Feasibility of Breath Condensate Lipids & Eicosanoids as Non-invasively Collected Biomarker Predictors of Pulmonary Pathobiology

Dr. David White

1 Center for Biomarker Analysis, University of Tennessee, Knoxville, TN 37932; 2 CIIT Centers for Health Research, Research Triangle Park, NC 27709-2137

Co-investigators : Sung Chan Jo 1 , James M. Cantu 1 , Edilberto Bermudez 2 , and Owen R. Moss 2

Final Report (Last updated - June 2007)

Breath aerosols (BA) have been collected non-invasively from 24 healthy smoking and 27 age-matched non-smoking volunteers to explore patterns of phosphatidylcholine (PC) derived lipids, eicosanoids, and cytokine regulatory biomarkers as monitors of inflammation utilizing the EcoScreen ® device (White et al.,2005b). The protocol for BA collection was approved by the University of Tennessee and the volunteers were paid for the inconvenience of BA collection. The BA was fractionated into a cytokine fraction with Amicon filters utilizing dyed microspheres conjugated with monoclonal antibodies specific for each target cytokine in a multiplex assay (BioPlex®). Antibody-conjugated beads react with the concentrated breath condensate sample and a secondary, detection, antibody in a microtiter well to form a capture immunoassay read in an array reader. They showed differences but were at the limits of detection so detailed analysis was not possible. BA eicosanoids are then recovered from the solid phase extractor. A new derivatization procedure to increase electrospray ionization MS/MS efficiency was required to achieve parts per trillion sensitivity for eicosanoid fatty acids. Eicosanoids are derivatized as positively charged N-methyl-2-picolinyl esters. These derivatives are separated by HPLC and detected after electrospray ionization with tandem mass spectrometry as the progenitor ions generating diagnostic product ions. Initial analyses show a complex hitherto undetected complement of eicosanoids and their metabolic products in BA. The phospholipids lipids are recovered from the solid phase extraction and the phosphocholine (PC)-based lipids separated by high performance liquid chromatography (HPLC). Phosphocholine containing lipids are identified as progenitors of positive product ions at m/z 158 with tandem mass spectrometry. Sensitivity at pg/ml BA showed no detectable phosphatidylcholines of platelet activation factors in healthy smokers or non-smokers.

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

Dr. Gerhard Scherer

ABF Analytisch-Biologisches Forschungslabor GmbH, Munich , Germany

Co-investigators : Dr. Heinz-Werner Hagedorn, Dr. Michael Urban, Dinamis Janket, Gerhard Gilch, Dr. Johannes Engl, Kirsten Riedel, Astrid Urban

Final Report (Last updated - June 2007)

Charcoal (CC) filters of cigarettes are known to reduce a series of volatile constituents in mainstream smoke. Many of these volatiles are cytotoxic. It has been also shown that a major part of the cytotoxic properties of cigarette smoke is related to compounds able to react with sulfhydryl (SH) groups and thus deplete cellular glutathione (GSH).

In this project, we developed a simple, cell-free assay, which detects the SH-reactivity of the gas phase, particulate phase and whole mainstream smoke of cigarettes. We compared the SH-reactivity of 25 commercial cigarette brands (11 brands with conventional cellulose acetate (CA) filter tips, 14 brands with CC filter tips). CC filter tipped cigarettes showed generally lower SH-reactivities compared to CA filter tipped cigarettes with similar tar yields. The differences between CC and CA filter tipped cigarettes were most pronounced for the gas phase. Four pairs of CA/CC filter tipped cigarettes with similar tar and nicotine yields but significantly different SH-reactivities (different by 26 - 59 %) of the gas phase were selected for use in a brand-switching study with 39 human smokers. Twenty (20) subjects smoked CA filter tipped cigarettes during the first week of the study, the remaining 19 subjects smoked CC filter tipped during the first week. In the second week, the subjects switched to the corresponding brand with the other filter type. Saliva samples were collected on each of the 14 study days, 24-h-urine samples were collected on Days 3, 6, 10 and 13. On average, smoking behavior, as determined by the daily cigarette consumption and butt length, did not significantly change when switching to the cigarettes with the other filter type. In general, the same was true for the carbon monoxide level in exhaled air, salivary cotinine and trans-3'- hydroxycotinine as well as the molar sum of the six major nicotine metabolites in urine (nicotine, cotinine, trans-3'-hydroxycotinine and their respective glucuronides). Urinary excretion rates of 3-hydroxypropyl-mercapturic acid (metabolite of acrolein), 3-hydroxy1-1- methylpropyl-mercapturic acid (metabolite of crotonaldehyde), monohydroxybutenylmercapturic acid (metabolite of 1,3-butadiene) and S-phenyl-mercapturic acid (metabolite of benzene) were significantly lower when smoking CC compared to CA filter tipped filter cigarettes. Other mercapturic acids and thioethers (a biomarker indicating the exposure to electrophilic compounds) were not or only slightly reduced upon smoking CC filter tipped cigarettes.

From these results we conclude that CC filter tipped cigarettes exhibit a lower exposure to toxicological relevant SH-reactive compounds. This was evident both from the results of a simple, cell-free assay and an in vivo study with human smokers.

Simultaneous Determination of Apurinic Sites, Endogenous DNA Adducts and 5-Methylcytidine by Capillary Electrophoresis and Laser-induced-fluorescence Detection

Dr. Oliver Schmitz

University of Wuppertal , Germany

Final Report (Last updated - June 2007)

A new method was developed for constructing double-tapered capillaries for capillary electrochromatography (CEC) with an optical-fiber splicer and was used to test the separation of fluorescence-labeled nucleotides on various new and familiar phases. In order to couple the laser-induced-fluorescence detector with the CEC, our detector was outfitted with an external pressure source to reduce air bubbles in the capillary. The system could be used very well to analyze uncharged compounds. Unfortunately, however, the negative charge and the resulting hydrophilicity of the labeled DNA adducts led to poor separatory power with all the combinations of stationary and mobile phases we tested. For this reason, micellar electrokinetic chromatography (MEKC) was used instead of CEC in further work on the project.

