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Research & development at British American Tobacco

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Date: 25/03/2013

Here is a list of our most recent open access manuscripts:-

In vitro and clinical studies examining the expression of osetopontin in cigarette smoke-exposed endothelial cells and cigarette smokers (2012) Emma Bishop, Eugenia H Theophilus and Ian M Fearon Full text: In vitro and clinical studies examining the expression... Opens new window

Cigarette smoking is a leading cause of mortality and morbidity and is associated with cardiovascular disease via contributory processes such as endothelial dysfunction, inflammation and thrombosis. Cigarette smoke both contains and stimulates the production of cellular oxidants and it may also promote vascular inflammation. Osteopontin is a non-collagenous matrix protein first identified in bone and there is increasing evidence for its role in inflammation and cardiovascular disease via its action as a soluble cytokine.

Real-time assessment of cigarette smoke particle deposition in vitro (2012) Jason Adamson, Sophie Hughes, David Azzopardi, John McAughey and Marianna D Gaça Full text: Real-time assessment of cigarette smoke particle deposition in vitro Opens new window

Recently 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. 

Model review – status wrt cigarette smoke testing - joint paper between Reynolds and BAT (2012) Ian M Fearon, Marianna D Gaça and Brian K. Nordskog Full text: Model review – status wrt cigarette smoke testing... Opens new window

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.

Arsenic Speciation in Tobacco and Cigarette Smoke (2012) Chuan Liu , Christopher G. Wright , Kevin G. McAdam, Sutthinum Taebunpakul, Julian Heroult, Julian Braybrook, and Heidi Goenaga-Infante Full text: Arsenic Speciation in Tobacco and Cigarette Smoke Opens new window  

Arsenic is one of the metals found in cured tobacco and mainstream cigarette smoke. Levels of arsenic in modern filtered cigarette smoke range from sub-ppm to a few tens of ppms. To enable accurate smoke toxicity assessment on arsenic in cigarette smoke, it is desirable to establish its chemical forms in addition to total quantities because different arsenic compounds possess different toxicological potentials. Progress has been made on measuring the arsenic speciation in tobacco and mainstream cigarette smoke by using a combination of synchrotron-based X-ray absorption spectroscopy and high-performance liquid chromatography- inductively coupled plasma mass spectrometry (HPLC-ICP-MS). In this paper, we describe the experimental procedures developed together with the main findings. A transient redox transformation between As(V) and As(III) was confirmed in freshly generated mainstream smoke. Potential areas for future research are highlighted in order to further our understanding of the speciation mechanism for arsenic in tobacco products.

γH2AX as a novel endpoint to detect DNA damage. Applications for the assessment of the in vitro genotoxicity of cigarette smoke (2012) Carolina Garcia-Canton, Arturo Anadón and Clive Meredith Full text: γH2AX as a novel endpoint to detect DNA damage... Opens new window

Histone H2AX is rapidly phosphorylated to become γH2AX after exposure to DNA-damaging agents that cause double-strand DNA breaks (DSBs). γH2AX can be detected and quantified by numerous methods, giving a direct correlation with the number of DSBs. This relationship has made γH2AX an increasingly utilised endpoint in multiple scientific fields since its discovery in 1998. Applications include its use in pre-clinical drug assessment, as a biomarker of DNA damage and in in vitro mechanistic studies. Here, we review current in vitro regulatory and non-regulatory genotoxicity assays proposing the γH2AX assay as a potential complement to the current test battery. Additionally, we evaluate the use of the γH2AX assay to measure DSBs in vitro in tobacco product testing.

Lack of effect of menthol level and type on smokers’ estimated mouth level exposures to tar and nicotine and perceived sensory characteristics of cigarette smoke (2012) Madeleine Ashley, Mike Dixon, Ajit Sisodiya and Krishna Prasad Full text: Lack of effect of menthol level and type on smokers’ estimated... Opens new window

Menthol can reduce sensory irritation and it has been hypothesised that this could result in smokers of mentholated cigarettes taking larger puffs and deeper post-puff inhalations thereby obtaining higher exposures to smoke constituents than smokers of non-mentholated cigarettes. The aim of our study was to use part-filter analysis methodology to assess the effects of cigarette menthol loading on regular and occasional smokers of mentholated cigarettes. We measured mouth level exposure to tar and nicotine and investigated the effects of mentholation on smokers’ sensory perceptions such as cooling and irritation. Test cigarettes were produced containing no menthol and different loadings of synthetic and natural l-menthol at 1 and 4 mg ISO tar yields. A target of 100 smokers of menthol cigarettes and 100 smokers who predominantly smoked non-menthol cigarettes from both 1 and 4 mg ISO tar yield categories were recruited in Poland and Japan. Each subject was required to smoke the test cigarette types of their usual ISO tar yield. There were positive relationships between menthol loading and the perceived ‘strength of menthol taste’ and ‘cooling’ effect. However, we did not see marked menthol-induced reductions in perceived irritation or menthol-induced increases in mouth level exposure to tar and nicotine.

