analyzed and a final selection of 52 stimuli used for the main experiment was performed (Data deposited in the figshare public repository).</p

Supplementary Table 1. Results of proteomics data including phosphoproteomics and cytokine level measurements for primary normal human bronchial epithelial cells (NHBE) and normal rat bronchial epithelial cells (NRBE) cells exposed to 52 stimuli.

<p>Supplementary Table 1. Results of proteomics data including phosphoproteomics and cytokine level measurements for primary normal human bronchial epithelial cells (NHBE) and normal rat bronchial epithelial cells (NRBE) cells exposed to 52 stimuli.</p> <p>The file contains the median of bead fluorescence intensities measured for each protein in every sample (cell lysate and corresponding supernatant for phosphoproteins and cytokines, respectively). For each stimulus, sample replicates have been extracted from 3 independent wells. The results are reported for (a) 19 phosphoproteins, with in addition the measurements for 2 control beads (Control A: Phycoerythrin-coated beads used as positive control bead; Control B: BSA-coated beads devoid of antibody used as negative control bead), and for the actin; (b) 22 cytokines.</p