In this project, syntheses were developed for 2'-deoxy-uridine-3'-monophosphate (dUMP), 2'-deoxy-thymine glycol-3'-monophosphate (dTgMP), 2'-deoxy-inosine-3'-monophosphate (dIMP) and 1,N 6 -etheno-2'-deoxyadenosine-3'-monophosphate (etheno-AMP). The products were thoroughly characterized for use as reference substances. After the separation parameters had been optimized, the method and the reference compounds were used for qualitative and quantitative determination of oxidative damage in mitochondrial DNA (mtDNA) from bovine liver and genomic DNA from patients with chronic lymphocytic leukemia (CLL).

Metabolism and DNA Adduct Formation of N '-nitrosonornicotine (NNN) in Rats: Interaction with Nicotinoids and other Tobacco Constituents

Dr. Wolfgang Zwickenpflug
Walther Straub Institute of Pharmacology and Toxicology, Munich , Germany

Co-investigators : Stefan Tyroller and Elmar Richter

Final Report (Last updated - June 2007)

N ? -nitrosonornicotine (NNN) was the first tobacco-specific nitrosamine (TSNA) identified as carcinogen in tobacco smoke, but no data exist on in vivo interactions between NNN and other tobacco alkaloids, TSNA or phenethyl isothiocyanate (PEITC) which have been demonstrated in various studies on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Acute effects on NNN metabolism were tested in male Fischer F344 rats injected s.c. with 30 nmol/kg body weight (bw) [5- 3 H]NNN either alone or simultaneously with 15 m mol/kg bw nicotine, nornicotine, anatabine, or anabasine, 150 m mol/kg bw cotinine, 3 m mol/kg bw myosmine, or 300 nmol/kg bw of either N ? -nitrosoanatabine or N ? -nitrosoanabasine. Another group of rats was fed a diet supplemented with PEITC at 1 m mol/g diet starting 24 h before NNN treatment. Within 24 h more than 80% and about 10% of the radioactivity was excreted with urine and feces, respectively. Urinary metabolites were separated by reversed-phase radio-HPLC and identified by co-chromatography with UV standards. In two sets of experiments with control rats treated with NNN only, 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxyl acid, 44.4/44.8%), 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 32.4/31.5%), NNN- N -oxide (5.0/3.8%), 4-(3-pyridyl)butane-1,4-diol (diol,1.1/1.0%) and norcotinine (2.3/1.0%) were consistently detected besides unmetabolised NNN (4.7/3.3%). Co-treatment with nicotine, cotinine, nornicotine and PEITC shifted the contribution of the two major metabolites significantly in favor of hydroxy acid (108-113% of control) as compared to keto acid (86-90% of control). The same treatments also increased norcotinine (135-170% of control). These changes are consistent with a decreased metabolic activation of NNN. In subacute studies rats received NNN in drinking water for 4 weeks at a daily dose of 30 nmol/kg bw with or without nornicotine at 15 m mol/kg bw or myosmine at 3 m mol/kg bw. On the last day of the experiment all rats received [5- 3 H]NNN at 30 nmol/kg bw with a contaminated apple bite followed by collection of urine and feces for 18 h. Most of the radioactivity, 87-96% of the dose, was recovered in urine and only minor amounts have been excreted in feces or persisted in blood. In urine of the NNN-control group keto acid (32.2%) and unmetabolised NNN (3.9%) were present in identical amounts as in the acute experiment whereas hydroxy acid (41.4% of total radioactivity in urine, 93% of acute NNN control) was reduced in expense of the minor NNN metabolites. Co-administration of nornicotine resulted in a small but significant rise of keto acid (107% of control) and a significant decrease in NNN- N -oxide (76% of control). After co-treatment with myosmine the increase of keto acid (104% of control) was even less but still significant whereas NNN- N -oxide and diol were significantly reduced to 72% and 79% of control, respectively.

Our experiments with rats indicate significant mutual effects of some of the major tobacco alkaloids and most relevant TSNA. Further studies on the impact on smokers and the inhibitory effects of isothiocyanates are needed for a final risk assessment.

Tobacco Smoke and Developmental Gene Expression

Dr. William Hanneman
Colorado State University, Ft. Collins, CO

Final Report (Last updated - June 2007)

The Surgeon General of the United States has identified the consumption of tobacco products as the number one cause of preventable disease in America . The list of those diseases includes cancer, heart disease, lung disease and birth defects. Understanding how tobacco products cause disease and addiction is one of the most important current medical research challenges.

Cigarette smoke contains over 4,000 known chemical compounds. Because of this, it's no wonder that exact cause of disease and addiction has never been determined. Some of theses known compounds have been previously shown to cause cancer in laboratory animals at very low doses. Moreover, at least one of the known compounds (nicotine) is associated with addiction to tobacco products. The fact that not all tobacco users develop cancer or heart disease is most likely due to the genetic diversity of the general human population. That is to say, for the same reason that each person looks, talks or acts differently, each person may respond differently to the complex mixture of chemicals in cigarette smoke or tobacco extract. This underlying difference between people is most often attributed to "their genetic make up" and is stored in DNA. Since the exact information (genes) contained in DNA is what differs between people, this information in part is what governs how a person responds to tobacco products and other poisons.