Mechanisms of genotoxicity of inhalable particles [Elektronische Ressource] : in vitro & in vivo investigations / vorgelegt von Anton Wessels
114 Pages
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Mechanisms of genotoxicity of inhalable particles [Elektronische Ressource] : in vitro & in vivo investigations / vorgelegt von Anton Wessels

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Mechanisms of genotoxicity of inhalable particles: in vitro & in vivo investigations Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Anton Wessels aus Papenburg Düsseldorf, Dezember 2009 aus dem Institut für umweltmedizinische Forschung an der Heinrich-Heine-Universität Düsseldorf gGmbH Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. P. Proksch Koreferent: PD Dr. W. Wätjen Tag der mündlichen Prüfung: "Alle Ding' sind Gift und nichts ohn' Gift; allein die Dosis macht, das ein Ding' kein Gift ist." Paracelsus (1493-1541) 1 Table of content List of abbreviations 3 Chapter 1 6 1. Introduction 6 1.1 Ambient particulate matter 7 1.1.1 Particle size distribution 7 1.1.2 Sources & composition 8 1.1.3 Particle deposition 9 1.1.4 Clearance & translocation 10 1.2 Health effects of particles 11 1.3 Mechanisms of toxicity and protection 12 1.3.1 Oxidative stress 12 1.3.2 Antioxidant defence 13 1.3.3 Oxidative DNA damage 14 1.3.4 DNA repair 15 1.3.5 Base excision repair 15 1.

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Published 01 January 2009
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Mechanisms of genotoxicity of inhalable particles:
in vitro & in vivo investigations







Inaugural-Dissertation



zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf


vorgelegt von

Anton Wessels
aus Papenburg






Düsseldorf, Dezember 2009





aus dem Institut für umweltmedizinische Forschung an
der Heinrich-Heine-Universität Düsseldorf gGmbH



























Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf




Referent: Prof. Dr. P. Proksch
Koreferent: PD Dr. W. Wätjen

Tag der mündlichen Prüfung:




"Alle Ding' sind Gift
und nichts ohn' Gift;
allein die Dosis macht,
das ein Ding' kein Gift ist."
Paracelsus (1493-1541)
1
Table of content

List of abbreviations 3

Chapter 1 6
1. Introduction 6
1.1 Ambient particulate matter 7
1.1.1 Particle size distribution 7
1.1.2 Sources & composition 8
1.1.3 Particle deposition 9
1.1.4 Clearance & translocation 10
1.2 Health effects of particles 11
1.3 Mechanisms of toxicity and protection 12
1.3.1 Oxidative stress 12
1.3.2 Antioxidant defence 13
1.3.3 Oxidative DNA damage 14
1.3.4 DNA repair 15
1.3.5 Base excision repair 15
1.4 Particle exposure, inflammation and genotoxicity 16
1.4.1 Redox active transition metals 17
1.4.2 Organic fraction 18
1.4.3 Surface area 18
1.4.4 Inflammation 19
1.5 Aims of the study 21
1.6 References 22

Chapter 2 29
Oxidant generation and toxicity of size-fractionated ambient particles in human lung
epithelial cells 29

2.1 Introduction 30
2.2 Materials and Methods 32
2.3 Results 35
2.4 Discussion 40
2.5 References 46

Chapter 3 49
Oxidative stress and DNA damage responses in rat and mouse lung to inhaled
carbon nanoparticles 49

3.1 Introduction 50
3.2 Materials and Methods 52
3.3 Results 59
3.4 Discussion 64
3.5 References 69




2
Chapter 4 74
Neutrophil-derived oxygen species contribute to oxidative stress and DNA damage
induction by respirable quartz particles 74

4.1 Introduction 75
4.2 Materials and Methods 77
4.3 Results 83
4.4 Discussion 90
4.5 References 94

Chapter 5 99
5. Summary and general discussion 99

6. Zusammenfassung 107

7. Summary 108

Curriculum Vitae 109

Danksagung 110

Eidesstattliche Erklärung 111


3
List of abbreviations

AM Alveolar macrophage
AP-1 Activator protein-1
APE-1 Apurinic/apyrimidinic endonuclease-1
a.u. Arbitrary unit
BAL Bronchoalveolar lavage
BALF BAL fluid
BER Base excision repair
BSA Bovine serum albumine
b.w. Body weight
cDNA complementary DNA
CNP Carbon nanoparticles
COPD Chronic Obstructive Pulmonary Disease
d Aerodynamic diameter ae
DEP Diesel exhaust particles
DMEM Dulbecco’s modified Eagle’s medium
DMPO 5,5-dimethyl-1-pyrroline-N-oxide
DMSO Dimethylsulfoxid
DNA Deoxyribonucleic acid
DQ 12 Dörentrup Quartz “ground product nr. 12”
EC Elemental carbon
EDTA Ethylenediaminetetraacetic acid
e.g For example
ESR Electron spin resonance
EPA Environmental Protection Agency
EU European Union
FCS Foetal calf serum
fpg Formamido-pyrimidine-DNA-glycosylase G
g Gram, gravity
GSH Glutathione
GV-SOLAS Guidelines provides for the Society for Laboratory Animals
Science

