European Environmental Mutagen Society
34th Annual Meeting
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European Environmental Mutagen Society
34th Annual Meeting
Program and Abstracts
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Contents
Introduction 5
Map 6
Programme 8
Abstracts 14
Poster presentations 45
Author Index 127
4
Local Organising committee
Joost van Delft (President)
Jan van Benthem (vice-President)
Peter Weterings (Treasurer)
Bert van der Horst
Bert van Zeeland,
Cyrille Krul
Els van Vliet
Frederik-Jan van Schooten
Frederique van Acker
Gerrit Alink
Harry van Steeg
Harry Vrieling
Ineke Verspeek
Jos Kleinjans
Madeleine Nivard
Rob Baan
Editorial Board
Jan van Benthem (vice-President)
Peter Weterings (Treasurer)
Frederique van Acker
Carmen Hermans (Conference and Events Office)
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Introduction
Dear participants,
Welcome to the 34th annual meeting of the European Environmental Mutagen Society with as central
theme “Genes and Environment, Bridging the Gap”. The Organising Committee is delighted to present to
you an exciting programme bringing many of the major scientific and regulatory topics and develop-
ments that currently are of interest for the Society’s members. DNA damage and repair, environmental
and dietary factors, genetic susceptibility, risk assessment and guidelines, are among them. To bridge the
gap between traditional and new technologies, special attention will be paid during the meeting to the
opportunities and achievements of genomics on R&D and on regulation.
Furthermore, to broaden the engagement of the Society as a whole and to specifically involve the new
members, the floor is given to many speakers that were selected on submitted abstracts. Hopefully, this
meeting will not only inspire your scientific life, but will also contribute to your social and Societal net-
work.
On behalf of the board of the Dutch EMS but also on behalf of all its members, I wish you a pleasant, fruit-
ful, informing and successful meeting. Furthermore, I hope you will enjoy your stay in Maastricht and
frequently bridge the gap between the two historic parts on both sides of the river Maas.
Finally, I would like to thank the Organising Committee and everyone else who was involved in settling
this meeting and making these days wonderful.
Joost van Delft
Chair of the Organising Committee
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Map
Sponsors
1. Gentronix Ltd 7. TNO Voeding
2. Elsevier Science 8. Research Tox Center S.p.A.
3. Biopredic International 9. NOTOX B.V.
4. PerkinElmer 10. COVANCE LTD.
5. Snijders Scientific/ORDINA Technical 11. Centre International de Toxicologie (CIT)
6. Perceptive Instruments Ltd 12. Safepharm Laboratories Ltd
Legenda Poster Presentation
Bordeaux Foyer: PW 1001 - PW 2018
Foyer D’Alsace: PW 2019 - PW 3009
Bourgogne: PW 3010 - PW 7007
6
Céramique Ground Floor
Céramique 3 Céramique 2 Céramique 1
Céramique foyer
1 2
3
coffee/tea buffet
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7
Bourgogne Souterrain
Beaune Pommard Macon
5
7
4
6
8 9 10
11
12
coffee/tea buffet
coffee/tea buffet
lunch buffet
lun
ch b
uff
et
Foyer D’Alsace
Bordeaux Ground Floor
Bordeaux Foyer
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Programme
Saturday 4 September
15.00 Registration
17.00 Welcome and Opening
Ceramic Hall
Joost van Delft, president of the Organizing Committee, President of the Dutch EMS
Jo Ritzen, President of the Executive Board of Maastricht University
Young Scientist Award Lectures
Ceramic Hall
Chair: Krzysztof Szyfter, President of the EEMS
17.15 Oxidatively damaged DNA: from genes to populations
Marcus Cooke, University of Leicester, United Kingdom
17.45 Cell-type and DNA damage-specific response of human epidermal cells.
Mariarosaria D’Erico, Italian National Health Institute, Rome, Italy
18.15 Welcome Party, Crowne Plaza Hotel
Sunday 5 September
08.00 Registration
09.00 Symposium 1 “DNA Damage, repair and cell cycle control”
Ceramic Hall
Chair: Micheline Kirsch-Volders and Harry Vrieling
09.00 The effect of p53 mutations on cell cycle control and carcinogen-induced tumorigenesis.
Annemieke de Vries, National Institute of Public Health and the Environment, Bilthoven,
The Netherlands.
09.30 Dynamic organization of DNA damage repair proteins and chromosomes.
Roland Kanaar, Erasmus Medical Center, Rotterdam, The Netherlands.
10.00 Translesion synthesis and the Y-family of DNA polymerases.
Alan Lehmann, University of Sussex, Brighton, United Kingdom.
10.30 ATM: mobilizing the cellular response to genotoxic stress.
Yoshi Shiloh, Tel Aviv University, Israel.
11.00 Coffee/tea break
11.20 Poster session 1
Bourgogne/Bordeaux Foyer/Foyer D’Alsace
13.00 Lunch
14.00 Workshop 1 “Nutrigenomics for healthy and safe foods”
Bordeaux Hall
Chair: Ben van Ommen and Cyrille Krul
14.00 Nutrigenomics and nutritional systems biology.
Ben van Ommen, TNO Nutrition and Food Research, Zeist, The Netherlands.
14.30 The effect of plant phenols on AP-1 expression in mouse epidermis.
Wanda Baer-Dubowska, University of Medical Sciences, Poznan, Poland.
14.50 Natural Ah receptor agonists in the human diet: beneficial food components or unpercieved
risk factors?
Pim de Waard, Maastricht University, The Netherlands.
15.10 Folic acid metabolism and gene polymorphisms in the risk of non-hodgkin’s lymphomas.
Fabio Coppedè, University of Pisa, Italy.
15.30 Altered vegetable intake affects pivotal carcinogenesis pathways in colon mucosa from
adenoma patients and controls.
Simone van Breda, Maastricht University, The Netherlands.
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14.00 Workshop 2A “DNA damage, repair and cell cycle control”
Ceramic Hall
Chair: Niels de Wind and Rob Baan
14.00 DNA translesion synthesis and mismatch repair in the response to endogenous and exoge-
nous DNA damage.
Niels de Wind, Leiden University Medical Center, The Netherlands.
14.30 Impact of mismatch repair on UVB-induced cell cycle arrest.
Gerdien Stout, Leiden University Medical Centre, The Netherlands.
14.50 Cadmium inhibits human DNA mismatch repair in vivo.
Lene Rasmussen, Roskilde University, Denmark.
15.10 Wild type p53 suppresses non-homologous end-joining of DSB but not overall repair profi-
ciency.
Jochen Dahm-Daphi, University of Hamburg, Germany.
15.30 Bystander apoptosis induced in non-irradiated cells needs of caspase 8 activity.
Mauro Grifalconi, University of Padova, Italy
15.50 Coffee/tea break
16.10 Workshop 3 “Influence of genetic variation on cancer”
Ceramic Hall
Chair: Ari Hirvonen and Radim Sram
16.10 Molecular epidemiology of sporadic breast cancer: role of polymorphisms in the xenobiotic
metabolism and DNA repair genes.
Ari Hirvonen, Finnish Institute of Occupational Health, Helsinki, Finland.
16.40 Gene environment interactions and genetic damage introduction by ETS: the importance of
exposure levels.
Soterios Kyrtopoulos, National Hellenic Research Foundation, Athens, Greece.
17.00 Polymorphisms in DNA repair genes and risk of lung cancer in a Danish prospective cohort.
Ulla Vogel, National Institute of Occupational Health, Copenhagen, Denmark.
Cytogenetic biomarkers and human cancer risk (cancer risk biomarkers).
Hannu Norppa, Finnish Institute of Occupational Health, Helsinki, Finland.
17.40 Influence of GSTM1, GSTT1 and NAT2 genotypes on p53 mutational spectrum in bladder
tumors.
Charlotta Ryk, Karolinska Institute, Huddinge, Sweden (provisional).
16.10 Workshop 4 “Oxidative damage”
Bordeaux Hall
Chair: Anthony Lynch and Bert van Zeeland
16.10 Gene expression changes as biomarkers of oxidative damage.
Anthony Lynch, Glaxo Smith Kline, Herts, United Kingdom.
16.40 The multiple physiological and pathological consequences of oxidative DNA damage.
Alberto Izzotti, University of Genoa, Italy.
17.00 DNA damage by respirable quartz particles in rat lung target cells: the role of mitochondria.
Hui Li, IUF, Düsseldorf, Germany.
17.20 An hypothesis on the role of oxidative damage in neurodegenerative diseases.
Lucia Migliore, University of Pisa, Italy.
17.40 PARP1 together with Csb and Xpa accelerates the global repair of oxidative DNA base
damage.
Claudia Flohr, University of Mainz, Germany.
19.30 Concert in the St. Jans Kerk, Vrijthof, Maastricht
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Monday 6 September
08:00 Registration
09.00 Symposium 2 “Toxicogenomics in genetic toxicology”
Ceramic Hall
Sponsored by LRI and organised by ECETOC.
chair: Awni Sarrif and Jos Kleinjans
09.00 Introduction.
Jos Kleinjans, Maastricht University, The Netherlands.
09.05 Experimental design, quality aspects of data collection and statistics.
Timothy Gant, University of Leicester, United Kingdom.
09.35 Discrimination of genotoxic from non-genotoxic carcinogens by expression profiling
in vitro.
Joost van Delft, Maastricht University, The Netherlands.
10.00 Toxicogenomic analysis of gene expression: an emerging approach for differentiating
genotoxic mechanisms.
Jiri Aubrecht, Pfizer Inc., Groton, USA.
10.25 Genotoxic and non-genotoxic carcinogens in rat liver.
Hans-Jurgen Ahr, Bayer Health Care AG, Wuppertal, Germany.
10.55 Final remarks.
Jos Kleinjans, Maastricht University, The Netherlands.
11.00 Coffee/tea break
11.20 Poster session 2
Bourgogne
13.00 Lunch
14.00 Minisymposium “Biological clocks, chronotoxicology and therapy”
Bordeaux Hall
Chair: Bert van der Horst
14.00 The circadian clockwork: a central regulator of behaviour, physiology and disease.
Michael Hastings, University of Cambridge, United Kingdom.
14.30 Implications of biological clocks for cancer processes and their treatments.
Francis Levi, Université Paris XI Paul Brousse Hôpital, Villejuif, France.
15.00 Workshop 2B “DNA damage, repair and cell cycle control”
Bordeaux Hall
Chair: Niels de Wind and Rob Baan
15.00 Gene expression profiles revealed p53R2 protein plays a role in radiation-induced mutagenesis.
Eric Chuang, NCI/NIH, Bethesda, USA.
15.25 Extent of p53 binding to target genes in vivo and their expression.
Paola Menichini, National Institute for Cancer Research, IST, Genova, Italy.
14.00 Workshop 5 “Hazard identification by omics technologies”
Sponsored by LRI and organised by ECETOC
Ceramic Hall
Chair: Timothy Gant and Els van Vliet
14.00 Introduction.
Awni Sarrif, Du Pont de Nemours and Co., Newark, USA.
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14.05 Use of genomics for regulatory science, industry perspective.
Silvio Albertini, Hoffmann-La Roche, Basel, Switzerland.
14.30 Identification of genotoxins by marker gene expression based on a supervised class predic-
tion approach.
Daniel Bauer, Novartis Pharma AG, Basel, Switzerland.
14.50 Effects of chlorpromazine with and without UV radiation on gene expression of HepG2
cells.
Roland Froetschl, Federal Institute for Drugs and Medical Devices, Bonn, Germany.
15.10 Rat liver transcriptomics after 28-day exposure to benzene and trichloroethylene (mix-
tures).
Wilbert Heijne, TNO Nutrition and Food Research, Zeist, The Netherlands.
15.30 Discussion and wrap up.
Silvio Albertini, Hoffmann-La Roche, Basel, Switzerland.
15.50 Coffee/tea break
16.10 Workshop 6 “Health risk assessment; guidelines, harmonisation, validation, etc.”
Bordeaux Hall
Chair: Stephan Pfuhler and Frederique van Acker
16.10 New strategies in toxicological risk assessments: transparent and efficient approaches in
determining hazard and risk.
Bas Blaauboer, University of Utrecht, The Netherlands.
16.40 Overview of validation and international acceptance of new or updated test methods for
hazard assessment in the OECD.
Drew Wagner, OECD, Paris, France.
17.10 Evaluation of the performance of a small battery of in vitro tests in detecting rodent and
human carcinogens.
David Kirkland, Covance Laboratories Ltd., Harrogate, United Kingdom.
17.40 Frontloading genetic toxicology evaluation of DEREK, AmesII and Greenscreen as screening
tools.
Jacky van Gompel, Johnson & Johnson Pharmaceutical Research, Beerse, Belgium.
16.10 Workshop 7 “Genomic stability and aging”
Ceramic Hall
Chair: Ian Hickson and Madeleine Nivard
16.10 Role of the Bloom’s syndrome helicase in maintenance of genome stability.
Ian Hickson, University of Oxford, United Kingdom.
16.40 The studies on localization of human RECQ helicases fused with fluorescent proteins.
Marek Rusin, Center of Oncology, Gliwice, Poland.
17.00 Age related genome instability in DNA repair deficient mice.
Martijn Dollé, National Institute of Public Health and the Environment, Bilthoven, The
Netherlands.
