Marianna Gaça

The role of oxidative stress in the biological responses of lung epithelial cells to cigarette smoke

The mechanism(s) by which cigarette smoke contributes to lung diseases, such as cancer, remains unclear. Recent developments in our knowledge of cell signalling events suggest that cigarette smoke causes oxidative stress and proinflammatory responses in cells of the lung. Cigarette smoke is a complex mixture of over 4000 compounds and high levels of oxidants and reactive oxygen species (ROS) have been detected in both mainstream and sidestream smoke. Oxidative stress that ensues, when the antioxidant defences are depleted, is accompanied by increases in ROS production in lung epithelial cells. Cigarette smoke-mediated oxidative stress produces DNA damage and activates survival signalling cascades resulting in uncontrolled cell proliferation and transformation. Intervention studies using antioxidants have provided compelling evidence that oxidative stress plays a critical role in the aetiology of smoking-related disorders.

Cigarette smoke total particulate matter increases mucous secreting cell numbers in vitro: a potential model of goblet cell hyperplasia.

Cigarette smoking is associated with chronic obstructive pulmonary disease (COPD)--a term encompassing chronic lung inflammation, chronic bronchitis and emphysema. Goblet cell hyperplasia is a characteristic feature of the lung epithelium in COPD patients contributing to the overproduction of airway mucus, including mucin MUC5AC. Using an in vitro model of differentiated lung epithelium we have investigated morphological and cellular changes in response to interleukin (IL)-13 (2.5-20 ng/ml), cigarette smoke total particulate matter (TPM; 0.31-20 microg/ml) and three mainstream cigarette smoke constituents: acrolein, formaldehyde and acetaldehyde (all at 0.001-1 microM) over 28 days during differentiation (agents replaced daily Monday-Friday). Control cultures of human bronchial epithelial cells (HBECs) underwent mucociliary differentiation producing a columnar epithelium containing goblet, ciliated and basal cells. Non-cytotoxic doses of IL-13 induced a significant increase in the percentage of MUC5AC positive cells. Using both flow cytometry and immunocytochemical techniques for identification of MUC5AC positive cells, TPM treatment induced an increase in MUC5AC positive cells, becoming maximal at 5 microg/ml. Acrolein treatment also increased the percentage of MUC5AC positive cells. However, formaldehyde or acetaldehyde had little effect. This study demonstrates that lung toxicants can induce a profound effect on cellular differentiation in an in vitro model of the human lung epithelium.

Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke

Abstract Electronic cigarettes (E-cigarettes) are a potential means of addressing the harm to public health caused by tobacco smoking by offering smokers a less harmful means of receiving nicotine. As e-cigarettes are a relatively new phenomenon, there are limited scientific data on the longer-term health effects of their use. This study describes a robust in vitro method for assessing the cytotoxic response of e-cigarette aerosols that can be effectively compared with conventional cigarette smoke. This was measured using the regulatory accepted Neutral Red Uptake assay modified for air–liquid interface (ALI) exposures. An exposure system, comprising a smoking machine, traditionally used for in vitro tobacco smoke exposure assessments, was adapted for use with e-cigarettes to expose human lung epithelial cells at the ALI. Dosimetric analysis methods using real-time quartz crystal microbalances for mass, and post-exposure chemical analysis for nicotine, were employed to detect/distinguish aerosol dilutions from a reference Kentucky 3R4F cigarette and two commercially available e-cigarettes (Vype eStick and ePen). ePen aerosol induced 97%, 94% and 70% less cytotoxicity than 3R4F cigarette smoke based on matched EC50 values at different dilutions (1:5 vs. 1:153 vol:vol), mass (52.1 vs. 3.1 μg/cm2) and nicotine (0.89 vs. 0.27 μg/cm2), respectively. Test doses where cigarette smoke and e-cigarette aerosol cytotoxicity were observed are comparable with calculated daily doses in consumers. Such experiments could form the basis of a larger package of work including chemical analyses, in vitro toxicology tests and clinical studies, to help assess the safety of current and next generation nicotine and tobacco products.

