Institute For Science And Health

Tobacco Science and Health Program Initiative - Projects from RFA 2002-A

Dr. William Hanneman

Project Title: Tobacco Smoke and developmental gene expression

Colorado State University, Ft. Collins, Colorado

Grant Award: $200,000
Duration: August 2003 - August 2005

Abstract:
Human exposure to chemicals is typically in the form of complex mixtures rather than as purified chemicals in isolation. Cigarette smoke is a prime example of this. It is comprised of nearly 3500 chemicals and is thought to contribute to approximately 40% of all cancers in the United States. In addition to the carcinogenic effects of tobacco there am unquestionably fetal effects of exposure to cigarette smoke. These effects include decreases in human fetal birth weight, intrauterine growth retardation, premature delivery, perinatal mortality, spontaneous abortion and fetal malformations including cleft palate. Exposure of children to cigarette smoke is also associated with increased rates of sudden infant death syndrome and respiratory illness including asthma. While all of the above health effects are well documented little is known about what genes regulate these pathologies. To address this problem, the goal of the study proposed here is to identify novel genes that are deregulated by exposure to Cigarette smoke condensates (CSC) using a relatively new technology called gene trapping.

Dr. Gerhard Scherer

Project Title: Influence of charcoal filters of cigarettes on a) the SH-reactivity of smoke and b) the urinary excretion of thioethers and mercapturic acids in smokers.

Analytisch-biologisches, Forschungslabor (ABF GmbH), Muenchen, Germany

Grant Award: $254,363
Duration: December 2003 - November 2004

Abstract:
Tobacco smoke contains electrophilic compounds which may react with cellular nucleophiles such as proteins, DNA, RNA and glutathione (GSH). Reaction with GSH is usually a detoxification mechanism and leads to the formation of mercapturic acids which are excreted in the urine. Earlier studies of the Leuchtenbergers have shown that SH-reactivity of tobacco smoke is mainly located in the gas phase and is strongly correlated with the cytotoxicity of the smoke. A direct or indirect role of inhaled cytotoxic (and ciliatoxic) compounds in the induction of smoking-related diseases such as COPD and also lung cancer must be assumed.

It has been shown earlier that charcoal filters of cigarettes may selectively decrease certain volatile agents from the smoke, some of which are irritating and/or ciliatoxic such as hydrogen cyanide, formaldehyde and acrolein. It has also been demonstrated in in vitro studies that smoke from cigarettes with charcoal filters is less cyto- and ciliatoxic than smoke from other filter cigarettes.

It is the aim of this study to compare the SH-reactivity of cigarettes with charcoal and cellulose acetate filters with a suitable in vitro assay. In the second part of this study, these cigarettes are smoked by smokers in a brand-switching study and suitable biomarkers, in particular thioethers and mercapturic acids, are determined. The cigarettes used in the switching study should be similar in their tar and nicotine yields.

Dr. Oliver Schmitz

Project Title: Simultaneous determination of apurinic sites, endogenous DNA adducts and 5-methyl-cytidine by capillary electrochromatography and laser-induced-fluorescence detection

Department of Analytical Chemistry, University of Wuppertal, Wuppertal, Germany

Grant Award: $67,609
Duration: December 2003 - November 2004

Abstract:
We have developed a DNA-modification analysis that involves the hydrolysis of DNA, fluorescence labeling of modified and unmodified nucleotides (as well as 2´-desoxyribose-3´-phosphate), micellar electrokinetic chromatography (MEKC) and laser-induced-fluorescence detection (CE-LIF) with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s -indacene-3-propionyl ethylenediamine (BODIPY â FL EDA) as fluorescence marker. So far we have explored the application to the detection of 5-methylcytidine and apurinic sites and to the determination of a number of endogenous and exogenous DNA adducts in oligonucleotides, calf-thymus DNA, human colon cells or human DNA (Figure 1). For the determination of the genomic methylation level in in vivo samples we need only 100 ng DNA, and for the analysis of apurinic sites 1 µg DNA is necessary. The limit of detection for exogenous DNA adducts in 10 µg DNA is 1 adduct in 10 6 to 10 7 unmodified nucleotides, so that the method is suitable for in vitro samples.

Dr. David White

Project Title: Feasibility of breath condensate lipids & eicosanoids as biomarker predictors of exposure and harm

Center for Biomarker Analysis, University of Tennessee, Knoxville, TN

Grant Award: $63,678
Duration: November 2003 - May 2005

Abstract:
Breath condensates collected from healthy smoking volunteers will be used to explore patterns of phosphatidylcholine (PC) derived lipids and eicosenoids. The critical components will be identified and used to compare pattern differences to matched non-smoking control volunteers. Breath condensate will be collected within an hour following cigarette smoking by breathing ambient air through a disposable mask attached to a piece of tubing cooled to 0 ° C for 15 -20 minutes or using ECoScreen ® , a non-invasive breath collecting system. The lipids extracted from breath condensate will be separated into polar lipids (mostly PC, lyso-PCs), platelet activation factors and eicosanoids. The polar lipids will be analyzed via HPLC/electrospray/tandem mass spectrometry. The eicosanoids will be derivatized to electron-withdrawing pentafluorobenzyl trimethyl silyl derivatives for separation by gas chromatography and analyzed by negative ion mass spectometry.

We expect, like asthma, that there are individuals whose lungs are hypersensitive to the stress of smoking, which should be reflected in the lipid/eicosanoid profile. Target eicosenoids include prostaglandins, (PGE 2 , PGD 2 , PCF 2a ) , thromboxane (TxB 2 ), and prostacyclins (6-Keto PGF 1a ), as they show differential responses to inflammation. Additionally, the leukotrienes (LTB 4 , LTC 4 , LTD 4 , LTE 4 ) show responses related to allergy, isoprostanes (8-iso PGF 2a ) are responsive to free radical oxidative stress and HETEs show differential lipo-oxygenase activities. Each of these compounds could play a significant role in establishing a lipid/eicosanoid profile that could be used to monitor the effects of smoking.

In this feasibility study we will correlate medical history of these healthy, smoking subjects to smoking subjects with a history of wheezing, pneumonias, or other manifestations of pulmonary hypersensitivity and peridontitis.

Dr. Wolfgang Zwickenpflug

Project Title: Metabolism and DNA adduct formation of N '-nitrosonornicotine (NNN) in rats: Interaction with nicotinoids and other tobacco constituents

Walther Straub Institute of Pharmacology and Toxicology, Munich, Germany

Grant Award: $81,200
Duration: February 2004 - June 2005

Abstract:
For a realistic health risk assessment of NNN in tobacco the simultaneous uptake from relevant TSNA and nicotinoids must be taken into account. Inhibition of metabolic activation of NNK and NNAL by nicotinoids and tobacco smoke exposure has been demonstrated both in vivo and in vitro. No such detailed data are available for NNN. Compared to NNK, the metabolism and adduct formation by NNN is less well investigated. Today, no specific biomarker is available for human NNN exposure. Using highly sensitive radio-HPLC methods the influence of nicotinoids and TSNA on urinary metabolite pattern of NNN at realistic low concentrations will be determined. After long-term exposure the relative contribution of NNN, NNK and myosmine to HPB-releasing adducts in hemoglobin DNA from target tissues could be investigated. The results will give a better insight in NNN metabolism and could help to find a specific biomarker. They will also be the basis for testing the impact of cigarette smoke exposure from new safer cigarettes under development.