17.20 DNA repair function, polymorphisms and genotoxicity in workers exposed to low dose
ionising radiation.
Peter van de Aka, Free University Brussels, Belgium.
17.20 DNA damage repair, apoptosis and necrosis in children, adults and old age humans.
Stelios Piperakis, NCSR Demokritos, Athens, Greece.
19.30 Reception at Town Hall, Maastricht
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Tuesday 7 September
08.00 Registration
09.00 Symposium 3 “Genomic stability and aging”
Ceramic Hall
Chair: Peter Stambrook and Harry van Steeg
09.00 Aging, cancer and cellular responses to DNA damage.
Judy Campisi, Lawrence Berkeley National Laboratory, Berkeley, USA.
09.30 Interplay between telomeres and DNA repair: implications for cancer and aging.
Maria Blasco, Spanish National Cancer Center, Madrid, Spain.
10.00 Genome instability, aging and cancer.
Jan Vijg, University Texas Health Science Center, San Antonio, USA.
10.30 Stress, mutations and aging.
Tom Kirkwood, University of Newcastle, United Kingdom.
11.00 Coffee/tea break
11.20 EEMS General Assembly
12.10 Fritz Sobels Award Lecture
How rare genetic diseases can help us understanding mutagenesis and carcinogenesis?
Alain Sarasin, Institut Gustave Roussy, Villejuif, France
Ceramic Hall
13.00 Lunch
14.00 Excursion to the caves of Maastricht and to the winery “Apostelhoeve”
19.30 Conference dinner at La Butte aux Bois, Belgium
Wednesday 8 September
08.00 Registration
09.00 Symposium 4 “Influence of genetic variation on cancer”
Ceramic Hall
Chair: Hannu Norppa and Frederik Jan van Schooten
09.00 Cancer: genes and the environment.
Kari Hemminki, German Cancer Research Center, Heidelberg, Germany.
09.30 Lung cancer and polymorphisms of genes involved in carcinogen metabolism and DNA
repair.
Federico Canzian, International Agency for Research on Cancer, Lyon, France.
10.00 Design and interpretation of studies on gene-environment interactions in carcinogenesis.
Paolo Vineis, University of Torino, Italy.
10.30 A significance of genetic factor in initiation and progression of laryngeal cancer.
Krzysztof Szyfter, Polish Academy of Sciences, Poznan, Poland.
11.00 Coffee/tea break
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11.20 Symposium 5 “Natural mutagens and carcinogens”
Ceramic Hall
Chair: Adela Lopez de Cerain and Gerrit Alink
11.20 Flavonoids and alkenylbenzenes; mechanisms of mutagenic action and carcinogenic risk.
Ivonne Rietjens, Wageningen University, The Netherlands.
11.50 Genotoxicity of phytoestrogens.
Helga Stopper, University of Würzburg, Germany.
12.20 Genotoxicology of heat processing of food.
Margaretha Jägerstad, Swedish University of Agricultural Sciences, Uppsala, Sweden.
12.50 Natural versus synthetic mutagens and carcinogens.
Lois Swirsky Gold, UC Berkeley and Lawrence Berkeley National Laboratory, Berkeley, USA.
13.20 Closure
Ceramic Hall
13.30 Lunch
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Abstracts
S1 - Symposium 1
Oral Presentation
OS1001
ATM: MOBILIZING THE CELLULAR RESPONSE TO
DOUBLE-STRAND BREAKS
Y Shiloh
Tel Aviv University, TEL AVIV, Israel
The intricate signaling network mobilized by DNA
damage spans numerous pathways linking DNA
repair, cell cycle checkpoints and stress responses
that affect various aspects of cellular metabolism. A
prominent inducer of this response is the double
strand break (DSB). Formation of DSBs in the DNA
leads to rapid recruitment of sensor/activator pro-
teins to the damaged sites. Among them are the MRN
complex whose core contains the Mre11, Rad50 and
Nbs1 proteins and the BRCT proteins Brca1, 53BP1 and
Mdc1/Nfbd1. This process is required for the next step
in the DSB response, the activation of the nuclear pro-
tein kinase ATM - the primary transducer of the DSB
alarm. ATM, a member of the PI3-kinase-like protein
kinase (PIKK) family, is activated via autophosphory-
lation, and a portion of it binds to the DSB sites.
Activated ATM then phosphorylates key players in a
wide range of processes. Repeated cycles of phospho-
rylation at the DSB sites, which include as target the
histone H2AX, lead to further recruitment of the sen-
sor/activator proteins and the formation of promi-
nent protein foci at these sites. Deficiencies of ATM or
components of the MRN complex lead to genomic
instability syndromes such as ataxia-telangiectasia
(A-T), A-T-like disease (A-TLD) and the Nijmegen
breakage syndrome (NBS). We are investigating the
early events that precede ATM activation and are
seeking novel downstream processes represented by
new ATM substrates. Two such substrates will be dis-
cussed: the COP9 signalosome (CSN) protein complex
and the co-repressor protein KAP-1. These new func-
tional links in the web of the DNA damage response
add further dimension to the increasing complexity
and richness of this process.
S1 - Symposium 1
Oral Presentation
OS1002
DYNAMIC ORGANIZATION OF DNA DAMAGE
REPAIR PROTEINS AND CHROMOSOMES
R Kanaar
Erasmus Medical Center, ROTTERDAM,
The Netherlands
Homologous recombination, the exchange of DNA
sequence between homologous DNA molecules, is
essential for accurate genome duplication and preser-
vation of genome integrity. DNA double-strand
breaks (DSBs) and single-stranded gaps are efficient
initiators of homologous recombination, which
results in their accurate repair using an intact homol-
ogous template DNA in the same cell. Homologous
recombination requires the co-ordinated action of the
RAD52 group proteins, including Rad51, Rad52 and
Rad54. Upon treatment of mammalian cells with ion-
izing radiation, these proteins accumulate into foci at
sites of DSB induction. We probed the nature of the
DNA damage-induced foci in living cells with the use
of photobleaching techniques. These foci are not stat-
ic assemblies of DNA repair proteins. Instead, they
are dynamic structures of which Rad51 is a stable core
component, while Rad52 and Rad54 reversibly inter-
act with the structure. Furthermore, even though the
RAD52 group proteins colocalize in the DNA damage-
induced foci, the majority of the proteins are not part
of the same multi-protein complex in the absence of
DNA damage. Executing DNA transactions through
dynamic multi-protein complexes, rather than stable
holo-complexes, allows greater flexibility during the
transaction. In case of DNA repair, for example, it
allows cross talk between different DNA repair path-
ways and coupling to other DNA transactions, such as
replication.
Interactions between ends from different DSBs can
produce tumorigenic chromosome translocations.
Two theories for the juxtaposition of DSBs in translo-
cations, the static ‘contact-first’ and the dynamic
‘breakage-first’ theory, differ fundamentally in their
requirement for DSB mobility. To test whether or not
DSBs are mobile, we introduced linear tracks of DSB-
containing chromosome domains in nuclei. We
observed changes in track morphology within min-
utes after DSB induction indicating movement of the
domains. In a sub-population of cells domains
formed clusters. Juxtaposition of different DSB-con-
taining chromosome domains through clustering,
which was most extensive in G1 phase cells, suggests
an adhesion process in which we implicate the Mre11
protein complex. Our results support the ‘breakage-
first’ theory to explain the origin of chromosomal
translocations.
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S1 - Symposium 1
Oral Presentation
OS1003
THE EFFECT OF P53 MUTATIONS ON CELL CYCLE
CONTROL AND CARCINOGEN-INDUCED TUMORI-
GENESIS
A de Vries
National Institute of Public Health and the
Environment, BILTHOVEN, The Netherlands
In vivo analysis of p53 function has been important to
confirm the importance of this gene in tumor sup-
pression. Homozygous p53 knockout mice develop
tumors with 100% incidence, primarily lymphomas.
As a more fine-tuned approach to study the role of
p53 in tumor suppression, we have generated
germline mutations in p53.
In the p53.R270H knock-in mouse model, an Arginine
to Histidine mutation was introduced in codon 270
(human 273) of the mouse p53 gene. This mutation is
a hot-spot mutation in several human and mouse
tumor types and is frequently found as a germ-line
mutation in the human Li-Fraumeni syndrome. In
addition to the mutation, a transcriptional stop-cas-
sette flanked by loxP sites was introduced in intron 1
of the p53 gene. As a result, expression of the R270H
protein can be induced through crosses with (tissue-
specific) Cre-transgenic mice. p53.R270H mice crossed
with mammary gland-specific WAP-Cre mice develop
mammary tumors with high incidence and short
latency time. In addition, chronic exposure of skin-
specific p53.R270H/ K14-Cre mice to UVB radiation
results in the same tumor phenotype. These experi-
ments show that the R270H mutation is an important
trigger for tumor development acting in a dominant-
negative manner.
Another mutation we generated is a Serine to
Alanine substitution at residue 389 (human 392).
Codon 389 is specifically phosphorylated after expo-
sure to UV radiation, whereas gamma radiation
involves phosphorylation of different residues. This
post-translational modification is envisioned to acti-
vate the p53 protein to exert its cellular functions.
Mice harboring the S389A mutation are sensitive to
UV- and 2-AAF induced tumor development, and
S389A cells display reduced p53-dependent cellular
responses. Additional in vivo and in vitro experi-
ments to further explore the phenotype of these p53
mutant mice are currently ongoing, and results
obtained will be presented.
Supported by Dutch Cancer Society and NIH/ NIEHS-
CMGCC.
S1 - Symposium 1
Oral Presentation
OS1004
TRANSLESION SYNTHESIS AND THE Y-FAMILY OF
DNA POLYMERASES
AR Lehmann, PL Kannouche
University of Sussex, BRIGHTON, United Kingdom
Most types of DNA damage block replication fork pro-
gression during DNA synthesis because replicative
DNA polymerases are unable to accommodate altered
DNA bases in their active sites. To overcome this
block, eukaryotic cells employ specialised translesion
synthesis (TLS) polymerases, which can insert
nucleotides opposite damaged bases. In particular,
TLS by DNA polymerase eta (pol eta) is the major
pathway for bypassing UV photoproducts. Pol eta is
deficient in individuals with the variant form of xero-
derma pigmentosum.
Pol eta is mostly localised uniformly in the nucleus,
but is associated with replication foci during S phase.
Following treatment of cells with UV-irradiation or
chemical carcinogens, the number of cells in which
pol eta accumulates into foci increases dramatically.
These foci are sites at which replication forks are
stalled at DNA damage. The C-terminal 120 aa are
needed for localisation in nuclei and into replication
foci. This region contains a nuclear localisation sig-
nal, a putative C2H2 zinc finger and a PCNA binding
domain, all of which are required for localisation of
pol eta into replication foci. Pol eta truncations lack-
ing the C-terminal 120 aa fail to correct the defects in
XP-variant cells.
How the cell switches from replicative to TLS poly-
merase at the site of blocked forks is unknown. In
human cells, PCNA becomes mono-ubiquitinated fol-
lowing UV-irradiation. Mono-ubiquitinated PCNA,
but not unmodified PCNA, specifically interacts with
pol eta and we have identified two motifs in pol eta
which are involved in this interaction. Our findings
provide an attractive mechanism by which mono-
ubiquitination of PCNA might mediate the switch
from replicative to TLS polymerase at the site of a
replication fork stalled at DNA damage.
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S2 - Symposium 2
Oral Presentation
OS2001
DISCRIMINATION OF GENOTOXIC FROM NON-
GENOTOXIC CARCINOGENS BY EXPRESSION PRO-
FILING IN VITRO.
JHM van Delft, E van Agen, SGJ van Breda, MH van
Herwijnen, DM van Leeuwen, YCM Staal,
JCS Kleinjans
Maastricht University, MAASTRICHT, The Netherlands
Two general mechanisms are implicated in chemical
carcinogenesis. The first involves direct damage to
DNA, referred to as genotoxic (GTX), to which the cell
responds by repair of the damages, arrest of the cell
cycle or induction of apoptosis. The second is non-
DNA damaging, non-genotoxic (NGTX), in which a
wide variety of cellular processes may be involved.
Therefore, it is likely that modulation of the underly-
ing gene expression patterns is distinct between GTX
and NGTX carcinogens, and thus that expression pro-
filing can classify chemical carcinogens as GTX or
NGTX.
We investigated this hypothesis by analysing gene
regulation induced by 20 chemical carcinogens in
HepG2 cells with microarrays that contain 597 toxico-
logically relevant genes. A training data set was gen-
erated consisting of 16 treatments (9 GTX and 7
NGTX) and the validation set of 6 treatments (3 and
3). Several class discrimination models were applied,
based on nearest shrunken neighbours analyses.
Models were developed with data from the training
set, where after they were tested with all data. The
correct classification of the carcinogens from the
training and validation set were grossly similar,
namely 88 and 83% respectively. Exclusion of the
treatments with only marginal effects on the expres-
sion profiles, improved the correct classification for
the training and validation sets both to 100%. The
prediction strongly depends on treatment period and
type of cells used. Building a prediction model based
on 24 h treatment data, poorly predicts classes fol-
lowing a 6 h treatment. Similarly, a model based
HepG2 data, poorly predicts classes generated in
human lymphocytes. Interestingly, many of the dis-
criminating genes are involved in apoptosis and cell
cycle control, some in DNA-damage response, but
none in DNA-repair.