Assessment of an in vitro whole cigarette smoke exposure system: The Borgwaldt RM20S 8-syringe smoking machine

BackgroundThere have been many recent developments of in vitro cigarette smoke systems closely replicating in vivo exposures. The Borgwaldt RM20S smoking machine (RM20S) enables the serial dilution and delivery of cigarette smoke to exposure chambers for in vitro analyses. In this study we have demonstrated reliability and robustness testing of the RM20S in delivering smoke to in vitro cultures using an in-house designed whole smoke exposure chamber.ResultsThe syringe precision and accuracy of smoke dose generated by the RM20S was assessed using a methane gas standard and resulted in a repeatability error of ≤9%. Differential electrical mobility particle spectrometry (DMS) measured smoke particles generated from reference 3R4F cigarettes at points along the RM20S. 53% ± 5.9% of particles by mass reached the chamber, the remainder deposited in the syringe or connecting tubing and ~16% deposited in the chamber. Spectrofluorometric quantification of particle deposition within chambers indicated a positive correlation between smoke concentration and particle deposition. In vitro air-liquid interface (ALI) cultures (H292 lung epithelial cells), exposed to whole smoke (1:60 dilution (smoke:air, equivalent to ~5 μg/cm2)) demonstrated uniform smoke delivery within the chamber.ConclusionsThese results suggest this smoke exposure system is a reliable and repeatable method of generating and exposing ALI in vitro cultures to cigarette smoke. This system will enable the evaluation of future tobacco products and individual components of cigarette smoke and may be used as an alternative in vitro tool for evaluating other aerosols and gaseous mixtures such as air pollutants, inhaled pharmaceuticals and cosmetics.

E-cigarette aerosols induce lower oxidative stress in vitro when compared to tobacco smoke

Abstract Tobacco smoking is a risk factor for various diseases. The underlying cellular mechanisms are not fully characterized, but include oxidative stress, apoptosis, and necrosis. Electronic-cigarettes (e-cigarettes) have emerged as an alternative to and a possible means to reduce harm from tobacco smoking. E-cigarette vapor contains significantly lower levels of toxicants than cigarette smoke, but standardized methods to assess cellular responses to exposure are not well established. We investigated whether an in vitro model of the airway epithelium (human bronchial epithelial cells) and commercially available assays could differentiate cellular stress responses to aqueous aerosol extracts (AqE) generated from cigarette smoke and e-cigarette aerosols. After exposure to AqE concentrations of 0.063–0.500 puffs/mL, we measured the intracellular glutathione ratio (GSH:GSSG), intracellular generation of oxidant species, and activation of the nuclear factor erythroid-related factor 2 (Nrf2)-controlled antioxidant response elements (ARE) to characterize oxidative stress. Apoptotic and necrotic responses were characterized by increases in caspase 3/7 activity and reductions in viable cell protease activities. Concentration-dependent responses indicative of oxidative stress were obtained for all endpoints following exposure to cigarette smoke AqE: intracellular generation of oxidant species increased by up to 83%, GSH:GSSG reduced by 98.6% and transcriptional activation of ARE increased by up to 335%. Caspase 3/7 activity was increased by up to 37% and the viable cell population declined by up to 76%. No cellular stress responses were detected following exposure to e-cigarette AqE. The methods used were suitably sensitive to be employed for comparative studies of tobacco and nicotine products.

