Identification of Intelligent Biomarkers of Exposure and Harm in the Respiratory Epithelia to Tobacco Smoke Components

Dr. Kelly A. BéruBé

Cardiff University , Wales UK

Co-investigators : Dominique Balharry, Keith Sexton, Tim Jones, Roy Richards

Final Report (Last updated - September 2009)

A novel toxicological tool, which consisted of a differentiated, 3-dimensional, in vitro model of human respiratory epithelia, EpiAirway-100 cells (MatTek Corp., USA), was utilized to identify intelligent biomarkers of exposure in human respiratory epithelia following exposure to selected particle and vapor phase tobacco smoke (TS) components. This approach provided a holistic approach towards the identification of biomarkers in the pulmonary epithelium. A range of TSC doses were used to elicit a classic dose response which was characterized by conventional toxicology techniques including transepithelial electrical resistance (TEER), cell viability (MTT) and protein assay. All four selected TS components appeared to induce similar stress responses (e.g. protective mechanisms) within the model system. Histological examination at the light microscopy level was utilized to investigate the morphological changes in cellular-tissue organization in the EpiAirway tissue model, following exposure challenges with the TS components. In conjunction, targeted immunohistochemistry assays were performed in order to better characterize the tissue response at the cellular level in basal, ciliated, intermediate, and Goblet cells. Three discrete zones of tissue injury and repair were delineated; (1) the apical region, (2) the suprabasal region, and, (3) the basal zone (where basal cells acted as progenitors or stem cells).

Validation of Biomarkers for the Smoking-related Airway Inflammation

Dr. Gerhard Scherer

ABF Analytisch-Biologisches Forschungslabor GmbH, Munich , Germany

Final Report ( Last updated - June 2007)

Background : Exhaled breath condensate (EBC) can be easily and non-invasively collected. Since EBC contains non-volatile and semi-volatile constituents from the epithelial lining fluid (ELF), it has high potential for investigating non-invasively molecular changes in the respiratory tract. Inflammation markers in EBC are determined for clinical purposes in patients with asthma, COPD, fibrosis and other lung diseases. The effect of tobacco smoking on EBC biomarkers has been also investigated with up to now controversial results.

Objectives : The purpose of our investigation was to establish, validate and apply methods for collecting EBC and measuring EBC biomarkers in order to determine the applicability of this technique for the evaluation of PREPs.

Methods : Two EBC sampling devices (ECoScreen ® and RTube T ) were evaluated. Analytical methods for the following EBC constituents were established and validated: Nitrite, nitrate, sulfate (ion chromatography); Na+, K+, Ca2+, Mg2+ (ion chromatography); urea (GC-MS after derivatization); a -amylase (EnzChek amylase kit); 3-nitrotyrosine (LC-MS/MS after derivatization); aldehydes (GC-MS after derivatization); total proteins (Bradford assay); interleukine-6 (EIA); tumor necrosis factor a (EIA); adenosine (LC-MS/MS), 8-isoprostane and leukotriene B4 (GC-MS). Cadmium in EBC was determined in the Institute of Occupational Medicine of the University of Munich . NOex was determined with an electrochemical sensor integrated in a hand-held instrument. Most of the EBC markers were applied semi-controlled pilot study with 12 volunteers (6 nonsmokers, 6 smokers). During the study, 11 EBC samples were collected from each subject. The tobacco smoke exposure dose was assessed by measuring COex as well as cotinine and trans-3'-hydroxycotinine in saliva.