S2 - Symposium 2
Oral Presentation
OS2002
GENE EXPRESSION PROFILING OF GENOTOXIC
AND NON-GENOTOXIC CARCINOGENS IN RAT
LIVER
J Ahr, H Ellinger-Ziegelbauer
Bayer Health Care AG, WUPPERTAL, Germany
Application of the recently developed gene expres-
sion techniques using microarrays in toxicological
studies (toxicogenomics) facilitates the interpreta-
tion of a toxic compound´s mode of action and may,
vice versa, allow the prediction of selected toxic
effects based on gene expression changes. In order to
test this hypothesis we investigated whether carcino-
gens at doses known to induce liver tumors in the
two year rat bioassay deregulate characteristic sets of
genes in a short time in vivo study. Male Wistar rats
were treated for up to 14 days with genotoxic and
non-genotoxic carcinogens. After 1,3,7, and 14 days the
livers were taken for histopathology and for analysis
of the gene expression profiles. By using statistical
and clustering tools, characteristically deregulated
genes were extracted and then functionally classi-
fied. Both common as well as compound specific
effect with regard to the functional classes of deregu-
lated genes were observed. Common to genotoxic car-
cinogens were a DNA damage response and the
activation of proliferative and survival signaling
pathways. Non-genotoxic carcinogens generally
showed induction of oxidative stress genes as well as
signs of DNA replication and cell cycle progression. In
addition many of the gene alterations found with
non-genotoxic carcinogens imply compound specific
mechanisms. Based on this mechanistic analysis it
became evident that common gene expression
responses for both classes of carcinogens may exist.
Therefore, with gene ranking and classification algo-
rithms discriminating marker genes were identified
and evaluated for their value to classify genotoxic
versus non-genotoxic carcinogens. The results sug-
gest, that it may be possible to identify a carcinogenic
potential and to discriminate genotoxic from non
genotoxic mechanisms by applying such algorithms
on expression profiles from short time studies.
16
abstractboek 20-10-2004 10:28 Pagina 16
S2 - Symposium 2
Oral Presentation
OS2003
TOXICOGENOMIC ANALYSIS OF GENE EXPRES-
SION: AN EMERGING APPROACH FOR DIFFEREN-
TIATING GENOTOXIC MECHANISMS
J Aubrecht
Pfizer, GROTON, CT, United States of America
During the safety evaluation process of new drugs, a
battery of in vitro genotoxicity tests is conducted.
Positive results are not uncommon and their biologi-
cal relevance needs to be determined. Without this,
the risk assessment of genotoxic agents is generally
based on linear extrapolation methods, though there
is substantial evidence that some chemicals may
exhibit a clear thresholded dose-response. Hence,
gaining an insight into genotoxic mechanisms, i.e.,
differentiation of DNA-reactive vs. DNA non-reactive,
is essential for risk assessment to humans. This
results in laborious and time consuming follow-up
strategies. Genotoxic stress triggers a variety of bio-
logical responses including the transcriptional acti-
vation of genes regulating DNA repair, cell survival
and cell death. Thus, we evaluated the utility of gene
expression profile analysis in cultured cells in vitro
for gaining an insight into genotoxic mechanisms
using a set of model agents. The gene expression
changes were compared with micronucleus induc-
tion and direct DNA damage. We investigated
whether gene expression profiles can differentiate
between DNA reactive and DNA non-reactive mecha-
nisms of genotoxicity including those associated
with general toxic stress. Although, more experimen-
tal work including evaluation of a large number of
compounds and stresses is necessary to refine and
enhance our understanding of gene expression pro-
files and toxic pathways, our results suggest the
potential utility of gene expression profile analysis
for evaluating molecular mechanisms of action of
genotoxicants.
S2 - Symposium 2
Oral Presentation
OS2004
MICROARRAY EXPERIMENTAL DESIGN, QUALITY
ASPECTS OF DATA COLLECTION AND STATISTICS
TW Gant, SD Zhang
Medical Research Council, LEICESTER, United Kingdom
Biological systems, and their reactions to xenobiotic
stress, are complex. Until recently our view of this
complexity was obscured by the simplicity of the
analysis tools available. For example prior to microar-
rays the analysis methods of gene expression would
allow the determination of only a few genes in any
one study. Then the genome sequencing and high
throughput library screening projects commenced
and produced gene clones and sequence data which
previously had not been available. To exploit this new
resource the microarray was invented (1). Further
development has expanded the number of clones
contained on any one microarray a point where now
an individual researcher can determine the expres-
sion of many thousands of genes in a system in a few
days. What these data are revealing is the full com-
plexity of the gene expression response to stimuli
such as xenobiotic exposure. Toxicogenomics is seek-
ing to use the complexity of this response as a finger-
print or signature characteristic of xenobiotic
exposure (2,3). Molecular toxicology uses the detail of
individual differentially expressed genes to identify
mechanisms of toxicity. The challenge for both analy-
ses though is data quality (4). Gene expression has
always been difficult to accurately determine, a
reflection of technique deficiencies and natural
intrinsic variability. Thus statistically relevant data of
differential gene expression has always been difficult
to obtain. For microarrays this problem is multiplied
by the number of genes on the microarray itself. To
overcome this technical and experimental variability
correct experimental design is critical (5). We have
recently produced a framework for the design of
microarray experiments and statistical analysis of
microarray data (Zhang and Gant, In Press). This pres-
entation will cover aspects of this design framework
and data analysis.
1. Nature Genetics 21,15(1999)
2. Molecular Carcinogenesis 24,153(1999)
3. TIPS 23,388(2002)
4. PNAS 99,12975(2002)
5. Nature Genetics 32,490(2002)
17
abstractboek 20-10-2004 10:28 Pagina 17
S3 - Symposium 3
Oral Presentation
OS3001
STRESS, MUTATIONS AND AGING
TBL Kirkwood
University of Newcastle, NEWCASTLE UPON TYNE,
United Kingdom
As predicted by the disposable soma theory of aging,
evidence from many lines of research confirms that
senescence is a process of gradual accumulation of
damage in cells and tissues of the body, leading even-
tually to frailty and increased risk from a spectrum of
age-associated diseases. Multiple kinds of stress-
induced damage affect cells, ranging from mutations
in DNA to oxidative attack on proteins. Some of our
recent work has shown how this damage impairs the
function of tissue stem cells – specifically stem cells
of intestinal epithelium – as ageing proceeds. The key
to understanding the factors that regulate aging and
longevity is to be found in the network of cell main-
tenance systems that slow the accumulation of dam-
age. For example, long-lived species carried out repair
better than short-lived species and are endowed with
grater capacity to withstand stressors. A major chal-
lenge is therefore to understand how the diverse
mechanisms which make up this network interact
with each other. Using mathematical and computer
modeling of molecular mechanisms of aging, we are
developing a Biology of Aging e-Science Integration
and Simulation system (BASIS) to support the devel-
opment of an integrated understanding of the under-
lying cell and molecular mechanisms of age-related
frailty and disease.
S3 - Symposium 3
Oral Presentation
OS3002
GENOME INSTABILITY, AGING AND CANCER
J Vijg
University Texas Health Science Centre,
SAN ANTONIO, United States of America
Genomic instability as a consequence of the natural
limitations of DNA maintenance and repair has been
implicated as a major causal factor in both cancer and
aging. Defects in genome maintenance lead to
increased tumor formation and the premature appear-
ance of various symptoms of aging, possibly through
increased genome instability. Using a transgenic
mouse model with a chromosomally integrated lacZ
mutational target gene, we demonstrated that muta-
tions accumulate with age in an organ- and tissue-
specific manner. Depending on the organ, many of
the mutations accumulating during aging were
genome rearrangements rather than point muta-
tions, some of them involving millions of basepairs.
Furthermore, while accelerated mutation accumula-
tion was observed in mouse models of premature
aging, long-lived mutant mice, i.e., Ames dwarf mice,
were characterized by a delayed age-related increase
in mutation frequency. How can randomly accumu-
lating somatic mutations cause organ dysfunction at
old age? While only 2% of the genomic sequence
encodes proteins, almost half of it is transcribed and
possibly plays a role in the regulation of gene expres-
sion. Hence, random mutations, especially large
genome rearrangements, can have adverse effects on
normal patterns of gene regulation, which we specu-
late can result in a mosaic of cells at various stages on
a trajectory of functional decline eventually resulting
in cell death or neoplastic transformation. To further
investigate this possibility we are presently studying
single cells, directly obtained from organs of young
and old mice through enzymatic disassociation, for
changes in gene expression profiles using real-time
PCR and microarrays.
18
abstractboek 20-10-2004 10:28 Pagina 18
S3 - Symposium 3
Oral Presentation
OS3003
CROSSTALK BETWEEN TELOMERES AND DNA
DAMAGE REPAIR
MAB Blasco
Spanish National Cancer Centre (CNIO), MADRID,
Spain
Telomeres are specialized regions of the chromatin
that cap linear chromosomes. Telomere dysfunction,
either due to critical telomere shortening, or due to
loss of a proper telomere capping structure, leads to
end-to-end chromosome fusions and loss of cell via-
bility. Significantly, a number of mammalian pro-
teins have evolved dual roles in DNA repair and
telomere function. In particular, several DNA repair
proteins are components of the telomeric chromatin,
as well as have a role in processing and signaling of
dysfunctional telomeres. On the other hand, dysfunc-
tional telomeres have been shown to interfere with
proper DNA double strand break (DSB) repair, thus
modifying the sensitivity to genotoxic agents.
Significantly, telomere shortening is accelerated in
many DNA repair human syndromes, such as
Fanconi´s anemia (FA), suggesting that the proteins
involved could also be constitutive telomeric proteins
and have a role in telomere metabolism. To evaluate a
telomeric function for different DNA repair proteins,
we have studied telomere length and telomere
capping in various mouse models deficient for these
proteins.
S3 - Symposium 3
Oral Presentation
OS3004
AGING, CANCER AND CELLULAR RESPONSES TO
DNA DAMAGE
J Campisi
Lawrence Berkeley National Laboratory, BERKELEY,
United States of America
Environmentally-induced damage, particularly dam-
age to nuclear DNA, can elicit either of two cellular
tumor suppressor responses – apoptosis or cellular
senescence. Both of these responses evolved to pro-
tect complex multi-cellular organisms from develop-
ing cancer. Apoptosis culminates in cell death, and
thus protects the organism by eliminating potential
cancer cells. The senescence response, by contrast,
prevents cancer by permanently arresting the growth
of cells at risk for neoplastic transformation. In addi-
tion to suppressing cancer, several lines of evidence
suggest that both the apoptotic and senescence
responses also contribute to organismal aging and
age-related pathologies. Thus, these cellular tumor
suppressor mechanisms may be examples of evolu-
tionary antagonistic pleiotropy. That is, while these
responses protect organisms from malignant tumori-
genesis early in life, later in life – as tissues lose cells
due to apoptosis or accumulate dysfunctional senes-
cent cells – apoptosis and cellular senescence may
contribute to the age-related loss of tissue structure
and function and the development of certain age-
related pathologies. We have found that senescent
stromal fibroblasts have a striking influence on the
behavior of premalignant and normal epithelial cells.
Using the mammary gland and three dimensional
cultures as a model system, we find that senescent
fibroblasts stimulate the proliferation of mammary
epithelial cells. In addition, the senescent stroma dis-
rupts normal functional and morphological differen-
tiation of mammary epithelial cells. Together, these
findings support the idea that cellular senescence is
antagonistically pleiotropic, and that senescent cells
can contribute to age-related tissue dysfunction as
well as certain age-related pathologies, including
cancer.
19
abstractboek 20-10-2004 10:28 Pagina 19
FSAL - Fritz Sobels Award Lecture
Oral Presentation
OS3FS1
HOW RARE GENETIC DISEASES CAN HELP US
UNDERSTANDING MUTAGENESIS AND CARCINO-
GENESIS ?
AS Sarasin
Institut Gustave Roussy, VILLEJUIF, France
Cancer is a genetic disease due to the accumulation of
numerous mutations rendering the tumour cell
insensitive to control by the local environment and
by the whole organism. Analysis of the frequency of
human cancer as a function of age shows that
between 5 and 7 mutations in key genes are usually
necessary to produce most human cancers.
The major demonstration bridging the lack of DNA
repair, mutation induction and cancer development
is the existence of DNA repair-deficient diseases asso-
ciated with a very high frequency of cancer. For exam-
ple, xeroderma pigmentosum (XP) is a rare hereditary
disease recessively-transmitted. XP patients are char-
acterized by an extreme photosensitivity and a high
predisposition to skin cancer. Nucleotide excision
repair (NER) of UV-induced DNA lesions is deficient in
XP cells due mutations in one of the seven XP genes.
XP variant is another form of XP associated with a
late onset of clinical symptoms, due to increased
error-prone translesion synthesis, leading therefore,
to higher level of targeted point mutations.
Besides XP, genetic disorders on the RB or p53 genes
allowed the discovery of tumour suppressor genes.
