Assessment of the impact of aerosol from a potential modified risk tobacco product compared with cigarette smoke on human organotypic oral epithelial cultures under different exposure regimens

Abstract

Cigarette smoke (CS) is affecting considerably the oral mucosa. Heating, instead of burning, tobacco reduces consistently the amount of toxic compounds and may exert a lower impact on oral health than combusted cigarettes. The carbon-heated tobacco product 1.2 (CHTP1.2) is a potential modified risk tobacco product (MRTP) based on heat-not-burn technology. Using a systems toxicology assessment framework, we compared the effects of exposure to CHTP1.2 aerosol with those of CS from a reference cigarette (3R4F). Human organotypic cultures derived from buccal and gingival epithelia were exposed acutely (28-min) or repeatedly (28 min/day for 3 days), respectively, to two matching concentrations of CHTP1.2 aerosol or 3R4F CS, and a non-diluted (100%) CHTP1.2 aerosol. The results showed an absence of cytotoxicity, reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP1.2 aerosol compared with 3R4F CS. Changes in mRNA and miRNA expression were linked by an integrative analysis approach, suggesting a regulatory role of miRNAs in several smoke/disease-relevant biological processes induced by 3R4F CS. The identification of mechanisms by which potential MRTPs can reduce the impact of tobacco use on biological systems is of great importance in understanding the molecular basis of the smoking harm reduction paradigm.

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).…”

“…MAP of inflammatory mediators secreted into the basolateral medium of cultures was performed using commercially available Milliplex panels (Merck Millipore, Burlington, MA, USA) with Luminex ® xMAP ® Technology (Luminex, Austin, TX, USA)-based analysis, according to the manufacturer's instructions and following previously published protocols (Iskandar et al 2018;Zanetti et al 2018). The samples were randomized prior to the analysis.…”

“…Total RNA, including miRNA, was isolated from the epithelial cultures (N = 9 independent exposure experiments) using a previously published method (Iskandar et al 2018;Zanetti et al 2018). Samples were randomized before processing.…”