Comparative toxicological assessment of cigarettes and new category products via an in vitro multiplex proteomics platform

Abstract

Cigarette smoking is a risk factor for several diseases such as cancer, cardiovascular disease (CVD), and chronic obstructive pulmonary diseases (COPD), however, the underlying mechanisms are not fully understood. Alternative nicotine products with reduced risk potential (RRPs) including tobacco heating products (THPs), and e-cigarettes have recently emerged as viable alternatives to cigarettes that may contribute to the overall strategy of tobacco harm reduction due to the significantly lower levels of toxicants in these products' emissions as compared to cigarette smoke. Assessing the effects of RRPs on biological responses is important to demonstrate the potential value of RRPs towards tobacco harm reduction. Here, we evaluated the inflammatory and signaling responses of human lung epithelial cells to aqueous aerosol extracts (AqE) generated from the 1R6F reference cigarette, the glo™ THP, and the Vype ePen 3.0 e-cigarette using multiplex analysis of 37 inflammatory and phosphoprotein markers. Cellular exposure to the different RRPs and 1R6F AqEs resulted in distinct response profiles with 1R6F being the most biologically active followed by glo™ and ePen 3.0. 1R6F activated stress-related and pro-survival markers c-JUN, CREB1, p38 MAPK and MEK1 and led to the release of IL-1α. glo™ activated MEK1 and decreased IL-1β levels, whilst ePen 3.0 affected IL-1β levels but had no effect on the signaling activity compared to untreated cells. Our results demonstrated the reduced biological effect of RRPs and suggest that targeted analysis of inflammatory and cell signaling mediators is a valuable tool for the routine assessment of RRPs.

Keywords: In vitro; aerosol; cigarette; electronic cigarette; heated tobacco product; lung toxicity.

Graphical abstract

Fig. 1

Cell viability following exposure to 1R6F AqE. Each graph corresponds to a different exposure time. Data from the CellTiter-Glo assay were first transformed into % viable cells by comparing the data from each 1R6F AqE concentration (% v/v from stock solution) to the untreated cells. Untreated cells were considered as 100% viable. Mean ± standard deviation from three independent replicates is presented. (.) p = 0.05, *p<0.05, **p<0.01, ***p<0.001.

Fig. 2

Optimized workflows used to assess inflammatory and cell signaling proteins in H292 cells exposed to AqEs from different tobacco product categories. A) Cells aimed for inflammatory protein analysis were exposed for 2 hrs to AqE from the test products, followed by recovery in complete culture medium for 48 hrs. At the end of the recovery period, the culture supernatant was kept for multiplex quantitative analysis. B) Cells aimed for phosphoprotein analysis were exposed for 2 hrs to AqE from the test products and the cell lysates were immediately harvested for multiplex semi-quantitative analysis. In both cases, cells were assessed for viability by CellTiter-Glo.

Fig. 3

Cell viability following exposure to 1R6F and RRP AqEs. Intracellular ATP levels measured by CellTiter-Glo immediately after 2 hrs exposure to 25% AqE of each test product (a) or after 2 hrs exposure and 48 hrs recovery (b) are presented as % relative to untreated cells. Relative ATP levels for each product AqEs are mean ± standard deviation from 3 independent replicates. **p<0.01, ***p<0.001.

Fig. 4

Inflammatory protein and cell signaling profiles of H292 cells exposed to 25% AqEs from 1R6F and RRP aerosols. (a) Heat maps showing MFI values of 20 inflammatory markers (left) and 17 phosphoproteins (right) normalized by subtracting background noise data from analysis of RPMI media or lysis buffer alone, respectively). (b) Volcano plots comparing MFI values between AqE-exposed and untreated cells for each test product category. Proteins showing significant fold-changes (higher than 1.5 or lower than −1.5) compared to the untreated control occur outside a pair of vertical black dotted lines and are highlighted in green and red to indicate increase and decrease, respectively. Proteins showing a tendency in fold change difference compared to the untreated control (higher than 1.2 or lower than −1.2) occur outside a pair of grey vertical dotted threshold lines and are highlighted in light green or light red respectively. Proteins with statistically significant changes compared to untreated control (p < 0.05, n=5, Mann-Whitney test) occur above the horizontal dotted black lines. Black dots correspond to proteins with statistically non-significant changes or with changes < 1.5 relative to untreated controls. (c) Concentration (pg/ml) of individual cytokines and MFI values of cell signaling phosphoproteins with > 1.5-fold change and p <0.05 from (b). Mean ± standard deviation from 5 independent replicates is presented. FC: fold change, *p<0.05, **p<0.01.