The Fanconi's anaemia, the ataxia telangiectasia and
the SCID-like patients allowed to better understand-
ing homologous and illegitimate recombination as
well as the cascades of DNA lesion signalisation.
HNPCC patients allowed the discovery of the role of
mismatch repair in human tumours as well as on the
instability of several other repair genes. Finally,
BRCA1/2 genes and Cockayne's syndrome patients
allowed us connecting cancer-proneness or neurolo-
gical disorders to transcription-coupled repair of
oxidative lesions.
Rare genetic diseases do not concern only few thou-
sands affected patients, but represent beautiful
examples of human 'mutants' very useful for under-
standing the normal mutagenesis and carcinogene-
sis pathways leading to cancer including in the
general population.
S4 - Symposium 4
Oral Presentation
OS4001
CANCER: GENES AND THE ENVIRONMENT
KH Hemminki
DKFZ, HEIDELBERG, Germany
In the search for cancer etiology, the contribution
by heritable and environmental causes has been
debated. Cancer geneticists have emphasized the for-
mer while epidemiologists traditionally argued for
the latter. The literature on heritable cancers is full of
overstatements regarding their prevalence but recent
data from twin studies may offer scientifically based
estimates on the etiological apportioning of cancer
causation. With the emergence of single nucleotide
polymorphisms (SNPs) as versatile tools, studies on
‘gene-environment interactions’ have become popu-
lar, however with many embedded controversial
issues. Mass publication is going on and results are
reported without consideration of the functionality
of SNPs or tissue of expression of the relevant genes
in subgroups lacking any biological rationale. It is
ironical that the historical roles of epidemiologists
and geneticists appear to be completely changed: the
proponents of gene-environment interactions appear
to trust on the overwhelming importance of heritable
factors when they act in concert with environmental
factors while geneticists are raising concerns. There is
a common failure to recognize that SNPs are inherited
and that the related studies focus essentially on
heritable effects. Thus any cancer with a small
familial effect cannot show large effects in genetic
association studies on common polymorphisms.
Analogously, nesting of an association study in famil-
ial cancers is beneficial for statistical power. We hope
that those who plan association studies would take
notice of the familial risks and proportions to be
found in the literature.
20
abstractboek 20-10-2004 10:28 Pagina 20
S4 - Symposium 4
Oral Presentation
OS4002
LOW PENETRATION GENES AS RISK FACTORS IN
TOBACCO SMOKE-ASSOCIATED LARYNGEAL CAN-
CER
K Szyfter, M Gajecka, M Rydzanicz, M Wierzbicka,
K Szymoniak
Institute of Human Genetics, Pol Acad Sc, POZNAN,
Poland
Incidence of laryngeal cancer is strongly associated
with tobacco smoking and abusing of strong alco-
holic beverages. However, only a fraction of exposed
persons develops laryngeal cancer.
The attempts to identify a significance of genetic fac-
tor concern genetic polymorhism in genes coding car-
cinogen activation, detoxication and DNA repair. A
distribution of polymorhic genes and alleles was
studied in relation to CYP1A1, CYP 2E1 (activation),
GSTM1, GSTT1, GSTM3, NAT2 (detoxication) and XPD,
XRCC1, XGCC3 and OGG1 genes. Out of 11 examined
polymorhisms significant differences were estab-
lished in distribution of polymorphic variants of
CYP1A1, GSTM1 and NAT2 genes. A co-incidence of
some cancer risk variants was found as a factor mul-
tiplying a risk. A comparison of the subjects with a
single primary tumour with subjects with multiple
primary tumour has shown an involvement of the
same genes in risk estimate but an association was
stronger for MPT.
S4 - Symposium 4
Oral Presentation
OS4003
LUNG CANCER AND POLYMORPHISMS OF GENES
INVOLVED IN CARCINOGEN METABOLISM AND
DNA REPAIR
F Canzian, P Boffetta, F Gemignani, J Hall, RJ Hung,
S Landi, P Brennan
International Agency for Research on Cancer, LYON,
France
Even though lung cancer is predominantly caused by
tobacco, only a minority of smokers will develop lung
cancer. A possible explanation for this is that the
metabolization of carcinogenic products, the level of
internal dose and subsequent DNA repair and cell
cycle control mechanisms vary widely between indi-
viduals because of genetic factors.
We are investigating the role of over 100 genes which
are potentially involved in the susceptibility to lung
cancer, by means of a study conducted in six coun-
tries of Central and Eastern Europe, involving 2300
lung cancer cases and 2800 controls. This is a multi-
centric study, conducted with an identical protocol
involving the collection of high quality detailed infor-
mation on lifestyle and occupational history, as well
as blood collection for DNA extraction.
The genes comprise those involved in the metabolism
of tobacco products and other potential carcinogens
(e.g. CYPs, GSTs, MPO), as well as genes involved in
DNA repair (e.g. XRCC1, XRCC3, XPD, XPF), tumor sup-
pression (p53, p16, CCND1 ) and nicotine addiction
(dopamine D2 and D4 receptor genes).
We are genotyping 250 polymorphisms by DNA
microarrays in the subjects with young age at onset,
and a subset of 100 polymorphisms by TaqMan in the
whole sample set. Using this large sample size, we
aim at accurately measuring the overall effect of each
gene in lung cancer. Subsequently, the effect of com-
binations of genes will be measured (gene-gene
interaction), as well as the effect of individual genes
in specific subgroups identified by tobacco consump-
tion and occupational history (gene-environment
interaction). Statistical techniques include haplotype
reconstruction, empirical Bayes and semi-Bayes
analysis to control for false positive results, and mod-
eling of complex pathways. A first set of preliminary
results will be presented.
21
abstractboek 20-10-2004 10:28 Pagina 21
S4 - Symposium 4
Oral Presentation
OS4004
DESIGN AND INTERPRETATION OF STUDIES ON
GENE-ENVIRONMENT INTERACTIONS IN CAR-
CINOGENESIS
P Vineis
Imperial College London, LONDON, United Kingdom
The purpose of the presentation is to describe a spe-
cific study on gene-environment interactions, nested
within a large cohort. I will describe the practical
problems associated with the processing, storage,
and analysis of blood samples, laboratory validation
studies nested within the GenAir investigation.
Several large prospective investigations are under
way or are planned in different parts of the world,
aiming at the investigation of gene-environment
interactions for chronic diseases. Such studies are
extremely complex, involving the collection of hun-
dreds of thousands of biological samples within the
general population (e.g. 500,000 each in EPIC and UK-
Biobank). Technical, practical and ethical issues are
raised by such large investigations. I wish to describe
how such issues were dealt with within a case-con-
trol study nested in EPIC, a large European cohort, and
the kind of validation studies that have been set up.
The GenAir investigation aimed to measure the
effects of air pollution and ETS on human health in
EPIC with a nested design and with biological meas-
ures. Validation studies included (a) comparisons
between cotinine measurements, hemoglobin adducts
and questionnaire data; (b) an analysis of the deter-
minants of DNA adduct concentration; (c) comparison
among different genotyping methods; (d) an analysis
of the determinants of plasma DNA amounts.
S5 - Symposium 5
Oral Presentation
OS5001
FLAVONOIDS AND ALKENYLBENZENES: MECHA-
NISMS OF MUTAGENIC ACTION AND CARCINO-
GENIC RISK
IMCM Rietjens, GM Alink
Wageningen University, WAGENINGEN,
The Netherlands
In spite of a long history of use botanical or herb-
based preparations may contain individual ingredients
known to be toxic and even genotoxic and carcino-
genic. The present lecture focuses on two categories
of botanical ingredients, the flavonoids with quer-
cetin as an important bioactive representative, and
the alkenylbenzenes including safrole, methyl-
eugenol and estragole.
For quercetin a metabolic pathway for activation to
DNA-reactive species includes enzymatic and/or
chemical oxidation to quercetin ortho-quinone, fol-
lowed by isomerisation of the ortho-quinone to
quinone methides. These quinone methides are sug-
gested to be the active alkylating DNA-reactive inter-
mediates. Recent insights suggest that the transient
nature of quercetin quinone methide adducts
observed in vitro, may affect extrapolation of the
genotoxicity to the carcinogenicity in vivo.
For alkenylbenzenes the ultimate electrophilic and
carcinogenic metabolite is their 1'-sulfooxy-deriva-
tive. Identification of the cytochrome P450 isoenzymes
involved in bioactivation of the alkenylbenzenes iden-
tifies the groups within the population at higher risk,
due to life style factors or genetic polymorphisms.
Furthermore, a relative decrease in conversion to the
carcinogenic metabolite at lower doses, may provide
another important argument that has to be taken
into account in the risk assessment of alkenylben-
zenes.
Together the results presented stress that mechanis-
tic data have to be taken into account and that new
mechanism- and toxicokinetic-based models are
required for cancer risk extrapolation from high dose
experimental data and in vitro data to low dose car-
cinogenic risks. Also, in vitro mutagenicity studies
should put more emphasis on the reversible nature of
the DNA adducts responsible for the mutagenicity in
vitro, since the stability of the formed DNA adducts
may play an essential role in whether the genotoxicity
observed in vitro will have any impact in vivo.
22
abstractboek 20-10-2004 10:28 Pagina 22
S5 - Symposium 5
Oral Presentation
OS5002
GENOTOXICITY OF PHYTOESTROGENS
H Stopper
University of Wuerzburg, WUERZBURG, Germany
Plant extracts containing phytohormones are mar-
keted as a 'natural' medicine for many kinds of dis-
eases. On the other side, the in vivo carcinogenicity
of certain estrogens such as diethylstilbestrol is well
known. Here we review data on in vitro genotoxicity
of phytohormones. Genistein, coumestrol, quercetin,
zearalenon, and resveratrol exerted genotoxic effects
in in vitro test systems. Other phytoestrogens such as
lignans, the isoflavones daidzein and glycetein,
anthocyanidins, and the flavonol fisetin were found
to have only weak or no effects in vitro. However,
some metabolites of daidzein showed a genotoxic
activity. In hormone-responsive cells, an additional
pathway for induction of genomic damage may be
exerted through the stimulation of enhanced cell pro-
liferation, yielding cell cycle checkpoints less exact.
This has been demonstrated for estradiol, and could
now also be detected after treatment of cells with the
phytoestrogen daidzein. In conclusion, the safety of
concentrated formulations of phytoestrogens needs
to be investigated in more detail. Some concerns that
have been raised for endogenous or synthetic estro-
gens may also be true for phytoestrogens. However,
this should not lead to a reduction of a high intake of
plant food, which is still one of the best cancer pre-
vention strategies.
S5 - Symposium 5
Oral Presentation
OS5003
GENOTOXICITY OF HEAT PROCESSED FOODS
I Jagerstad
Swedish University of Agricultural Sc., UPPSALA,
Sweden
One of the first reports of food carcinogens generated
by cooking was reported in 1939 by a Swedish scien-
tist (Widmark), who prepared organic solvent extracts
of grilled horse meat and repeatedly painted them on
the skin of mice, resulting in tumours in the mam-
mary glands. During the 1960s and 1970s, much inter-
est was focussed on two new classes of chemicals
producing tumours in long-term animal studies -
polycyclic aromatic hydrocarbons (PAHs) and
N-nitrosocompounds. Since the early 1970s genotoxi-
city of cholesterol oxides also have been investigated.
In the late 1970s, a new, highly mutagenic class of
compounds, heterocyclic amines (HAs) was identified
in meat extract and grillled or broiled meat and fish.
More recently, reports on the presence of acrylamide
in a range of fried and oven-cooked foods have caused
worldwide concern because this compound has been
classified as probably carcinogenic in humans. The
occurrence in food of these mutagens/carcinogens is
very much dependent on the cooking condions and
the presence of precursors and modifiers. Following a
number of evaluations, the International Agency for
Research on Cancer (IARC) has come to the conclusion
that several of these food-borne genotoxic com-
pounds are probably or possibly carcinogenic to
human, based on both high-dose, long-term animal
studies and in vitro genotoxicity tests.Yet, there is
insufficient scientific evidence that these mutagens
really cause human cancer, and no limits have been
set for their presence in cooked foods. However, the
relevant authorities in most Western countries rec-
ommend minimising their occurrence. This presenta-
tion proviedes a brief overview of the state-of-the-art
research on some of these compounds, particularly
heterocyclic amines and acrylamide with respect to
their formation, occurrence, exposure, risk evaluation
and ways of minimising their levels in foods using
improved cooking methods and developments in
food technology.
23
abstractboek 20-10-2004 10:28 Pagina 23
S5 - Symposium 5
Oral Presentation
OS5004
NATURAL VS. SYNTHETIC CARCINOGENS: MAR-
GIN OF EXPOSURE BETWEEN RODENT CARCINO-
GENIC DOSE AND HUMAN EXPOSURE
LS Gold
UC Berkeley & LBNL, BERKELEY, United States of
America
The focus of cancer testing and regulatory policy is
synthetic chemicals; however, 99.9% of the chemicals
humans ingest are naturally-occurring. The human
diet contains an enormous background of natural
chemicals, e.g. plant pesticides and the products of
cooking, that are not a focus of carcinogenicity test-
ing. In the Carcinogenic Potency Database, half the
chemicals tested, whether natural or synthetic, are
carcinogenic; this high proportion may be due in part
to effects that are unique to high doses. Human expo-
sures to mutagens and rodent carcinogens are ubiq-
uitous.
