Comet assay and air–liquid interface exposure system: A new combination to evaluate genotoxic effects of cigarette whole smoke in human lung cell lines

BackgroundTobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity.FindingsThe findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference.ConclusionsWe have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.

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“…Theoretical percentage of cigarette smoke was calculated from Webber et al,2013 [18] using the following equation (Figure 2). …”

Section: Methodsmentioning

confidence: 99%

Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system

et al. 2014

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“…These have used both cell lines (Weber et al, 2013) and primary cell cultures (Mathis et al, 2013) in conjunction with various smoking machines to deliver aerosols from combustible and non-combustible products. In the present study we used the NCI-H292 bronchial cell culture system (Phillips et al, 2005) because of its ease of cell culture, its high throughput relative to primary cell cultures and because it allows for the standardisation of the complete cell culture and exposure system.…”

Section: Inflammatory Mediator Exposure Group Seeding Density and Pasmentioning

confidence: 99%

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

Azzopardi

Haswell

Foss-Smith

et al. 2015

Toxicology in Vitro

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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.

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“…It has been reported, although under rather different smoke exposure conditions (1-min exposure and extremely high smoke concentration), that the level of DNA strand breaks in the lung returns to control values already after 1 hour (69). In vitro, cigarette smoke is clearly active (70). In peripheral lymphocytes of smoke-exposed rats the assay shows, at best, a weak response (71).…”

Section: Discussionmentioning

confidence: 97%

Cigarette-Smoke- and Age-Dependent Oxidative Stress Effects in Rats

Meisgen

Roemer

Conroy

et al. 2014

Beiträge Zur Tabakforschung International/Contributions to Tobacco Research

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SUMMARYOxidative stress is a basic mechanism involved in both ageand smoking-related diseases. To test whether smoking affects young, old, and calorie-restricted organisms to the same extent, we assessed oxidative stress parameters in the lung, heart, and liver of male Fischer 344 rats (4 months old and 19-22 months old) exposed to air or cigarette mainstream smoke. Smoke-related effects were seen for parameters of DNA damage, lipid peroxidation, protein oxidation, and glycoxidation. No smoke-related effects were observed for DNA damage in the lung and heart (Comet assay) and for malondialdehyde in the lung. The old rats showed higher smoke-related responses than the young rats for 8-hydroxy-desoxyguanosine (8-OHdG) in the heart and liver, DNA damage in the liver, and protein carbonyls in the lung; however, there was little evidence for an overadditive effect of smoking on aging. Caloric restriction, which is known to retard aging effects, also had little impact on smoke-related oxidative changes. [Beitr. Tabakforsch. Int. 26 (2014) 109-120]

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Comet assay and air–liquid interface exposure system: A new combination to evaluate genotoxic effects of cigarette whole smoke in human lung cell lines

Cited by 38 publications

References 24 publications

Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system

Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system

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

Cigarette-Smoke- and Age-Dependent Oxidative Stress Effects in Rats

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