Comparative biological impacts of an aerosol from carbon-heated tobacco and smoke from cigarettes on human respiratory epithelial cultures: A systems toxicology assessment

Abstract

The biological impact of an aerosol of a potential modified-risk tobacco product, carbon heated tobacco product 1.2 (CHTP1.2), was comprehensively assessed for the first time in vitro using human small airway and nasal epithelial models following a systems toxicology approach. The potentially reduced effects of CHTP1.2 aerosol exposure were benchmarked against those of 3R4F cigarette smoke at similar nicotine concentrations. Experimental repetitions were conducted for which new batches of small airway and nasal cultures were exposed to CHTP1.2 aerosol or 3R4F smoke for 28 minutes. The biological impacts were determined based on a collection of endpoints including morphology, cytotoxicity, proinflammatory mediator profiles, cytochrome P450 1A1/1B1 activity, global mRNA and microRNA changes and proteome profiles. Alterations in mRNA expression were detected in cultures exposed to CHTP1.2 aerosol, without noticeable morphological changes and cytotoxicity, and minimal impact on proinflammatory mediator and proteome profiles. The changes linked to CHTP1.2 aerosol exposure, when observed, were transient. However, the impact of 3R4F smoke exposure persisted long post-exposure and greater than CHTP1.2 aerosol. Morphological changes were observed only in cultures exposed to 3R4F smoke. The lower biological effects of CHTP1.2 aerosol than 3R4F smoke exposure were observed similarly in both small airway and nasal epithelial cultures.

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

“…† For the 3D buccal epithelial cultures, PRM was generated from samples collected at 24 h post-exposure. G, glycerol; p2HAX, phosphorylated H2A histone family member X; PG, propylene glycol; ROS, reactive oxygen species (Iskandar et al 2018;Zanetti et al 2018). After exposure, the medium was not changed until the cultures were collected for various endpoint measurements (up to 48 h post-exposure [PE]).…”

“…Doses of 3R4F CS, MESH Classic Tobacco aerosol, and Base aerosol applied to the cultures are summarized in Table 3. The concentrations of 3R4F CS (112-puff exposure = 28-min exposure) were chosen based on the observations in our previous studies, where we could detect a wide range of toxic response, from subtoxic to toxic (Iskandar et al 2018;Zanetti et al 2018). Because this is a comparative assessment in which the impact of MESH Classic Tobacco or Base aerosols was compared with that of 3R4F CS, the doses chosen for MESH Classic Tobacco and Base aerosols were those resulting in a similar and/or higher nicotine concentrations deposited in the exposure chamber compared with 3R4F CS exposure (Table 3).…”

“…Histological sections were obtained from cultures harvested 48 h PE, because morphological alterations would occur at later timepoints following exposure and after molecular changes took place (Castro et al 2008). The processing of the organotypic cultures was conducted following previously published protocols (Iskandar et al 2018;Zanetti et al 2018).…”

“…A luminescence signal was measured using a FLUOStar Omega reader (BMG LABTECH GmbH, Ortenberg, Germany). For each of the three experimental repetitions/phases (i.e., each culture batch), the values of the luminescence signal were normalized to the mean value of the positive (Triton X-100-treated cultures) and negative (untreated) control samples as previously described (Iskandar et al 2018). The mean values (from a total of 1 3 9 independent exposure experiments) of the normalized relative luminescence units were then reported in figures as percentages.…”

Applying knowledge-driven mechanistic inference to toxicogenomics

“…The raw human microarray data ( Fig. 1.d) used as input was sourced from the Open TG-Gates [35] database, the carcinoGENOMICS [36] project and Netherlands Toxicogenomics Centre projects available in the diXa Data Warehouse [37], as well as other publicly available tobacco-related human microarray datasets [38][39][40][41] in ArrayExpress [42] that used human nasal, buccal and bronchial epithelial tissue. These sources provided a variety of time series gene expression experiments, at different time points and using different human cell types.…”