A broad perspective on possible cancer hazards is pro-
vided by comparing the Margin of Exposure (MOE)
for a variety of humans exposures: MOE is the ratio of
the rodent carcinogenic dose of a chemical to the
human exposure (in mg/kg/day). The lower MOE, the
closer the human exposure is to the rodent carcino-
genic dose. Of the 100 human exposures investigated,
half are to chemicals that occur naturally in food.
MOE ranges a billion-fold across human exposures.
Several categories of exposure to both mutagenic and
nonmutagenic chemicals are investigated: occupa-
tional, pharmaceuticals and herbal supplements, nat-
ural pesticides, synthetic pesticide residues, cooking
and preparation of food, food additives, and environ-
mental contaminants. The lowest margins of expo-
sure are for some historically high exposures in the
workplace and some pharmaceuticals. Exposures to
natural chemicals occur throughout the range of
MOE, and pesticide residues have large MOEs. The
background of natural chemicals casts doubt on the
relative importance of low-dose exposures to syn-
thetic chemicals.
An increasing literature on mode of carcinogenic
action at the high doses used in animal cancer tests,
suggests that there are dose-dependent transitions in
the carcinogenic process and no-observed–adverse-
effect-levels for some chemicals, indicating a lack of
relevance of rodent results for assessing carcinogenic
hazards to humans at low dose.
MS - Minisymposium Biological clocks, chrono-
toxicology and therapy
Oral Presentation
OS6001
IMPLICATIONS OF BIOLOGICAL CLOCKS FOR CAN-
CER PROCESSES AND THEIR TREATMENTS
F Levi
INSERM E 0354 and Chronotherapy Unit, VILLEJUIF,
France
Genetic mutations and polymorphisms account for
differences in the susceptibility of malignant tumours
and normal tissues for anticancer medications. In
addition, most cellular and biochemical functions are
regulated at transcriptional and/or post transcrip-
tional levels by a molecular clock. As a result, 1) the
tolerability and the efficacy of anticancer agents can
vary as a function of circadian dosing time and 2) the
disruption of the circadian system can accelerate
malignant growth. Cancer chronotherapeutics involves
the adaptation of treatment delivery to circadian
rhythms and/or the administration of clock-targeted
therapies.
Circadian rhythms characterize many determinants
of anticancer drug pharmacology in bone marrow,
intestine or liver of rodents. Large 24-hour changes
were found for cell cycle, apoptosis (BCL-2 and BAX),
detoxication (dehydropyrimidine dehydrogenase,
glutathione) or DNA repair (O6 -alkylguanine-DNA
alkyltransferase) and partly related to the expression
patterns of clock genes.
The clinical relevance of cancer chronotherapeutics
has been investigated in over 2000 patients, regis-
tered in Phase I, II or III trials. Oxaliplatin safety and
antitumor activity against colorectal cancer were ini-
tially established based upon drug chronopharmacol-
ogy. In randomised clinical trials, we demonstrated
that the mucosal tolerability of 5-fluorouracil and the
neurological tolerability of oxaliplatin were improved
5-fold and 2 fold respectively, whereas the antitumor
activity was nearly doubled with chronotherapy as
compared with constant rate infusion. In two recent
EORTC trials, we confirmed the improved tolerability
of chronomodulated 5-fluorouracil and Platinum
complex as compared to constant rate infusion, and
found that 2-day infusions could ameliorate tolerabil-
ity as compared to more protracted infusional sched-
ules.
Conclusions: The circadian system plays a clinically
relevant role in the therapeutic index of cancer
treatments. Further development will stem out of a
circadian-based dynamic modelling approach of treat-
ment schedules.
Support: ARTBC, Hôpital Paul Brousse, Villejuif,
France.
24
abstractboek 20-10-2004 10:28 Pagina 24
MS - Minisymposium Biological clocks, chrono-
toxicology and therapy
Oral Presentation
OS6002
CIRCADIAN CLOCKS: NEURAL AND PERIPHERAL
PACEMAKERS
H Hastings, A Reddy, E Maywood
MRC Laboratory of Molecular Biology, CAMBRIDGE,
United Kingdom
Effective adaptation to the solar day is accomplished
by the action of internal timers; circadian clocks that
orchestrate an internal daily programme of physiolo-
gy and behaviour. The principal pacemaker is the
suprachiasmatic nuclei (SCN) of the hypothalamus.
This is entrained to the solar cycle by retinal innerva-
tion, but its ability to oscillate with a near 24-hour
period is an intrinsic property of its neurons. Signals
emanating from the SCN, both neural and neuroen-
docrine, control the activity of sub-ordinate oscilla-
tors in a wide range of peripheral tissues. The genes
encoding the SCN and peripheral oscillators have been
identified, and the core timing mechanism charac-
terised as an auto-regulatory, transcriptional/post-
translational negative feedback loop. This molecular
mechanism drives daily waves of gene expression
involving about 10% of the transcriptome of any one
tissue. The targets of the daily clock inclide cell cycle
factors, through which cell division is synchronised to
solar time.
W1 - Workshop 1
Oral Poster Presentation
OW1001
ALTERED VEGETABLE INTAKE AFFECTS PIVOTAL
CARCINOGENESIS PATHWAYS IN COLON MUCOSA
FROM ADENOMA PATIENTS AND CONTROLS
SGJ van Breda1, E van Agen1, LGJB Engels2,
JC Moonen1, JHM van Delft1
1 Maastricht University, MAASTRICHT,
The Netherlands2 Maasland Hospital, SITTARD, The Netherlands
Globally, cancer of the colon and rectum is the fourth
most common incident cancer and cause of death
from cancer. The evidence from epidemiological
and experimental studies that vegetables reduce the
risk of colorectal cancer is convincing. However, the
involved genes and genetic pathways are not clear.
Microarray technology makes it possible to assess the
effect of a specific vegetable diet on the expression of
multiple genes. Therefore, a human dietary interven-
tion study was carried out to identify the genes the
expression of which is modified in vivo in normal
human colorectal mucosa by vegetables. Twenty
female adenoma patients and eight healthy controls
were randomly split into two groups of ten and four
persons respectively, receiving either a decreased
(= 75 g/day) or increased (= 300 g/day) amount of veg-
etables for a period of two weeks. In order to assess
effects on gene expression at target level, before and
after the intervention colorectal biopsies from
healthy mucosa tissue were collected. Total RNA was
isolated from the biopsies to measure gene expres-
sion of 597 toxicologically relevant genes by means of
microarrays. Comparison of pre- and post-interven-
tion samples shows that 20 genes were modulated
which are related to (colon)carcinogenesis, including
c-fos proto-oncogene, ornithine decarboxylase and
cyclooxygenase-2. An increased intake of vegetables
resulted in down-regulation of genes promoting cell
proliferation and bioactivation of procarcinogens,
and in up-regulation of genes involved in cell growth
arrest; in contrast, a decreased intake of vegetables
resulted in down-regulation of genes inhibiting cell
growth and up-regulation of genes promoting cellu-
lar differentiation and bioactivation of procarcino-
gens. Some of the genes play a role in known colon
carcinogenic pathways and could thus be new targets
for chemoprevention.
25
abstractboek 20-10-2004 10:28 Pagina 25
W1 - Workshop 1
Oral Presentation
OW1002
THE EFFECT OF PLANT PHENOLS ON AP-1 EXPRES-
SION IN MOUSE EPIDERMIS
W Baer-Dubowska, V Krajka-Kuzniak, H Szaefer
Poznan University of Medical Sciences, POZNAN,
Poland
A wide array of phenolic substances present in edible
plants, have been reported to possess anticarcino-
genic and antimutagenic properties. These include
also polyphenol, tannic acid (TA), simple phenol, pro-
tocatechuic acid (PCA) and chlorogenic acid (CHA)
and phytoalexin 3,5,4’-trihydroxystilbene, resveratrol
(RES). In our previous studies we showed that these
potential chemopreventive compounds modulate the
12-O-tetradecanoylphorbol 13-acetate (TPA) - stimulat-
ed expression of inducible nitric oxide synthase and
cyclooxygenase-2. As the one of possible mechanism
of these activity, modulation of transcription factor,
the activator protein - 1 (AP-1) was postulated. AP-1
plays an important role as a mediator of tumour pro-
motion. AP-1 is a heterodimer formed by c-Jun and c-
Fos proteins. In the present study we evaluated the
expression of these proteins after topical application
of TPA (10 nmol per mice). Maximal expression of c-
Jun and c-Fos measured by immunoblot analysis was
observed 4 h after TPA application. Pretreatments
with TA, PCA, CHA and RES at a dose of 16 mmoles per
mice 15 minutes before the TPA application decreased
c-Jun and c-Fos expression. TA and PCA were the most
potent inhibitors of TPA – induced AP-1 activation.
These data suggest that modulation of AP-1 proteins
expression may be involved in antipromotional activ-
ity of the investigated plant phenols.
W1 - Workshop 1
Oral Poster Presentation
OW1003
FOLIC ACID METABOLISM AND GENE POLYMOR-
PHISMS IN THE RISK OF NON-HODGKIN’S LYM-
PHOMAS
F Coppedè1, CF Skibola2, MT Smith2, MS Forrest2,
L Agana2, PM Bracci2, EA Holly2
1 University of Pisa, PISA, Italy2 University of California, BERKELEY, United States of
America
Folic acid is an important nutrient required for both
DNA synthesis and methylation. Impairments in the
synthesis of DNA could be responsible for mutations
critical for cancer outcome in rapidly dividing cells
such lymphocytes. We performed this study to evalu-
ate the role of several folate gene polymorphisms to
the risk of Non-Hodgkin’s Lymphomas (NHL). DNA
was extracted from 400 patients with NHL, and 800
healthy individuals, both groups collected in the San
Francisco Bay Area. The analysis of DNA polymor-
phisms was performed by real-time PCR for allelic
discrimination using an ABI PRISM 7700 sequencer
system. Among genes under study, thymidilate syn-
thase (TS) gene codes for an enzyme that catalyzes
the conversion of dUMP + 5,10- methylenetetrahydro-
folate (MTHF) to dTMP + dihydrofolate. A 28-bp tan-
dem repeat sequence upstream the TS gene initiation
start site is polymorphic, containing either two (2R) or
three repeats (3R); the 3R allele resulting in an in vitro
2.6-fold greater TS expression than the 2R. We also
studied a 6-bp deletion in the 3’UTR of TS gene, a
recently identified polymorphisms supposed to affect
the mRNA levels. We found that the TS 6bp++ geno-
type is associated with an increased risk of NHL.
Linkage disequilibrium was observed between the TS
28-bp 2R/3R tandem repeat and the 3’UTR 6bp dele-
tion polymorphisms (p<.001). Moreover interaction
was observed between the TS 28-bp 2R/3R tandem
repeat and the TS 6-bp deletion polymorphisms. The
TS 6bp++/3R3R genotype was associated with a
reduced risk of NHL while the 6bp--/3R3R genotype
with an increased risk. MTHF is reduced by methyl-
enetetraifolate-reductase (MTHFR) in the DNA
methylation pathway. The MTHFR C667T polymor-
phism was studied, and the 677C genotype was asso-
ciated with a reduced risk of NHL. Our data indicate
that the folic acid metabolic pathway plays an essen-
tial role in modulating the interindividual suscepti-
bility to NHL.
26
abstractboek 20-10-2004 10:28 Pagina 26
W1 - Workshop 1
Oral Poster Presentation
OW1004
NATURAL AH RECEPTOR AGONISTS IN THE HUMAN
DIET: BENEFICIAL FOOD COMPONENTS OR UNPER-
CEIVED RISK FACTORS?
WJ de Waard1, LAP Hoogenboom2, A Peijnenburg2,
JMMJG Aarts3, TH de Kok1, FJ van Schooten1
1 Maastricht University, MAASTRICHT,
The Netherlands2 RIKILT, WAGENINGEN, The Netherlands3 Wageningen University, WAGENINGEN,
The Netherlands
Activation of the omnipresent cytoplasmatic aromat-
ic hydrocarbon receptor (AhR) is thought to mediate
the toxic effects of dioxins like 2,3,7,8-tetrachloro-p-
dibenzodioxin (TCDD). Some fruits and vegetables
contain natural compounds which are also agonists
of the AhR, like indole-3-carbinol (I3C) in cruciferous
vegetables and furanocoumarins in citrus fruits.
However, some of these compounds are claimed to
be beneficial for human health, which promotes
increased consumption of food or pills containing
these compounds.
The objective of the project is to perform a food safe-
ty analysis of Natural AhR Agonists (NAhRA’s) in the
human diet through a genomics approach. Bioassays
measuring AhR activation (CALUX, EROD) were used
to determine the exposure level of some NAhRA’s pro-
ducing near maximum AhR activation. Microarray
gene expression profiles induced at these concentra-
tions in the human colon Caco-2 cell line by citrus
pulp and grapefruit juice extracts, and indolo[3,2-
b]carbazole (I3C metabolite) are compared to the
expression profile induced by TCDD. Benzo[a]pyrene
(B[a]P) is tested as positive control with a higher
metabolic rate compared to TCDD.