Results : The ECoScreen device yielded by about 30 % higher EBC volumes (2 - 3 ml/15 min) and also higher EBC marker concentrations compared to the RTube system. For practical reasons (multiple systems, disposable part, easier to clean), the RTube device was used in the pilot study. All EBC markers measured in the pilot study showed high (to extremely high) intra - and inter -individual variations. In particular, urea, the cations and sulfate, which were intended to be used as EBC dilution markers possessed high variability, precluding their application for this purpose. In the very low level range of EBC markers, there was significant interference from background contamination. No effect of smoking was observed for the EBC inflammation markers adenosine, IL-6 and TNF- a . Nitrite and NOex were inversely associated with smoking, whereas nitrate was not affected by smoking. 3-Nitrotyrosine tended to be elevated in smokers and showed a week association with the smoking dose as determined by COex, salivary cotinine and trans-3'-hydroxycotinine as well as daily cigarette consumption. Acrolein, crotonaldehyde and hexanal were significantly higher in EBC of smokers compared to nonsmokers. These aldehydes significantly correlated with the smoking dose. The effect of smoking was weaker and not significant for heptanal and malondialdehyde and almost absent for nonanal. Cadmium tended to be elevated in EBC of smokers and there was also a weak association with the smoking dose.

Conclusions : Aldehydes (in particular crotonaldehyde) and 3-nitrotyrosine show some potential for biomarkers of smoking-related effects in the lung. NOex is a suitable medium- to long-term biomarker for smoking related changes in the respiratory tract which can easily and reliably be measured. EBC technology requires further improvements in terms of contaminant-free sampling and determination at the extremely low biomarker levels before it can be generally applied for PREP testing.

Measurement and Assessment of Polonium 210 and Lead 210 as Biomarkers of Actual Dose of Inhaled Cigarette Smoke

Dr. Beverly Cohen

New York University School of Medicine, Tuxedo, New York

Co-investigators : Qingsham Qu, Naomi Harley

Final Report ( Last updated - October 2009)

Although cigarette smoking continues to occur worldwide, there are few methods available to assess a person's retrospective exposure to mainstream smoke. The tobacco of cigarettes contains trace quantities of 210Pb and 210Po, which are volatilized and inhaled when a cigarette is smoked. It was hypothesized that urinary 210Pb and 210Po activity concentrations could be used as biomarkers of smoking intensity.

Human subjects (n=250) were recruited from Beijing , China and reported their smoking habits. Each subject provided a 24-hour urine sample, which was assayed for its 210Pb and 210Po activity concentrations. Although the urinary 210Po activity from smoking was very low compared to background levels, the urinary 210Pb activity correlated with the number of cigarettes smoked per day (CPD, ?=0.38, p<0.001) and the urinary cotinine concentration (?=0.52, p<0.001). In a linear regression model, a 1-unit increase in CPD was associated with an increase of 0.13 mBq in urinary 210Pb activity. In a logistic regression model, a 1-unit increase in urinary 210Pb activity was associated with an estimated 25% increase in the odds of being a smoker.

DNA Repair Activity in Human Lung Tissue as a Possible Marker for Individual Response to Tobacco Smoke Constituents

Dr. Heidi Foth

Martin Luther University , Halle/Saale, Germany

Co-Investigators : Abdel-Rahman Torky, Felix Glahn

Final Report ( Last updated - June 2007)

Poly(ADP-ribose)polymerase -1 (PARP-1), plays a primary role in the process of poly(ADP-ribosy)lation mainly via its activation in response to DNA damage reflecting the severity of genotoxic stress.

We have examined PARP activity in primary human lung cells under exposure to heavy metals, cigarette smoke condensate (CSC) and prolonged cultivation.

Normal human bronchial epithelial (NHBEC) and peripheral lung cells (PLC) from lung cancer patients were grown as explant cultures and were seeded on glass cover-slides. PARP-1 expression was proved by Western Blotting in (NHBEC) and tumor lung cell A549.

To study PARP-1 activity in NHBEC a functional assay has been used. Induction of DNA damage by H 2 O 2 (100 µM) in NHBEC led to formation of ADP-ribose polymers which were detected by immunofluorescence. Copper(II) (0.05 mM - 24h) did not trigger PARP-1 activity in NHBEC but decreased PARP-1 activity induced by H 2 O 2 (100 µM). Similarly mercury (II) (0.03 mM-24h) exerted no effect on PARP-1 activity and decreased the activity induced by H 2 O 2 (0.1 mM). Treatment of NHBEC with CSC (0.5 mg/L) for 24h induced PARP-1 activity by factor 1.3 compared to the control (basal cellular PARP activity) and increased H 2 O 2 (100 µM) -induced PARP-1 activity from 1.4 fold to 1.8 fold.

