Whole Smoke Research Articles

Electronic Nicotine Delivery Systems Exhibit LowerToxicity Compared to Cigarettes: “The Replica Study Experience”

Electronic nicotine delivery systems (ENDS) can reduce the health risks associated with chronic smoke exposure, and their potential benefits are the subject of intense scientific debate. The international “Replica Study Group” replicated independently three relevant studies from the tobacco industry on the cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol. Our primary goal was to establish the reliability of the results and the robustness of the conclusions.In order to assess cytotoxicity of smoke and aerosol, we exposed human bronchial epithelial cell (H292) to cigarette smoke and to ENDS aerosol at air‐liquid interface (ALI). Moreover, we aimed to assess different inflammatory and remodeling mediators release (IL‐6, IL‐8 and MMP‐1) from cells exposed to whole smoke (WS) and to smoke deprived of total particulate matter (vapor phase; VP).We were able to replicate the results obtained in the original studies on cytotoxicity confirming that almost 80% of the cytotoxic effect of smoke is due to the vapor phase of smoke. Moreover, our results substantiated the significantly reduced cytotoxic effects of ENDS aerosol vs. cigarette smoke. However, our data were notably different in terms of inflammatory and remodeling activity triggered by smoke.Altogether, the data obtained independently in different laboratories clearly confirm the reduced toxicity of ENDS products compared to smoke, thus providing a valuable tool to the harm reduction strategies in smokers. Otherwise, we were not able to replicate the results on inflammatory and remodeling mediators released from smoke exposed cells. This could be due to the lack of normalization of the quantity of cytokines released, with the number of viable cells, in the original paper, since the production of cytokines itself requires the metabolic capability of cells.

Determination of eight carbonyl compounds in aerosols trapped in phosphate buffer saline solutions to support in vitro assessment studies

When investigating the toxicological impact of aerosols using in vitro systems like cell cultures, it is essential to have a quantitative measurement of the chemicals that the cells are exposed to.Carbonyl compounds represent an important class of marker compounds for in vitro and in vivo exposure to different toxicological agents, including cigarette smoke (CS). A new LC-MS/MS method that quantifies eight of these analytes in aerosols trapped in phosphate-buffered saline solutions has been developed to measure exposure. During the method development phase, particular attention has been paid to the efficient derivatization of the target compounds in the trapped aerosols and to avoid the formation of poly-derivatized molecules, which could lead to inaccurate quantifications.The method has been successively validated using the accuracy profile procedure. Selectivity, detection limits, precision, and accuracy have been evaluated for Vitrocell®, Gas Vapor Phase (GVP), and Whole Smoke (WS) matrices of smoke generated by 3R4F cigarettes and aerosol generated by the Tobacco Heating System (THS) 2.2, a heat-not-burn tobacco product developed by Philip Morris International (Smith et al., 2016) [1]. Validation results confirmed that the established working ranges also allow the analysis of THS aerosols, where the concentrations of carbonyl compounds are substantially lower than those generated by 3R4F cigarettes. Moreover, data gathered on 3R4F aerosol samples trapped with DNPH in acetonitrile solutions have been compared to the quantification given by an in-house UHPLC-MS/MS and reference values from the literature.

Development, qualification, validation and application of the Ames test using a VITROCELL® VC10® smoke exposure system.

The Ames test has established use in the assessment of potential mutagenicity of tobacco products but has generally been performed using partitioned exposures (e.g. total particulate matter [TPM], gas vapor phase [GVP]) rather than whole smoke (WS). The VITROCELL®VC10® smoke exposure system offers multiple platforms for air liquid interface (ALI), or air agar interface (AAI) in the case of the Ames test exposure to mimic in vivo-like conditions for assessing the toxicological impact of fresh WS in in vitro assays. The goals of this study were to 1) qualify the VITROCELL®VC10® to demonstrate functionality of the system, 2) develop and validate the Ames test following WS exposure with the VITROCELL®VC10® and 3) assess the ability of the Ames test to differentiate between a reference combustible product (3R4F Kentucky reference cigarette) and a primarily tobacco heating product (Eclipse). Based on critical function assessments, the VITROCELL®VC10® was demonstrated to be fit for the purpose of consistent generation of WS. Assay validation was conducted for 5 bacterial strains (TA97, TA98, TA100, TA1535 and TA102) and reproducible exposure-related changes in revertants were observed for TA98 and TA100 in the presence of rat liver S-9 following exposure to 3R4F WS. In the comparative studies, exposure-related changes in in vitro mutagenicity following exposure of TA98 and TA100 in the presence of S9 to both 3R4F and Eclipse WS were observed, with the response for Eclipse being significantly less than that for 3R4F (p < 0.001) which is consistent with the fewer chemical constituents liberated by primarily-heating the product.