Gene expression profiles of the NAhRA’s showed
remarkable resemblance with that of TCDD and B[a]P.
Of the 29 genes up- or down-regulated more than 1.5
times by TCDD after 24 hours exposure, most genes
were regulated in the same direction by the NAhRA’s
and none in the opposite direction, and many are pos-
sibly involved in the toxic effects of TCDD.
Biomarkers will be chosen and a human intervention
study with diets of cruciferous vegetables and/or cit-
rus juices will be carried out.
W1 - Workshop 1
Oral Presentation
OW1005
NUTRIGENOMICS AND NUTRITIONAL SYSTEMS
BIOLOGY
B van Ommen
TNO Nutrition and Food Research, ZEIST,
The Netherlands
Nutritional sciences are discovering the application
of the so-called 'omics' sciences. Driven by the unrav-
elling of the human genome and its related techno-
logical developments, genotyping, transcriptomics,
proteomics and metabolomics are now available to
nutritional research. Screening tools can be devel-
oped for the selection of bioactive nutrients. New bio-
markers for the in vivo efficacy of nutrients in terms
of health promotion or disease prevention are aris-
ing. Better insights will be obtained on the influence
of genetic polymorphisms on nutrient metabolism.
However, are these promises just based on a biotech-
nological hype, or is a real fundamental change in
human nutritional sciences at hand? A new concept
of systems biology based biomarker is presented,
fully exploiting the multiple minor changes in
genomic responses (captured in patterns, profiles and
complex data sets) related to nutrition and health,
instead of single 'target' gene responses common in
drug therapy. It is concluded that systems biology
clearly provides new insights into the molecular
action of nutrients, without the need for a priori
knowledge on any mechanisms. Implications for the
development of biomarkers to predict human health
effects of nutrients are discussed.
27
abstractboek 20-10-2004 10:28 Pagina 27
W2A - Workshop 2A
Oral Poster Presentation
OW2001
CADMIUM INHIBITS HUMAN DNA MISMATCH
REPAIR IN VIVO
LJR Rasmussen, A Lützen, SE Liberti
Roskilde University, ROSKILDE, Denmark
The heavy metal Cadmium (Cd) is a human carcino-
gen that inhibits DNA repair activities. We show that
DNA mismatch repair (MMR)-mediated cell cycle
arrest after alkylation damage is suppressed by expo-
sure to Cd and that this effect is reversed by preincu-
bation with excess of zinc (Zn). We show that
Cd-mediated inactivation of MMR activity is not
caused by disruption of complex formation between
the MMR proteins hEXO1-hMutSa and hEXO1-hMutLa
nor does Cd inhibit 5’-exonuclease activity of hEXO1 in
vitro. Thus, our studies show that exposure of human
cells to Cd suppresses MMR activity, a repair activity
known to play an important role in colon cancer and
that this effect can be reversed by Zn treatment.
W2A - Workshop 2A
Oral Poster Presentation
OW2002
WILD-TYPE P53 SUPPRESSES NON-HOMOLOGOUS
END-JOINING OF DSB BUT NOT OVERALL REPAIR
PROFICIENCY
X Dahm-Daphi1, P Hubbe1, RA El-Awady1, SN Powell2,
H Willers2
1 University of Hamburg, HAMBURG, Germany2 Harvard Medical School, BOSTON, United States of
America
Non-homologous end-joining (NHEJ) of DNA double-
strand breaks (DSBs) can be an error-free or error-
prone repair process depending the structure of DNA
ends created. Complementary ends can be rejoined
by precise ligation (error-free or high-fidelity repair).
All non-compatible ends require DNA end modifica-
tion and hence sequence alteration prior to ligation
(error-prone). This error-prone repair may promote
chromosomal rearrangements and genomic instabil-
ity. The DNA-Pk dependent repair pathway is known
to largely control NHEJ. In this study, we asked
whether p53 can also affect NHEJ by dictating the
choice between error-free and error-prone mecha-
nisms. We applied a novel plasmid-based assay to
monitor the repair of chromosomal DSBs created by
the I-SceI endonuclease in isogenic mouse fibroblast
lines. Expression of wild-type p53 mediated a striking
inhibition of error-prone NHEJ events by > 3 orders of
magnitude compared to p53-null and mutated cells.
On the other hand, p53 appear to promote the precise
ligation of I-SceI breaks (error-free rejoining). p53 pre-
sumably exert a direct inhibitory effect through
restricting DNA end modification by blocking the
annealing of mismatched DNA strands along flank-
ing microhomologies. Importantly, p53 did not reduce
the overall levels of repair of I-SceI- or ionizing-radia-
tion-induced DSBs. We therefore conclude that p53
enhances repair fidelity without compromising
repair capacity. Our data support a model in which
p53 acts as a central regulator of recombinational
processes that maintains genomic stability by restrict-
ing both erroneous NHEJ and homologous recombina-
tion, as shown previously.
28
abstractboek 20-10-2004 10:28 Pagina 28
W2A - Workshop 2A
Oral Poster Presentation
OW2003
IMPACT OF MISMATCH REPAIR ON UVB-INDUCED
CELL CYCLE ARREST
GJ Stout1, M van Oosten1, N de Wind1, FR de Gruijl1,
CMP Backendorf2, LHF Mullenders1
1 Leiden University Medical Center, LEIDEN,
The Netherlands2 Leiden Institute for Chemistry, LEIDEN,
The Netherlands
Nucleotide excision repair (NER), apoptosis and cell
cycle regulation are important defence mechanisms
against the carcinogenic effects of UVB radiation. The
impact of NER pathways on cell cycle progression and
apoptosis in the epidermis of UVB-exposed mice was
investigated previously in our laboratory. We then
found an UVB-induced G2 arrest in epidermal cells of
Xpc-/- mice (no GGR), but not in Xpa-/- (no TCR and no
GGR), nor wild type mice, which resolved without
apoptosis. We hypothesised that the observed G2
arrest in Xpc-/- mice is caused by replication of
genomic sequences containing large numbers of
lesions, giving rise to compound lesions ((mis-)incor-
porated bases opposite a lesion). We suggested that
compound lesions could be recognised by the mis-
match repair (MMR) giving rise to futile cycles of
breakage and resynthesis, which might be the signal
for a G2 arrest. Cell cycle checkpoint kinases may well
be involved in this UV-induced cell cycle arrest.
The present experiments show that the MMR system
indeed plays a role in the generation of the UVB-
induced G2-arrest in keratinocytes of Xpc-/- mice; in
cell cycle analysis with two independent we observed
17% G2-phase cells in Xpc-/-, compared to 11% in Xpc-
/-Msh2-/- mouse epidermis. We confirmed these
results in a recently developed in vitro system
analysing independent Xpc-/- and Xpc-/-Msh2-/-
deficient keratinocyte cell lines. Additional analysis
of artificially (calyculin A) induced premature chro-
mosome condensation of the cultured Xpc-/- ker-
atinocytes indicated that the delayed arrest of 4N
cells occurred in late-S instead of G2-phase.
Furthermore, in vitro pulse-chase experiments
revealed that, unlike Xpc-/-, Xpc-/-Msh2-/- cells finish
the cell division and re-enter G1. More interestingly,
Xpc-/-Msh2-/- cells display less UV sensitivity as com-
pared to Xpc-/- cells.
Based on these findings we suggest that mismatch
repair plays a role in the DNA damage S-phase check-
point.
W2A - Workshop 2A
Oral Poster Presentation
OW2004
BYSTANDER APOPTOSIS INDUCED IN NON-IRRA-
DIATED CELLS NEEDS OF CASPASE 8 ACTIVITY
L Celotti, M Grifalconi, S Canova, M Mognato
University of Padova, PADOVA, Italy
Non-targeted responses to ionising radiation include
the bystander response, where cells neighbouring
those, which have been irradiated, respond to signals
released from the irradiated cells. Our study focused
on the apoptotic response induced in non-irradiated
TK6 cells by the medium from irradiated cells, with
the aim to analyse the mechanisms by which the
response has been activated. Culture medium was
renewed immediately after g-ray irradiation. From
irradiated and unirradiated cells the medium was
poured off donor flasks 6 h after irradiation, filtered
through a 0.22-mm filter and used to treat unirradiat-
ed TK6 cells. For detection of apoptotic morphology,
fixed cells were stained with DAPI 3.5 µg/µl in an
antifade solution which prevents fluorescence decay.
At least 2000 cells were scored for each time- and
dose-point by using fluorescence microscopy. Control
samples were treated with medium from unirradiat-
ed cells. Caspase-8 inhibitor (z-IETD-fmk) was added
to unirradiated cells to evaluate the involvement of
caspase-8 activity into inducing bystander apoptosis.
A significant increase of apoptotic index was observed
in TK6 cells after addition of medium from irradiated
cells, as well as in directly irradiated cells. No or little
increase of apoptotic index was measured when the
conditioned medium was added together with cas-
pase-8 inhibitor. z-IETD-fmk has no effect on apopto-
sis induction when added together with medium
g-irradiated without cells.
Our data suggest that the bystander signal, which
induces the apoptotic response in unirradiated cells,
needs of procaspase 8 activation. Moreover, the
bystander signal does not consist in reactive oxygen
species directly generated by irradiation, but derives
from the metabolism of irradiated cells.
As many indications exist on the bystander respons-
es in cellular models after exposure to low-LET radia-
tion, it is very important to take into consideration
this effect when radiation risk in environmental and
therapeutic exposures was evaluated.
29
abstractboek 20-10-2004 10:28 Pagina 29
W2B - Workshop2B
Oral Poster Presentation
OW2005
GENE EXPRESSION PROFILES REVEALED P53R2
PROTEIN PLAYS A ROLE IN RADIATION-INDUCED
MUTAGENESIS
EY Chuang1, M-H Tsai1, X Chen1, CVR Gadisetti1,
CN Coleman1, JB Mitchell1, Y Chen2, HL Liber3, H Yan1,
S Zhao1
1 NCI/NIH, BETHESDA, United States of America2 NHGRI/NIH, BETHESDA, United States of America3 Colorado State University, FORT COLLINS, United
States of America
The p53 protein has been implicated in multiple cel-
lular responses related to DNA damage, including
apoptosis, cell cycle control, as well as DNA replica-
tion, transcription, and repair. Alterations in any of
these processes could be related to increased genomic
instability. Our previous study indicated that the lack
of wild-type p53 does not lead to increased mutabili-
ty. To investigate further how p53 is involved in regu-
lating mutational processes, we used 8K cDNA
microarrays to compare the patterns of gene expres-
sion among three closely related human cell lines
including TK6 (wild-type p53), NH32 (p53-null), and
WTK1 (mutant p53). Total RNA samples were collected
at different time points (1, 3, 6, 9, and 24h) after 10Gy
gamma-ray. After template-based clustering analysis
of the gene expression over the time course, our
results showed that the gene expression profiles
among these three cell lines revealed distinct pat-
terns after 10Gy irradiation. Furthermore, we found
several genes associated with DNA repair such as
DDB2, p53R2, XPC, PCNA, BTG2 and MSH2 were highly
induced in TK6 compared to WTK1 and NH32. Among
these DNA repair related genes, p53R2 showed the
highest expression level in TK6 cells. p53R2, which is
regulated by tumor suppressor p53, is a small subunit
of ribonucleotide reductase. To determine whether it
is involved in radiation-induced mutagenesis, p53R2
protein was knocked down by siRNA in TK6 cells fol-
lowing by 2Gy gamma-ray irradiation. The mutation
frequencies of siRNA transfected TK6 cells were
approximately equal as untreated TK6 cells at the TK
locus. Whereas, the siRNA transfected TK6 cells after
2Gy radiation were much more sensitive to gamma-
ray-induced mutation than the irradiated TK6 cells
without p53R2 knock down. Our result indicated that
p53R2 is induced by p53 protein and plays a pivotal
role in mutagenesis by repairing damaged DNA in
the nucleus.
W2B - Workshop2B
Oral Poster Presentation
OW2006
EXTENT OF P53 BINDING TO TARGET GENES IN
VIVO AND THEIR EXPRESSION
T Menichini, R Magrini, D Russo, P Tamagno,
G Fronza
National Institute for Cancer Research, IST, GENOVA,
Italy
P53 is found mutated in about 50% of all tumor types.
Since p53 plays a crucial role in the activation of genes
important for cell cycle arrest, DNA repair and apop-
tosis, the question of which functions are affected
and which are retained by any p53 mutant protein is
an important issue in terms of the significance of p53
mutations in cancer.
Our aim was to determine the extent of binding of
wild type and p53 mutant proteins to p21, bax and
mdm2 promoters in vivo by Chromatin Immuno-
precipitation (ChIP) and, then, make a correlation with
the expression of the same genes by RT-PCR. Human
tumor cell lines carrying p53WT (A549), p53R280K
(SKMes1) and p53R273H (LX1) were used. Wild type p53
was induced by UV irradiation.
Our results show that in cells carrying p53WT the
binding to p21 and mdm2 promoters increased after
UV irradiation. For p21 there was correlation between
binding and expression. For mdm2 we did not observe
a relative increase in gene expression. The two mutat-
ed p53 proteins were not able to significantly bind p21
and mdm2 promoters in vivo and the low expression
of these genes correlated with binding data. Clearly,
for all cell lines the expression of bax was not p53-
binding dependent.