We have analyzed PARP-1 activity in cultures of lung cells for 10 weeks. In higher passages and generations H 2 O 2 -induced PARP activity decreased, this reflects an adaptation of cells to cultivation. The expression of PARP-1 was almost the same in different generations of the same patient. This means that PARP-1 expression is not affected by prolonged cultivation and is stable.

Validation of Biomarkers of Exposure and Host Response

Dr. Stephen Rennard

University of Nebraska Medical Center, Omaha, NE

Final Report (Last Updated - January 2008)

This study evaluated the feasibility, reliability and validity of exhaled breath condensate (EBC) as means to assess toxin exposure. These goals were approached by measuring selected markers in EBC believed related to the pathogenesis of lung disease before and after a smoking cessation intervention. The markers were to be evaluated in four ways. First, multiple measures made before and after cessation was performed to permit evaluation of the reproducibility of the markers. Second, the sensitivity of the markers to change was assessed by evaluating the changes associated with smoking cessation. Third, the markers were to be validated by relating them to a suite of previously validated biochemical and clinical measures. Finally, in order to assess the potential of the markers in evaluating harm reduction strategies, individuals who do not quit smoking, but who are likely to achieve varying degrees of reduction, were also be evaluated. This was to permit an estimate of the usefulness of the measures as measures in harm reduction. This study will, therefore, establish the feasibility and validity of EBC biomarkers for use in studies designed to evaluate harm reduction strategies.

The study was a prospective, open-label, clinical trial. After consent and screening, all individuals underwent a battery of initial evaluations. They were then treated with nicotine replacement therapy and attempted cessation. Failing cessation, subjects attempted to reduce their smoking as much as possible. All individuals were reassessed at regular intervals both during and after discontinuation of nicotine replacement therapy.

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

Dr. Thorsten Streibel

University of Augsburg , Augsburg , Germany

Final Report (Last updated - January 2008)

In this study the general applicability of online photo-ionisation mass spectrometry for various tobacco related matrices has been demonstrated, going all the way from pure tobacco analyses by means of thermal treatment and subsequent analysis of the evolved off-gases via machine mainstream measurements using different puff parameter protocols and human breath analysis to the investigation of the inhaled smoke of actual human smokers. By doing this, a bridge is spanned between exemplary approaches such as thermal desorption and pyrolysis of tobacco, standardised machine smoking of research cigarettes and real life smoking of commercially available cigarettes utilizing the same measurement principle for the investigation of toxic compounds in matrices such as pyrolysis off gases, smoke and breath.

For the thermal desorption/pyrolysis experiments, three different tobacco samples (Burley, Virginia, and Oriental) were thermally desorbed at varying temperatures (190, 250, and 310 °C) and the evolved gas phase at every temperature step has been analyzed applying single photon ionisation (SPI) - time of flight mass spectrometry (TOFMS) and resonance enhanced multiphoton ionisation (REMPI) - time of flight mass spectrometry. Many oxygen containing compounds could be detected such as phenol, hydroquinone, and (vinyl)guiacol along with unsaturated hydrocarbons and nicotine. In addition, with REMPI a large variety of PAH are accessible. Different tobacco types yield slightly distinguished mass spectra. By utilizing statistical tools (Fisher criterion and Principle Component Analysis) the three tobaccos can be discerned rather rapidly from each other and possible marker compounds for a given tobacco can be identified.

