Linsey Haswell

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.

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.

Targeted omics analyses, and metabolic enzyme activity assays demonstrate maintenance of key mucociliary characteristics in long term cultures of reconstituted human airway epithelia

3D reconstituted respiratory epithelia have emerged as better in vitro models for toxicological testing compared to cell lines due to the conservation of key morphological features and functions. MucilAir™ is a commercially available human airway epithelia system that can potentially maintain functional attributes for up to a year, however, detailed mucociliary characteristics and xenobiotic metabolism relevant to inhaled pro-toxicant bioactivation is lacking. Here, we assessed in MucilAir™ some key biomarkers that are characteristic of the respiratory epithelia including morphology, function and xenobiotics metabolism. The end points that were measured included targeted proteomics using a panel of 243 airway surface liquid (ASL) proteins, cilia beat frequency (CBF), a qRT-PCR screen of xenobiotic metabolizing enzymes, and CYP2A6/13, CYP1A1/1B1 activity. Comparison of ASL proteomics with human sputum identified key proteins common to both matrices, but present at different levels. Xenobiotic metabolism gene profiling demonstrated strong similarities with the normal human lung and did not reveal any consistent changes when assessed over a 6 month period. Inducibility and activity of CYP1A1/1B1 and activity of CYP2A6/2A13 were present at one month in culture and maintained in one tested MucilAir™ donor for several months. In conclusion, MucilAir™ presented important morphological and metabolic characteristics of a mucociliary epithelium in short and long term culture. MucilAir™ is therefore a potentially useful model to test repeated sub-cytotoxic doses of toxicants.

Changes in levels of biomarkers of exposure and biological effect in a controlled study of smokers switched from conventional cigarettes to reduced-toxicant-prototype cigarettes

BACKGROUND Development of cigarettes that reduce exposure to harmful smoke constituents is a suggested tobacco harm reduction strategy, but robust methods for measurement of change are required. We investigated whether changes in biomarkers of exposure (BoE), effective dose (BoED) and biological effect (BoBE) could be detected after switching from conventional cigarettes to a reduced-toxicant-prototype cigarette (RTP). METHODS Regular smokers of 6-8mg ISO tar yield cigarettes were recruited in Hamburg, Germany, and supplied with a conventional 7mg ISO tar yield cigarette for 2weeks then switched to the same cigarette with a different tipping paper (control) or the RTP for 6months. Subjects smoked mostly at home and attended five residential clinic visits where urine and blood samples were collected for analysis. Primary endpoints were changes in specific biomarker levels compared with non-smoker background levels. Changes in daily cigarette consumption were also investigated. RESULTS BoE levels in controls generally increased over the study period, whereas most BoE and all BoED significantly declined in RTP smokers. Most BoBE data were similar across groups and/or too variable within individuals to detect changes. Increased daily cigarette consumption was affected by supply of free cigarettes, perceived shorter smoking time per cigarette than usual brands, and perceived reduced harm. CONCLUSIONS Despite increased cigarette consumption, reductions in BoE and BoED were detectable.

A cross-sectional analysis of candidate biomarkers of biological effect in smokers, never-smokers and ex-smokers.

Abstract Context: Biomarkers of biological effect (BOBE) have been proposed as potential tools to assess tobacco product use, toxicity and disease risk. Objective: To determine if candidate BOBE can distinguish between smokers, never-smokers and former smokers. Methods: Biomarker levels were compared from 143 smokers, 61 never-smokers and 61 ex-smokers. Results: In total, 27 candidate biomarkers were assessed, 14 were significantly different between smokers and never-smokers (p < 0.01) and of these 14 biomarkers, 12 were able to distinguish between smokers and former smokers (p < 0.05), which indicates the potential for reversibility. Conclusions: A total of 12 of 27 BOBE are potentially useful tools for future product assessment.

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.

The cytotoxic and inflammatory response of bronchial epithelial cells to cigarette whole smoke and vapour phase at the air-liquid interface

INTRODUCTION Prolonged cigarette smoke exposure is known to be a causative factor in the development of cardiovascular disease, chronic obstructive pulmonary disease (COPD) and cancer (1). Cigarette whole smoke (WS) is a complex aerosol, made up of approximately 5700 identified constituents distributed in the particulate and vapour phases (VP), with the latter constituting ~95% by weight (2). The role of each component of cigarette smoke in the development of smoking related diseases is largely unknown, however, exposure to both phases and the retention of smoke particulate has been implicated to contribute to smoking related injury and disease (1,3).

The inflammatory response of the H292 lung epithelial model to cigarette smoke particulate generated using different smoking regimes and reduced toxicant prototypes

Ar se n ic B e n zo (a )p yre n e Cd m u m Ch V I The inflammatory response of the H292 lung epithelial model to cigarette smoke particulate generated using different smoking regimes and reduced toxicant prototypes Sarah Corke, Geoff Foss-Smith, Katherine Hewitt, Linsey Haswell, David Azzopardi and Gary Phillips British American Tobacco, Group Research & Development, Regents Park Road, Southampton, SO15 8TL, UK Corresponding author: Sarah_Corke@bat.com