4
h Hour
HBSS Hanks’ Buffered Salt Solution
HO-1 Heme Oxygenase-1
H O Water 2
H O Hydrogen peroxid 2 2
HPLC High performance liquid chromatography
IARC International Agency Research on Cancer
IL-8 Interleukin-8
iNOS inducible Nitric Oxide Synthase
IVCAB In vivo comet assay buffer
l Liter
LDH Lactate dehydrogenase
LMP Low melting point
m Meter, milli
M Molar
MGG May-Grünwald / Giemsa
min Minute
MPO Myeloperoxidase
mRNA messenger RNA
n Nano
NADPH Nicotinamide adenine dinucleotide phosphate
NER Nucleotide excision repair
NFKB Nuclear factor kappa B
NO Nitric oxid
NOX NADPH oxidase
OC Organic carbon
Ogg-1 8-oxoguanine glycosylase
OH Hydroxyl radical
PAH Polyaromatic hydrocarbons
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PM Particulate matter
PMA Phorbol-12-myristate-13-acetate
PMNs Polymorphonuclear neutrophils

5
POL Polymerase ß
PTFE Polytetrafluoroethylene
qRT-PCR quantitative Real Time -PCR
RNA Ribonucleic acid
RNS Reactive Nitrogen Species
ROS Reactive Oxygen Species
RTLS Respiratory tract lining fluid
SD Standard deviations
SEM Standard error of mean
SOD Superoxide dismutase
TNF- Tumour necrosis factor-
UFP Ultrafine particle
vs. Versus
WHO World Health Organisation
XRCC1 X-ray Repair Complementing defective repair
γ -GCS Gamma-glutamylcysteine synthetase
8-OHdG 8-hydroxydeoxyguanosine
µ Micro
°C Degree celsius








α α
βChapter 1 6


Chapter 1
1. Introduction

The human lung has a large surface area (40-120 m²) and is exposed to between
10,000 and 20,000 litre of ambient air each day (1). Thus, air pollutants easily have
access to the human body. According to an assessment from the World Health
Organisation (WHO), more than 2.4 million premature deaths each year are related
to the effects of urban outdoor- and indoor air pollution (2).
That particulate matter (PM) can lead to adverse health effects is described since the
late middle ages. Agricola (1494-1555) reported the association between mining and
illness. Paracelsus (1493-1541) related the work in mines to lung diseases (3).
Historically, particle research focussed on high occupational exposures during work.
Coal miners and workers in coking industries were exposed in former days to dust-
3concentrations up to 40 mg/m , whereas nowadays in mining an exposure situation
3of 2-3 mg/m can occur. Nowadays, general workplace limit values are introduced.
thUntil the mid 20 century no great effort was undertaken to monitor/restrict air
pollution for protection of the general population. This thinking mainly changed, after
the London smog episode in 1952. It has been estimated that the smog caused
12,000 deaths and is one of history's most important air pollution episodes in terms of
its impact on science, public perception of air pollution, and government regulation
(4). Since this time air pollution is also of public concern and many epidemiological
studies have been performed. Dockery and co-workers in 1993 showed that per 10
3 µg/m increase in annual PM concentration mortality is increased by 1.4% and
respiratory diseases by 4 % (5). These investigations and observations led to a
European directive (1999/30/EG), which regulates the ambient PM concentration. 10
st 3Since 1 January 2005 the daily limit value is 50 µg/m , which is not to be exceeded
more than 35 times per calendar year. The acceptable annual mean of PM is 40 10
3µg/m . In 2008 the directive was updated and provides additional standards for PM 2.5
(2008/50/EC).
In the following chapter the composition of particles, reactive oxygen species (ROS)
generation and particle-related health effects are described to understand the need
of limit values and to provide a useful basis for further reading of this thesis.
Chapter 1 7


1.1 Ambient particulate matter

1.1.1 Particle size distribution

Particulate matter (PM) is a heterogeneous mixture of solid and liquid particles
present in the air. The size distribution of particles in the urban air is conventionally
characterised by three parameters according to the aerodynamic diameter of the
particles (figure 1).

Number

Surface area
Mass







0.001 0.01 0.1 1 10 100
Particle diameter, µm

Nano/ultrafine Fine Coarse

Figure 1. Particle size distribution of ambient PM by three characteristics: mass, surface area
and number (adapted from Finlayson-Pitts, 2000).
The black curve represents the distribution pattern by number of particles and includes mainly
ultrafine particles (< 0.1 µm). Distribution by surface area constitutes fine particles from 0.1 -1.0
µm (dotted curve). Mass distribution is both fine and coarse particles from 0.1-100 µm (dashed
curve). ultrafine particle: < 0.1 µm, fine particle: < 2.5 µm, coarse: > 2.5 µm

3The aerodynamic diameter is calculated as the diameter of a unit density (1 g/cm )
sphere having the same settling speed as the specified particle of whatever shape
and density. Thus, particles with identical inertial properties have the same
aerodynamic diameter irrespective of their actual size, shapes or densities (6). For
standard classification of PM the American EPA (Environmental Protection Agency,
1997) introduced a “national air quality standard for particulate matter” and defined
for ambient particulate matter different size-fractions according to the aerodynamic
diameter (table 1), which have been taken over by the EU (1999) and WHO (2000).