The effect of a novel tobacco process on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter (2012) R. Combes, K. Scott , D. Dillon, C. Meredith, K. McAdam and C. Proctor Full text: The effect of a novel tobacco process on the in vitro cytotoxicity... Opens new window

Some of the toxic effects of smoking have been attributed to the combustion of nitrogenous protein in tobacco. The effects of a treatment which reduces tobacco’s protein nitrogen level, on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter (PM), were measured. PMs were tested in the Neutral Red Uptake (NRU) test; the Salmonella mutagenicity assay (SAL); the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT). PMs from all of the cigarettes were cytotoxic and genotoxic. PM obtained from smoking treated tobacco, showed a small, consistent and statistically significant reduced mutagenicity (revertants/μg) in TA98 with post-mitochondrial supernatant (S9). No consistent quantitative or qualitative differences were detected in the other tests. The data are discussed in relation to published information on smoke chemistry obtained from cigarettes made of tobacco treated using this technique. The observations confirm that the method did not give rise to any new qualitative or quantitative cytotoxic or genotoxic effects, and may have reduced PM’s bacterial mutagenicity in TA98 with S9. Further toxicity testing is warranted, to investigate the effects of the tobacco treatment in more detail and add to the data already obtained.

Determination of Nicotine Absorption from Multiple Tobacco Products and Nicotine Gum (2012) Helena Digard,Christopher Proctor, Anuradha Kulasekaran, Ulf Malmqvist and Audrey Richter Full text: Determination of Nicotine Absorption from Multiple Tobacco Products and Nicotine Gum Opens new window

Copper manganese oxides (CMOs) were synthesized using a co-precipitation method with different precursors and precipitants for carbon monoxide oxidation. The as-synthesized catalysts were characterized by powder X-ray diffraction (XRD), low temperature N2 sorption, Fourier transform-infrared spectroscopy (FT-IR), H2-temperature programmed reduction (H2-TPR), and thermal gravimetric analysis (TGA). Their catalytic activities for CO oxidation were tested by temperature programmed reaction. The results showed that the activity of CO oxidation strongly depended on the combination of precipitant and precursor anions, ranking in the order (Ac + CO32−) > (NO3 + CO32−) > (Ac + OH) > (NO3 + OH). The crystalline phase of copper manganese oxides obtained using strong electrolyte (OH) as the precipitant were mainly spinel Cu1.5Mn1.5O4, while the catalysts prepared with weak electrolyte (CO32−) as the precipitant mostly comprised of MnCO3, Mn2O3 and CuO, and showed a much higher CO oxidation activity than that of the Cu1.5Mn1.5O4. Keeping the same precipitant while changing the precursor caused a change in the H2 consumption which influenced the CO oxidation activity. A suitable combination of precipitant and precursor resulted in the most efficient CO oxidation catalyst.

Design and chemical evaluation of reduced machine-yield cigarettes
(2012) K.G. McAdam, E.O. Gregg , M. Bevan, D.J. Dittrich, S. Hemsley, C. Liu, C.J. Proctor Full text: Design and chemical evaluation of reduced machine-yield cigarettes Opens new window

Experimental cigarettes (ECs) were made by combining technological applications that individually reduce the machine measured yields of specific toxicants or groups of toxicants in mainstream smoke (MS). Two tobacco blends, featuring a tobacco substitute sheet or a tobacco blend treatment, were combined with filters containing an amine functionalised resin (CR20L) and/or a polymer-derived, high activity carbon adsorbent to generate three ECs with the potential for generating lower smoke toxicant yields than conventional cigarettes. MS yields of smoke constituents were determined under 4 different smoking machine conditions. Health Canada Intense (HCI) machine smoking conditions gave the highest MS yields for nicotine-free dry particulate matter and for most smoke constituents measured. Toxicant yields from the ECs were compared with those from two commercial comparator cigarettes, three scientific control cigarettes measured contemporaneously and with published data on 120 commercial cigarettes. The ECs were found to generate some of the lowest machine yields of toxicants from cigarettes for which published HCI smoke chemistry data are available; these comparisons therefore confirm that ECs with reduced MS machine toxicant yields compared to commercial cigarettes can be produced. The results encourage further work examining human exposure to toxicants from these cigarettes, including human biomarker studies.

Pyrolysis and combustion of tobacco in a cigarette smoking simulator under air and nitrogen atmosphere (2012) Christian Busch & Thorsten Streibel & Chuan Liu & Kevin G. McAdam & Ralf Zimmermann Full text: Pyrolysis and combustion of tobacco in a cigarette smoking... Opens new window

A coupling between a cigarette smoking simulator and a time-of-flight mass spectrometer was constructed to allow investigation of tobacco smoke formation under simulated burning conditions. The cigarette smoking simulator is designed to burn a sample in close approximation to the conditions experienced by a lit cigarette. The apparatus also permits conditions outside those of normal cigarette burning to be investigated for mechanistic understanding purposes. It allows control of parameters such as smouldering and puff temperatures, as well as combustion rate and puffing volume. In this study, the system enabled examination of the effects of “smoking” a cigarette under a nitrogen atmosphere. Time-of-flight mass spectrometry combined with a soft ionisation technique is expedient to analyse complex mixtures such as tobacco smoke with a high time resolution. The objective of the study was to separate pyrolysis from combustion processes to reveal the formation mechanism of several selected toxicants. A purposely designed adapter, with no measurable dead volume or memory effects, enables the analysis of pyrolysis and combustion gases from tobacco and tobacco products (e.g. 3R4F reference cigarette) with minimum aging. The combined system demonstrates clear distinctions between smoke composition found under air and nitrogen smoking atmospheres based on the corresponding mass spectra and visualisations using principal component analysis.

