Dr. Beverly S. Cohen

Project Title: Pololnium-210 and lead-210 as biomarkers of actual dose of inhaled cigarette smoke

Nelson Institute of Environmental Medicine, New York University School of Medicine
(A portion of this project will be conducted on mainland China)

Grant Award: $400,000
Duration: July 2005 - June 2008

Abstract:
Evaluation of the risk of disease from cigarette smoking requires valid estimates of inhaled dose, for both current and past smoking. This is important because reductions in tar levels have not led to the subsequent reduction in mortality that was anticipated several decades ago. We hypothesize that direct estimates of the inhaled dose of cigarette smoke are available via Pb-210 and Po-210 concentrations measured in the urine of cigarette smokers. When a cigarette is smoked about 5 mBq of these radioactive elements are volatilized per gram of tobacco. Some is inhaled in the smoke particles and deposited in the lungs. They are subsequently distributed to various body compartments, where some are sequestered (e.g. Pb in bone) and some excreted. Bone and lung concentrations of Pb-210 in smokers, and some former smokers, are about double that in non-smokers (Holtzman 1966, Cohen 1979, NCRP 1987). There is clear evidence that both of these radioactive isotopes are elevated in the urine of cigarette smokers as compared with those in non-smokers (Azerado and Lipsztein 1991, Santos 1994). As yet no one has examined whether the amount of Po-210 and Pb-210 excreted in urine correlates with the intensity of smoking. The complex relationship between metabolic pathways and radiological half-lives of Pb-210 and its offspring Po-210 present an opportunity for assessing inhaled dose of cigarette smoke and for distinguishing between inhaled dose from current and long term cigarette smoking. We propose to establish dose-response relationships between surrogate indicators of exposure (e.g. smoking history) and measured concentrations of Po-210 and Pb-210, and then construct a mathematical model that uses urinary excretion data to estimate the dose of inhaled cigarette smoke. Evaluation of urinary concentrations would then allow direct estimate of inhaled dose regardless of individual smoking patterns, which are known to vary when a smoker is presented with cigarettes designed to reduce inhaled tar.

Dr. Heidi Foth

Project Title: DNA repair activity in human lung tissue as a possible marker for individual response to tobacco smoke constituents

Institute for Environmental Toxicology (IUT), Martin Luther University, Germany

Grant Award: $70,000
Duration: July 2005 - June 2006

Abstract:
Epidemiological data indicate that tumor development by tobacco smoke cannot be explained only by data on exposure (dose-effect relationship). There is still a considerable lack of information on the mechanisms of toxicity by tobacco smoke constituents with respect to the importance of DNA repair in human lung. Other cooperating factors which are pertinent to the individuum must exist and are decisive. Lung tissue from smokers should contain markers which help to understand why only a fraction of smokers develop cancer. The experimental design of this seed project aims to clarify whether cell culture from normal, perhaps already pre-transformed lung tissue of smokers reflect the functional activity of DNA repair. Poly(ADP)ribosylation of nuclear protein (PARP activity) is an early key event within of other important factors. Distinct samples from the upper and lower respiratory tract will be isolated from lung cancer patients when they undergo lung resection. Stressors in cigarette smoke initiate aberrant expression of cancer related genes in lung cancer patients The putative role of triggers for impaired DNA repair should be clarified in in-vitro models of lung tissue obtained from smokers (cancer and non cancer cases). Long term cultures from human lung should be proven as models for control of differentiation, apoptosis and anti-apoptosis. In particular, transfection assays may help to understand the complex interplay of gene expression and activity of gene products related to tumor formation, DNA repair and longevity of cells.