Real-time assessment of cigarette smoke particle deposition in vitro

BackgroundRecently there has been a rapid increase in approaches to assess the effects of cigarette smoke in vitro. Despite a range of gravimetric and chemical methods, there is a requirement to identify simpler and more reliable methods to quantify in vitro whole smoke dose, to support extrapolation and comparisons to human/in vivo dose. We have previously characterised an in vitro exposure system using a Borgwaldt RM20S smoking machine and a chamber exposing cellular cultures to whole smoke at the air-liquid interface. In this study we demonstrate the utility of a quartz crystal microbalance (QCM), using this exposure system, to assess real-time cigarette smoke particulate deposition during a 30 minute smoke exposure. Smoke was generated at various dilutions (1:5–1:400, smoke:air) using two cigarette products, 3R4F Kentucky reference and 1 mg commercially available cigarettes. The QCM, integrated into the chamber, assessed particulate deposition and data generated were compared to traditional chemical spectrofluorometric analysis.ResultsThe QCM chamber was able to detect mass differences between the different products within the nanogram range. 3R4F reference cigarette smoke deposition ranged from 25.75 ±2.30 μg/cm2 (1:5) to 0.22 ±0.03 μg/cm2 (1:400). 1 mg cigarette smoke deposition was less and ranged from 1.42 ±0.26 μg/cm2 (1:5), to 0.13 ±0.02 μg/cm2 (1:100). Spectrofluorometric analysis demonstrated statistically significant correlation of particulate deposition with the QCM (p < 0.05), and regression R2 value were 97.4 %. The fitted equation for the linear model which describes the relationship is: QCM = −0.6796 + 0.9744 chemical spectrofluorescence.ConclusionsWe suggest the QCM is a reliable, effective and simple tool that can be used to quantify smoke particulate deposition in real-time, in vitro and can be used to quantify other aerosols delivered to our chamber for assessment.

Evaluation of precision and accuracy of the Borgwaldt RM20S® smoking machine designed for in vitro exposure

The Borgwaldt RM20S® smoking machine enables the generation, dilution, and transfer of fresh cigarette smoke to cell exposure chambers, for in vitro analyses. We present a study confirming the precision (repeatability r, reproducibility R) and accuracy of smoke dose generated by the Borgwaldt RM20S® system and delivery to exposure chambers. Due to the aerosol nature of cigarette smoke, the repeatability of the dilution of the vapor phase in air was assessed by quantifying two reference standard gases: methane (CH4, r between 29.0 and 37.0 and RSD between 2.2% and 4.5%) and carbon monoxide (CO, r between 166.8 and 235.8 and RSD between 0.7% and 3.7%). The accuracy of dilution (percent error) for CH4 and CO was between 6.4% and 19.5% and between 5.8% and 6.4%, respectively, over a 10–1000-fold dilution range. To corroborate our findings, a small inter-laboratory study was carried out for CH4 measurements. The combined dilution repeatability had an r between 21.3 and 46.4, R between 52.9 and 88.4, RSD between 6.3% and 17.3%, and error between 4.3% and 13.1%. Based on the particulate component of cigarette smoke (3R4F), the repeatability (RSD = 12%) of the undiluted smoke generated by the Borgwaldt RM20S® was assessed by quantifying solanesol using high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Finally, the repeatability (r between 0.98 and 4.53 and RSD between 8.8% and 12%) of the dilution of generated smoke particulate phase was assessed by quantifying solanesol following various dilutions of cigarette smoke. The findings in this study suggest the Borgwaldt RM20S® smoking machine is a reliable tool to generate and deliver repeatable and reproducible doses of whole smoke to in vitro cultures.

Assessing modified risk tobacco and nicotine products: Description of the scientific framework and assessment of a closed modular electronic cigarette