Characterization and Application of the VITROCELL VC1 Smoke Exposure System and 3D EpiAirway Models for Toxicological and e-Cigarette Evaluations

Abstract The VITROCELL VC1® smoke exposure machine is an aerosol exposure system that has been used to conduct toxicological assessments with cigarettes and e-cigarettes and can be used with the human 3D EpiAirway™ tissue model (MatTek Corporation). The goals of this study were to assess exposure parameters of the VC1, and evaluate donor-to-donor variability of three EpiAirway tissue donors with the following endpoints: viability, barrier integrity, and gene promoter/expression regulation. Additionally, we applied the EpiAirway model to assess the effects of aerosol from two reference combustible cigarettes and two e-cigarettes. Whole smoke (WS) exposures with Kentucky Reference 3R4F cigarettes and Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Monitor were conducted under International Organization for Standardization or Health Canada Intense (HCI) conditions, and e-cigarette exposures were conducted under modified HCI conditions. Particulate deposition and collection were consi...

Evaluation of an air–liquid interface cell culture model for studies on the inflammatory and cytotoxic responses to tobacco smoke aerosols

In vitro toxicological studies for tobacco product assessment have traditionally been undertaken using the particulate phase of tobacco smoke. However, this does not truly reflect exposure conditions that occur in smokers. Thus in vitro cell culture systems are required in which cells are exposed to tobacco whole smoke (WS) at the air–liquid interface (ALI). In this study bronchial epithelial cells were cultured on semi-permeable membranes, transitioned to the ALI and the robustness and sensitivity of the cells to tobacco WS and vapour phase (VP) assessed. Although no effect of air exposure was observed on cell viability, IL-6 and IL-8 release was increased. Exposure to WS resulted in a significant dose dependent decrease in cell viability and a significant non-dose dependent increase in inflammatory mediator secretion. The VP was found to contribute approximately 90% of the total cytotoxicity derived from WS. The cell culture system was also able to differentiate between two smoking regimens and was sensitive to passage number with increased inflammatory mediator secretion and lower cell viability observed in cell cultures of low passage number following WS exposure. This simple cell culture system may facilitate studies on the toxicological impact of future tobacco products and nicotine delivery devices.

Evaluation method for the cytotoxicity of cigarette smoke by in vitro whole smoke exposure

An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effects of fresh cigarette smoke using the VITROCELL® system associated with the neutral red uptake (NRU) cytotoxicity assay. The VITROCELL® system is a newly representative culture and exposure system for in vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements of cigarette smoke were investigated using this WS exposure system. The factors include synthetic air exposure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic air exposure used in the system did not significantly alter cell survival; 24h after smoke exposure appeared to be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose–response relationship between smoke exposure and cell viability was demonstrated using this system, and the evaluation method was sensitive to distinguish the differences in smoke-induced cytotoxic effects from different cigarettes. In addition, we tried converting the values of EC50 from WS exposure testing into the values in unit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate that the cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.

