Jason Adamson

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.

An inter-machine comparison of tobacco smoke particle deposition in vitro from six independent smoke exposure systems.

There are several whole smoke exposure systems used to assess the biological and toxicological impact of tobacco smoke in vitro. One such system is the Vitrocell® VC 10 Smoking Robot and exposure module. Using quartz crystal microbalances (QCMs) installed into the module, we were able to assess tobacco smoke particle deposition in real-time. We compared regional deposition across the module positions and doses delivered by six VC 10s in four independent laboratories: two in the UK, one in Germany and one in China. Gauge R&r analysis was applied to the total data package from the six VC 10s. As a percentage of the total, reproducibility (between all six VC 10s) and repeatability (error within an individual VC 10) accounted for 0.3% and 7.4% respectively. Thus Gauge R&r was 7.7%, less than 10% overall and considered statistically fit for purpose. The dose-responses obtained from the six machines across the four different locations demonstrated excellent agreement. There were little to no positional differences across the module at all airflows as determined by ANOVA (except for one machine and at three airflows only). These results support the on-going characterisation of the VC 10 exposure system and suitability for tobacco smoke exposure in vitro.

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.

Quantification of Cigarette Smoke Particle Deposition In Vitro Using a Triplicate Quartz Crystal Microbalance Exposure Chamber

There are a variety of smoke exposure systems available to the tobacco industry and respiratory toxicology research groups, each with their own way of diluting/delivering smoke to cell cultures. Thus a simple technique to measure dose in vitro needs to be utilised. Dosimetry—assessment of dose—is a key element in linking the biological effects of smoke generated by various exposure systems. Microbalance technology is presented as a dosimetry tool and a way of measuring whole smoke dose. Described here is a new tool to quantify diluted smoke particulate deposition in vitro. The triplicate quartz crystal microbalance (QCM) chamber measured real-time deposition of smoke at a range of dilutions 1 : 5–1 : 400 (smoke : air). Mass was read in triplicate by 3 identical QCMs installed into one in vitro exposure chamber, each in the location in which a cell culture would be exposed to smoke at the air-liquid interface. This resulted in quantification of deposited particulate matter in the range 0.21–28.00 μg/cm2. Results demonstrated that the QCM could discriminate mass between dilutions and was able to give information of regional deposition where cell cultures would usually be exposed within the chamber. Our aim is to use the QCM to support the preclinical (in vitro) evaluation of tobacco products.

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.

In Vitro Models of Chronic Obstructive Pulmonary Disease (COPD)

1.1 The lung The lungs are situated at the air-blood interface and are a crucial boundary between the organism and the environment, protecting the host from a battery of potential insults such as inhaled particles, pollutants, carcinogens and infectious agents that deposit on airway surfaces during normal tidal breathing. The upper or conducting airways (tracheo-bronchial region) are covered with a columnar epithelium composed of ciliated cells and mucus producing goblet cells (Figure 1A). The apical surface of the epithelium is covered by a surface liquid which is comprised of two distinct layers. The outer mucus layer provides a physical barrier that traps inhaled particles. The underlying periciliary fluid is a low viscosity liquid which allows cilia to beat and continually move the mucus layer towards the pharynx. Thus inhaled particles trapped in the mucus are cleared from the airways. Under normal conditions mucus protects the lung airway epithelium; however abnormalities in mucus hypersecretion or clearance can lead to respiratory disease (Rogers 2007). In the lower bronchioles, the epithelium is simple columnar, containing secretary Clara cells and has progressively fewer ciliated cells. The alveolar epithelium is composed primarily (95%) of flattened alveolar type I (AT-I) cells that form a thin barrier for gas exchange. These cells are interspersed with rounded alveolar type II (AT-II) cells that secrete pulmonary surfactant to decrease the surface tension within the alveoli and prevent alveolar collapse during expiration (Figure 1B).

In vitro RNA-seq-based toxicogenomics assessment shows reduced biological effect of tobacco heating products when compared to cigarette smoke

The battery of regulatory tests used to evaluate the risk of novel tobacco products such as heated tobacco products (THPs) presents some limitations including a bias towards the apical endpoint tested, and limited information on the mode of action. This is driving a paradigm shift to more holistic systems biology approaches. In this study, we used RNA-sequencing to compare the transcriptomic perturbations following acute exposure of a 3D airway tissue to the aerosols from two commercial THPs and a reference 3R4F cigarette. 2809 RNAs were differentially expressed for the 3R4F treatment and 115 and 2 RNAs for the two THPs (pFDR < 0.05, FC > 1.5), respectively. The relationship between the identified RNA features and gene ontologies were mapped showing a strong association with stress response, xenobiotics metabolism, and COPD-related terms for 3R4F. In contrast, fewer ontologies were found enriched for the THPs aerosols. “Response to wounding” was a common COPD-related term over-represented for the two THPs but at a reduced significance. Quantification of a cytokine panel post-exposure confirmed a pro-inflammatory effect of cigarette smoke but not for THPs. In conclusion, THPs have a reduced impact on gene expression compared to 3R4F.

Extreme testing of undiluted e-cigarette aerosol in vitro using an Ames air-agar-interface technique

There is a growing consensus that e-cigarettes hold the potential for reducing the harm associated with cigarette smoking. Recently published studies have reported in vitro testing of e-cigarettes, demonstrating reduced toxicological and biological effects. Few studies however have reported the use of e-cigarettes under extreme testing conditions. To assess the full mutagenic potential of a commercially available electronic-cigarette (Vype ePen), this study investigated the delivery of aerosol under extreme conditions, using a scaled-down 35 mm plate Ames bacterial reverse mutagenicity assay. S. typhimurium strains TA98, TA100, TA97, TA104 and E. coli WP2 uvrA pKM101 with or without metabolic activation (S9), were employed. Using a modified Vitrocell VC 10 exposure system 0, 180, 360, 540, 720 or 900 puffs of undiluted e-cigarette aerosol was generated and delivered to bacterial cultures aligned to reported human consumption data. The results demonstrate that no mutagenic activity was observed in any strain under any test condition even when exposed to 900 puffs of undiluted e-cigarette aerosols +/- S9. Positive control responses were observed in all strains +/- S9. Nicotine assessments demonstrated an increased and consistent aerosol delivery, with calculated maximum doses of ∼1 mg/mL delivery of nicotine. These data demonstrate the validity of this unique testing approach and adds further information to the growing weight of evidence that e-cigarettes offer substantially reduced exposure when compared to conventional cigarette smoke. For future in vitro assessments of next generation tobacco and nicotine products, the generation, delivery and testing of undiluted aerosols can now be considered.