It has been shown that when p53 binds to the respon-
sive element of p21, the acetylation of histones bound
to its promoters, may influence p21 induction. Thus, to
measure histone acetylation at the p21 and mdm2
promoters before and after UV irradiation, we are
using ChIP with antibodies that recognize multiple
acetylated forms of H3 and H4 histones. Preliminary
results seem to indicate that the acetylation of H3 and
H4 histones is different at each promoter.
30
abstractboek 20-10-2004 10:28 Pagina 30
W2A - Workshop 2A
Oral Presentation
OW2007
DNA TRANSLESION SYNTHESIS AND MISMATCH
REPAIR IN THE RESPONSE TO ENDOGENOUS AND
EXOGENOUS DNA DAMAGE
N de Wind, V Borgdorff, JG Jansen, A Shtylik
Leiden University Medical Center, LEIDEN,
The Netherlands
The control of mutagenesis is seminal for the mainte-
nance of genomic integrity and therefore for averting
cancer and inherited disease. In mammals, two path-
ways determine DNA damage-induced and ‘sponta-
neous’ mutagenesis. DNA translesion synthesis (TLS)
polymerases introduce misincorporations while
replicating DNA damage. DNA mismatch repair
(MMR) recognizes and removes nucleotides, misin-
corporated by replicative DNA polymerases.
We have generated mouse cells and mice, mutated for
TLS polymerases and/or for MMR and have used these
to investigate interactions between MMR and TLS in
DNA damage responses including spontaneous and
induced mutagenesis.
Embryonic stem (ES) cells carrying a deletion in the
Rev1 TLS polymerase display reduced spontaneous
and short-wave Ultraviolet C (UVC)-induced point
mutagenesis, implying a role for the protein in repli-
cating both endogenous and exogenous (UVC-
induced) DNA damage. In contrast, exposure of ES
cells, deficient for the Msh2 MMR gene, to UVC results
in significantly higher mutation induction than in
wildtype ES cells. This suggests that MMR counter-
acts misincorporations introduced by TLS opposite
UVC-induced pyrimidine dimers. Truncation of the
Rev3 TLS polymerase results in embryonic growth
retardation, apoptosis and midterm lethality indicat-
ing an essential role of Rev3 in replicating endoge-
nous DNA damage. Remarkably, introduction of MMR
deficiency results in significant alleviation of the
Rev3 embryonic phenotype demonstrating that
MMR-dependent processing of DNA damage, rather
than the Rev3 deficiency itself, causes the apoptotic
response. We infer that the absence of Rev3 results in
replication of endogenous DNA damage by another,
more error-prone TLS polymerase. We hypothesize
that processing of these so-called compound lesions
by MMR results in either the repair of the TLS poly-
merase-induced misincorporations, or in provoking
an apoptotic response. We conclude that the action of
both MMR and TLS is essential in determining cellu-
lar responses to endogenous and exogenous DNA
damage.
W3 - Workshop 3
Oral Poster Presentation
OW3001
GENE-ENVIRONMENT INTERACTIONS AND GENET-
IC DAMAGE INDUCTION BY ETS: THE IMPORTANCE
OF EXPOSURE LEVELS
SA Kyrtopoulos1, P Georgiadis1, J Topinka2,
D Vlachodimitropoulos3, G Stephanou1, M Stoikidou4,
H Autrup5, N Demopoulos3, K Katsouyianni4,
RJ Sram2
1 National Hellenic Research Foundation, ATHENS,
Greece2 Institute of Experimental Medicine AS CR, PRAGUE,
Czech Republic3 University of Patras, PATRAS, Greece4 Univ. of Athens Med. School, ATHENS, Greece5 University of Aarhus, AARHUS, Denmark
Current evidence regarding the influence of genetic
variation on genetic damage associated with environ-
mental tobacco smoke is contradictory. In the context of
the AULIS project, we examined the effects of different
CYP1A1, CYP1B1, GSTM1, GSTP1 and mEH genotypes on
lymphocyte bulky DNA adducts and % aberrant cells in
194 non-smokers, in whom recent personal exposure to
airborne PAH and ETS were also measured.
The levels of both biomarkers were found to respond, in
a parallel fashion to changes in exposure/ CYP1A1*2A or
CYP1A1*2B genotype combinations: Specifically, regard-
less of season, in subjects exposed to ETS for more than
3.25 hours during the last 4 days and to airborne B[a]P
within the range 0.28-0.93 ng/m3, biomarker levels
were increased (as compared to CYP1A1*1 carriers) in
CYP1A1*2A and combined CYP1A1*2A/ *2B carriers.
Outside these exposure limits, the differential effect in
CYP1A1*2 variants was lost. On the other hand, in the
same B[a]P exposure range and in subjects with ETS
exposure below the abovementioned level, biomarker
levels were consistently reduced in CYP1B1*2 carriers,
and enhanced in with CYP1B1*3/*4 carriers.
Genetic variations in mEH, GSTM1 and GSTP1 individual-
ly had no or limited effects on biomarker levels.
However, they enhanced significantly the effects of
CYP1A1 or CYP1B1 polymorphisms described above.
These results are interpreted as indicating
a) differential induction of CYP1A1 expression, and asso-
ciated DNA and chromosome damage, in CYP1A1*2A and
CYP1A1*2A/*2B carriers, by components of ETS-polluted
air at levels readily encountered by large sections of the
general population,
b) that, under conditions where CYP1A1 induction is
unlikely, CYP1B1-mediated metabolism dominates the
induction of genetic damage, and
c) that subjects with CYP1A1*2A and CYP1A1*2A/*2B car-
riers may have increased susceptibility to the genotoxic
effects of ETS.
The AULIS project was supported by the European
Union (ENV4V-CT96-0203 and IC20-CT96-0063). 31
abstractboek 20-10-2004 10:28 Pagina 31
W3 - Workshop 3
Oral Poster Presentation
OW3002
POLYMORPHISMS IN DNA REPAIR GENES AND
RISK OF LUNG CANCER IN A DANISH PROSPEC-
TIVE COHORT
U Vogel1, BA Nexo2, H Autrup3, M Sorensen4, H Bak4,
H Wallin1, K Overvad5, A Tjønneland5, O Raaschou-
Nielsen4
1 National Institute of Occupational Health, COPEN-
HAGEN, Denmark2 Institute of Human Genetics, AARHUS, Denmark3 University of Aarhus, AARHUS, Denmark4 The Danish Cancer Society, COPENHAGEN,
Denmark5 Aalborg Hospital, AALBORG, Denmark
We have investigated the occurrence of lung cancer in
relation to polymorphisms in DNA repair genes in
case-cohort study nested in the prospective cohort
‘Diet, Cancer and Health’. Among 54,220 members of
the Danish prospective cohort study aged 50 to 65 at
entry, 265 lung cancer cases were identified and a
sub-cohort, matched by age, sex and duration of
smoking, comprising of 272 individuals was used for
comparison. 90 % of both cases and participants in
the comparison group were ever-smokers. The poly-
morphism XPA A-23G, XPC Lys939Gln and XPD
Lys751Gln in NER genes were associated with risk of
lung cancer. Carriers of risk alleles in two NER genes
were at higher risk of lung cancer than homozygous
carriers of the wild type alleles. The polymorphism
XRCC3 Thr241Met in the double strand DNA repair
gene XRCC3 was also associated with risk of lung can-
cer, whereas no associations were found with poly-
morphisms in the BER genes XRCC1 and OGG1. DNA
adduct levels were quantified by 32P-postlabelling
and associations between polymorphisms and DNA
adduct levels will be presented at the meeting.
W3 - Workshop 3
Oral Poster Presentation
OW3003
CYTOGENETIC BIOMARKERS AND HUMAN CAN-
CER RISK (CANCERRISKBIOMARKERS)
H Norppa1, S Bonassi2, M Fenech3, I-L Hansteen4,
L Hagmar5, P Rössner6, C Lindholm7, M Kirsch-Volders8,
S Gundy9, J Lazutka10, A Cebulska-Wasilewska11,
E Fabiánová12, RJ Sram13, LE Knudsen14, R Barale15,
GJ Köteles16, E Mirkova17, A Fucic18, P Boffetta19
1 Finnish Institute of Occupational Health,
HELSINKI, Finland2 National Cancer Research Institute, GENOA, Italy3 CSIRO, ADELAIDE, Australia4 Telemark Hospital, SKIEN, Norway5 Lund University, LUND, Sweden6 Natl Inst of Public Health, PRAGUE, Czech Republic7 STUK, HELSINKI, Finland8 Vrije Universiteit Brussels, BRUSSELS, Belgium9 National Institute of Oncology, BUDAPEST, Hungary10 Vilnius University, VILNIUS, Lithuania11 Jagiellonian Univ Med College, KRAKOW, Poland12 Sate Health Institute, BANSKA BYSTRICA, Slovak
Republic13 Institute of Experimental Medicine AS CR,
PRAGUE, Czech Republic14 University of Copenhagen, COPENHAGEN, Denmark15 University of Pisa, PISA, Italy16 NRIRR, BUDAPEST, Hungary17 NCHMEN, SOFIA, Bulgaria18 Institute for Med Res Occup Health, ZAGREB,
Croatia19 International Agency for Research on Cancer,
LYON, France
Cytogenetic analyses of peripheral blood lympho-
cytes have been used for decades to show the geno-
toxic effects of chemical and radiation exposure in
humans. Nordic-Italian studies have revealed that a
high frequency of chromosome aberrations (CAs), but
not of sister chromatid exchanges (SCEs) or micronu-
clei (MN), in lymphocytes is predictive of an increased
cancer risk. Individual exposure assessment of the
cancer cases and their matched healthy controls indi-
cated that the association between CAs and cancer
risk was not explained by smoking or known occupa-
tional exposure to carcinogens. High CA level was
associated with increased cancer risk also in non-
smokers and in subjects with no history of occupa-
tional contact with carcinogens. Thus, high CA
frequency appeared to predict increased cancer risk
regardless of the reason for the elevated CAs.
Furthermore, the time between CA analysis and can-
cer outcome did not influence the association, sug-
gesting that the findings are not explained by
32
abstractboek 20-10-2004 10:28 Pagina 32
undetected cancer. An international collaborative
study (CancerRiskBiomarkers, QLK4-CT-2000-00628)
is presently going on to further characterise the rela-
tionship between cytogenetic biomarkers and cancer.
The project aims at clarifying, e.g., the cancer risk
predictivity of cytogenetic biomarkers in several new
European cohorts, the role of genetic polymorphisms
of carcinogen metabolism, DNA repair and folate
metabolism, the classes of chromosome alterations
having cancer predictive value, and the types of can-
cer predicted. The project also cooperates with the
International Collaborative Project on Micronucleus
Frequency in Human Populations (HUMN), to assess
the possible cancer risk predictivity of the cytokine-
sis-block MN assay.
W3 - Workshop 3
Oral Poster Presentation
OW3004
INFLUENCE OF GSTM1, GSTT1, GSTP1 AND NAT2
GENOTYPES ON P53 MUTATIONAL SPECTRUM IN
BLADDER TUMOURS
CM Ryk1, P Berggren1, R Kumar2, P Larsson3,
G Steineck3, B Lambert1, S Hou1
1 Karolinska Institute, HUDDINGE, Sweden2 German Cancer Research Center, HEIDELBERG,
Germany3 Karolinska Hospital, STOCKHOLM, Sweden
Genetic polymorphisms affecting expression or activity
of the corresponding enzymes can influence the risk
of acquiring gene mutations and various cancers. We
have studied 327 bladder cancer patients with regard
to the functionally related polymorphisms of GSTM1,
GSTT1, GSTP1 and NAT2, and analysed the p53 muta-
tional status of their tumours. 50 p53 mutations, 26%
transversions and 74% transitions, were detected in
44 patients. P53 mutation frequency was significantly
higher in advanced than in less advanced tumours
(P<0.002). Also, patients with at least one variant
GSTP1 allele (Ile105Val) tended to have more advanced
tumours (P=0.057, Fisher’s exact test). Overall, there
was no significant difference in frequency of p53
mutation among patients with different genotypes.
With regard to the possible influence of genetic poly-
morphisms on the p53 mutational spectrum, several
trends were observed. Among patients with p53
mutation, transversions were significantly more fre-
quent in GSTM1 negative as compared to GSTM1 posi-
tive individuals (OR 5.18, CI 1.07-25.02). With one
exception, all tumours with the most common type
of transversion, G:C-C:G, occurred in GSTM1 negative
patients. Among smokers, carriers of the GSTP1 vari-
ant allele had a higher proportion of transversions
than non-carriers (P=0.022, Fisher’s exact test).
Samples carrying at least one variant GSTP1 allele had
more transitions at CpG sites than wild type samples
(OR 4.61, CI 0.82-26.04). No significant associations
were found for the NAT2 gene. Our results suggest
that impaired glutathione conjugation may affect the
mutation spectrum in critical target genes.