An ex vivo approach to the differential parenchymal responses induced by cigarette whole smoke and its vapor phase

Using a rat lung slice model, this study compared the stress responses induced by cigarette whole smoke (WS) to that induced by the vapor phase (VP) of the smoke. Following a 3-day exposure, lung slices exposed to 4, 10 and 20% WS retained 85, 42 and 16% relative survival respectively in comparison to the air-exposed ones. Consistently, histological observations revealed concentration-related alveolar damages in the lung slices. Expression of 5 stress–response genes was examined following a single 30min exposure to 4% WS or VP. WS exposure resulted in 4, 11 and 50-fold induction of IL-1β, kinin type I receptor (B1R) and CYP1A1 genes, respectively, while CYP1B1 and TNF-α genes expression was found only two times higher in comparison to VP group. Since cigarette WS consists of particulate and vapor phases, these results highlight the preferential or synergistic role of the particulate phase in the induction of IL-1β, B1R and CYP1A1 genes and that VP did not have comparable effects on expression of these genes. However, both phases fairly contributed to the induction of CYP1B1 and TNF-α genes. VP was the fraction responsible for the toxic effect since WS did not produce further toxicity. The 4% whole smoke deposited about 7.1μg/cm2 of total particulate matter (TPM) to the exposure chamber which may account for observed differential stress responses in the lung slices.

Effects of smoking regimens and test material format on the cytotoxicity of mainstream cigarette smoke

The purpose of this study was to evaluate the effects of test material format and smoking regimens on comparative toxicity testing of cigarette smoke. Total particulate matter (TPM) or whole smoke (WS) generated from three test cigarettes under International Organization for Standardization (ISO) or Health Canada Intensive (HCI) regimens were assessed for cytotoxicity using the neutral red uptake (NRU) cytotoxicity assay. Under both ISO and HCI regimens, the relative differences of cytotoxicity among the test cigarettes indicated by the EC50 values in WS were significantly higher than those in TPM. For TPM testing, cytotoxicity was decreased going from ISO regimen to HCI regimen, consistent with the reported reductions of toxicant output on a per unit of TPM basis under the HCI regimen. For WS, cytotoxicity increased for the two lower TPM cigarettes, and decreased for the higher TPM cigarette going from HCI regimen to ISO regimen. Results from this study demonstrated WS should be the preferable test material format for smoke toxicity testing whenever possible. Intensive smoking regimens, such as HCI, are less likely to underestimate smoke toxicant intakes by smokers, and should be included in the comparative toxicological testing strategy.

Nr1d1, an Important Circadian Pathway Regulatory Gene, Is Suppressed By Cigarette Smoke in Murine Lungs

Nuclear receptor subfamily 1, group D member 1 (Nr1d1), also known as Rev-erb-alpha, belongs to the family of "orphan receptors" and functions as a member of clock gene family. In addition to being an important member of clock circuitry, Nr1d1, also regulates cell proliferation, lipid metabolism, and inflammation and is also touted as a tumor suppressor. Our focus on Nr1d1 was stimulated by data from a genome-wide search for mRNA correlates of cigarette smoke (CS) sensitive--whole smoke (WS) and filtered smoke (FS)--lung transcriptomes in tumor-resistant C57BL6 and tumor-susceptible AJ mice strains. Differential analysis of approximately 15,000 genes using Affymetrix 430A 2.0 high-density oligonucleotide arrays identified modulation of genes related to circadian pathways by CS in lungs of both mouse strains. Nr1d1 expression was downregulated by both WS and FS irrespective of mouse strain as compared to respective air-breathing controls. WS was more effective than FS on decreasing Nr1d1 expression. The present data suggest that transcriptional regulation of Nr1d1 by CS may affect circadian rhythmicity and thus may play a complementary role in CS-induced lung respiratory tract pathobiology and/or lung tumorigenesis.

In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens

Previous studies on the biological assessment of cigarette smoke (CS) mainly focused on the total particulate matter (TPM) collected using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS). To study the effects of native CS in vitro, direct exposure methods have been developed. Meanwhile, in vitro micronucleus (MN) assays have been reported to evaluate the mutagenicity of CS. The objective of this research is to investigate the MN-inducing activity of whole smoke (WS) and GVP using a whole smoke exposure system, CULTEX ®, which allows direct exposure of cultured cells to native CS at the air/liquid interface (ALI). CS was generated according to the International Organization for Standardization (ISO; 35 ml, 2 s, once per 60 s) or the Health Canada Intensive (HCI; 55 ml, 2 s, once per 30 s, with complete ventilation block) regimens and Chinese hamster lung (CHL/IU) cells were then exposed to this smoke. Dosages were adjusted according to the amount of smoke entering the actual exposure position. Under both smoking regimens, WS and GVP from 2R4F reference cigarettes induced MN responses. The concept of the dosage and similar dose–response relationships between theoretical and monitored dosage values under the two regimens enabled us to compare the MN-inducing activities of cigarettes in the direct exposure assay, even in the case of various experimental settings or different TPM amounts. MN-inducing activities of 2R4F under the ISO regimen seemed to be higher than those under HCI estimated by the TPM equivalent calculated values.