Advances in Plant Senescence (2012) Kieron D. Edwards,Matt Humphry and Juan Pablo Sanchez-Tamburrino Full text: Advances in Plant Senescence Opens new window

Senescence is an integral component of a plant’s lifecycle, which refers to changes that take place as the plant matures. A general distinction between plant senescence and animal senescence is the events observed in the animal kingdom typically steer growth while plant senescence orchestrates a massive shutdown or coordinated cell death in response to various stimuli designed to facilitate survival of the plant itself or the plant species. In order to assist in the survival of the plant species, a sequence of tightly regulated genetic events efficiently governs a plant’s death. These events are observable in a variety of plant models and in the different plant parts such as leaves, petals, reproductive organs (stamens and style), root cap, cortex and germinating seed. Leaf senescence will be the primary focus of this chapter. A popular aspect of leaf senescence is the bright hues that can be observed on trees and plants during Autumn. The brilliant burst of colour that precedes the browning of leaves is an indication of active metabolic changes that result in the recycling or redistribution of nutrients to other parts of the plant. Evidence indicates the primary purpose of senescence in plants is for mobilization and recycling, a phenomenon that has tremendous implications for crop growth and food production.

The use of a novel tobacco treatment process to reduce toxicant yields in cigarette smoke (2011) Liu, C., DesGrandpre, Y., Porter, A., Griffiths, A., McAdam, K., Voisine, R., Cote, F., Proctor, C. Full text: Food and Chemical Toxicology. 4 (9) 1904-1917  Opens new window

The US Institute of Medicine has encouraged the pursuit and development of potential reduced-exposure products (PREPs) – tobacco products that substantially reduce exposure to one or more tobacco toxicants and can reasonably be expected to reduce the risk of one or more specific diseases or other adverse health effects. One potential approach is to reduce levels of some smoke toxicant precursors, such as proteins and polyphenols, in tobacco. We describe a treatment process involving aqueous tobacco extraction and treatment with protease; filtration of the extract to remove peptides, amino acids and polyphenols, and recombination of extract and treated tobacco. The process reduced levels of protein nitrogen (59%), polyphenols (33–78%) and nicotine (12%) while sugars increased 16%.

Use of classical adsorption theory to understand the dynamic filtration of volatile toxicants in cigarette smoke by active carbons (2011) Branton, P., McAdam, K., Duke, M., Liu, C., Curle, M., Mola, M., Proctor, C., Bradley, R. Full text: Adsorption Science and Technology Opens new window

The ability of two very different active carbons, a polymer-derived carbon (with ultramicropores and supermicropores, and a large volume of “transport” pores) and a coconut shell-derived carbon (predominantly ultramicroporous), to reduce the levels of volatile toxicants in cigarette smoke has been measured and compared. The polymer-derived carbon was found to be approximately twice as effective in removing the majority of measured smoke vapour-phase toxicants compared to the coconut shell-derived carbon in three different cigarette formats and with two different smoking regimes. Single- component dynamic breakthrough experiments were conducted with benzene, acrylonitrile and 2-butanone at 298 K for beds of each carbon under dry (0% RH) and wet (60% RH) conditions. Longer breakthrough times were found with the polymer-derived carbon, and breakthrough times recorded under wet conditions were found to be up to 20% shorter than those obtained under dry conditions. Correlations between micropore volume, dynamic adsorption volume and filter bed breakthrough time have been demonstrated.

An inter-laboratory comparison of urinary 3-hydroxypropylmercapturic acid measurement demonstrates good reproducibility between laboratories (2011) Minet, E., Errington, G., Scherer, G., Newland, K., Sharifi, M., Bailey, B., McEwan, M., Cheung, F. BMC Research Notes, 4: 391 Full text: An inter-laboratory comparison of urinary 3-hydroxypropylmercapturic... Opens new window

Biomarkers have been used extensively in clinical studies to assess toxicant exposure in smokers and non-smokers and have recently been used in the evaluation of novel tobacco products. The urinary metabolite 3-HPMA, a metabolite of the major tobacco smoke toxicity contributor acrolein, is one example of a biomarker used to measure exposure to tobacco smoke. A number of laboratories have developed liquid chromatography with tandem mass spectrometry (LC-MS/MS) based methods to measure urinary 3-HPMA; however, it is unclear to what extent the data obtained by these different laboratories are comparable.