Dr. Stefan Tyroller

Project Title: 3'-Hydroxymyosmine as specific urinary biomarker for myosmine exposure

Walther Straub-Institute of Pharmacology and Toxicology (WSI), Germany

Grant Award: $70,000
Duration: May 2005 - April 2005

Abstract:
The risk of cancer after tobacco consumption has been associated with metabolic activation of TSNA derived from major tobacco alkaloids nicotine and nornicotine, namely NNN ( N' -nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The minor tobacco alkaloid myosmine has been proposed as a marker of environmental tobacco smoke (ETS) exposure but its toxicological impact has been neglected for a long time. Studies on the occurrence of myosmine demonstrated not only its presence in tobacco and tobacco smoke but also its widespread appearance in various foods. Therefore, myosmine exposure is no longer limited to the consumption of tobacco or tobacco products. Myosmine is easily nitrosated yielding NNN and to a much higher extent HPB (4-hydroxy-1-(3-pyridyl)-1-butanone) without participation of any metabolic activation. In contrast, both NNN and NNK require metabolic activation forming an instable intermediate with the potency to generate a common hemoglobin and DNA adduct releasing HPB upon hydrolysis. Major urinary metabolites after p.o. administration of myosmine to rats are HPB and keto acid (4-(3-pyridyl)-4-oxobutanoic acid). Covalent DNA binding has been shown for myosmine under nitrosation conditions in vitro and HPB-releasing DNA adducts were increased in different rat tissues including esophagus after p.o. administration of myosmine. HPB-releasing DNA adduct levels of human esophagus and cardia did not correlate with the individual smoking status but a strong correlation was found with increasing body mass index. Therefore, dietary myosmine could be mainly responsible for these adducts and not TSNA and myosmine from tobacco. In the course of studies on myosmine metabolism in rats 3'-hydroxymyosmine has been identified as urinary metabolite. This compound results exclusively from myosmine and is consequently suitable as a specific biomarker for myosmine exposure. Recording and quantification of urinary 3'-hydroxymyosmine could help to estimate the total amount of incorporated myosmine. In combination with specific nicotinic biomarkers like urinary cotinine we might be able to differentiate the origin of incorporated myosmine. The results will allow a better risk assessment of myosmine exposure through tobacco and tobacco smoke or diet.

Dr. Gerhard Scherer

Project Title: Validation of biomarkers for the smoking-related airway inflammation

Analytisch-biologisches Forschungslabor (ABF), Germany

Grant Award: $209,700
Duration: April 2005 - October 2006

Abstract:
Smoking is causally related to major chronic diseases such as cancer, cardiovascular diseases and chronic obstructive lung disease. Although the pathological mechanisms by which smoking can induce these diseases is not completely understood, chronic irritation and inflammation provoked by smoking might be a common early event in the pathogenesis of these diseases. A tissue response to irritation and inflammation is the formation of reactive oxygen species (ROS) such as hydroxyl radicals (.OH) and hydrogen peroxide (H 2 O 2 ) as well as nitrogen species (RNS) such as nitrogen oxide (NO) and peroxynitrite (ONOO - ) which can react with cellular components like DNA, RNA, lipids and proteins. In addition, a number of cellular reaction cascades are initiated by the inflammation process giving rise to changes in various cellular components such as cytokines and leukotrienes. The reaction products and cellular components can be used as biomarkers of effect for inflammatory processes.

For obvious reasons, inflammation processes in the lung are of primary interest as a source for biomarkers of early biological effects induced by smoking. Exhaled breath condensate (EBC), which can be collected non-invasively by breathing into a cooling system for 10 - 15 min, has increasingly been used in the last 5 years for lung diagnostic purposes. Reports from the literature indicate that smokers might exhibit increased levels of inflammation biomarkers compared to non-smokers.

In the first step of this project, analytical methods for known and new biomarkers of inflammation will be established and validated. These biomarkers will then be applied in several studies with human subjects. It is expected that a series of inflammation-related biomarkers in EBC can be identified, which show clear relationships to the smoking dose.

Dr. Thorsten Streibel

Project Title: Characterization of Selected Hoffmann Analytes in Smoke before and after Inhalation in real time (Mouthspace), Main- and Side-stream Smoke as well as Tobacco Pyrolysis Gases with sub Puff Time Resolution Using Laser Mass Spectrometry Methods based on Soft Ionization Techniques

GSF Research Center, Ins. for Ecological Chemistry, Neuherberg, Germany

Grant Award: $145,000
Duration: July 2005 - June 2007

Abstract:
The objective of the proposed work is to develop and demonstrate a methodology for a rational approach for determination of the exposure of human smokers with smoke components believed to be related to smoking-related diseases (Hoffmann analytes). The exposure of human smokers with some selected Hoffmann analytes during smoking will be investigated and compared with results obtained from machine smoking experiments. The amounts of selected Hoffmann analytes in the mouth before and after inhalation will be determined. The influence of cigarette type (low-tar and full flavour) and smoker type (heavy, medium and social smoker etc.) on the human smoking profile will be studied. Furthermore the time resolved formation and release of selected Hoffmann analytes from pyrolysis experiments and smoking machine experiments (side stream smoke and main stream smoke in sub puff resolution) will be investigated. A newly developed on-line real-time monitoring technology (REMPI/SPI-TOFMS) which allows characterizing organic compounds from tobacco smoke with 100 ms time resolution and faster will be applied for this purpose.