ABSTRACT Cigarette smoking causes many human diseases including cardiovascular disease, lung disease and cancer. Novel tobacco products with reduced yields of toxicants compared to cigarettes, such as tobacco‐heating products, snus and electronic cigarettes, hold great potential for reducing the harms associated with tobacco use. In the UK several public health agencies have advocated a potential role for novel products in tobacco harm reduction. Public Health England has stated that “The current best estimate is that e‐cigarettes are around 95% less harmful than smoking” and the Royal College of Physicians has urged public health to “Promote e‐cigarettes widely as substitute for smoking”. Health related claims on novel products such as ‘reduced exposure’ and ‘reduced risk’ should be substantiated using a weight of evidence approach based on a comprehensive scientific assessment. The US FDA, has provided draft guidance outlining a framework to assess novel products as Modified Risk Tobacco Products (MRTP). Based on this, we now propose a framework comprising pre‐clinical, clinical, and population studies to assess the risk profile of novel tobacco products. Additionally, the utility of this framework is assessed through the pre‐clinical and part of the clinical comparison of a commercial e‐cigarette (Vype ePen) with a scientific reference cigarette (3R4F) and the results of these studies suggest that ePen has the potential to be a reduced risk product. HighlightsDescription of a scientific framework to assess the risk profile of next generation products relative to cigarettes.Multi‐disciplinary studies comparing a commercial e‐cigarette (Vype ePen) with a scientific reference cigarette (3R4F).The test e‐cigarette aerosol is compositionally simpler, containing reduced levels of toxicants relative to cigarette smoke.Results from a range of multi‐disciplinary studies showed reduced responses for the test e‐cigarette relative to cigarettes.

In vitro models for assessing the potential cardiovascular disease risk associated with cigarette smoking

Atherosclerotic cardiovascular disease is a prevalent human disorder and a significant cause of human morbidity and mortality. A number of risk factors may predispose an individual to developing atherosclerosis, and of these factors, cigarette smoking is strongly associated with the development of cardiovascular disease. Current thinking suggests that exposure to toxicants found in cigarette smoke may be responsible for this elevated disease likelihood, and this gives rise to the idea that reductions in the levels of some smoke toxicants may reduce the harm associated with cigarette smoking. To assess the disease risk of individuals who smoke cigarettes with altered toxicant levels, a weight-of-evidence approach is required examining both exposure and disease-related endpoints. A key element of such an assessment framework are data derived from the use of in vitro models of cardiovascular disease, which when considered alongside other forms of data (e.g. from clinical studies) may support evidence of potential reduced risk. Importantly, such models may also be used to provide mechanistic insight into the effects of smoking and of smoke toxicant exposure in cardiovascular disease development. In this review the use of in vitro models of cardiovascular disease and one of the contributory factors, oxidative stress, is discussed in the context of assessing the risk potential of both conventional and modified cigarettes. Practical issues concerning the use of these models for cardiovascular disease understanding and risk assessment are highlighted and areas of development necessary to enhance the power and predictive capacity of in vitro disease models in risk assessment are discussed.

Assessment of tobacco heating product THP1.0. Part 5: In vitro dosimetric and cytotoxic assessment

ABSTRACT Tobacco heating products (THPs) represent a subset of the next‐generation nicotine and tobacco product category, in which tobacco is typically heated at temperatures of 250–350 °C, thereby avoiding many of the harmful combustion‐related toxicant emissions of conventional cigarettes. In this study, we have assessed aerosol generation and cytotoxicity from two commercially available THPs, THP1.0 and THS, relative to tobacco smoke from 3R4F reference cigarettes, using an adapted Borgwaldt RM20S Smoking Machine. Quantification of nicotine in the exposed cell‐culture media showed greater delivery of nicotine from both THPs than from the cigarette. Using Neutral Red Uptake assay, THPs demonstrated reduced in vitro cytotoxicity in H292 human bronchial epithelial cells as compared with 3R4F cigarette exposure at the air–liquid interface (p < 0.0001). Both THPs demonstrated a statistically similar reduction in biological response, with >87% viability relative to 3R4F at a common aerosol dilution (1:40, aerosol:air). A similar response was observed when plotted against nicotine; a statistical difference between 3R4F and THPs (p < 0.0001) and no difference between the THPs (p = 0.0186). This pre‐clinical in vitro biological testing forms part of a larger package of data to help assess the safety and risk reduction potential of next‐generation tobacco products relative to cigarettes, using a weight of evidence approach. HighlightsTHP and 3R4F aerosols were assessed using the Borgwaldt RM20S smoking machine.In vitro cytotoxicity from two THPs were compared to a 3R4F reference cigarette.THPs demonstrated significantly reduced cytotoxicity compared to 3R4F exposure.Nicotine delivery to the culture media was greater with the two THPs than 3R4F.