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Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, ...

Philip Morris Products S.A.

Monocyte adhesion and migration to the subendothelial space represent critical steps in atherogenesis. Here, we investigated whether extracts from the aerosol of a prototypic modified risk tobacco product (pMRTP), based on heating rather than combusting tobacco, exhibited differential effects on the migratory behavior of monocytes compared with that from the reference cigarette, 3R4F. THP-1 cells, a monocytic cell line, and human coronary arterial endothelial cells (HCAECs) were used to investigate chemotaxis and transendothelial migration (TEM) of monocytes in conventional and impedance-based systems. THP-1 cells migrated through a monolayer of HCAECs in response to C-X-C motif ligand 12 (CXCL12), a chemokine involved in diverse cellular functions including chemotaxis and survival of stem cells. Treatment of THP-1 cells with extracts from 3R4F or pMRTP induced concentration-dependent increases in cytotoxicity (7-aminoactinomycin D), and inflammation (IL-8 and TNF-α). CXCL12-mediated chemotaxis and TEM were decreased in extract-treated THP-1 cells. Extracts from 3R4F were ~21 times more potent than those from pMRTP in all examined endpoints. Extracts from 3R4F and pMRTP induced concentration-dependent responses in assays of inflammation, cytotoxicity, chemotaxis, and TEM. Furthermore, our findings indicate that extracts from a pMRTP are significantly less cytotoxic and induce less inflammation than those from the reference cigarette, 3R4F.

A prototypic modified risk tobacco product exhibits reduced effects on chemotaxis and transendothelial migration of monocytes compared with a reference cigarette.

Cigarette smoking causes serious and fatal diseases. The best way for smokers to avoid health risks is to quit smoking. Using modified risk tobacco products (MRTPs) may be an alternative to reduce the harm caused for those who are unwilling to quit smoking, but little is known about the toxic effects of MRTPs, nor were the molecular mechanisms of toxicity investigated in detail. The toxicity of an MRTP and the potential molecular mechanisms involved were investigated in high-content screening tests and whole genome transcriptomics analyses using human bronchial epithelial cells. The prototypic (p)MRTP that was tested had less impact than reference cigarette 3R4F on the cellular oxidative stress response and cell death pathways. Higher pMRTP aerosol extract concentrations had impact on pathways associated with the detoxification of xenobiotics and the reduction of oxidative damage. A pMRTP aerosol concentration up to 18 times higher than the 3R4F caused similar perturbation effects in biological networks and led to the perturbation of networks related to cell stress, and proliferation biology. These results may further facilitate the development of a systems toxicology-based impact assessment for use in future risk assessments in line with the 21st century toxicology paradigm, as shown here for an MRTP. 

Biological impact of cigarette smoke compared to an aerosol produced from a prototypic modified risk tobacco product on normal human bronchial epithelial cells.

Reduction of harmful constituents by heating rather than combusting tobacco is a promising new approach to reduce harmful effects associated with cigarette smoking. We investigated the effect from a new candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, on the migratory behavior of monocytes in comparison with combustible 3R4F reference cigarettes. The monocytic cell line (THP-1) and human coronary arterial endothelial cells (HCAECs) were used to analyze chemotaxis and transendothelial migration (TEM). To assess the influence of aerosol extract from THS2.2 and smoke extract from 3R4F on toxicity and inflammation, flow cytometry and ELISA assays were performed. The results show that treatment of THP-1 cells with extract from 3R4F or THS2.2 induced concentration-dependent increases in cytotoxicity and inflammation. The inhibitory effects of THS2.2 extract for chemotaxis and TEM were ∼18 times less effective compared to 3R4F extract. Furthermore, extract from 3R4F or THS2.2 induced concentration-dependent decreases in the integrity of HCAEC monolayer. For all examined endpoints, the extract from 3R4F showed more than one order of magnitude stronger effects than that from THS2.2 extract. These data indicate the potential of a heat not burn tobacco product to reduce the risk for cardiovascular disease compared to combustible cigarettes.

Aerosol from a candidate modified risk tobacco product has reduced effects on chemotaxis and transendothelial migration compared to combustion of conventional cigarettes.

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.

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

Cigarette smoking causes cardiovascular diseases. Heating tobacco instead of burning it reduces the amount of toxic compounds in the aerosol and may exert a reduced impact on health compared with cigarette smoke. Aqueous extract from the aerosol of a potential modified risk tobacco product, the Carbon Heated Tobacco Product (CHTP) 1.2, was compared in vitro with aqueous extract from the smoke of a 3R4F reference cigarette for its impact on the adhesion of monocytic cells to artery endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated for 4 h with conditioned media from human monocytic Mono Mac 6 (MM6) cells exposed to CHTP1.2 or 3R4F extracts for 2 h or directly with those extracts freshly generated. In vitro monocyte-endothelial cell adhesion was measured concomitantly with inflammatory, oxidative stress, cytotoxicity, and death markers. Furthermore, transcriptomics analyses enabled to quantify the level of perturbation in HCAECs, and provide biological interpretation for the underlying molecular changes following exposure to 3R4F or CHTP1.2 extract. Our systems toxicology study demonstrated that approximately 10-15-fold higher concentrations of the CHTP 1.2 aerosol extract were needed to elicit similar effects as the 3R4F smoke extract on cardiovascular disease-relevant inflammation and cytotoxicity-related mechanisms and markers investigated in vitro.

In vitro systems toxicology-based assessment of the potential modified risk tobacco product CHTP 1.2 for vascular inflammation- and cytotoxicity-associated mechanisms promoting adhesion of monocytic cells to human coronary arterial endothelial cells.

Didier Goedertier

PMI R&D

Philip Morris Products S.A.

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies