untitled - dlr

NANOPARTICLE WORKSHOP

COMBUSTION GENERATED NANOPARTICLES AND THEIR HEALTH EFFECTS:

MOLECULAR AND CELLULAR BASICS

1-4 May 2005 Bonn, Germany

PROGRAM AND ABSTRACTS

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Location of the Nanoparticle Workshop:

Gustav-Stresemann-Institut e.V. (GSI) Europäische Tagungs- und Bildungsstätte Bonn Langer Grabenweg 68 53175 Bonn - Bad Godesberg

Germany

Phone: +49 (0) 228 / 8107-0 Fax: +49 (0) 228 / 8107-197

Organizing Committee:

Co-Directors:

Dr. med. vet. Christine E. Hellweg Deutsches Zentrum für Luft- und Raumfahrt (DLR) Institut für Luft und Raumfahrtmedizin Linder Höhe 51170 Köln Germany Phone: +49 (0) 2203 / 601-3243 Fax: +49 (0) 2203 / 61970

Dr. rer. nat. Christa Baumstark-Khan Deutsches Zentrum für Luft- und Raumfahrt (DLR) Institut für Luft und Raumfahrtmedizin Linder Höhe 51170 Köln Germany Phone: +49 (0) 2203 / 601-3140 Fax: +49 (0) 2203 / 61970

Dr. rer. nat. Horst-Henning Grotheer Deutsches Zentrum für Luft- und Raumfahrt (DLR) Institut für Verbrennungstechnik, Stuttgart Pfaffenwaldring 38-40 70569 Stuttgart Germany Phone: +49 (0) 711 / 6862-378 Fax: +49 (0) 711 / 6862-578

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Welcome Welcome to Bonn for the Nanoparticle Workshop on Combustion Generated Nanoparticles and their Health Effects: Molecular and Cellular Basics. The workshop will provide the platform for (i) learning about the current state of knowledge and the most up-to date research in the fundamental processes that result from interaction of combustion generated nanoparticles with living matter, and the necessary concepts of risk estimation, (ii) presenting your own results and ideas that are relevant to the theme of the workshop, and (iii) formulating recommendations for future research work needed to provide the necessary information for the assessment of risks from increased levels of nanoparticles from combustion processes to human health. Some of you might be of the opinion that each area of concern, nanoparticle formation in combustion as well as medical aspects of nanoparticle exposure, deserves a workshop on its own. You may be a competent expert in one area, e.g. nanoparticle detection, but don't feel quite familiar with the other area, e.g. molecular effects of nanoparticles, or vice versa. It may be the special challenge of this workshop to identify common interests and to benefit from mutual exchange of information and experience from physical and life sciences. Let's try! There will be ample opportunity of holding discussions with a wide variety of colleagues from different fields in nanoparticle research, from engineering via molecular biology to medicine. The international nature of the workshop will also foster contacts between colleagues from many countries, which include Austria, Belgium, China, France, Germany, Italy, Netherlands, Norway, Sweden, Switzerland, United Kingdom, and United States of America. It is an explicit goal of our workshop to promote close working relations between scientists from different countries with different professional experience. With our best wishes to what will certainly be a highly interesting and enjoyable week in Bonn, Ludwig van Beethoven’s native city. Christine Hellweg Christa Baumstark-Khan Horst-Henning Grotheer Co-Directors

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THE ORGANIZING COMMITTEE IS DEEPLY GRATEFUL TO THE FOLLOWING ORGANIZATIONS AND COMPANIES FOR THEIR SPONSORSHIP DLR Institute of Aerospace Medicine

Executive Board

DEUTSCHE LUFTHANSA AG

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Program and Time Schedule

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Sunday, 1 May 2005

16:00-19:00 REGISTRATION

19:00 DINNER

20:00 INFORMAL GET-TOGETHER

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Monday, 2 May 2005

Opening of the Nanoparticle Workshop

Co-chair: Christine Hellweg, Christa Baumstark-Khan and Horst-Henning Grotheer, Germany

08:30 Hellweg C.E., Baumstark-Khan C., Grotheer H.H. INTRODUCTION TO THE NANOPARTICLE WORKSHOP

08:35 WELCOME ADDRESSES

Institute of Aerospace Medicine, Köln, Germany. Prof. Dr. R. Gerzer

Radiobiology Unit, Institute of Aerospace Medicine, Köln, Germany. Dr. G. Reitz

Session 1: Nanoparticle Formation in Combustion

Co-chair: Horst-Henning Grotheer, Germany and Frederik Ossler, Sweden

08:45 Grotheer, H.H., Germany Keynote Lecture BRIEF REVIEW ON NANOPARTICLES IN COMBUSTION PROCESSES

09:30 Mauss, F., Sweden DETAILED KINETIC MODELS FOR SOOT FORMATION IN COMBUSTION

10:00 Ossler, F., Sweden IN-SITU STUDIES OF NANOPARTICLE FORMATION IN FLAMES BY THE USE OF SYNCHROTRON X-RAY SCATTERING

10:30 COFFEE BREAK AND POSTER VIEWING

11:00 Thierley, M., Germany THE FAST COAGULATION OF COMBUSTION GENERATED NANOPARTICLES (NOC)

11:25 Lanzuolo, C., Italy A MODELLING STUDY OF THE COAGULATION PROCESS OF COMBUSTION GENERATED NANOPARTICLES

11:50 Reynaud, C., PHYSICOCHEMICAL PROPERTIES OF CARBON NANOPARTICLES FORMED BY LASER PYROLYSIS OF HYDROCARBONS

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Posters:

P-1 Szymczak, W., Germany EVALUATION OF TOF-SIMS FOR CHARACTERISING ULTRAFINE PARTICLES USED IN HEALTH-EFFECT STUDIES

P-2 Lanzuolo, G., Italy A MODELLING STUDY OF THE COAGULATION PROCESS OF COMBUSTION GENERATED NANOPARTICLES

12:15 LUNCH

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Monday, 2 May 2005

Session 2: Nanoparticle Emissions from Combustion Engines and Other Sources

Co-chair: Marcus Thierley, Germany and Fabian Mauss, Sweden

13:45 Netzell, K., Sweden ASPECTS OF MODELING SOOT FORMATION IN COMBUSTION

14:10 Minutolo, P., Italy NANOPARTICLES EMITTED BY DIESEL AND SPARK-IGNITED ENGINES: CHEMICAL-PHYSICAL CHARACTERIZATION AND IN VITRO BIOLOGICAL EFFECTS

14:35 Gonzalez Baquet, T., Germany

NANOPARTICLE MEASUREMENTS BEHIND VEHICLE ENGINES

15:00 Charwath, M., Germany IN-SITU CHARACTERIZATION OF NANOSCALED PARTICLE SIZE DISTRIBUTIONS IN COMBUSTION DEVICES

Posters:

P-3 Commodo, M., Italy UV LASER INDUCED FLUORESCENCE FOR THE DETECTION OF NANOSIZED ORGANIC CARBON (NOC) AND SOOT PARTICLES IN LAMINAR PREMIXED FLAMES.


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Monday, 2 May 2005

Session 3: Nanoparticles in the Environment and their Measurement

Co-chair: Tania Gonzalez Baquet, Germany and Wolfram Birmili, Germany

16:00 D’Alessio, A., Italy (presented by Sgro, LA, Italy) Keynote Lecture NANOPARTICLES OF ORGANIC CARBON SMALLER THAN 3 NM: DIAGNOSTICS, FORMATION AT HIGH TEMPERATURE, EVOLUTION IN THE ATMOSPHERE, EFFECTS ON HUMAN HEALTH

16:45 Krinke, T., Germany, NOVEL INSTRUMENTS TO DETECT AND CHARACTERIZE NANOPARTICLES FROM COMBUSTION SOURCES

17:10 Birmili, W., Germany MONITORING THE NON-VOLATILE RESIDUES OF AMBIENT PM (3 NM < DP < 900 NM) AT A RURAL, AN URBAN BACKGROUND, AND A STREET CANYON SITE USING THERMODESORPTION AND ELECTROMOBILITY CLASSIFICATION

Posters:

P-4 Mülhopt, S., Germany SAMPLING, CONDITIONING AND EXPOSURE TO LUNG CELLS OF FLY ASH OF A MUNICIPAL WASTE INCINERATION

P-5 Gerhart, C., Germany SIZE AND CONCENTRATION RANGES OF A HIGHLY SENSITIVE ELECTROMETER AND NANO-DMA COMBINATION

19:00 DINNER

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Tuesday, 3 May 2005

Session 4: Medical Aspects of Nanoparticle Exposure – Epidemiological

Evaluations (Acute, Chronic and Late Effects), Biomarkers

Co-chair: Christine Hellweg, Germany and Philip Demokritou, USA

08:45 Peters, A., Germany Keynote Lecture EPIDEMIOLOGICAL EVIDENCE ON HEALTH EFFECTS OF ULTRAFINE PARTICLES

09:30 Riediker, M., Switzerland CARDIOVASCULAR EFFECTS IN PATROL OFFICERS ARE ASSOCIATED WITH FRESH TRAFFIC PARTICLES

10:00 Stölzel, M., Germany PARTICULATE MATTER IN SEVERAL SIZE CLASSES AND DAILY MORTALITY IN ERFURT, GERMANY


11:00 Gupta, T., USA (presented by Demokritou, P.) A NOVEL TECHNIQUE FOR CONDUCTING IN-VIVO AND IN-VITRO EXPOSURES WITH CONCENTRATED AMBIENT ULTRAFINE PARTICLES

11:30 Zhao, Y., China TOXICOLOGICAL STUDIES ON NANOPARTICLES IN CHINA

12:00 Borken, J., Germany COPING WITH PARTICULATE EMISSIONS BY TRAFFIC MANAGEMENT - POSSIBILITIES AND CONSTRAINTS

Posters:

P-6 Stampfl, A., Germany ULTRAFINE CARBON PARTICLES INCREASE THE HEART RATE

P-7 Vigotti, M.A., Italy CARDIOVASCULAR CONDITIONS AND AIR POLLUTION IN PISA, ITALY, 1998-2003

12:30 LUNCH

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Tuesday, 3 May 2005

Session 5: Effects of Nanoparticles on Cellular Level

(Inflammatory and Toxic Effects)

Co-chair: Andrea Arenz, Germany and Günther Oberdörster, USA

13:45 Donaldson, K., UK Keynote Lecture CURRENT HYPOTHESES ON THE MECHANISMS OF TOXICITY OF ULTRAFINE PARTICLES

14:30 Oberdörster, G., USA NANOMEDICINE AND NANOTOXICOLOGY – APPLICATIONS AND IMPLICATIONS

15:00 Beck-Speier, I., Germany THE SPECIFIC SURFACE AREA OF ULTRAFINE CARBONACEOUS PARTICLES DETERMINES GENERATION OF LIPID MEDIATORS AND OXIDATIVE STRESS IN ALVEOLAR MACROPHAGES.


16:00 Duschl, A., Austria DIESEL SOOT EXPOSURE MODULATES FUNCTIONAL MATURATION OF BONE MARROW-DERIVED DENDRITIC CELLS

16:30 Stöger, T., Germany SPECIFIC SURFACE AREA ABOVE A CERTAIN THRESHOLD DETERMINES THE ACUTE INFLAMMATORY RESPOND OF INSTILLATED CARBONACEOUS NANOPARTICLES IN MICE

17:00 Hellweg, C.E., Germany DETECTION OF CYTOTOXIC AND INFLAMMATORY EFFECTS BY MEANS OF STABLY TRANSFECTED LUNG CELLS

Posters:

P-8 Dopp, E., Germany CYTO- AND GENOTOXIC EFFECTS OF NATURAL AND SURFACE-TREATED ULTRAFINE TITANIUM DIOXIDE PARTICLES IN MAMMALIAN CELLS

P-9 Rumelhard, M., France INVOLVEMENT OF AMPHIREGULIN IN AIRWAY EPITHELIAL CELL RESPONSES TO URBAN PARTICULATE MATTER

P-10 Ramgolan, K., France EFFECT OF URBAN PARTICULATE MATTER ON AIRWAY EPITHELIAL CELLS IN VITRO: ROLE OF PARTICLE COMPOSITION AND SIZE

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P-11 Nelissen, I., Belgium FLOW CYTOMETRIC CHARACTERIZATION OF ANTIGEN-PRESENTING DENDRITIC CELLS AFTER IN VITRO EXPOSURE TO DIESEL EXHAUST PARTICLES

20:00 WORKSHOP DINNER

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Wednesday, 4 May 2005

Session 6: Molecular Effects of Nanoparticles

(DNA damage induction, signalling pathways, gene expression)

Co-chair: Christa Baumstark-Khan, Germany and Roel Schins, Germany

08:45 Borm, P.J.A., The Netherlands Keynote Lecture TRANSLOCATION AND KINETICS OF NANOPARTICLES: IMPLICATIONS FOR RISK ASSESSMENT

09:30 Unfried, K., Germany SIGNALLING EVENTS RELEVANT FOR APOPTOSIS AND PROLIFERATION INDUCED BY NANOPARTICLES IN RAT LUNG EPITHELIAL CELLS

10:00 Baumstark-Khan, C., Germany FROM SPACE RESEARCH TO NANOPARTICLE EFFECTS: BIODIAGNOSTICS AT THE GERMAN AEROSPACE CENTRE


11:00 Arenz, A., Germany GENE EXPRESSION MODULATION IN A549 LUNG CELLS IN RESPONSE TO COMBUSTION GENERATED NANOPARTICLES

11:30 Schins, R., Germany INFLAMMATORY AND GENOTOXIC EFFECTS OF COMBUSTION GENERATED NANOPARTICLES WITH AND WITHOUT BENZO(A)PYRENE AND/OR IRON SULPHATE

12:00 Chen, M., Germany ALTERED NUCLEAR STRUCTURE AND FUNCTION IN RESPONSE TO SILICA NANOPARTICLES

Posters:

P-12 Sydlik, U., Germany APOPTOSIS AND PROLIFERATION INDUCED BY DIFFERENT TYPES OF NANOPARTICLES IN RAT LUNG EPITHELIAL CELLS

P-13 Diabaté, S., Germany EFFECTS OF FLY ASH ON THE LEVEL OF GLUTATHIONE AND THE EXPRESSION OF HEME OXYGENASE-1

P-14 Langenbach, F, Germany EFFECTS OF COMBUSTION GENERATED NANOPARTICLES ON BACTERIAL SOS

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RESPONSE CONTROLLED GENE EXPRESSION

P-15 Simonelli, A., Italy COMBUSTION GENERATED NANOPARTICLES: MUTAGENICITY AND CHEMICAL REACTIVITY

12:30 Round table discussion FUTURE RESEARCH ON HEALTH EFFECTS OF NANOPARTICLES

13:15 CLOSING REMARKS

13:20 LUNCH

14:00 DEPARTURE

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Monday, 2 May 2005


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K-1

GENERATION OF NANOPARTICLES IN COMBUSTION PROCESSES

Horst-Henning Grotheer Deutsches Zentrum für Luft- und Raumfahrt (DLR), Institut für Verbrennungstechnik, Stuttgart,

Germany

Combustion generated nanoparticles with sizes between 2 and 6 nm are precursors to soot and have been found in flames by a variety of measurement methods. The transformation to soot is, however, not necessarily complete so that nanoparticles can be emitted along with soot or even under non-sooting conditions. This applies in particular to vehicle engines. Indeed, nanoparticles could be measured behind currently used car and bike engines by spectroscopic methods and mass spectrometry. Since nanoparticles could be also shown to be highly toxic, i.e cytotoxic as well as genotoxic, they may form a serious hazard associated with the motorization of modern societies. In the paper it is attempted to summarize the current knowledge on nanoparticles, to point out conflicting views and to address needs for further research.

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L-1

DETAILED KINETIC MODELS FOR SOOT FORMATION IN COMBUSTION

Fabian Mauss Division of Combustion Physics, Lund University, P.O. Box 118, SE-22100 LUND, Sweden, e-mail:

[email protected], fax: +46 (0)46 222 9757

In this presentation an overview on detailed kinetic models for soot formation in combustion processes will be given. The presentation is divided into three chapters.

1) The kinetic modeling of soot formation

Soot formation in practical combustion systems is in interaction with the kinetics of the oxidation process for the hydrocarbon fuel. In theory there are a high number of reaction pathways from simple carbon fuel molecules with up to 20 carbon atoms to complex soot particles with millions of carbon atoms possible. However, most of the soot is formed at temperatures above 1500 K, in a gas environment with a high number of radical species. This causes that a radical abstraction carbon addition process becomes dominant in soot formation. However, it is observed that the dominant surface growth mechanism in premixed flames and in diffusion flames is different. In premixed flames surface growth is dominated by heterogeneous surface reactions, while in diffusion flames surface growth is often dominated by condensation of PAH on the particle surface.

2) The physical modeling of soot formation

There are a number of physical processes that are sensitive on the particle formation processes in flames. First of all particle inception or nucleation, and particle coagulation. Combustion processes in technical applications can involve different pressure regimes. In Diesel engines pressures up to 200 bar occur. At the same time particle sizes in the range between 1 – 150 nm, and both spherical particles and agglomerates are observed. It follows that the particulate movement changes from free molecular to continuum regime, during the combustion process. A further important physical process under investigation is thermophorezes.

3) The mathematical description of the population balance equation.

There are a number of mathematical descriptions for the population balance equations available in the literature. For application of the particulate model with CFD it is important that a transport equation can be formulated for the unknowns, which are introduced by the mathematical description. Therefore the usage of the method of moments, and of the sectional method is mostly found in the literature. The convective and diffusive transport terms are not yet developed for stochastic methods to describe the population balance equation. However these methods have the advantage that it is “more easy” to describe two- or more-dimensional balance equations; i.e. size and shape, or size and surface site distributions.

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L-2

IN-SITU STUDIES OF NANOPARTICLE FORMATION IN FLAMES BY THE USE OF SYNCHROTRON X-RAY SCATTERING

Frederik Ossler 1 and Jörgen Larsson 2

1 Division of Combustion Physics, Lund Institute of Technology, Box 118, S-221 00 LUND, [email protected], Fax 46-46-2224542, 2 Department of Physics, Lund Institute of

Technology, Box 118, S-221 00 LUND.

The chemical and physical properties of nanoparticles are strongly related to their size and structure, which in turn are highly dependent on combustion conditions. Studies based on molecular dynamics have shown that particle structure can change rapidly at high temperatures [1,2]. Unfortunately, many polyatomic structures are hard to measure at high temperatures with laser diagnostic tools. There is a need to study and understand particle dynamics from an experimental point of view. Therefore new diagnostic methods, which allow the measurement of particle structure in-situ need to be developed. We use synchrotron radiation to study the X-ray scattering in the wide-angle region (WAXS) [3] to obtain properties of nanoparticles directly while combustion is taking place. These studies (at MAXLAB, beamline D 611, LUND) have revealed the formation of nanometer-sized fullerene-like and graphitic-like structures in atmospheric pressure flames. Our technique has a higher sensitivity to particle structure than the previously developed size-distribution measurement techniques based on synchrotron radiation Small-Angle X-ray Scattering (SAXS) [4,5] and therefore would be an important complement to nanoparticle measurements in flames. Results from the ongoing project will be presented and discussed.

1. F. Fugaciu, H. Hermann, G. seifert, Phys. Rev. B. 60 (1999) 10711. 2. A. I. Melker, S. N. Romanov, D. A. Kornilov, Mater. Phys. Mech. 2 (2002) 42. 3. F. Ossler, J. Larsson, Chem. Phys. Letters 387 (2004) 367. 4. W. A. England, Combust. Sci. Technol. 46 (1986) 83. 5. J. P. Hessler, S. Seifert, R. E. Winans, Proc. Combust. Inst. 29 (2002) 308.

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L-3

THE FAST COAGULATION OF COMBUSTION GENERATED NANOPARTICLES (NOC)

Marcus Thierley, Heinz Pokorny, Horst-Henning Grotheer, Manfred Aigner DLR Institute of Combustion Technology, Pfaffenwaldring 38, 70569 Stuttgart, Germany, e-mail:

[email protected], Fax: ++49 711-6862-578

A hybrid setup consisting of a movable low presssure burner (C2H4/O2 flame, 130 mbar) coupled to a fast flow reactor was used to monitor combustion generated nanoparticles using a photo-ionisation mass spectrometer and to study the coagulation of primary nanoparticles in the flow reactor. For these particles two modifications could be shown. Primary nanoparticles coagulate rapidly with a preliminary value for the room temperature rate coefficient of kc (350 K) = 3.5·10-10 cm³/s. At higher temperatures they react even faster and this suggests to consider nanoparticles as intermediates in models of soot formation.

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L-4

A MODELLING STUDY OF THE COAGULATION PROCESS OF COMBUSTION GENERATED NANOPARTICLES

Gianluca Lanzuolo, Antonio D’Alessio and Andrea D’Anna Dipartimento di Ingegneria Chimica - Università degli Studi di Napoli “Federico II”, Piazzale V.

Tecchio,80 - 80125 Napoli, Italy, e-mail [email protected], FAX +39 081 593 6936

The coagulation of combustion formed nanoparticles in the size range between 1 and 10 nm is modelled in the framework of gas kinetic theory by evaluating Van der Waals interactions between particles in terms of Lennard-Jones potentials. Different physical (shape) and chemical (molecular structure) characteristics of the particles are considered.

The model predicts that collision efficiency of nanoparticles in the free molecular regime (Kn → ∞) is very low. Indeed, the attractive part of the interaction potential of 1-5nm nanoparticles is very low and the kinetic energy of their relative motion may exceed the depth of the potential minimum. Therefore, all particles with a thermal kinetic energy in excess of the potential minimum will escape and rebound elastically if their residence time in the potential well is also short with respect to the time of energy accommodation between the colliding particles.

Model results are compared with coagulation efficiencies of nanoparticles measured “in situ” in premixed ethylene-air flames employing multi-wavelength extinction and light scattering methods. The “sticking“ properties of nanoparticles on mica substrates are evaluated by “ex-situ” Atomic Force Microscopy (AFM) and “in-situ” scattering/extinction measurements. Agreement of the simple model with experimental data is surprising; combustion formed nanoparticles which are rapidly produced in locally rich combustion regions have unique transport properties, because the sticking coefficient for coagulation and adhesion is orders of magnitudes lower than that expected for larger aerosols like soot particles, also of nano-scale. The transition of the regime is quite sharp yielding non-linear behaviour which is sometime overlooked.

The model is used to analyse the emission of nanoparticles from engine exhausts as a function of operating conditions. The same model is also used to study the evolution of nanoparticles in the atmosphere in order to find the critical size and concentration which causes a rapid evolution of nano-sized particles into micrometer-sized particles. The latter are easily measured in the atmosphere.

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L-5

PHYSICOCHEMICAL PROPERTIES OF CARBON NANOPARTICLE FORMED BY LASER PYROLYSIS OF HYDROCARBONS

Cécile Reynaud Laboratoire Francis Perrin, CEA-CNRS URA 2453, Service des Photons, Atomes et Molécules,

DSM, CEA Saclay, 91191 Gif/Yvette Cedex, France, [email protected], fax +33 1 69 08 87 07

Laser pyrolysis in the gas phase is a versatile method for the synthesis of nanoparticles [1]. The interaction of an infrared laser with a flow of molecular precursors leads to the formation of ultra fine particles with sizes typically in the 10-50 nm range and a narrow distribution. Chemical composition, structure and size of the nanoparticles are sensitive to the precursor mixture, the laser power and the gas flow.

In this paper, the properties of the carbon nanoparticles obtained by pyrolysis of hydrocarbon compounds will be discussed. These properties are very sensitive to the physical and chemical conditions of the pyrolysis process.

With a pure hydrocarbon precursor, aromatic nanoparticles are obtained with structure dependant on the laser power. As the flame temperature in the interaction zone increases, the nanoparticles evolve drastically from poorly organized, hydrogenated samples toward turbostratic concentric particles of carbon. The multi-scale organization of the samples and its evolution with the synthesis parameters are quantitatively determined and correlated to infrared spectroscopy through HRTEM image analysis [2].

Fullerene-rich soots can be obtained when an oxygen-rich precursor mixture is pyrolysed. The production yield of fullerene is very sensitive to the C/O ratio as observed in combustion [3]. The C60 yield is comparable to the highest yields found in the literature. The residence time of the reactants in the pyrolysis flame influences also dramatically the fullerene formation [4].

Finally, precursor mixtures including hetero-atoms such as nitrogen [5] of iron are shown to influence noticeably the growth and the structure of the carbon nanoparticles.

1. N. Herlin-Boime, M. Mayne-L'Hermite, C. Reynaud, Synthesis of covalent nanoparticles by CO2 laser, in “Encyclopedia of Nanoscience and Nanotechnology », Vol 10, H. S. Nalwa Ed., American Scientific Publishers (2004)

2. A. Galvez, N. Herlin-Boime, C. Reynaud, J.-N. Rouzaud, C. Clinard, Carbon Nanoparticles from laser Pyrolysis, Carbon 40, 2775 (2002)

3. F. Ténégal, S. Sidis-Petcu, N. Herlin-Boime, X. Armand, M. Mayne, C. Reynaud, Effect of the C/O ratio on the C60 and C70 formation in soot synthesized by laser pyrolysis of benzene based mixtures, Chem. Phys. Lett. 335, 155 (2001)

4. F. Ténégal, I. Voicu, X. Armand, N. Herlin-Boime, C. Reynaud, Residence time effect on fullerene yield in butadiene based laser pyrolysis flame, Chem. Phys. Lett. 379, 40-46 (2003)

5. E. Marino, B. Bouchet-Fabre, D. Porterat, C. Reynaud, FTIR and NEXAFS study of carbon nitride nanoparticles synthesised by laser pyrolysis, Diamond & Related Materials, in press (2005)

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P-1

EVALUATION OF TOF-SIMS FOR CHARACTERISING ULTRAFINE PARTICLES USED IN HEALTH-EFFECT STUDIES

W. Szymczak 1, W. G. Kreyling 2,3 and K. Wittmaack 1

GSF-National Research Centre for Environment and Health, 1Institute of Radiation Protection, 2Institute of Inhalation Biology, 3Focus Network Aerosols and Health, 85758 Neuherberg,

Germany, email: [email protected], fax: 089 – 3197 2942

Health effects due to the inhalation of combustion generated nanoparticles are often explored using ultrafine particles produced in a spark source generator. The relevance of such studies rests on the assumption that the employed particles can be used as a surrogate for ambient particles like soot. To substantiate this assumption, a detailed analysis of the surface and of the bulk composition of the artificial particles is mandatory.

A base material particularly well suited for radioactive labelling is metallic iridium which allows ultrafine particles to be produced fairly easily by spark source erosion. The generated atoms, molecules and clusters, however, are ejected in a rather hot state. Hence they are prone to heavy reactions with residual gases inevitably present in the generator. The previously unknown chemical composition of the produced iridium and carbon – iridium particles was analysed in detail by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The instrument was modified to allow operation with either short (∼ 2 ns) or long (up to 200 ns) primary ion pulses so that surface analysis as well depth profiling could be performed routinely with the same beam. Valuable additional information about the morphology and the extent of agglomeration was achieved by scanning electron microscopy.

The investigations revealed a very complex picture of particle composition. The contributions of different molecules to isobaric mass interferences had to be evaluated by a dedicated fitting routine. Each spectral group representing clusters containing iridium monomers, dimers and trimers were described by a sequence of up to 48 molecules, including the respective isotopic abundance. Within the limits of accuracy of SIMS analysis, iridium was identified only in the form of oxygen, nitrogen and carbon compounds, not in pure metallic form. Particularly remarkable was the size dependence of composition. The smallest particles (< 10 nm) consisted of carbon containing iridium oxides, medium-size particles (ca. 10-20 nm) were predominantly made of carbon-rich iridium compounds, and the largest particles were composed of iridium with roughly equal fractions of carbon and nitrogen. The results imply that the spark erosion process gives rise to a broad spectrum of reactions of the ejected matter with residual gases. The complexity of the composition is further aggravated by pronounced agglomeration of the generated nanoparticles. Prior to further use of the particles in animal studies it appears desirable to reduce the chemically and morphologically unexpectedly wide band of the particle spectrum by thermal methods of aftertreatment or other means.

30


P-2

A MODELLING STUDY OF THE COAGULATION PROCESS OF COMBUSTION GENERATED NANOPARTICLES

Gianluca Lanzuolo, Antonio D’Alessio and Andrea D’Anna Dipartimento di Ingegneria Chimica - Università degli Studi di Napoli “Federico II”, Piazzale V.

Tecchio,80 - 80125 Napoli, Italy, e-mail [email protected], FAX +39 081 593 6936

The coagulation of combustion formed nanoparticles in the size range between 1 and 10 nm is modelled in the framework of gas kinetic theory by evaluating Van der Waals interactions between particles in terms of Lennard-Jones potentials. Different physical (shape) and chemical (molecular structure) characteristics of the particles are considered.

The model predicts that collision efficiency of nanoparticles in the free molecular regime (Kn → ∞) is very low. Indeed, the attractive part of the interaction potential of 1-5nm nanoparticles is very low and the kinetic energy of their relative motion may exceed the depth of the potential minimum. Therefore, all particles with a thermal kinetic energy in excess of the potential minimum will escape and rebound elastically if their residence time in the potential well is also short with respect to the time of energy accommodation between the colliding particles.

Model results are compared with coagulation efficiencies of nanoparticles measured “in situ” in premixed ethylene-air flames employing multi-wavelength extinction and light scattering methods. The “sticking“ properties of nanoparticles on mica substrates are evaluated by “ex-situ” Atomic Force Microscopy (AFM) and “in-situ” scattering/extinction measurements. Agreement of the simple model with experimental data is surprising; combustion formed nanoparticles which are rapidly produced in locally rich combustion regions have unique transport properties, because the sticking coefficient for coagulation and adhesion is orders of magnitudes lower than that expected for larger aerosols like soot particles, also of nano-scale. The transition of the regime is quite sharp yielding non-linear behaviour which is sometime overlooked.

The model is used to analyse the emission of nanoparticles from engine exhausts as a function of operating conditions. The same model is also used to study the evolution of nanoparticles in the atmosphere in order to find the critical size and concentration which causes a rapid evolution of nano-sized particles into micrometer-sized particles. The latter are easily measured in the atmosphere.

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Monday, 2 May 2005

Session 2: Nanoparticle Emissions from Combustion Engines

and Other Sources

33


L-6

ASPECTS OF MODELING SOOT FORMATION IN COMBUSTION

Karl Netzell, Fabian Mauss and Harry Lehtiniemi Division of Combustion Physics, Lund University, P.O. Box 118, SE-22100 LUND, Sweden, e-mail:

[email protected], fax: +46 (0)46 222 0885

In this work we discuss soot formation in a turbulent diffusion flame and in a Diesel engine from a modeling viewpoint. We follow the detailed kinetic soot model proposed by Frenklach and Wang, and Mauss. During the oxidation of the hydrocarbon fuel, aromatic species are formed and grow further to poly-aromatic hydrocarbon (PAH) structures. From a size of four aromatic rings the PAH may collide and build three-dimensional structures of a size of about 1 nm, these being the first soot particles. The soot particles grow by condensing larger PAH molecules onto the soot surface or by heterogeneous surface reactions as described by the HACARC (hydrogen-abstraction-acetylene-addition-ring-closure) mechanism, or by coagulation with other soot particles. Soot is oxidized by reactions with molecular oxygen on radical sites on the soot surface, or by hydroxyl radicals on neutral sites on the soot surface. The particle inception, condensation and coagulation processes are described by the Smoluchowski equation. Agglomeration can be modeled assuming coalescent growth or by applying an agglomeration model allowing the fractal dimension of soot to be considered.

The Smoluchowski equation can be solved using the method of moments or by the sectional method. In the method of moments, the moments of the particle size distribution function are transported. This method is computationally very efficient, but does not give any direct information on the amount of particles in different size classes. In order to obtain such information, the sectional method is used. In the sectional method the soot size distribution is discretized into a given number of sections. A transport equation is solved for each section, thereby providing detailed information on the appearance of the particle size distribution function.

It is possible to extend the above soot model to describe arbitrary soot surface chemistry. Transport equations for the mole fractions of the chosen active site classes are solved.

Soot volume fraction in a turbulent ethylene fueled jet diffusion flame was calculated using the unsteady flamelet method. We found that by considering maximum mixture fraction in the model for scalar dissipation rate, and by conditioning the scalar dissipation rate on the location in mixture fraction space on which maximum scalar dissipation rate occurs, and by applying an agglomeration model good agreement between experiments and calculations was obtained without considering preferential diffusion.

In Diesel engine CFD calculations information on the soot formation processes is obtained using a pre-calculated flamelet library for the source terms of soot formation and by transporting the soot mass fraction. In a more advanced setting, the moments or size classes of the particle size distribution function are transported. Good agreement between engine experiments and calculations is obtained, and effects depending on the amount of exhaust gas recirculation are predicted correctly.

34


L-7

NANOPARTICLES EMITTED BY DIESEL AND SPARK-IGNITED ENGINES: CHEMICO-PHYSICAL CHARACTERIZATION AND IN VITRO BIOLOGICAL EFFECTS

A. Brun 1, A. D’Alessio 2, C. De Lisi 1, D Guarnieri . 3, M. Iuori 4, P. Minutolo 5, P A. Netti . 3,6, L.A. Sgro 2

(1)Dipartimento di Scienze Fisich, (2)Dipartimento di Ingegneria Chimica, (3)Dipartimento Ingegneria dei Materiali e della Produzione(DIMP), -Universita`Federico II; (4) Istituto Materiali Compositi e

Biomedici(IMCB), (5) Istituto di Ricerche sulla Combustione(IRC),- CNR, Napoli; (6)Centro Ricerche Interdipartimentali sui Biomateriali (CRIB)

The health effects of nanoparticles emitted into the atmosphere by engines and practical combustion is an increasing public health concern worldwide. While the toxicological mechanism for how particles affect human health is not yet known, recent studies show growing evidence for an inverse dependency of health effects on particle size and that number concentration and surface area are possibly more important characteristic for health effects than mass concentration.

We characterized nanoparticles emitted from two last-generation vehicles, one powered by direct injection, spark-ignited engine fuelled with unleaded gasoline and the other equipped with a high-pressure injection (“common rail”) diesel engine, fuelled with commercial, low-sulfur light oil. Nanoparticles were sampled from the exhausts into water suspensions by floating the vehicle exhausts through a heat exchanger in order to condense and collect the water vapour produced in combustion, along with any hydrophilic matter trapped in the liquid phase. After separating soot and gaseous compounds, nanoparticles contained in the water samples were subjected to chemico-physical characterization and in vitro biological assays.

Fluorescence Correlation Spectroscopy (FCS) and Time Resolved Fluorescence Polarization Anisotropy (TRFPA) techniques were used to determine translational and rotational diffusion coefficients of the nanoparticles and their characteristic dimensions. The mean sizes determined by FCS and TRFPA were on the order of 2 nm, and are in good agreement with the particle’s size distribution function measured by an Electrospray-nano Differential Mobility Analysis (E-DMA) system designed to classify and count singly charged particles smaller than 3 nm. Information on the chemical structure of sampled material was determined by measuring absorption and fluorescence spectra, fluorescence lifetime, and the concentration of total organic carbon (TOC) contained in the collected water suspension with a TOC analyzer.

For the biological tests 105 mouse embryo fibroblasts NIH3T3 were seeded in a 24-well with different suspensions of particles in DMEM W/O phenol red and 10% FBS, incubated at 37°C. We measured cytotoxicity with a colorimetric assay (AlamarBlue, BioSource). Dramatic cell morphology changes were observed after incubating the cells with particle suspensions for 1 and 3 hours. Cells treated with particles were rounded with great vacuoles inside, compared to well-spreaded control cells. In addition, treated cells appeared more fluorescent than the controls by confocal microscopy, indicating cellular uptake. The Alamar blue assay revealed significant dose-response cytotoxicity above a critical concentration of 21 ppm for nanoparticles collected from diesel-powered vehicle exhausts and 18 ppm for those from gasoline-powered vehicle exhausts. In particular, particles produced by the gasoline engine were the most cytotoxic.

35


L-8

DETECTION OF COMBUSTION GENERATED NANOPARTICLES (NOC) BEHIND VEHICLE ENGINES USING MASS SPECTROMETRY

Tania Gonzalez Baquet, Karl-Ludwig Barth, Horst-Henning Grotheer, and Manfred Aigner DLR Institute of Combustion Technology, Pfaffenwaldring 38, 70569 Stuttgart, Germany, e-mail:

[email protected], Fax: ++49 711-6862-578

The ability to detect combustion generated nanoparticles through photoionisation mass spectrometry was recently explored using premixed low pressure flames. In a second step this technique was extended to flames and other practical combustion systems operating at atmospheric pressure by using a novel fast flow probe designed as to minimize subsequent reactions of the reactive sample gas which contains the particles. The present paper deals with the application of the new method to vehicle engines. In first qualitative tests we measured nanoparticles behind a motor scooter and a motor bike. The next step was to measure nanoparticles in the exhaust train of a direct injection gasoline research engine. Through variation of the operation conditions a close correlationship of the nanoparticle signals to the smoke number could be established.

36


L-9

IN-SITU CHARACTERIZATION OF NANOSCALED PARTICLE SIZE DISTRIBUTIONS IN COMBUSTION DEVICES

M. Charwath, T. Lehre, R. Suntz and H. Bockhorn Engler-Bunte-Institut/Bereich Verbrennungstechnik, and Institut für Chemische Technik und

Polymerchemie, Universität Karlsruhe (TH)

Many years it was assumed that soot particles are harmless for human health. Several studies revealed that soot and especially soot from diesel engines are a potential health hazard. Soot is emerging mainly from incomplete combustion procedures of solid or liquid carbonaceous, organic materials. Due to its small diameters ultra-fine soot particles can be ingested deep into human lungs. It was used to believe that only the on the surface of the soot particles adsorbed polycyclic aromatic hydrocarbon could cause several diseases. New perceptions due to animal experiments revealed that the core of soot particles (pure carbon) has a strong effect on causing tumours. Hence, the legislative releases restrictions concerning the total emitted mass of soot from diesel engines, which could be easily realized by filtering the bigger particles off the exhaust gases while the smaller ones are still emitted in the atmosphere. Thus the characterization of soot particles size distribution found in exhaust gases from technical combustion devices is an important task. Therefore, measurements techniques with a high time und spatial resolution are required. However, nonintrusive optical measurement systems are an adequate solution.

A method to determine particle size distribution of nanoscaled soot particles relies on the simultaneous detection of the laser-induced incandescence signal (LII) at two different wavelengths. Absorbing intense laser light pulses (~10ns) these particles are heated up to temperatures far above the ambient gas or flame temperature. The LII-signal due to the absorption of a short laser pulse describes the temporal evolution of the enhanced thermal radiation of the particles. The particle temperature can be calculated from the ratio of the LII-signal intensities at two wavelengths. An improved LII-model with prior validated parameters was used to assess parameters of a lognormal size distribution from measured temperature curves using multidimensional non-linear regression. The possibility to characterize particle size distributions independent of the optical particle properties is a big advantage of this measuring system.

The developed 2 colour LII-technique was used to determine spatially resolved local particle size distributions from single shot measurements. Distinct zones of particle formation, growth and oxidation can be located in the investigated flames. The developed measuring system can be used in turbulent, non-stationary, technical systems and it can be a powerful tool for air pollutions control tasks. Furthermore it assists to design processes of combustion devices with lower particle emissions or gas-to-particle conversion reactors for the synthesis of nanoscaled particles.

37


P-3

UV LASER INDUCED FLUORESCENCE FOR THE DETECTION OF NANOSIZED ORGANIC CARBON (NOC) AND SOOT PARTICLES IN LAMINAR PREMIXED FLAMES

M. Commodo 1, C. Allouis 2, P. Minutolo 2, A. D’Anna 1, A. D’Alessio 1

1 Dipartimento di Ingegneria Chimica, Università di Napoli “Federico II”, Napoli, ITALY, 2 Istituto di Ricerche sulla Combustione, CNR, Napoli, ITALY, e-mail: [email protected] Fax: +390815936936 Nano-sized organic carbon (NOC) particles, with typical size lower than 3 nm, are produced in rich premixed hydrocarbon flames as well as in practical systems [1]. “In situ“ optical techniques, such as elastic light scattering and UV-visible absorption spectroscopy, have been extensively employed for the characterisation of NOC both in non-sooting and sooting flames [2,3]. Nevertheless, extinction measurements, requiring a line-of-sight measurement, suffer a limited space resolution. Laser Induced Incandescence (LII) has been proposed to determine soot particles volume fractions in practical systems, where high space resolution is necessary [4], but such technique fails to detect NOC particles which are much smaller and cannot be treated as solid particles [5]. Previous works have showed that the interaction of UV laser beam with the particulate produced in flames brings to the emission of a broadband fluorescence in the UV which was attributed to NOC, and the emission from laser-produced C2 radicals for high laser fluences which is due to the fragmentation of soot and carbonaceous structures [5,6]. The aim of this work is to exploit broadband UV fluorescence and the emission from laser-produced C2 radicals for high laser fluences for the determination of the volume fractions of NOC and soot particles by single shot measurement. We explore the interaction of the fifth harmonics of a pulsed Nd-YAG laser at 213nm with ethylene-air flames with C/O ratio ranging from the stoichiometric value, 0.33, to the fully-sooting regime, 0.92. The excitation wavelength was selected in order to enhance the fluorescence of NOC particles, which exhibit a large absorption band between 200 and 250 nm [1], and the fluence of the laser beam was high enough to produce C2 emission. NOC and soot volume fractions and mean particle sizes are determined by light absorption and light scattering measurements. In rich but non sooting flames, a broadband UV fluorescence is emitted with a maximum at 300-350 nm and it is proportional to the volume fraction of NOC particles. In such flames also C2 signal is emitted delayed respect to the fluorescence and present a higher lifetime. In sooting flame conditions there is a linear correlation between C2 emission and the total extinction coefficient in the visible and therefore the soot volume fraction. The different rate of C2 production determined in sooting respect to non sooting flames allow to discriminate the effect of soot and NOC particles. The analysis of both temporal and integrated UV-excited spectra let us to hope that UV fluorescence with C2 emission may be employed to follow the volume fraction of NOC and soot particles and the coupling with the scattering coefficient measurement might allow to evaluate their average sizes.

1. L.A. Sgro, G. Basile, A.C. Barone, A. D'Anna, P. Minutolo, A. Borghese, A. D'Alessio, Chemosphere 51 (2003) 1079-1090

2. D’Alessio, A. D’Anna, P. Minutolo, L.A. Sgro, A. Violi, Proc. Combust. Inst. 28 (2000) 2547-2554. 3. G. Basile, A. Rolando, A. D’AlessioA. D’Anna, P. Minutolo, Proc. Combust Inst. 29 (2002) 2391-2397. 4. T. Lehre , H. Bockhorn, B. Jung.eisch, R. Suntz Chemosphere 51 (2003) 1055–1061 5. P.E. Bengtsson, M. Alden, Combust. Flame 80 (1990) 322-8. 6. A. D'Alessio G. Gambi, P. Minutolo S. Russo and A. D'Anna Proc. Combust. Inst., 25, 645-651, 1994

mailto:[email protected]

39

Monday, 2 May 2005

Session 3: Nanoparticles in the Environment and their

Measurement

41


K-3

NANOPARTICLES OF ORGANIC CARBON SMALLER THAN 3 NM: DIAGNOSTICS, FORMATION AT HIGH TEMPERATURE, EVOLUTION IN THE ATMOSPHERE, EFFECTS ON HUMAN HEALTH

Antonio D’Alessio Università degli Studi di Napoli Federico II, Italy, [email protected]

This communication deals with the properties and fate of nanoparticles of organic carbon (NOC) which are formed in the fuel rich combustion of hydrocarbons. In the first place, the different diagnostics employed for their detection will be concisely addressed, consisting of both optical methods, such as elastic and inelastic light scattering, fluorescence and UV absorption, and intrusive techniques such as mass spectrometry, differential mobility analysis, size exclusion chromatography, electron microscopy and atomic force microscopy. The formation process in flames will be considered mostly with relationship to laminar premixed and diffusion flames and the results are interpreted in terms of the kinetics of elementary reactions. Regimes where NOC are formed in the absence and in the presence of soot particles will be discussed. Furthermore, the coagulation and deposition rates of NOC will be examined both experimentally and theoretically. Typical results obtained in practical combustion systems are addressed with particular emphasis on the measurements inside and at the exit of diesel and gasoline engines. NOC exhibit a higher affinity for water than soot particles. Therefore, their interactions with rain and their role in the formation of clouds will be object of speculation. Also the question of NOC concentration in the atmosphere and their role in the formation of “fine particles” in urban atmospheres will be addressed. Finally, the potential health effects of NOC will be discussed by considering both the extremely small size of these nanostructures and their observed interactions with cellular models.

42


L-10

NOVEL INSTRUMENTS TO DETECT AND CHARACTERIZE NANOPARTICLES FROM COMBUSTION SOURCES

Thomas Krinke, Oliver F. Bischof TSI GmbH, Particle Instruments ([email protected], Fax: 0241-523 03 49)

Nanoparticles from combustion sources can best be characterized by their number concentration, size distribution, shape, and chemical composition. A high time resolution of the measurement is required in cases when the particulate emission is changing rapidly over time. This presentation will describe a number of novel instruments developed for the characterization of airborne nanoparticles, suitable for combustion generated ones in particular.

The characterization of emissions with strong time dependence, like emissions from Diesel engines, requires the capability to determine particle size distributions with real-time resolution. This is made possible with an instrument designed for dynamic measurements of combustion generated nanoparticles that can obtain ten complete size distributions per second. This technique allows the visualisation of transient processes in the exhaust gas occurring during engine load change or diesel filter loading and subsequent regeneration. Another application is the characterization of exhaust gas during chasing experiments or close to highways.

The concentration measurement of particles in the size range below 100 nm often requires that particles are made detectable. The detection technique employed by condensation particle counters (CPC) is based on exposing the nanoparticles to a supersaturated vapour so they form larger droplets and their subsequent counting. For many years CPCs using butanol as the working fluid have been available. A novel technique permits the use of water as working fluid and allows the detection and counting of nanoparticles down to 2.5 nm. In order to determine the size distribution of these nanoparticles, their electrical mobility - which is mainly a function of particle size and number of charges on the particle - can be measured with a differential mobility analyzer (DMA). Due to their high mobility diffusion losses play an important role, which are minimized in a low-loss, high flow Nano-DMA. In combination with an ultrafine water-based CPC the size distribution of nanoparticles can be measured with very high resolution in the size range between 2.5 nm and 150 nm. In addition to the size distribution measurement, the investigation of nanoparticles with SEM, TEM and EDX gives valuable information on chemical composition and shape. For this purpose a deposition of the particles on appropriate substrates like TEM-grids or silicon wafers is necessary. Electrostatic precipitation in a nano-aerosol sampler is an appropriate way to collect charged nanoparticles by means of a homogeneous electric field. The benefit of this technique is that it can be easily coupled to the analysis with a Nano-DMA.

43


L-11

MONITORING THE NON-VOLATILE RESIDUES OF AMBIENT PM (3 NM < DP < 900 NM) AT A RURAL, AN URBAN BACKGROUND, AND A STREET CANYON SITE USING THERMODESORPTION

AND ELECTROMOBILITY CLASSIFICATION

Wolfram Birmili, Christa Engler, Diana Rose, Thomas Tuch, Birgit Wehner, Alfred Wiedensohler Leibniz Institute for Tropospheric Research, Permoserstrasse 15, 04303 Leipzig, Germany, phone:

++49 341 2352412, fax: ++49 341 2352361, e-mail: [email protected], internet: http://www.tropos.de/eng/PHYSICS/aerosol/aerosol.html

The carbonaceous fraction of inhaled particulate matter (PM) has been recognised as a potential risk for human health, due partially to its reduced solubility and probability of clearance in the lung, but also to toxins residing on the surface of combustion-generated particles. Volatility analysis has proved to be a useful tool in previous work on ambient, source, or test aerosols with regard to a rapid quantification of the physico-chemical fractions associated with soot or elemental carbon.

For this work we have analysed 1 year of continuous volatility measurements of atmospheric particles at three different exposure sites (rural, urban background, street canyon) in the Leipzig region, thereby assuming that the non-volatile material identified in fine and ultrafine PM is mainly associated with elemental carbon. During the experiments both, polydisperse and monodisperse ambient aerosols were heated to 300°C while passing through a thermodenuder. Volatilised material was removed from the gas phase before the residual, non-volatile particle number size distributions (maximum range: 3-900 nm) were determined at time resolution of approximately 10 minutes using electromobility classification.

Over the period of 1 year, the abundance of non-volatile particles was found to depend most strongly on the proximity to traffic sources – including significant diurnal effects, but also on synoptic scale meteorological influence. The non-volatile mass fraction of 150 nm particles was estimated to be 30 % in the street canyon, 15 % in urban background air, and 10 % in rural air. Volatility analysis of monododisperse particles showed that in urban air the major mass fraction of elemental carbon exists as a distinct, externally mixed particle population. The number fraction of these almost entirely non-volatile particles at 150 nm particles was estimated to be 50 %, 30 %, and 20 % in the three environments. A further combination of the volatility analysis of polydisperse and monodisperse particle number distributions suggested that the ambient size distribution of non-volatile (~ elemental carbon) particles have a number concentration maximum around 80 nm, which is highly consistent with measured emission profiles of motor vehicles, especially diesel cars.

Although more specific tests on the morphology and the chemical structure of the thermo-analysed particles will be necessary in the future, the body of these observations shows that thermodesorption in combination with electromobility classification is a technique suitable to monitor non-volatile residues of PM over short and long-term periods.

44


P-4

SAMPLING, CONDITIONING AND EXPOSURE TO LUNG CELLS OF FLY ASH OF A MUNICIPAL WASTE INCINERATION

S. Mülhopt, H-R. Paur, H. Seifert Forschungszentrum Karlsruhe, Institut für Technische Chemie, Bereich Thermische

Abfallbehandlung, Postfach 3640, 76021 Karlsruhe, [email protected], +49-7247-82 3807, Fax +49-7247-82 4332

Particle emissions from industrial combustion processes and traffic are the main sources of anthropogenic airborne particles in the environment. In recent years epidemiological studies have shown that there is an association between the concentration of fine particles (PM10, PM2.5) in the atmosphere and the rate of mortality or morbidity due to respiratory and cardiovascular disease (1). The causes of the toxicological effects of fine particles to the human organism are yet unknown. Besides the chemical composition, the physical properties of the particles seem to be of particular importance for the effects. For the quantitative assessment of the toxicity of industrial ultra fine particles the dose-response relationship is essential. A technique was developed, to expose human lung cells in vitro to assess industrial particle emissions with respect to their effects on human health.

For a realistic determination of the toxicity the cells, cultured on Transwell plates, are subjected to constant flow of the aerosol. By this procedure an exposure at the air liquid interface is achieved, which resembles the conditions in the human lung. A modified exposure system is set up to assure reproducible exposure conditions. This system consists of an isokinetic sampling unit to collect the aerosol from the flue gas of for example a combustion process. The particles bigger than 1 µm are removed by means of a cyclone. In the next step the aerosol is humidified with respect to the conditions in a human throat. Then the aerosol flow (100-1000 ml/min) is directed into a CULTEX® exposure system (2), which contains six Transwell plates with human lung cells. The aerosol flows perpendicular onto the surface of the cell culture. Typical exposure times range from 0.5 to 2 hours. After the exposure the aerosol laden Transwell plates are subjected to analysis. This is done by the Institute for Toxicology and Genetics of FZK who are developing the corresponding bioassay (3, 4).

Exposure experiments with the ultra fine fraction of fly ash of the municipal waste incinerator of Mannheim have shown a deposition efficiency of the particles of 16 % in the exposure system. The grade efficiency was determined by measuring the particle size distribution upstream and downstream the exposure system. It shows the characteristics of a filter with a high efficiency for very small and the bigger particles and a minimum in the size range of 200 – 400 nm.

1. Peters, A., Wichmann, H.E., Tuch, T., Heinrich, J., Heyder, J. (1997). Am. J. Respir. Crit. Care. Med. 155: 1376-1383.

2. Aufderheide, M. and Moor, U. (2000). Exp. Toxic. Pathol. 52, 265-270. 3. Diabaté, S., Mülhopt, S., Paur, H.-R., Krug, H.F. (2001). Arch. Pharmacol. 363, p. 585. 4. Wottrich, R., Diabaté S., Krug H.F. (2004): Int. J. Hyg. Environ. Health 207:353-361

45


P-5

SIZE AND CONCENTRATION RANGES OF A HIGHLY SENSITIVE ELECTROMETER AND NANO-DMA COMBINATION

C. Gerha tr 1 , T. Rettenmoser1, M. Richter2 and H. Grimm1 1 GRIMM AEROSOL Technik GmbH, Dorfstrasse 9, D-83404 Ainring, Germany, chg@grimm-

aerosol.com, Fax +49 8654-578-35; 2 G.I.P GmbH, Research Department, Mühlbecker Weg 38, 0671 Pouch, Germany.

In the recent years small particles and formation or nucleation of new particles in the range of 1 to 50 nm became more into the focus of atmospheric aerosol research. Therefore also the development of new techniques and instrumentation has been increased. Up to now mainly sensitive Condensation Particle Counters (CPC) are used for the range down to a few nanometre. The main disadvantages of CPCs are: 1. limitation to a d50 cut-off as 2-5 nm, 2. a relatively slow response time, 3. operation: due to the operation principle of nucleous condensation a defined temperature difference and a condensation liquid and supply is required. An alternative to the condensation particle counting is the direct counting of charged particles (or ions) by electrometers. Faraday Cup Electrometer (FCE), require no liquids and supply, no optical and laser parts and are theoretical not limited in the particle size. The only limitation is given by the number of charged particles and the necessity to combine it with a Differential Mobility Analyser (DMA) to cut out adequate charged particles. The presented new GRIMM FCE 5.700 is highly sensitive to low concentration but on the other side robust enough to mechanical shocks and pressure fluctuations. The FCE in combination with the GRIMM Nano S-DMA (a Vienna type DMA), which is able to measure even below 1 nm, is the proper solution for nucleation and particle birth measurements. With this system it is possible to measure already larger ion clusters around 0.8 nm and up to about 50 nm [1]. There are three important advantages of this system (fig 1): 1. The DMA has an active length of just 15 mm, short inlet distances and avoids electrical fields at the isolator section. This leads to comparatively very low losses of fine particles or ions. 2. The FCE has a sensitivity down to 0.1 fA (1 Hz, ~ 600 charged particles), an automatic zero point adjustment, changeable concentration ranges and sheath airflow to reduce the humidity. 3. The connection of both devices is very simple. The DMA outlet is directly connected with the FCE inlet. The DMA is directly screwed into the FCE inlet section, so that the classified particles are directly introduced to the FCE. The distance to the FCE filter is less than 20 mm, which also leads to a reduced particle loss. The nano-DMA is a short version of the new Vienna Type DMA 5.500 of GRIMM, which allows flexible length combinations by an easy alteration [2]. Both units are controlled and supplied by a separate DMA-controller. This controller supplies the system with sheath air (5 – 20 l/min) and sample air (5 – 20 l/min), controls the high-voltage power supply for the DMA and has a data acquisition system and interface integrated. [1] J. M. Mäkelä, G. P. Reischl and J. Necid „Ion Spectra of Organic Vapours as a Mobility Reference for DMA Testing Purposes“ J. Aerosol Sci., Vol 28, Suppl. 1, pp S705 - S706, 1997 [2] Reischl, G. P., Mäkelä, J. M. and Necid, J. "Performance of Vienna Type DMA at 1.2 - 20 Nanometer" Aerosol Sci. and Technol., Vol. 27, pp. 651 – 672 (1997).

47

Tuesday, 3 May 2005

Session 3: Medical Aspects of Nanoparticle Exposure –

Epidemiological Evaluations, Biomarkers

49

Session 4: Medical Aspects of Nanoparticle Exposure – Epidemiologic Evaluations, Biomarkers

K-4

EPIDEMIOLOGICAL EVIDENCE ON HEALTH EFFECTS OF ULTRAFINE PARTICLES

A. Peters , H.E. Wichmann

GSF-Forschungszentrum für Umwelt und Gesundheit, Ingolstädter Landstr. 1, 85764 Neuherberg, Gemany, fax: 089 3187 3380, e-mail: [email protected]

Ambient fine particle mass has been consistently linked with acute and chronic health effects. The health effects include exacerbation of respiratory and cardiovascular diseases by daily variations in fine particle concentrations and regional differences in annual mean concentrations of fine particles.

Ultrafine particles with a diameter below 100 nm have been implicated as being an independent class of particles. They have higher variation in daily averages than fine particles in urban areas. The correlation between the ultrafine number concentrations and the fine particle mass concentrations varies between different regions, but rarely exceeds a correlation coefficient of 0.5. Data for example from Helsiniki indicated that the ultrafine particles were generated locally and are fresh and potentially highly biologically active particles while the fine particle mass is dominated by regionally transported particles containing secondary particles such as sulphates as well as aged soot components.

Health effects of ultrafine particles were studied so far only for short-term health effects. Independent effects of ultrafine particles were documented for daily variations in all cause mortality. In addition, asthmatics might have decreases in lung function, a higher frequency of respiratory symptoms and of medication use on days with high ultrafine number concentrations than on days with low ultrafine number concentrations. Furthermore, evidence was found that ultrafine particle may induce ischemia during sub-maximal exercise in patients with coronary artery disease. A European multi-centre study indicated that hospital admissions for cardiovascular diseases in myocardial infarction survivors may be associated with ultrafine particle number concentrations.

The evidence from these studies suggests that ultrafine particle may have independent effects in addition of the effects of fine particle mass. Further epidemiological studies characterising the aerosol with respect to the ultrafine particle fraction are needed to evaluate the role of freshly and locally produced ambient particles in the ultrafine mode.

50

Session 4: Medical Aspects of Nanoparticle Exposure: Epidemiologic Evaluations, Biomarkers

L-12

CARDIOVASCULAR EFFECTS IN PATROL OFFICERS ARE ASSOCIATED WITH FRESH TRAFFIC PARTICLES

Michael Riediker

Institute of Occupational Health Sciences, Rue du Bugnon 19, 1006 Lausanne, Switzerland, Phone: ++41-21-314-7421, Fax: ++41-21-314-7430, e-mail: [email protected]

Exposure to fine and ultrafine particulate matter is associated with increased risks for cardiovascular morbidity and mortality. People driving in a vehicle might receive an enhanced dose of mobile source pollutants. This study investigated cardiovascular and haematological parameters in highway patrol troopers who work all day on busy roads.

Nine non-smoking, healthy, young, male North Carolina patrol trooper were monitored, each on four consecutive days while working a 3 PM to midnight shift. The exposure to air pollution was determined inside the vehicles, while a global positioning system and a diary provided location and activity information. Troopers refrained from alcohol, caffeine and medication. Blood was drawn 14 hours after each shift and the troopers wore ambulatory electrocardiogram monitors throughout the shift and the following night. Potential sources of in-vehicle particles were identified with variance-maximizing rotational principal factor analysis. Associations of health parameters with particles, components and sources were analysed using mixed models.

Average pollutant levels inside the cars were relatively low: carbon monoxide 2.7 ppm, nitrogen dioxide 41.7 µg/m3, ozone 11.7 ppb, fine particulate matter (PM2.5) 24 µg/m3, and a number of hydrocarbons and aldehydes were in the ppb-range. Elevated pollutant levels were related to locations with high traffic volumes. Four sources of particles were identified: 1) crustal material, 2) wear of steel automotive components, 3) gasoline combustion, 4) speed-changing traffic with emissions from load-changing engines and brake wear. Two source factors were significantly associated with health parameters: the “crustal” factor with increased mean heart cycle length (MCL) and serum uric acid concentration; and the “speed-change” factor with increased MCL, heart rate variability, supraventricular ectopic beats, blood urea nitrogen, red blood cell volume MCV, % neutrophils, von Willebrand Factor, and decreased % lymphocytes and protein C. These associations were little affected by stress estimates based on activity parameters.

These results suggest that mostly fresh traffic particles originating from load changing combustion engines and from brake wear were responsible for the observed associations with the physiological endpoints. The changes in the heart rate variability suggest an increased parasympathetic input to the heart with an associated increase in arrhythmic events, possibly in response to lung inflammation and endothelial activation. These findings suggest the hypothesis that pollutants emitted during speed-changing traffic conditions negatively impact the health risks. A long-term cardiovascular risk to the troopers or otherwise frequent vehicle drivers and passengers, or people living near such roads cannot be excluded.

51


L-13

PARTICULATE MATTER IN SEVERAL SIZE CLASSES AND DAILY MORTALITY IN ERFURT, GERMANY

M. S ölzelt 1, . A. Peters 1, J. Cyrys 2, M. Pitz 3, G. Wölke 1, W.G. Kreyling 4, J. Heinrich 1, H.EWichmann 1,2

1 Institute of Epidemiology, GSF Research Center, Neuherberg, Germany, [email protected], Fax +49-89-3187-3380; 2 University of Munich, 3 University of Augsburg, 4 Institute of Inhalation

Biology, GSF Research Center, Neuherberg, Germany

While the link between PM10 (particulate matter < 10 µm diameter) or PM2.5 (fine particles (FP), diameter < 2.5 µm) with mortality is well established the role of ultrafine particles (UFP, diameter < 0.1 µm) is not yet fully investigated. In an earlier analysis for the years 1995-1998 (Wichmann et al. 2000) borderline significant associations of FP (lag 0) and UFP (lag 4 days) with daily mortality were observed in Erfurt, Germany. The purpose of this analysis is to explore the role of several particle size fractions ranging from 10 nm to 2.5 µm for an extended period of observation. The particle size distribution was measured continuously from September 1995 to August 2001 by a combination of a differential mobility analyzer and an optical laser aerosol spectrometer. Daily means of particle number concentrations were computed for three ultrafine size ranges: 0.01-0.03 µm, 0.03-0.05 µm, and 0.05-0.10 µm. Mean daily mass concentrations were calculated for a size range from 0.01 to 2.5 µm (PM2.5) assuming spherical particles of an apparent mean density of 1.5 g cm-3. Death certificates were obtained from local health authorities and aggregated to daily total counts. The data were analyzed using Poisson regression techniques in Generalized Additive Models. We adjusted for trend, seasonality, influenza epidemics, and meteorology using penalized splines and for day of week using an indicator variable. The decrease of FP mass concentrations during the study period is 37% compared to 28% for UFP number concentrations (table 1). The regression results for the whole study period (1995-2001) again show a borderline significant increase in the number of daily deaths in association with increased UFP number concentrations, delayed with a lag of 4 days (table 1). This seems to be mainly due to the associations observed from 1995-98. Positive associations of FP mass concentrations with daily mortality were neither observed during the whole study period nor during the period from 1999-2001. In a polynomial distributed lag model for 1995-2001 a statistically significant increase in the number of daily deaths of 3.7 % (95% CI: 0.9-6.5%) was observed in association with increased UFP number concentrations combined from lag 0 to lag 4 days. This analysis shows that the magnitude of associations of particle measures with daily mortality may change with changing air pollution levels.

Table 1: Regression results for UFP number and FP mass concentrations. RR=relative risk per interquartile range, CI=confidence interval. Sep 1995-Dec 98 Jan 1999-Aug 2001 1995-2001 mean 5-95 %

percentile Mean 5-95 %

percentile Mean 5-95 %

percentile

UFP concentration (cm-3) 15329 4284-36228 11051 4107-23160 13488 4217-31246 FP concentration (µg m-3) 26.7 6.6-71.6 16.8 5.1-38.6 22.3 5.9-62.3

RR 95 % CI RR 95 % CI RR 95 % CI UFP, lag 4 days 1.045 0.996-1.096 1.002 0.960-1.046 1.025 0.999-1.053 FP, lag 0 1.030 0.999-1.062 0.966 0.930-1.003 1.006 0.983-1.029

52


L-14

A NOVEL TECHNIQUE FOR CONDUCTING IN-VIVO AND IN-VITRO EXPOSURES WITH CONCENTRATED AMBIENT ULTRAFINE PARTICLES

Tarun Gupta, Philip Demokritou and Petros Koutrakis Exposure Epidemiology and Risk Program, School of Public Health, Harvard University, Boston,

Massachusetts, USA.

Several epidemiological studies have consistently shown combustion-related particles as being causally linked to increased morbidity and mortality. Also, recent toxicological studies have suggested that ultrafine particles may play an important role in explaining the underlying biological mechanism for the adverse health effects from particulate matter. Artificial particles used in previous toxicological studies do not adequately represent the heterogeneous mixture of ambient particles, as the components of ambient particles may synergistically interact to produce toxic health effects, not seen with artificially generated atmospheres. Hence, recent toxicological studies employ ultrafine particle concentrators to conduct in-vivo exposures using real ambient aerosol.

In an ultrafine particle concentrator, ambient ultrafine particles are condensationally grown to super-micrometer sizes and then are concentrated using a two-stage virtual impaction technology. Subsequently, original ultrafine particle size distribution is restored, using a thermal method. The performance of the Harvard ultrafine concentrated ambient particle system (HUCAPS) was tested by challenging it with a variety of artificially generated aerosols of different chemistry, solubility, and hygroscopicity as well as by ambient origin ultrafine particles. The HUCAPS ability to concentrate these different particles was evaluated for a wide range of supersaturation ratios.

The hygroscopic behavior of atmospheric aerosols is a complex function of chemical composition and surface properties of individual particles and it considerably affects the final droplet size to which particles grow and hence the overall concentration enrichment factors. It was found that hygroscopic particles grow and are concentrated more efficiently than hydrophobic ones. The effect of aerosol-hygroscopicity and water solubility on the overall aerosol concentration enrichment factor diminishes with increasing supersaturation ratio. Results indicated that a supersaturation ratio higher than that predicted by theory, with a value of about 3, is needed to assure that all particles grow and efficiently concentrated by the same enrichment factor, regardless of particle hygroscopicity and solubility. This is particularly important because, typically about 50-70 % of atmospheric ultrafine mass consists of hydrophobic carbonaceous materials. Under such an optimum supersaturation ratio, the performance of the HUCAPS was evaluated using ambient origin ultrafine particles. The results show that the HUCAPS concentrates ambient ultrafine particles by a factor of 40-50 with insignificant size distortion. Also, operating HUCAPS for long durations of time tested the stability of the overall system and the concentration enrichment factor remained relatively constant throughout this time period (< 10 % variation). Its overall low pressure drop (2.2 kPa), an output flow of 58 LPM, and its high concentration enrichment factor, presents HUCAPS as a versatile device suitable for in-vitro and in-vivo inhalation toxicological studies.

53


L-15

TOXICOLOGICAL STUDIES ON NANOPARTICLES IN CHINA

Yuliang Zhao 1,2, Gengmei Xing 1, Chunying Chen 1, Guang Jia 1,3, Haifang Wang 2, Yufang Liu 2, Zhifang Chai 1

1 Lab for Bio-Environmental Health Sciences of Nanoscale Materials, Institute of High Energy Physics, The Chinese Academy of Sciences, Beijing 100049, China, 2 College of Chemistry and

Molecular Engineering, Peking University, Beijing 100871, China, 3 Department of Occupational and Environmental Health, Peking University, Beijing 100083, China

We summarize the experimental results of the recent studies on biological activities and toxicological effects of nanoparticles (carried out in Lab. for Bio-Environmental Health Sciences of Nanoscale Materials, China). As nanoparticles of unique physical and chemical properties that are clearly different from the micro-sized particulates may lead to unpredictable distribution and effects within biological systems, the Chinese Academy of Sciences has established the Nanosafety Lab studying biological activities /effects of nanoparticles, such as recognization, identification and quantification of hazards resulting from exposure to nanoparticles and the public health aspects of nanoparticles in air, water, other parts of the environment, foods, and nanodrugs.

In the presentation, we will discuss the nuclear analytical techniques of high sensitivity developed for detection and quantification of nanoscale particles in biological systems; the cytotoxicity test protocol for studying the interaction between carbon nanoparticles and living cells; the real-time and on-line techniques to visualize the activities of magnetic nanoparticles in vivo, etc. We are studying not only the toxicological effects of nanoparticles but also their reverse-utilization in clinic diagnoses and therapy by assimilating knowledge and techniques of nanoscience, medicine, life sciences, chemistry and physics, etc.

54


L-16

COPING WITH PARTICULATE EMISSIONS BY TRAFFIC MANAGEMENT - POSSIBILITIES AND CONSTRAINTS

Jens Borken, Astrid Gühnemann und Reinhart Kühne DLR, Institute of Transportation Research, Rutherfordstr. 2, 12489 Berlin, Fax: +49-30-67055-

202, [email protected]

An important way to reduce particulate emissions from road vehicles is of course to limit their creation and emission, be it by tuning the combustion conditions or the exhaust. However, a complementary strategy to limit the both emission and impact can be traffic management. First, traffic management has the potential to increase the smoothness of the traffic and hence to offer speed-acceleration conditions more favourable to a complete combustion in the engine itself. Second, vehicle specific traffic management, e.g. sensitive to the emission level – which could even be coupled to on-board diagnosis systems – could selectively select high emitting vehicles, responsible for a disproportionately high share. The third and additive possibility would be, accepting the emissions, to direct the main part of the traffic along routes such that the exposure is contained as much as possible, such that the impact is minimal, or where the ventilation and dilution is maximal. Technically, these possibilities exist and e.g. combined with routing information to the vehicle drivers in exchange, the measures could also find acceptance. However the immediate impact of these measures need not be over-estimated, given the high background levels. Nonetheless, exactly because quick successes do not seem at hand, all and particularly fast and cost effective measures should be explored. Furthermore, dynamic traffic management not only offers the possibility of a particle sensitive guidance, but to integrate the air pollutants into a whole-scale strategy to reduce the transport related health risks in urban areas while maintaining mobility as much as possible. And last but not least, dynamic traffic charging would offer the real possibility to finance reducing emissions at the source, wherever this might be most efficient. The goal of our research is to design an intelligent decision support system that accomplishes this task. For this purpose, we combine dynamic microscopic traffic simulation tools with an assessment model in order to evaluate environmental as well as economic impacts of new management strategies and to generate optimal solution for a desired state of transport system and environment.

55


P-6

ULTRAFINE CARBON PARTICLES INCREASE THE HEART RATE

R. Radykewicz 1., V. Harde r .2, H. Schulz 2, A Stampfl 1

GSF – National Research Center for Environment and Health, 1) Institute for Toxicology, 2) Institute of Inhalation Biology, Neuherberg/Munich, Germany, [email protected], FAX: 089/31873449

Epidemiological studies have shown that air pollution in particular ultrafine particles (UFP; diameter less than 100 nm) can cause diseases especially in the cardiovascular system.

It is still unknown if these impacts have a systemic background as inflammatory or metabolic effects or if they are evoked by a direct action on the heart itself.

Air pollution consists of many different UFP with a wide range of physical and chemical properties. The aim of our investigation was to analyze the influence of UFP on the heart without systemic interactions. For our studies on isolated perfused hearts of guinea-pigs we used Printex 90 (black carbon) with a nominal diameter of 14 nm as a model particle because it has a low oxidative potential.

However, the particles agglomerate in Krebs Henseleit Buffer (KHB). By adding 1% bovine serum albumin to KHB, sonication and filtration we had the ability to prepare solutions with particle and agglomeration sizes < 100 nm diameter. To verify the quality of our particle suspensions we used a Malvern HPPS particle sizer.

At first the isolated heart was eluted with KHB for one hour to get rid of all stress hormones, remains of blood or contaminations gained from the preparation. Then the KHB was recirculated. After reaching a stable state of heart frequency we perfused the heart with KHB containing UFP for one hour and for the following hour we perfused it with the doubled concentration of UFP. After that the particles were washed out with normal KHB. During the whole experiment we recorded the ECG signal, blood pressure and the flow rate of our perfusion solutions.

We found changes in ECG and an increase of heart frequency by 8% at 8*108 UFP/ml and by 10% at 1.6*109 UFP/ml (n=5). During the 30 min wash out period the beating rate decreased nearly to the rate before adding UFP.

Further investigations are necessary to clarify the mechanisms and elucidate if chemical properties of particles evoke these effects.

56


P-7

CARDIOVASCULAR CONDITIONS AND AIR POLLUTION, IN PISA, ITALY, 1998-2003.

Maria Angela Vigotti ,1 Clara Carpeggiani 2

1 Dip.Scienze Uomo e Ambiente, Universita’ di Pisa, Italy; 2 IFC, CNR. Pisa Italy.

Various epidemiological studies have demonstrated an increase of cardiovascular events in relation to air pollution levels in urban areas. Since many years the CNR Institute of Clinical Physiology of Pisa have organized a database containing major clinical parameters of all patient admitted to the cardiological Department of the Institute. Moreover Pisa has been included in the national study on short term effects of air pollution on health (MISA-2), so a daily data set of pollutants levels from 1998 up to 2003 have been organized and validated for this study. We have started a study in order to evaluate the possible association among air pollution levels and clinical parameters collected the day of admission to the hospital in cardiac patients resident in town. Pollutant data come from the local regional network (ARPA-T) of monitoring stations located in town and daily values have been computed after a standardized choice of the stations. Meteorological data come from Pisa Airport.

The association will be considered using the case-crossover design.

We are also considering the possibility to extract data from patients, resident in Pisa, with implanted defibrillators and to evaluate the associations between defibrillator discharge interventions and air pollutant levels. The study is on a very preliminary phase and it is supposed to last for at least one year, after which the possibility of a prospective study will be considered.

57

Tuesday, 3 May 2005


59


K-5

CURRENT HYPOTHESES ON THE MECHANISMS OF TOXICITY OF ULTRAFINE PARTICLES

Ken Donaldson University of Edinburgh, Edinburgh, Scotland, e-mail: [email protected], Fax: 0131 651

1606

Combustion has been recognised as a potential source of harmful gaseous and particulate matter (PM) air pollution. Epidemiological studies do not readily allow associations of adverse effects with sub-components of PM, dependent as they usually are on mass measures of PM. However several epidemiological studies have been able to identify combustion-derived particles as an important component in driving adverse effects of PM. Toxicology can more readily study the components of PM and there has been considerable amount of research on the combustion-derived particles component. The talk will focus on this toxicology and describe how primary combustion-derived nanoparticles (PCDNP) produced in a range of situations, produce adverse health effects. Nanoparticles have the ability to cause inflammation and also, in the case of insoluble PCDNP, they have potential to escape from the site of deposition in the lungs and translocate to the blood and to other target organs. The exemplar PCDNP discussed will include welding fume and nanoparticulate carbon black, which are both occupational hazards, coal fly-ash which is an environmental hazard and diesel soot which is both an environmental and an occupational hazard. The large surface area of PCDNP maximises dissolution of soluble species in the lungs For insoluble NP, the large surface area provides a surface on which catalytic chemistry can occur that favours the formation of free radicals. These free radicals are responsible for driving oxidative stress, the underlying mechanism that promotes an inflammatory response to PCDNP. Large surface areas in the lungs has been shown to correlate well with the ability of a range of low toxicity, low solubility nanoparticles to induce inflammation in the rat lung. PCDNP may be soluble and release transition metals as their primary pro-inflammatory mechanism or may be insoluble and cause inflammation because of their surfaces; some types of PCDNP have both soluble components and an insoluble core. Exposure to PCDNP of various types is associated with a range of adverse health effects including fibrosis, bronchitis, metal fume fever, airways disease and cancer. These endpoints are found across a number of exposure conditions and to different kinds of PCDNP. The ability of PCDNP and their associated metals to translocate to the blood and the brain will be discussed. These unusual toxic properties unite these materials and suggest that they can usefully be seen as a group of particulate lung toxins that act through similar pathways.

60


L-17

NANOMEDICINE AND NANOTOXICOLOGY – APPLICATIONS AND IMPLICATIONS

Günther Oberdörster University of Rochester, Dept. of Environmental Medicine Rochester, NY, USA, e-mail:

[email protected]

Nanotechnology is defined as research and technology development at the atomic, molecular or macromolecular levels, in the length scale of ~1-100 nm range. One of the many promising applications of engineered nanoparticles (NP) is in the area of medicine, for example, targeted drug delivery as aerosols and to tissues which are difficult to reach. The discipline of nanomedicine has arisen to develop, test, and optimize these applications. However, the same properties that make NP attractive for development in nanomedicine and for specific industrial processes could also prove deleterious when NP interact with cells. An emerging discipline – nanotoxicology, which can be defined as safety evaluation of engineered nanostructures and nanodevices, is gaining increased attention. Nanotoxicology research will not only provide information for risk assessment of NP based on data for hazard identification, dose response relationships and biokinetics but will also help to advance further the field of nanomedicine by providing information to alter undesirable NP properties. This presentation summarizes results of studies with nano-sized combustion derived and engineered nanoparticles, with a focus on the respiratory tract and the skin as portals-of-entry. Results demonstrate that their potential to induce toxicity, as well as their desirable properties, which makes them attractive as medicinal aerosols, depend not only on their size but on a variety of surface properties. To establish the principles which govern nanoparticle-cell interactions will be a major challenge for the fields of nanomedicine and nanotoxicology, requiring a well coordinated cross-disciplinary approach.

61


L-18

THE SPECIFIC SURFACE AREA OF CARBONACEOUS ULTRAFINE PARTICLES DETERMINES GENERATION OF LIPID MEDIATORS AND OXIDATIVE STRESS IN ALVEOLAR MACROPHAGES.

I. Beck-Speier, . N Dayal, E. Karg, K.L. Maier, M. Semmler, S. Takenaka, K. Stettmaier a, W. Bors a, A. Ghio b, . J.M Samet b, H. Schulz

GSF-Research Center for Environment and Health, Institute for Inhalation Biology, aInstitute for Radiation Biology, D-85758 Neuherberg/Munich, Germany, bEnvironmental Protection Agency,

Chapel Hill, USA. E-mail: [email protected]; Fax number: 089/31872809

Acute exposure to inhaled ambient particles is found to be associated with adverse health effects worldwide. Ultrafine particles (UFP) represent a special fraction of the ambient aerosol, because they are very small in diameter (< 0.1 µm), occur in high number concentrations and possess very large specific surface areas. Together with their agglomerates, they are considered as important factors contributing to adverse health effects including pulmonary and cardiovascular diseases. However, the molecular mechanisms underlying these particle-induced effects are still not well known.

The reactivity of ultrafine carbonaceous particles such as agglomerated UFP of carbon (EC), Printex 90, Printex G and diesel exhaust particles (DEP; standard reference material 1650a) was investigated in i) a cell-free in vitro system to determine their capacity to oxidize methionine as measurement of their innate oxidative potential, and ii) a cellular system consisting of canine and human alveolar macrophages to evaluate their effect on synthesis of lipid mediators such as arachidonic acid (AA), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4), generation of reactive oxygen species (ROS) and induction of oxidative stress marker 8-isoprostane.

In the cell-free system EC exhibited the highest oxidative potential among the particles and showed an ESR signal for an organic carbon-centered free radical. In the cellular system, EC with the highest oxidative potential induced synthesis of AA, PGE2 and LTB4, and produced ROS and 8-isoprostane (P<0.05). Printex 90, Printex G and DEP with weak oxidative capacities enhanced generation of AA, PGE2 and ROS (P<0.05) but not that of LTB4 and 8-isoprostane. EC, aged for 24 h by incubation in water, exhibited a drastic loss of its oxidative potential and a significant reduction of its cellular induced LTB4 and 8-isoprostane levels, whereas cellular AA and PGE2 formation were still elevated (P<0.05).

In conclusion, all types of particles studied were able to activate formation of AA, PGE2 and ROS independent of their oxidative capacity. However, induction of cellular LTB4 formation and oxidative stress, indicated by 8-isoprostane, were strongly dependent on the oxidative potential of the particles. The synthesis of PGE2 and AA correlated with the particulate specific surface area (r = 0.99) but not with particle mass. Both, the particulate innate oxidative potential (r = 0.94) and the particle-induced cellular oxidative stress indicated by 8-isoprostane (r = 0.99) correlated also with the surface area. This indicates that the particulate surface area is an important parameter for the biologic reactivity of the particles.

62


L-19

DIESEL SOOT EXPOSURE MODULATES FUNCTIONAL MATURATION OF BONE MARROW-DERIVED DENDRITIC CELLS

Yvonne Gruijthuijsen, Ines Grieshuber, Claudia Reitinger, Jutta Horejs-Höck, and Albert Duschl Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria, Tel: +43 662 8044 5731, Fax: +43 662 8044

5751, Email: [email protected]

Exposure to environmental pollutants, such as diesel exhaust particles, has been associated with the establishment of dysfunctional immune responses. One of the main goals of the European 5th framework MAAPHRI (Multidisciplinary Approaches to Airborne Pollutant Health-Related Issues) project is to evaluate the immunomodulatory potential of diesel soot and exhaust, by analysing the effects of diesel particle exposure on cytokine secretion patterns in various cell lines. In addition, the MAAPHRI-project focusses on the effects of diesel particle exposure of primary cells which are key regulators of the immune system.

Dendritic cells (DCs) tightly orchestrate immune responses. Due to their presence at exposed locations such as the lungs, and their phagocytic potential, immature DCs (iDCs) get loaded with high levels of particles. Therefore, the effects of diesel soot exposure on the functional maturation of iDCs to mature DCs (mDCs) were investigated in more detail. Diesel soot was generated by using a typical on-road European diesel car engine, with Euro3-compliant fuel and regular semi-synthetic oil. Particles were collected with a silicone carbide monolith particle trap and counterblowed with clean compressed air. Murine iDCs were generated by culturing of Balb/c mice-derived bone marrow (BM) cells with GM-CSF for 9 days, in absence or presence of the diesel soot. Subsequently, iDCs were matured with the biological stressor lipopolysacharid (LPS).

Both in absence and presence of diesel soot, BM-precursor cells efficiently differentiated into iDCs (CD11c+ cells). In contrast to non-exposed iDCs however, soot-exposed iDCs were unable to upregulate maturation-associated cell surface markers (MHCII, CD40, CD80, CD86) in response to LPS. Moreover, soot-exposed DCs were impaired in their functional responses as demonstrated by a dramatic reduction in LPS-induced cytokine secretion (IL-10, TNFα, IL-6) and NO production. Additionally, diesel soot exposure of naive iDCs resulted in the formation of oxygen radicals. Taken together, these data clearly show that diesel soot does not only induce cellular stress in exposed iDCs, but also impairs the functional maturation of these cells to mDCs in response to biological stressors. It is therefore very likely that diesel soot exposure may have severe consequences for the proper induction and regulation of immune reponses. These studies concerning the immunomodulatory effects of diesel soots and other particle mixtures are at present continued within the European 6t framework NOMIRACLE (Novel Methods for Integrated Risk Assessment of Cumulative Stressors in Europe) project.

63


L-20

SPECIFIC SURFACE AREA ABOVE A CERTAIN THRESHOLD DETERMINES THE ACUTE INFLAMMATORY RESPOND OF INSTILLATED CARBONACEOUS NANOPARTICLES IN MICE

Tobias Stoeger, l C audia Reinhard, Shinji Takenaka, Andreas Schroeppel, Erwin Karg, Baerbel Ritter, Joachim Heyder, and Holger Schulz

GSF - National Research Center for Environment and Health, Institute of Inhalation Biology, Ingolstaedter Landstrasse 1, D-85764 Muenchen-Neuherberg (Germany), mailto:

[email protected], Fax: +49 89 3187-2400

Ambient fine-mode particulate matter mainly consists of carbonaceous particles derived from combustion processes. In fact the fraction of smaller sized ambient particles contributes less to mass but high to particle number concentrations and is characterized by a highest specific surface area. Current epidemiological and toxicological research suggests the sub-100-nm nanoparticles to be more harmful per unit mass than larger particles. Nanoparticles are found in particular to deposit efficiently in the respiratory tract, translocate efficiently into the interstitium and largely evade macrophage clearance.

In the current study we aimed a quantitative comparison of different carbonaceous soot particles, characterized by different physical/chemical properties, in their potency to cause acute inflammatory effects in the lung of healthy Balb/cJ mice. Six different particles with a primary particle size from 10 to 50 nm, a organic mass content from 1 to 20 % and a specific BET surface of 30 to 800 m2/g were used: two commercial pigments, PrintexG and Printex90, two laboratory made flame soot particles with different organic content (Soot-L/H), one spark generated soot (ufCP) and the reference diesel exhaust SRM1650a. To enable a confrontation of the particle specific dose-respond relationships, 3 different doses (5, 20 and 50µg) were instilled. 24h after instillation bronchoalveolar lavage was performed and markers of cytotoxicity, the influx of inflammatory cells and proinflammatory mediators were quantified.

At respective mass-doses, particle caused detrimental effects could be ranked in the following order: ufCP > SootL = SootH > Printex90 > PrintexG > SRM1650a. Spark generated ufCPs at 20µg caused for all investigated endpoints higher levels of inflammation than 50µg SRM1650a or PrintexG, demonstrating that particle mass by its one is only a weak predictor for acute inflammation. Surprisingly a similar weak relation turned out for the particle characteristic organic content (OC). Even for the close related flame soot particles, a lower inflammatory respond was caused by soot-H (OC: 19%) compared to soot-L (OC: 7%). Additional heavily with OC loaded (OC: 20%) diesel exhaust showed a weaker inflammatory respond, than the Printex particles with less than 2% OC. Otherwise primary particle size showed a strong relation to the particle caused detrimental effects, with smallest particles causing the highest inflammatory effects. In agree to that, the best correlation (r = 0.9) was found between the surface area of the instilled particles and the generated inflammatory respond. No particle related inflammatory effect could be detected below the instilled surface threshold dose of 20cm2.

64


L-21

DETECTION OF CYTOTOXIC AND INFLAMMATORY EFFECTS BY MEANS OF STABLY TRANSFECTED LUNG CELLS

C. E Hellweg. 1, C. Schmitz1, A. Arenz 1, . r C Baumstark-Khan 1, H.H. Grothee 2

1) DLR, Institut für Luft- und Raumfahrtmedizin, Strahlenbiologie, Köln; 2 DLR, Institut für Verbrennungstechnik, Stuttgart; Germany ([email protected], Fax: +49-2203-61970)

High levels of ambient air pollution are associated with aggravation of asthma, respiratory morbidity, and cardiopulmonary morbidity; long-term exposures to PM have been linked to possible increases in lung cancer risk, chronic respiratory disease, and death rates. The effects of the smallest of these particles on human and animal health are not yet understood. Nanoparticles or nanosized organic carbon (NOC) are generated by combustion processes as soot precursors. These are transparent and made up of clusters of polyaromatic structures with mass numbers ranging from 1 000 to 40 000 amu (ø 1.5 to 5 nm). Nanoparticle toxicity displays a special risk for humans, notably as these respirable and water soluble particles can penetrate lung mucosa. Cell response to different kinds of environmental challenge is complex and involves the participation of different classes of genes for different cellular outcomes (DNA repair, cell cycle control, signal transduction, inflammation, apoptosis, necrosis and oncogenesis). Cytoxicity and inflammatory effects of NOC were examined by mammalian cellular bioassays developed by the Cellular Biodiagnostics group at the German Aerospace Center (DLR). Cytotoxic effects of condensation water samples collected after combustion of gaseous fuels (propane, ethylene) under laboratory conditions were determined by monitoring growth of the lung carcinoma cell line A549. For this purpose, the A549 cells were stably transfected with a plasmid carrying the gene for Enhanced Green Fluorescent Protein (EGFP) under control of the strong constitutive CMV promoter. A clone with strong EGFP expression was selected for cytotoxicity tests. Incubation of these stably transfected cells with the condensation water samples resulted in complete growth inhibition in dilution ratios from 1:5 to 1:160, showing high cytotoxicity in this concentration range for all samples. Due to the autofluorescence of the samples in the green range of the spectrum overlapping with EGFP fluorescence, untransfected A549 cells incubated with the condensation water samples were used for background fluorescence correction. A sample of polynuclear aromatic hydrocarbons showed only minor effects on cell growth. In-vitro experiments have revealed that diesel exhaust and PM10 are capable to induce the release of proinflammatory cytokines such as interleukin (IL)-6 and IL-8 from bronchial epithelial cells mediated by the trans-cription factor Nuclear Factor κB (NF-κB). Thus, an important aspect of this work was to determine whether NOC causes activation of NF-κB in lung epithelial cells. In the past, monitoring of NF-κB activation by a NF-κB-responsive plasmid stably transfected into a human embryonic kidney cell line (293) has been shown to be useful for several cellular stressors. A549 cells were stably transfected with this vector carrying EGFP under control of a synthetic promoter containing four copies of the NF-κB response element. The successful integration of the plasmid in several clones was examined by treating the clones with the known NF-κB activator tumor necrosis factor α. Cells with activated NF-κB showed an increase in the expression of EGFP which was quantified by flow cytometry. The positive clones showed NF-κB activation also after treatment with IL-1β. In future, the inflammatory potential of NOC will be tested using this stable transfected cell line.

65


P-8

CYTO- AND GENOTOXIC EFFECTS OF NATURAL AND SURFACE-TREATED ULTRAFINE TITANIUM DIOXIDE PARTICLES IN MAMMALIAN CELLS

E. Dopp 1; H. Crame r .2; K. Bhattacharya 1,3; S Yadav 3; S. Geh 1;T. Shi 4; B. Shokouh 1

1 Institute of Hygiene and Occupational Medicine, University Hospital, Hufelandstrasse 55, 45122 Essen, Germany ([email protected], Fax: 0049 201 723 4546); 2 Institute of Environmental

Process Engineering and Plant Design, University of Duisburg-Essen, Germany; ³ Industrial Toxicology Research Centre, Division of Fibre Toxicology, 226 001 Lucknow, India; 4 Institute of

Environmental Health Research (IUF), Düsseldorf, Germany

Titanium dioxide (TiO2) is a naturally occuring mineral found in the form of Rutile, Anatase, Brookite and as the iron containing mineral Ilmenite (FeTiO3). TiO2 is widely used in various industries such as cosmetics and paints, while surface treated TiO2-particles with vanadium pentoxide (V2O5) are used more often commercially for selective catalytic reactions such as the removal of nitrous oxide from exhaust gases of combustion power plants by addition of ammonia (SCR-Process). Anatas particles with a specific area of approx. 70m2/g are coated for this purpose with catalytic active components such as V2O5. In the present study, we have tested untreated ultrafine TiO2-particles (Anatas, size: 30-50 nm) and vanadium pentoxide (V2O5) treated TiO2-particles for their biological effects in V79 cells (hamster lung fibroblasts). Cytotoxic effects of the particles were assessed by trypan blue extrusion test while, genotoxic effects were investigated by micronucleus assay. Additionally, we measured the generation of reactive oxygen species (ROS) by the acellular method of electron spin resonance technique (ESR) and by the cellular technique of determination of thiobarbituric acid-reactive substances (TBARS). Our results demonstrate that V2O5-Anatas induces stronger cyto- and genotoxic effects than untreated Anatas. Also, the acellular and the cellular radical formation are more pronounced with V2O5-Anatas than with untreated Anatas. Thus, it can be concluded that V2O5-treated Anatas was more cyto- and genotoxic than natural Anatas and was capable of inducing DNA-damage through production of free radicals. Thus, it may be recommended that commercial use of ultrafine V2O5- treated Anatas particles should be handled properly and with utmost care. Also, we have to consider that ultrafine particles may exert their genotoxic effects in serveral organs of the body because of their ability to penetrate membranes and to accumulate in the body.

66


P-9

INVOLVEMENT OF AMPHIREGULIN IN AIRWAY EPITHELIAL CELL RESPONSES TO URBAN PARTICULATE MATTER

Mélina Rumelhard, Kiran Ramgolam, Francelyne Marano, Armelle Baeza Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris7, Denis Diderot, case

7073, 2 place jussieu, 75 251 Paris cedex 05, France, [email protected], Fax : 33 1 44 27 69 99

Exposure to ambient particulate matter (PM) is known to be associated with airway inflammation, and chronic exposure may also induce airway remodelling (Churg et al., EHP, 2003). Bronchial epithelial cells respond to PM by increased production of pro-inflammatory cytokines such as GM-CSF and a ligand of the epidermal growth factor receptor (EGFR), amphiregulin. The aim of this study was to determine whether amphiregulin is involved in pro-inflammatory and proliferation responses induced by urban particulate matter.

For this purpose human bronchial epithelial cells (16HBE cell line) and primary human nasal epithelial cells were exposed to Paris PM2.5. Fifty percent of these particles have a diameter below 260 nm (Baulig et al., EST, 2005).

PM2.5 induced amphiregulin mRNA expression (measured by macroarray and RT-PCR) and a dose-dependent amphiregulin secretion (measured by Elisa) by epithelial cells (16HBE cells and primary cells) treated for 24hrs. Human recombinant amphiregulin used from 5 to 50 ng/mL (a concentration found in the culture medium of PM-treated 16HBE cells), induced a dose-dependent release of GM-CSF. The PM2.5-induced GM-CSF secretion was repressed in the presence of an inhibitor of EGFR tyrosine kinase (AG1478, 1µM) and attenuated in the presence of a neutralizing anti-amphiregulin antibody (5 µg/mL), suggesting the involvement of EGFR activation by amphiregulin in the GM-CSF release.

Furthermore PM2.5 induced 16HBE proliferation assessed by the increased incorporation of 3H thymidine and the increase of cell number after 48h of exposure. This mitogenic effect was abrogated by AG1478.

To conclude, we showed that amphiregulin secretion induced by PM2.5 in epithelial cells contributes to GM-CSF release and could be involved in the PM2.5 mitogenic effect. These two amphiregulin-mediated responses may reflect an important mechanism for PM induced airway remodelling.

This work was supported by Renault, ADEME, INSERM and PRIMEQUAL

67


P-10

EFFECT OF URBAN PARTICULATE MATTER ON AIRWAY EPITHELIAL CELLS IN VITRO: ROLE OF PARTICLE COMPOSITION AND SIZE

Kiran Ramgolam, Mélina Rumelhard, Augustin Baulig, Laurent Martinon *, Servanne Chevaillier *, Francelyne Marano, A melle Baeza r

Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris7, Denis Diderot, case 7073, 2 place jussieu, 75 251 Paris cédex 05, *LEPI, 7 rue George Eastman, 75013 Paris.

[email protected], Fax : 33 1 44 27 69 99

Epidemiological investigations have associated exposure to ambient particulate matter (PM) to adverse respiratory effects such as airway inflammation and remodeling. PM is a complex mixture of particles that in urban areas contains nanoparticles produced by combustion.

Using in vitro models of airway epithelial cells we have previously shown that PM2.5 and DEP (Diesel exhaust particles) induce a proinflammatory response characterized by the release of the GM-CSF cytokine and the amphiregulin EGFR ligand (Boland et al. AJP, 2000, Blanchet et al., AJRCMB, 2004).

The aim of the present study was to investigate which particle component is responsible for this proinflammatory response and whether this response differs according to particle size.

For this purpose we used (1) Paris PM2.5 sampled in a kerbside site in which 50% of the particles have a diameter ≤ 260nm. The content of metals and polycyclic aromatic hydrocarbons has been characterized (Baulig et al, Environ. Sci. Technol, 2005) and the contribution of the different components has been analysed by testing either the native particles or their organic or aqueous extracts. (2) Paris size-fractionated PM sampled with a 13 stages low pressure impactor (10 µm to 0.03 µm) in an urban background site. Three size classes (PM1-2.5, PM0.1-1, PM0.03-0.1) were tested for their biological reactivity towards airway epithelial cells.

PM2.5 (from 1 to 30 µg/cm²) induces after 24 h of exposure a dose-dependent GM-CSF and amphiregulin release in both human bronchial (16HBE cell line) and nasal (primary cultures) epithelial cells. The epithelial secretions induced by native PM are as important as those induced by washed PM (PM devoid of hydrosoluble compounds) but lower than those induced by PM-organic extract (used at the concentration they are on native PM). By contrast PM-aqueous extract and carbon black particles (Ø 95 nm) have no effect suggesting that soluble metals and the carbonaceous core are not involved in these biological responses. Preliminary experiments performed with size-fractionated PM revealed that GM-CSF secretion is principally induced by the smallest PM whereas the amphiregulin secretion is mainly induced by PM1-2.5.

To conclude, chemical fractionation of PM2.5 permitted us to demonstrate a strong implication of organic compounds in the proinflammatory response of airway epithelial cells. In addition size fractionation of PM showed that the two exposure biomarkers studied were differentially induced according to particles size. PM physico-chemical characteristics and the involved cellular signaling pathways are under investigations.

68


P-11

FLOW CYTOMETRIC CHARACTERIZATION OF ANTIGEN-PRESENTING DENDRITIC CELLS AFTER IN VITRO EXPOSURE TO DIESEL EXHAUST PARTICLES

S. Verstraelen, R. Van Den Heuvel, I. Nelissen, H. Witters, G. Verheyen, G. Schoeters Vito, Centre of Expertise for Environmental Toxicology, Mol, Belgium, E-mail:

[email protected], Fax: ++32 14 58 26 57

Exposure to particulate matter air pollution has been associated with damage to the immune and respiratory system and subsequently with increased incidence of respiratory health effects, such as asthma. Pulmonary dendritic cells are professional antigen-presenting cells that recently have been implicated in allergic diseases of the respiratory tract.

In this study more insight was obtained into the effect of diesel exhaust particles (DEP) on the activation and maturation of primary human dendritic cells. Monocyte-derived dendritic cells (Mo-DC) from seven different donors were exposed to different DEP concentrations (0.2, 2, 20, 200 and 2000 ng/ml) in the absence or presence of lipopolysaccharide (LPS). Changes in the cell surface expression of HLA-DR, CD86 and CD83 were examined.

Exposure of Mo-DC to DEP alone could not alter the expression levels of any of the markers. Treatment with LPS alone upregulated the expression levels of all three surface markers, although the levels were not significantly different compared to untreated DC. The LPS-induced marker expression was further enhanced by co-stimulation of the cells with DEP. Statistical significantly increased levels of CD83 expression were observed after exposure to 0.2 (p = 0.018), 20 (p = 0.010) and 200 ng/ml (p = 0.047) DEP combined with LPS.

It is concluded that DEP synergize with LPS in inducing maturation of Mo-DC.

69

Wesnesday, 4 May 2005


71


K-6

TRANSLOCATION AND KINETICS OF NANOPARTICLES: IMPLICATIONS FOR RISK ASSESSMENT

Paul J.A Borm. , James Justus Meiring Centre of Expertise in Life Sciences (CEL), Hogeschool Zuyd, Heerlen (NL), and IUF at the

University of Düsseldorf (Germany)

Nanotechnology uses components that are smaller than 100 nm in a wide variety of applications. In this size range materials elicit unique properties that allow or forward application in medical diagnostics, drug delivery, filters, membranes, electronics, optics and many others. Common tools in these applications are engineered nanoparticles (NP) and nanotubes (NT). The current discussion on risks of NP and NT is corroborated by the small-data base on toxicological hazards, generated with a confined set of bulk (TiO2, CB) and combustion (diesel) NP (Colvin, 2004; Borm & Kreyling, 2004). A conceptual understanding of the properties of NP that are crucial in toxicity is lacking, including the meaning of their translocation after uptake and distribution to secondary organs. Upon inhalation NP can translocate from the nose to the olfactory bulb in the brain, and through the pulmonary interstitium to the blood stream. Drug delivery has engineered NP to carry drugs through the blood-brain barrier (Kreuter, 2004). Upon dermal exposures NP do not seem to cross the upper horny layer, although the published data set is small, and no studies have been reported in diseased skin. Gastro-intestinal uptake and storage of NP occurs readily through the Peyer’s patches, and is actually a pathway used to increase bioavailability of food constituents in nutraceuticals. Finally, NP are being used for intravenous administration in drug delivery systems to reach specific targets for drug treatment. A central question is under which conditions this translocation occurs and which disease conditions can affect their uptake and distribution, affect organ homeostasis and eventually cause toxicity. In this presentation the author will review the data sets that have generated this know-how and try to define the particle properties and potential mechanisms that play a role.

1. Borm PJA & Kreyling W (2004) Toxicological hazards of inhaled Nanoparticles- potential implications for drug delivery, J. Nanosci. Nanotech. 4(5) 521-531.

2. Colvin V (2003) The potential environmental impact of engineered nanomaterials. Nature Biotechnology 21 (10) 1166-1170.

3. Kreuter J (2004) Influence of the surface properties on nanoparticle-mediated transport of drugs to the brain, J. Nanosci. Nanotechnol. 4(5) 484-488.

72


L-22

SIGNALLING EVENTS RELEVANT FOR APOPTOSIS AND PROLIFERATION INDUCED BY NANOPARTICLES IN RAT LUNG EPITHELIAL CELLS

K. Unfried 1, U. Sydlik 1, K. Bierhals 1, R. Duffin 2, C. Albrecht 1, and R. Schins 1

1 Institut für umweltmedizinische Forschung (IUF), Düsseldorf, Germany; 2 ELEGI/Colt Laboratories, Centre for Inflammation Research, Medical School, University of Edinburgh, UK.

Inhalation of environmental nanoparticles is associated with adverse health effects due to the deregulation of the cell cycle resulting in altered cell proliferation, apoptosis, and senescence. Besides indirect inflammatory effects which are mainly mediated by the activation of the transcription factor NFκB, direct effects of particles on target cells as lung epithelial cells have to be considered.

We were able to show that both apoptosis as well as proliferation are induced by different types of nanoparticles after application to lung epithelial cells (RLE-6TN). Further investigations of these endpoints suggest that proliferation in these cells is induced independently and should not be considered as compensation of particle induced apoptosis.

As molecular events mediating particle induced effects several signalling pathways which are known to be activated upon extracellular stress have been investigated. These studies demonstrate the activation of a MAP-kinase pathway via extracellular regulated kinases 1 and 2 (Erk1/2). Erk1/2 which are known to induce proliferation via the activation of the transcription factor AP-1 are phosphorylated in a specific time and dose dependent manner after treatment of epithelial cells with nanoparticles. Moreover, the activation of the anti-apoptotic protein kinase B (AKT) pathway was observed with striking similarities to Erk1/2 phosphorylation in time and dose dependence, suggesting a common upstream pathway. In order to identify triggering events of these signalling cascades membrane located receptors have been investigated. Using specific inhibitors, the involvement of epidermal growth factor receptor (EGF-R) as well as of extracellular matrix binding integrin receptors was observed.

We therefore hypothesise that proteins of the extracellular matrix, or the receptors themselves, are target molecules for particle cell interactions which are crucial for induction of nanoparticle effects.

73


L-23

FROM SPACE RESEARCH TO NANOPARTICLE EFFECTS: BIODIAGNOSTICS AT THE GERMAN

AEROSPACE CENTRE

C. Baumstark-Khan 1, A. Arenz 1, C. E. Hellweg 1, H.H. Grothee r 2


The research activities of the DLR Institute of Aerospace Medicine and especially of the Radiation Biology Unit are focussed on the central task of providing benefit for the health and performance of persons involved in space flight. This includes, amongst radiation health and protection issues for astronauts and aircraft crews, monitoring of biological consequences of environmental radiation and genotoxic conditions by biological dosimetry, biosensors and bioassays. The Biodiagnostics Group develops cellular test systems capable of monitoring the biological consequences of such conditions on humans already on cellular and molecular level. Bioassays devepoped so far relay on the reporter-receptor principle well known for many bacterial assays. The sensing system consists of specially designed plasmids where promoters which are dependent on certain environmental conditions act as sensing receptors. For reporter components fuorescent or bioluminescent proteins are valuable tools. Such receptor-reporter assays allow the detection of cellular signal transduction events dependent on special environmental parameters. For determination of genotoxic and cytotoxic environmental influences on living cells the bacterial SWITCH-Test and the mammalian test systems µFADU and A549-NF-κB-EGFP-Test have been developed. The SWITCH-Test (Salmonella Weighting of Induced Toxicity and Cytotoxicity for Human Health) measures in paralell the genotoxic (SOS-Lux-Test) and cytotoxic (Lac-Fluoro-Test) potency of agents. The SOS-Lux-Test is based on the bacterial SOS-response which is induced in presence of DNA-damaging agents. It uses the strong SOS-dependent ColD-promoter as receptor component and the promoterless luxCDABFE genes of Photobacterium leiognathi as reporter component. With this system dose-dependent light yields can be recorded in recombinant Salmonella typhimurium treated with genotoxic agents. The Lac-Fluoro-Test uses constitutive green fluorescent protein (GFP from Aequoria victoria) expression mediated by a bacterial protein expression vector to accentuate cellular response to cytotoxins by dose-dependent reduction of GFP-expression. The Switch-Test was used to analyse the geno- and cytotoxic potency of a set of nanoparticle samples generated by combustion processes. Condensation water samples from combusted gaseous fuels (propane, ethane, ethylene, acetylene) and exhaust gases from combustion vehicles operated under laboratory conditions were obtained. Results show genotoxic and cytotoxic properties of nanoparticle samples generated by condensation of model flame exhaust to dependent on the C/O relationship: the higher the C/O value, the less material of the samples was needed to display the toxic effects. The sample with the higher C/O relationship expresses genotoxicity and cytotoxicity at greater dilution, implying a higher concentration of nanoparticles The results for two motor-scooters show toxicity levels comparable to model conditions. Genotoxicity can not be related to the formation of polynuclear aromatic hydrocarbons (PAHs) during combustion as PAH preparations need to be metabolically activated to display geno- and cytotoxic effects.

74


L-24

GENE EXPRESSION MODULATION IN A549 LUNG CELLS IN RESPONSE TO COMBUSTION GENERATED NANOPARTICLES

A. Arenz 1, . r C Baumstark-Khan 1, C. E. Hellweg 1, H.H. Grothee 2


Cell response to different kinds of environmental challenge is complex and involves the participation of different classes of genes for different cellular outcomes (DNA repair, cell cycle control, signal transduction, inflammation, apoptosis and oncogenesis). Ambient particulate matter (PM) is a complex mixture of chemicals and particles that may be compositionally diverse depending on geography and season. High levels of ambient air pollution are associated with aggravation of asthma, respiratory morbidity, and cardiopulmonary morbidity; long-term exposures to PM have been linked to possible increases in lung cancer risk, chronic respiratory disease, and death rates. Nanometer sized particles (∅ 1.5-5 nm) which are generated by combustion processes as soot precursors show surprising features, such as water solubility and transparency, contrary to the better known soot particles. The damaging effects of nanoparticles are attributed to the fact that they are respirable and water soluble. Such particles can penetrate lung mucosa. Additionally, these particles are less readily cleared than fine particles, thereby prolonging interaction with the lung epithelium and possibly potentiating cellular damage. Each of the individual components of particulate matter has been shown to invoke inflammatory responses after exposure in animal models. In-vitro experiments have revealed that diesel exhaust and PM10 are capable to induce the release of proinflammatory cytokines such as IL-6 and IL-8 from bronchial epithelial cells mediated by the transcription factor NF-κB through a mechanism partially involving TNF-α. Thus, an important aspect of our work was to determine whether nanoparticles cause activation of NF-κB and expression of NF-κB dependent genes by pulmonary epithelial cells. Recombinant A549 lung cells (A549-NF-κB-EGFP) were incubated with different concentrations of condensation water samples collected after combustion of gaseous fuels (propane: sample 003/004; ethylene: sample 015 and 025/026) under laboratory conditions. TNF-α treated cells were used for comparison. RNA was extracted from exposed cells after various recovery times and a real-time QRT-PCR assay was applied, which employs relative quantification of candidate mRNA biomarkers. The expressions of different DNA damage inducible genes (GADD45ß, p21) and NF-κB dependent genes (NFκBIA, IL-6, “B-EGFP”) were analysed. The results show a reproducible up-regulation for the NF-κB dependent genes IL-6 (32x) and NFκBIA (13x) with maximal values for 1 and 2 hrs treatment with TNF-α, while the DNA damage inducible genes are not induced. For nanoparticle treated cells, a down-regulation of NF-κB dependent genes, especially for IL-6, could be shown for high particle concentrations (Dilution ratios 1:8 to 1:20). For intermediate particle concentrations (Dilution ratio 1:100), the DNA damage inducible gene GADD45ß is up-regulated (~5x) for up to 4 hrs and for lower particle concentrations (Dilution ratio 1:200) the maximal induction value for GADD45ß is about 3. NF-κB dependent gene expression is down-regulated for the first hours of nanoparticle incubation for NFκBIA (-3x, 4 hrs). For IL-6 a first down-regulation (-6.5, 30 min) is followed by a significant up-regulation for incubation times of 2 and 4 hrs.

75


L-25

INFLAMMATORY AND GENOTOXIC EFFECTS OF COMBUSTION GENERATED NANOPARTICLES WITH AND WITHOUT BENZO(A)PYRENE AND/OR IRON SULPHATE

R. Schins 1, R. Duffin 2, P Voss . 1, C. Albrecht 1, F.J. van Schooten 3, and K. Unfried 1

1 Institut für umweltmedizinische Forschung (IUF) at the Heinrich Heine University, Düsseldorf, Germany; 2ELEGI/Colt laboratories, Centre for Inflammation Research, Medical School, University of Edinburgh, UK; 3Department of Health Risk Analysis and Toxicology, University of Maastricht,

The Netherlands, Email: [email protected]; Phone: 0211-3389-269; Fax: 0211-3389-331

There is conflicting evidence in the literature as to the compositional element(s) which crucially drive the adverse health effects of ambient particulate matter (PM10), including inflammatory lung disease and cancer. It has been suggested that combustion-generated nanoparticles as well as constituents adsorbed onto their high surface area such as transition metals and polycyclic aromatic hydrocarbons may play a dominant role in the molecular pathways implicated in these diseases. Therefore, we have investigated the inflammatory and mutagenic effects in rat lungs of carbonaceous nanoparticles in the presence or absence of iron sulphate (FS) or benzo(a)pyrene (BaP). Rats were instilled intra-tracheally with toluene-extracted carbon black (CB, 14nm) and CB which had been reconstituted with BaP (30 mg/g) with or without FS (2.8 mM). Bronchoalveolar lavage (BAL) was performed and analysed for biomarkers of lung toxicity and inflammation, and in vivo mutagenesis was evaluated in lung tissue DNA using the transgenic rat lacI-system and concomitant analysis of BaP-diolepoxide (BPDE) adducts (32P-postlabelling) as well as 8-hydroxydeoxyguanosine (HPLC-ECD). All four particle treatments, i.e. CB, CB+FS, CB+BaP, and CB+BaP+FS, caused a marked neutrophilic inflammation, but this effect was significantly less for the CB+BaP and the CB+BaP+FS group, compared to both the CB and CB+FS group. In line with these observations, various markers of pulmonary inflammation and toxicity were affected only in the CB and CB+FS group, including the BAL expression of myeloperoxidase (MPO), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), monocyte chemotactic protein-1 (MCP-1), as well as the lung tissue expression of the Nuclear Factor kappa B inhibitor protein IκB-alpha. Interestingly, the inflammatory effects, as induced by the different nanoparticle preparations, were found not to correlate with an induction of oxidative DNA-damage, whereas the nanoparticle-associated BaP was found to induce significantly higher persisting BPDE-DNA adducts in the rat lung tissue than (free) BaP instilled at the same dose. The different nanoparticle treatments also showed significant contrasts in the mutation frequencies as well as in mutation spectra. In conclusion our data show that the carrier function of combustion-generated nanoparticles may have considerable impact on their inflammatory, toxic and mutagenic effects within the respiratory tract.

This work is supported by the German Research Council (DFG-SFB503).

76


L-26

ALTERED NUCLEAR STRUCTURE AND FUNCTION IN RESPONSE TO SILICA NANOPARTICLES

Min Chen and Anna von Mikecz Institut für umweltmedizinische Forschung (IUF) at Heinrich-Heine-University Düsseldorf, Auf´m Hennekamp 50, D-40225 Düsseldorf, Germany, Fax: +49-211-3389245; E-mail: chenmin@uni-

duesseldorf.de

Despite of their exponentially growing use, little is known about cell biological effects of nanoparticles. Here, we report uptake of silica (SiO2) nanoparticles to the cell nucleus where they induce aberrant clusters of topoisomerase I (topo I) in the nucleoplasm that additionally contain signature proteins of nuclear domains, and protein aggregation such as ubiquitin, proteasomes, cellular glutamine repeat (polyQ) proteins, and huntingtin. Formation of intranuclear protein aggregates (1) inhibits replication, transcription, and cell proliferation; (2) does not significantly alter proteasomal activity or cell viability; and (3) is reversible by Congo red and trehalose. Since SiO2 nanoparticles trigger a subnuclear pathology resembling the one occurring in expanded polyglutamine neurodegenerative disorders, we suggest that integrity of the functional architecture of the cell nucleus should be used as a read out for cytotoxicity and considered in the development of safe nanotechnology.

77


P-12

APOPTOSIS AND PROLIFERATION INDUCED BY DIFFERENT TYPES OF NANOPARTICLES IN RAT LUNG EPITHELIAL CELLS

U. Sydlik, J. Abel and K. Unfried Institut für umweltmedizinische Forschung an der Heinrich-Heine-Universität Düsseldorf gGmbH,

Düsseldorf, Germany, e-mail: [email protected], Fax: 0211-3389-331

Nano-sized particles are known to induce various adverse health effects in humans, including fibrosis and lung cancer. Neoplasia as well as tumor progression are generally associated with deregulation of cell proliferation and suppression of apoptosis. However several observations indicate that apoptosis may be considered as active process during the initial steps of carcinogenesis. To investigate the molecular mechanisms induced by particle-cell interaction leading to these endpoints, a rat lung epithelial cell line (RLE-6TN) was treated with non-cytotoxic amounts of different types of nanoparticles. Caspase-3 activity assays demonstrated that carbon black (printex 90) as well as amorphous silica (SiO2) are able to induce apoptosis in a dose dependent manner with a maximum after 8 h of exposure. Additionally, both samples applied in similar dose ranges induced a delayed proliferation with a maximum after 24 h detected by BrdU-incorporation. In order to investigate molecular events involved in particle – cell interaction integrins have been tested for their involvement in the induction of particle specific apoptosis and/or cell proliferation. This family of heterodimeric transmembrane receptors specifically binds to amino acid sequences of the extracellular matrix proteins. Pre-incubation of the cells with specifically integrin blocking peptides (RGD) prior to particle treatment resulted in marked effects on proliferation, whereas control peptides had virtually no effect. Apoptosis was not effected using RGD peptides. Pre-treatment of the cells with an inhibitor of epidermal growth factor receptor (EGFR) phosphorylation (AG1478) caused significant decreases in apoptosis and proliferation. These results indicate the importance of signalling pathways using integrin receptors as well as EGFR for regulation of particle induced apoptosis and proliferation.

78


P-13

EFFECTS OF FLY ASH ON THE LEVEL OF GLUTATHIONE AND THE EXPRESSION OF HEME OXYGENASE-1

S. Diabaté, D. Ettehadieh, H.F. Krug Forschungszentrum Karlsruhe, [email protected], Fax 07247-82-3557

There is increasing evidence that environmental particulate air pollutants contribute to adverse health effects concerning respiratory and cardiovascular diseases. In vivo inhalation studies with model particles result in oxidative lung damage and inflammation, however, the underlying mechanisms are largely unknown. Anthropogenic derived particulate pollutants mainly origin from combustion of fossil fuel for energy generation. Here we use fly ash from a municipal waste incineration facility as a model for environmental particles.

It has been shown earlier that fly ash induces oxidative stress as measured by oxidation of the fluorescent dye H2DCF. This response was reduced be preincubation of the cells with the antioxidant N-acetyl cysteine (NAC) or by preincubation of the particles with the iron chelator desferrioxamine. Only the insoluble fraction of the fly ash was effective in inducing oxidative stress, not the water soluble fraction. The aim of this study was to determine other parameters of oxidative stress such as the cellular glutathione content and the induction of the antioxidative enzyme heme oxygenase-1 (HO-1) induced by fly ash at non-cytotoxic concentrations. These effects were studied in BEAS-2B human bronchial epithelial cells and RAW264.7 mouse macrophages.

The cellular content of glutathione (GSH) and glutathione disulfide (GSSG) was measured by the recycling method with 5,5’-dithio-bis(2-nitrobenzoic acid) (DNTP) und glutathione reductase. It is demonstrated that fly ash induces a transient decline in the cellular glutathione content in the first hours after the beginning of exposure which is followed by a progressive increase up to the 2-fold of the original level after 24 hours. The increased glutathione content is only induced by the insoluble fraction of the fly ash, not by the water soluble fraction, and it can be inhibited by NAC.

The decline in the cellular glutathione content in the first hours after the beginning of exposure is accompanied by a rapid induction of the antioxidant enzyme heme oxygenase-1 which was proved by western blot. HO-1 is only induced by the insoluble fraction of the fly ash, not by the water soluble fraction, and it can be inhibited by NAC. These effects were observed at lower fly ash concentration as compared to oxidation of H2DCF or change in the glutathione level.

These data demonstrate that induction of the antioxidant enzyme heme oxigenase-1 in epithelial cells and macrophages is a sensitive marker for fly ash-induced oxidative stress and that changes in the cellular glutathione content might be involved in this response.

79


P-14

EFFECTS OF COMBUSTION GENERATED NANOPARTICLES ON BACTERIAL SOS RESPONSE CONTROLLED GENE EXPRESSION

F. Langenbach 1, R. Huxoll, C. Baumstark-Khan 1, C. E. Hellweg 1, H.H. Grothee r 2

1) DLR, Institut für Luft- und Raumfahrtmedizin, Strahlenbiologie, Köln; 2 DLR, Institut für Verbrennungstechnik, Stuttgart; Germany ([email protected], Fax: +49-2203-

61970)

Nanoparticles are generated by combustion processes as soot precursors. These are made up of clusters of polyaromatic structures with typical diameters of 1.5 to 5 nm and mass numbers ranging from 1000 to 40000 amu. Nanoparticle toxicity displays a special risk for humans, especially as these respirable and water soluble particles can penetrate lung mucosa. In order to differentiate between genotoxicity and cytoxicity of nanoparticles, the recombinant SWITCH test (SWITCH: Salmonella Weighting of Induced Toxicity and Cytotoxicity for Human Health) was applied on a set of 14 condensation water samples from combusted gaseous fuels (propane, ethane, ethylene, acetylene). This luminescent/fluorescent bioassay makes use of two sensing and reporting systems for the two biological endpoints under investigation: the SOS-Lux test and the LAC-Fluoro test. The SWICH plasmid carries the promoterless lux operon of Photobacterium leiognathi as reporter element under the control of the DNA-damage dependent SOS promoter of ColD as sensor element (for genotoxicity testing) and the sequences for a hybrid protein consisting of ß-galactosidase and GFPuv of Aequorea victoria as reporter element under the control of the (in Salmonella constitutively active) LAC promoter of Escherichia coli as sensor element (for cytotoxicity testing). The system has worked properly for model compound of known geno- and cytotoxic potency. First experiments show the nanoparticle preparations to be positive genotoxins for the recombinant Salmonella strain.

80


P-15

COMBUSTION GENERATED NANOPARTICLES: MUTAGENICITY AND CHEMICAL REACTIVITY

Simonelli, A., Miraglia, N., Acampora A., Sannolo, N., Pascarella, L., D’Anna, A., D’Alessio, A., Sgro, L.A.

Seconda Università degli Studi di Napoli, Italy, [email protected], fax: +39 (0)81 5469185

Two types of carbonaceous particles are formed in fuel rich combustion of hydrocarbons, and have been detected in vehicle exhausts, suggesting their presence also in urban atmospheres. Nanoparticles of Organic Carbon (NOC) and soot particles. Epidemiological studies show that some causal relationships exist between particle concentration in the air and a wide range of health effect, but no toxicological studies are reported on the potential health risk of particles smaller than 4 nm.

The present study investigated the mutagenicity and the reactivity of NOC collected in water samples from premixed laboratory ethylene air flames, and from the exhausts of diesel and gasoline engines. Mutagenicity was tested following the widely used Ames test, with and without S-9. Reactivity was investigated by using a new approach aimed to identify electrophilic agents present in the sample material, which if introduced into the organism, could interact with nucleophilic sites of biological macromolecules (DNA and proteins), forming adducts. Adducts can be used as genotoxicity indicators and/or exposure biomarkers associated with xenobiotic substances. Given the large number of nucleophilc sites within biological macromolecules, the complexity of NOC, and the inexact knowledge of its chemical structure, this approach was simplified by examining in vitro interactions between NOC particles and model peptides.

For Salmonella tester strain TA 98, samples from gasoline engines were positive both with and without S-9, while diesel exhausts resulted mutagenic only when metabolic activation occurs. The mutagenic responses were about 4-fold when compared to negative controls. Whereas, when tested with the TA 100 strain, even if both exhausts samples showed positive linear dose-response curves, their response was less than double the control case in the examined dose ranges (3.75-280 and 2.5-145 µg/plate, NOC diesel and gasoline, respectively) except for the higher concentration. Flame samples presented lower particle concentrations (giving doses of 1.1-41 µg/plate). Linear dose responses were observed for both strains without S9, but most of them were lower than twice the control response, except for the highest dose in the TA98 strain. Testing of higher concentrations is needed to verify mutagenicity of flame generated NOC.

NOC samples from diesel and gasoline engines were analysed by LC/MS before and after the incubation with four different model peptides. Chromatographic profiles of NOC samples revealed numerous peaks with a 44Da mass increment. After the addition of peptides, in most cases, LC/MS analyses showed the disappearance of peptide and NOC particles signals, and the appearance of a number of co-eluting and unresolved peaks at high retention times. These substances had high molecular weights, not corresponding to a simple NOC-peptide interaction, and hence they were not identified yet. Nevertheless, the disappearance of peptides evidenced the high reactivity of NOC particles and showed that peptide-NOC interactions cause the sample material to increase significantly in size, due to chemical and/or physical bonds.

81

Author Index

A Abel, J. ..........................................77 Acampora, A. ................................80 Aigner, M. ...............................26, 35 Albrecht, C. .............................72, 75 Allouis, C.......................................37 Arenz, A. ...............17, 19, 64, 73, 74

B Baeza, A. .................................66, 67 Barth, K.-L.. ...................................35 Baulig, A........................................67 Baumstark-Khan, C...4, 5, 12, 19, 64,

73, 74, 79 Beck-Speier, I...........................17, 61 Bhattacharya, K. ............................65 Bierhals, K. ....................................72 Birmili, W.................................15, 43 Bischof, O. F. .................................42 Bockhorn, H. ...........................36, 37 Borken, J. ................................16, 54 Borm, P. J. A............................19, 71 Bors, W. ........................................61 Brun, A..........................................34

C Carpeggiani, C. .............................56 Chai, Z. .........................................53 Charwath, M. ..........................14, 36 Chen, C.........................................53 Chen, M..................................19, 76 Chevaillier, S..................................67 Commodo, M..........................14, 37 Cramer, H. ....................................65

Cyrys, J. ........................................ 51

D D’Alessio, A. .. 15, 27, 30, 34, 37, 41,

80 D’Anna, A................... 27, 30, 37, 80 Dayal, N. ....................................... 61 De Lisi, C. ..................................... 34 Demokritou, P......................... 16, 52 Diabaté, S. ........................ 19, 44, 78 Donaldson, K. ......................... 17, 59 Dopp, E. ................................. 17, 65 Duffin, R. ................................ 72, 75 Duschl, A. ............................... 17, 62

E Engler, C....................................... 43 Ettehadieh, D. ............................... 78

G Geh, S. ......................................... 65 Gerhart, C. ............................. 15, 45 Gerzer, R. ..................................... 12 Ghid, A. ........................................ 61 Gonzalez Baquet, T. .......... 14, 15, 35 Grieshuber, I. ................................ 62 Grimm, H...................................... 45 Grotheer, H. H. .. 4, 5, 12, 23, 26, 64,

73, 74, 79 Grotheer, H.-H. ............................. 35 Gruijthuijsen, Y. ............................ 62 Guarnieri, D. ................................. 34 Gühnemann, A. ............................ 54 Gupta, T. ................................ 16, 52

82

H Harder, V.......................................55 Heinrich, J....................44, 51, 75, 76 Hellweg, C. E......4, 5, 12, 16, 17, 64,

73, 74, 79 Heyder, J. ................................44, 63 Horejs-Höck, J................................62 Huxoll, R........................................79

I Iuori, M. ........................................34

J Jia, G.............................................53

K Karg, B. ...................................61, 63 Karg, E. ...................................61, 63 Koutrakis, P. ..................................52 Kreyling, W. G. ..................29, 51, 71 Krinke, T..................................15, 42 Krug, H. F. ...............................44, 78 Kühne, R. ......................................54

L Langenbach, F. ........................19, 79 Lanzuolo, C. ..................................12 Lanzuolo, G. ......................13, 27, 30 Larsson, J.......................................25 Lehre, T. ..................................36, 37 Lehtiniemi, H. ................................33 Liu, Y.............................................53

M Maier, K. L.....................................61 Marano, F................................66, 67 Martinon, L....................................67

Mauss, F. .................... 12, 14, 24, 33 Meiring, J. J. ................................. 71 Minutolo, P. ...................... 14, 34, 37 Miraglia, N. ................................... 80 Mülhopt, S.............................. 15, 44

N Nelissen, I. .............................. 18, 68 Netti, P. A. .................................... 34 Netzell, K. ............................... 14, 33

O Oberdörster, G........................ 17, 60 Ossler, F. ................................. 12, 25

P Pascarella, L. ................................. 80 Paur, H-R. ..................................... 44 Peters, A. .................... 16, 44, 49, 51 Pitz, M. ......................................... 51 Pokorny, H. ................................... 26

R Radykewicz, R. .............................. 55 Ramgolan, K. .................... 17, 66, 67 Reinhard, C................................... 63 Reitinger, C................................... 62 Reitz, G......................................... 12 Rettenmoser, T.............................. 45 Reynaud, C. ............................ 12, 28 Richter, M..................................... 45 Riediker, M. ............................ 16, 50 Ritter, ........................................... 63 Rose, D. ........................................ 43 Rumelhard, M. .................. 17, 66, 67

S Samet, J. M................................... 61 Sannolo, N. ................................... 80

83

Schins, R............................19, 72, 75 Schoeters G. ..................................68 Schroeppel, A. ...............................63 Schulz, H. ..........................55, 61, 63 Seifert, H. ................................25, 44 Semmler, M...................................61 Sgro, L. A. .....................................80 Sgro, L.A. ..........................15, 34, 37 Shi, T. ............................................65 Shokou, B......................................65 Simonelli, A. ............................20, 80 Stampfl, A. ..............................16, 55 Stettmaier, K. ................................61 Stoeger, T......................................63 Stöger, T. ......................................17 Stölzel, M. ...............................16, 51 Suntz, R...................................36, 37 Sydlik, U. ...........................19, 72, 77 Szymczak, W. ..........................13, 29

T Takenaka, S. ............................61, 63 Thierley, M. .......................12, 14, 26 Tuch, T. ...................................43, 44

U Unfried, K....................19, 72, 75, 77

V Van Den Heuvel, R. ....................... 68 van Schooten, F. J. ........................ 75 Verheyen, G.................................. 68 Verstraelen, S................................ 68 Vigotti, M. A. .......................... 16, 56 von Mikecz, A. .............................. 76 Voss, P.......................................... 75

W Wang, H. ................................ 33, 53 Wehner, B. ................................... 43 Wichmann, H. E. ............... 44, 49, 51 Wiedensohler, A. .......................... 43 Witters, H. .................................... 68 Wittmaack, K. ............................... 29 Wölke, G. ..................................... 51

X Xing, G. ........................................ 53

Y Yadav, S. ...................................... 65

Z Zhao, Y................................... 16, 53

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