Dr. Kelly A. BéruBé

Project Title: Identification of intelligent biomarkers of exposure and harm in the respiratory epithelia to tobacco smoke components

Cardiff University, Wales UK

Grant Award: $420,880
Duration: June 2005 - May 2008

Abstract:
A novel toxicological tool, which consists of a differentiated, 3-D, in vitro model of human respiratory epithelia, i.e. EpiAirway-100 cells (MatTek Corp., USA), will be utilized to examine the early gene response(s) following exposure to tobacco smoke components (TSC). EpiAirway-100 cells will be exposed at the air/liquid interface to representative particle and vapor phase components of cigarette smoke. The TSC selected represent an overview of the compounds found in cigarette smoke that induce thrombogenic events (e.g. nicotine), cell toxicity (e.g. cadmium) and produce reactive metabolites during xenobiotic metabolism (e.g. formaldehyde and urethane). Surrogate solutions of TSC will be tested for their capacity to up- and/or down-regulate genes in the respiratory epithelia following acute exposure, as a means to identifying "intelligent biomarkers" of exposure and harm.

Conventional toxicological analysis will be used, at the first instance, to establish the dose of the various TSC needed to cause changes in epithelial resistance, secreted surface proteins and release of inflammatory markers. It will be imperative that the final working doses used are subtoxic, since the objective is to identify the important biomarkers involved with toxicant stress and avoid studying dead or dying cells. Following the establishment of the dose required to achieve these different biological endpoints, the toxicogenomic investigations will be initiated.

Toxicogenomic experiments will be designed to identify early molecular markers for events in pulmonary injury and thus, it will be necessary to optimize the exposure time to the TSC to be between 1 and 24 hours. Macroarray technology will be employed to compare the patterns of mRNA expression of human genes associated with stress, simultaneously from control and TSC treated lung tissue. The major candidate genes will be classified (e.g. growth factors, inflammation, and xenobiotic metabolism) and associated with the biological endpoints. It is anticipated that stringent lists of candidate genes associated with these changes will be generated, thereby providing data on the mechanisms of the biological endpoints, i.e. intelligent biomarkers.

Finally, mRNA levels do not necessarily predict the levels of the corresponding proteins in a cell. Consequently, proteomic analysis will be used to correlate candidate gene response with a specific protein. The interest will be to study the proteins involved in the early stress phase of toxicant challenge. Once protein function has been identified, this will provide insight into the mechanism of action of the responsive genes and hence, the "intelligent biomarkers" of TSC exposure in lung epithelia.

Dr. Stephen Rennard

Project Title: Validation of biomarkers of exposure and host response

University of Nebraska Medical Center

Grant Award: $190,000
Duration: June 2005 - May 2007

Abstract:
The proposed study will evaluate biomarkers measured in exhaled breath condensate (EBC) for the purpose of assessing clinical strategies of harm reduction. It will take advantage of a recently developed device that permits collection of exhaled breath condensate reproducibly, with minimal subject effort and with no oral contamination. The major goal of the trial is to provide evidence to validate biomarkers in EBC. This will be accomplished by collecting samples from asymptomatic smokers before and after inducing a change in their smoking habit (cessation or reduction) with the aid of partial nicotine replacement.

Measures to be made in EBC include H2O2, the most widely explored biomarker in this "body" fluid. Methods that reliably can quantify levels in normal non-smokers and in asymptomatic smokers will be used. Two fold increases in smokers have been reported by in several reports and confirmed in preliminary data by the investigators. In addition, other biomarkers of oxidant stress: TBARs, 8-isoprostane and nitrotyrosine will be quantified using standard methods.