Effects of mainstream cigarette smoke on the global metabolome of human lung epithelial cells.

Metabolomics is a technology for identifying and quantifying numerous biochemicals across metabolic pathways. Using this approach, we explored changes in biochemical profiles of human alveolar epithelial carcinoma (A549) cells following in vitro exposure to mainstream whole smoke (WS) aerosol as well as to wet total particulate matter (WTPM) or gas/vapor phase (GVP), the two constituent phases of WS from 2R4F Kentucky reference cigarettes. A549 cells were exposed to WTPM or GVP (expressed as WTPM mass equivalent GVP volumes) at 0, 5, 25, or 50 microg/mL or to WS from zero, two, four, and six cigarettes for 1 or 24 h. Cell pellets were analyzed for perturbations in biochemical profiles, with named biochemicals measured, analyzed, and reported in a heat map format, along with biochemical and physiological interpretations (mSelect, Metabolon Inc.). Both WTPM and GVP exposures likely decreased glycolysis (based on decreased glycolytic intermediaries) and increased oxidative stress and cell damage. Alterations in the Krebs cycle and the urea cycle were unique to WTPM exposure, while induction of hexosamines and alterations in lipid metabolism were unique to GVP exposure. WS altered glutathione (GSH) levels, enhanced polyamine and pantothenate levels, likely increased beta-oxidation of fatty acids, and increased phospholipid degradation marked by an increase in phosphoethanolamine. GSH, glutamine, and pantothenate showed the most significant changes with cigarette smoke exposure in A549 cells based on principal component analysis. Many of the changed biochemicals were previously reported to be altered by cigarette exposure, but the global metabolomic approach offers the advantage of observing changes to hundreds of biochemicals in a single experiment and the possibility for new discoveries. The metabolomic approach may thus be used as a screening tool to evaluate conventional and novel tobacco products offering the potential to reduce risks of smoking.

Mutagenicity of native cigarette mainstream smoke and its gas/vapour phase by use of different tester strains and cigarettes in a modified Ames assay

The “Bacterial Reverse Mutation Assay” is generally accepted to analyse the genotoxic capacity of single compounds or complex mixtures such as cigarette-smoke condensates. With an adapted and modified Ames assay, the mutagenicity of native cigarette mainstream whole smoke (WS) and its gas/vapour phase (GVP) was studied. The bacteria were directly exposed to the smoke in a CULTEX ® 1 1 Patent No. DE 19801763/PCT/EP99/00295. system closely connected to a smoking robot (VC10). A variety of standard tester strains (TA98, TA100, TA1535, TA1537, TA1538, TA102, WP2 uvrApKM101) and descendants of TA98 (YG1021, YG1024, YG1041) and TA100 (YG1026, YG1029 and YG1042) were exposed to whole and filtered smoke of the research cigarette K2R4F to find the most sensitive strains for analysing the mutagenic activity of these test atmospheres. Mutagenicity of WS was detected by TA98, TA100 and their YG descendant strains as well as by WP2 uvrApKM101 in the presence of S9 mix. The GVP induced a mutagenic signal in TA100, YG1029 and YG1042 and WP2 uvrApKM101 only in the absence of S9 mix. To detect mutagenicity in WS the presence of the plasmid pKM101 is required and a frame-shift mutation is more effective than a missense mutation. To detect mutagenicity in GVP, the presence of the plasmid pKM101 and a missense mutation are required. The differentiating capacity of this modified Ames assay was demonstrated by exposing strain TA98 to WS and TA100 to the GVP of cigarettes with different tar content. The mutagenic activity of WS and the GVP increased with rising tar content of the cigarettes with two exceptions in WS. Thus, the concept of tar content alone is misleading and does not reflect the mutagenic activity of a cigarette.