The use of SPI-TOFMS for tobacco smoke analysis could be demonstrated for mainstream and sidestream smoke, where a puff-by-puff analysis is possible for a broad range of health-related compounds. Advancing to a increased time resolution showed no significant differences in the concentration profiles of single substances, thus sticking to puff resolved analysis is sufficient. Usually the mainstream and sidestream yields of such compounds are determined by conventional off-line methods by smoking the cigarettes under standard ISO conditions. In general, SPI is very well suited for the analysis of several low volatile compounds, such as aldehydes, ketones, alkenes, and amines, as well as several other compound classes. In a first step towards the investigation of human smoking behaviour, different, more intense, machine smoking conditions were used to examine the influence of filter ventilation hole blocking, puff duration and puff volume on the chemical composition of mainstream tobacco smoke on a puff-by-puff basis. Throughout the studies it was discovered that puff parameters such as puff volume and frequency seem to have significant influence on the yields of certain compounds and compound-classes. However, this study can only represent a preliminary step, as a much larger scale investigation has to be carried out.

During the investigation of sidestream smoke new details about the dynamics of sidestream smoke formation were revealed. It could be demonstrated that the emissions have a distinguished puff behaviour which also is related to mainstream smoke in case of the post-puff emissions. As already shown, the use of different puff parameters has a vast influence on the chemical composition of mainstream smoke. Therefore, especially considering the role of sidestream smoke and environmental tobacco smoke (ETS) and its possible health effects on second-hand smokers, a further analysis of sidestream composition changes with different smoking behaviour will be of interest.

As a brief intermediate step from machine smoking to human smoking, human breath analysis has been carried out to detect differences in the breath of a smoker and a non-smoker, respectively. Several well known biomarkers for smoke could thereby be identified in the smoker's breath.

Since in this work the first puff by puff resolved measurements of organic compounds in inhaled smoke of human smokers in real time were performed, special emphasis had to be placed on the experimental setup of the system. As a consequence, only a limited number of actual measurements could be carried out. Nevertheless, the feasibility of the method was proven and, despite it is very hard to make any statistically secured conclusions, some trends became clearly evident. Moreover, the first puff resolved quantified yields of benzene and toluene in inhaled human smoke could be achieved.

It could be shown that the ISO standard conditions for smoking of cigarettes are not realistic for nowadays smokers. This may be caused by the change in cigarettes, as current cigarettes have other paper, better filters and restricted maximum tar yields, thus they are not as harsh as the cigarettes 30 years ago. The ISO machine conditions were chosen in 1936 arbitrarily. Studies of this time showed a puff volume between 27 and 61 mL. But at that time a non-filter cigarette with a much higher yield of tar and nicotine was common, what is antiquated today. Other standards such as the Canadian intense smoking regime with increased puff volumes and frequencies seem to be closer to real smoking conditions.

Furthermore, it could be seen that the contents of cigarette smoke change significantly with the individual puffing behaviour of the smoker. Substance yields in inhaled smoke depend strongly on the flow rate and volume of each puff.

However, to go into more detail, it is necessary to conduct much more experiments with a larger number of human smokers and cigarette types in the future, since the here presented experiments featuring two subjects are clearly too few to reach general conclusions. Nevertheless, the presented measurement principle would provide a valuable tool fur such investigations. In doing so, an important point would be the refinement and optimisation of the flow measurement for the human smokers. Furthermore, implementation of an automatic purge gas mechanism with a special valve would be a great advancement towards a more realistic smoking environment. Finally, the application of electron beam pumped excimer rare gas lamps (EBEL) as alternative beam sources for the generation of VUV light for SPI-TOFMS could enhance the setup with respect to compactness and simplification of the experimental setup.

Measurements of this work do not cover information of the retention of substances in the human body, since it was concentrated on the investigation of inhaled smoke. Hence, the experimental system should be extended to enable monitoring of the substance concentrations in the exhaled volume with the final objective to carry out quantified puff resolved retention measurements of toxic species. This was achieved until today only without puff by puff resolution in offline methods. Further research with this technique could make human smoking behaviour and the chemistry as well as the intake of hazardous substances connected to it better understood.

3'-Hydroxymyosmine as Specific Urinary Biomarker for Myosmine Exposure

Dr. Stefan Tyroller

Walther Straub Institute of Pharmacology and Toxicology, Munich , Germany

Final Report ( Last updated - June 2007)