33
abstractboek 20-10-2004 10:28 Pagina 33
W3 - Workshop 3
Oral Presentation
OW3005
MOLECULAR EPIDEMIOLOGY OF SPORADIC
BREAST CANCER: POLYMORPHISMS IN THE
XENOBIOTIC METABOLISM AND DNA REPAIR
GENES
A Hirvonen
Finnish Institute of Occupational Health, HELSINKI,
Finland
The major known risk factors for female breast can-
cer are associated with prolonged exposure to
increased levels of oestrogen. The predominant theory
relates to effects of oestrogen on cell growth. Enhanced
cell proliferation, induced either by endogenous or
exogenous oestrogens, increases the number of cell
divisions and thereby the possibility for mutation.
However, current evidence also supports a role for
oxidative metabolites, in particular catechol oestro-
gens, in the initation of breast cancer. As observed in
drug and chemical metabolism, there is considerable
interindividual variability (polymorphism) in the
conjugation pathways of both oestrogen and catechol
oestrogens. These person-to-person differences,
which are attributed to polymorphisms in the genes
encoding for the respective xenobiotic metabolizing
enzymes, might define subpopulations of women
with higher lifetime exposure to hormone-depend-
ent growth promotion, or to cellular damage from
particular oestrogens and/or oestrogen metabolites.
Such variation could explain a portion of the cancer
susceptibility associated with reproductive effects
and hormone exposure. On the other hand, DNA
repair plays a central role in protecting the genome
against insults by genotoxic cancer-causing agents
such as tobacco smoke. Accordingly, it has been sug-
gested that individuals with reduced DNA repair
capacities might have altered risk of developing envi-
ronmentally induced malignancies, including breast
cancer. This presentation will give an overview of the
hitherto performed studies on the role of polymor-
phisms in genes coding for enzymes involved in
xenobiotic metabolism and DNA repair in individual
susceptibility to sporadic breast cancer.
W4 - Workshop 4
Oral Poster Presentation
OW4001
DNA DAMAGE BY RESPIRABLE QUARTZ PARTICLES
IN RAT LUNG TARGET CELLS: THE ROLE OF MITO-
CHONDRIA
H Li, R Duffin, C Albrecht, T Shi, J Eicker, D Höhr,
PJA Borm, RPF Schins
IUF GmbH Düsseldorf, DUSSELDORF, Germany
Respirable quartz dust has been classified as human
carcinogen by the International Agency for Research
on Cancer (IARC), and chronic inhalation studies in
rats indicate that alveolar type II cells are key target
cells for quartz carcinogenesis. The aim of our studies
is to investigate the mechanism of DNA damage by
quartz. Quartz particles were found to elicit a dose-
dependent increase in DNA strand breakage (comet
assay) in primary rat lung epithelial cells as well as in
the rat lung cell line RLE. Using electron spin reso-
nance (ESR) with spin trapping we demonstrated that
quartz particles can generate hydroxyl-radicals and
immunohistochemical analysis showed that quartz
induces the formation of 8-hydroxydeoxuanosine.
However, transmission electron microscopy analysis
did not reveal quartz particles within the nucleus, e.g.
in close proximity with the nuclear DNA, indicating
that the observed DNA damage does not result form
particle surface-derived hydroxyl radicals directly.
Interestingly, DNA strand breakage by quartz could
be reduced in the presence of the mitochondrial
inhibitors rotenone, antimycin-A or tenoyltrifluo-
roacetone (TTFA). These inhibitors did not affect DNA
damage, but led to a reduced oxygen consumption by
the cells. Our observations suggest a role for the res-
piratory chain function in quartz-induced DNA dam-
age, but the exact mechanism remains to be
elucidated. This study is supported by the DFG
International Graduate College 738.
34
abstractboek 20-10-2004 10:28 Pagina 34
W4 - Workshop 4
Oral Poster Presentation
OW4002
AN HYPOTHESIS ON THE ROLE OF OXIDATIVE
DAMAGE IN NEURODEGENERATIVE DISEASES
M Migliore, I Fontana, R Colognato, F Coppedè,
G Tognoni, G Siciliano
University of Pisa, PISA, Italy
Oxidative stress plays a key role in the degenerative
neuronal death and progression of many neurode-
generative diseases, such as Alzheimer’s disease (AD)
and Parkinson's disease (PD), although it is not clear if
it is the primary triggering event in the pathogenesis
of these disorders.
We have previously found chromosomal, primary
and oxidative DNA damage in peripheral lympho-
cytes of PD patients (Migliore et al., 2002). The present
study was performed on a group of AD patients and a
group of mild cognitive impairment (MCI) patients,
beeing the latter a clinical condition between normal
aging and AD, characterized by a memory deficit
without loss of general cognitive and functional abil-
ities. We performed this study by comet assay to eval-
uate the level of oxidative DNA damage in two groups
of MCI and AD patients, respectively, compared to
healthy controls. Data showed a significantly higher
level of primary DNA damage in leukocytes of AD and
also of MCI patients than in control individuals (aver-
age: 2.09±0.79 and 2.47±1.01, respectively for AD and
MCI, vs. 1.04±0.31). Moreover the amount of oxidised
DNA bases (either purines and pyrimidines) was
higher in the two groups of patients (AD and MCI)
compared with controls (p< 0.002 and p< 0.001 for
purines and pirimidines, respectively). Our results
give a further indication that oxidative stress, at least
at the DNA level, is an earlier event in the pathogene-
sis of AD.
W4 - Workshop 4
Oral Poster Presentation
OW4003
THE MULTIPLE PHYSIOLOGICAL AND PATHOLOGI-
CAL CONSEQUENCES OF OXIDATIVE DNA DAMAGE
AI Izzotti, C Cartiglia, M. Longobardi, A Camoirano, M
Bagnasco, E Tampa, A Merello, S de Flora
University of Genoa, GENOA, Italy
We evaluated the consequences of oxidative DNA-
damage (ODD) in various experimental conditions by
monitoring 8-hydroxy-2’-deoxyguanosine by 32P-
postlabelling and gene-expression by cDNA array.
In mouse foetal liver, the low basal level of ODD was
increased following transplacental exposure to ciga-
rette smoke. The foetus counteracted ODD by increas-
ing the expression of genes inhibiting cell replication
and triggering apoptosis (FASEB J.,17:1127-9,2003).
Accordingly, smoke-induced ODD in foetus resulted
in growth retardation.
During the foetus-newborn transition, the acquisi-
tion of independent respiratory function triggered the
expression of genes involved in the removal of oxi-
dized proteins and detoxification of oxidative species
in mouse lung (Mutat.Res.Rev.,544:441-9,2004). These
alterations were attenuated by administering the
antioxidant N-acetyl-L-cysteine to the dams during
pregnancy.
A main problem of postgenomic analysis is whether
or not gene expression reflects protein amounts. We
demonstrated that the relationship between cDNA-
and antibody-microarray data in rat lung is not sig-
nificant under basal conditions, but becomes highly
significant in case of exposure to chromium(VI), an
ODD-inducing agent (Int.J.Oncol.,24,2004, in press).
In humans, we found that ODD is consistently
detectable in the aorta of atherosclerotic patients
(FASEB J.,11:1021-31,1997), being 4-fold higher in the
endothelium than in the medium layer.
To substantiate the hypothesis that ODD is related to
various chronic-degenerative diseases, we analyzed
the level of ODD in patients affected by primary open
angle glaucoma, the main cause of irreversible blind-
ness worldwide. We found that ODD is significantly
increased in the trabecular meshwork, the specialized
epithelium regulating the intraocular pressure, of
glaucoma patients as compared to unaffected con-
trols (Am.J.Med,114:638-46,2003). This situation leads
to an increase of the intraocular pressure resulting in
optic nerve alterations and visual field defects.
Altogether, these data support the hypothesis that
ODD is involved in a variety of physiological process
(e.g., birth) and pathological conditions (e.g., cancer,
atherosclerosis, glaucoma).
35
abstractboek 20-10-2004 10:28 Pagina 35
W4 - Workshop 4
Oral Poster Presentation
OW4004
PARP1 TOGETHER WITH CSB AND XPA ACCELER-
ATES THE GLOBAL REPAIR OF OXIDATIVE DNA
BASE DAMAGE
CF Flohr1, I Schulz1, A Bürkle2, JP Radicella3, B Epe1
1 University of Mainz, MAINZ, Germany2 University of Konstanz, KONSTANZ, Germany3 CEA, FONTENAY-AUX-ROSES, France
In mammalian cells, oxidative DNA base modifica-
tions are repaired by the base excision repair (BER)
pathway. Whereas, in vitro only a few proteins are
necessary to accomplish BER, several other 'accessory'
proteins seem to be important for efficient DNA
repair in vivo. To elucidate the functions of those
accessory proteins on the repair of oxidative DNA
base modifications, we studied the repair rates of
oxidative purine modifications including 8-hydrox-
yguanine (8-oxoG) in cell lines defective in various
proteins, i.e. poly(ADP-ribose)polymerase 1 (PARP1),
Cockayne syndrome B (CSB), Xeroderma pigmentosum
A protein (XPA) and XRCC1. Low levels of 8-oxoG and
other oxidative purine modifications were induced
by the photosensitizer Ro 19-8022 plus visible light,
and their removal was followed by means of a modi-
fied alkaline elution assay in combination with the
repair glycosylase Fpg protein as a probe.
Repair rates of the induced oxidative DNA base modi-
fications were significantly slower in PARP-deficient,
CSB-deficient and XPA-deficient cells compared to
wild type cells. This retardation of BER could not be
observed in a cell-free assay measuring the incision
at an oligonucleotide containing a single 8-oxoG.
Interestingly, in cells that are deficient in CSB and
XPA, an additional inhibition of PARP activity did not
further reduce the repair rates. The loss of the plat-
form protein XRCC1 did not influence the repair rates
of oxidative base modifications, although the reseal-
ing of induced SSB was significantly slowed down.
Furthermore, in XRCC1 deficient cells the repair of
oxidative base modifications was reduced when PARP1
was inhibited.
The results suggest that PARP, CSB and XPA are
involved in a repair mechanism that stimulates BER
in vivo, possibly initiated by interaction with the
stalled transcription machinery. Details of the novel
pathway remain to be established.
W4 - Workshop 4
Oral Presentation
OW4005
GENE EXPRESSION CHANGES AS BIOMARKERS OF
OXIDATIVE DAMAGE
AM Lynch
GlaxoSmithKline, WARE, HERTS, United Kingdom
The differential gene expression (DGE) of selected
genes as part of the cellular response to oxidative
stress may be exploited to provide novel biomarkers
which may be predictive of developing toxicity with-
in a wide variety of tissues and organs. To test this
hypothesis, we have measured gene expression
changes in response to oxidative stress in vitro and in
vivo following treatment with various, well charac-
terised, reference compounds, using:
1. Real Time RT-PCR (TaqMan) in cultured Hep-G2
cells (Morgan et al, Toxicol Pathol. 30: 435-451,
2002),
2. Biophotonic imaging in HO-1.luc mice, transgenic
for the murine haem oxygenase-1 (HO-1) promoter
coupled to a luciferase reporter gene (Speight et al,
Mutagenesis 17: 569, 2002) and
3. RT-PCR to measure hepatic DGE in Sprague Dawley
rats.
In addition, traditional endpoints of toxicity were
assessed in parallel. These included the measurement
of the ratio of reduced (GSH) to oxidized (GSSG) glu-
tathione (GSH:GSSG ratio) in HepG2 cells and Sprague
Dawley rats. Clinical chemistry and pathology end-
points were also assessed in Sprague Dawley rats and
transgenic HO-1.luc mice along with RT-PCR analysis
of endogenous HO-1. The results of these studies con-
firm that real-time site-specific gene expression (or
expression of the transgene in the mouse model) may
be used as predictors of developing toxicity.
36
abstractboek 20-10-2004 10:28 Pagina 36
W5 - Workshop 5
Oral Poster Presentation
OW5001
RAT LIVER TRANSCRIPTOMICS AFTER 28-DAY
EXPOSURE TO BENZENE AND TRICHLOROETHYL-
ENE (MIXTURES)
WHM Heijne, B van Ommen, JP Groten, M Bart,
D Jonker, AP Freidig, RH Stierum
TNO Nutrition and Food Research, ZEIST,
The Netherlands
Trichloroethylene (TCE) is hepatotoxic, nephrotoxic,
and a suspected carcinogen. Benzene (B) is hemato-
toxic and myelotoxic, and can provoke leukemia. The
response to benzene, trichloroethylene (TCE) and
binary mixtures was analyzed in detail at the gene
expression level in liver. In the first study, rats were
dosed with B or TCE at three dose levels for 28-days.
Both compounds exerted (modest) toxic effects in
liver, which is not the primary target organ for toxic-
ity. Besides routine toxicological examinations,
oligonucleotide microarrays were used to find more
sensitive markers of effects and elucidate mecha-
nisms of response to B and TCE. Additionally, metabo-
lite profiling was used to analyse compound and dose
specific metabolite contents in urine. Transcriptome
data revealed compound-specific effects that enabled
to hypothesise on the mechanisms of (combined)
action of B and TCE. Drug-metabolizing enzymes and
genes involved in other biological processes like gly-
colysis and fatty acid metabolism were induced or
repressed by B, TCE or by both. Possible implications
of liver gene expression for toxic and carcinogenic
effects in other organs are discussed.
In a subsequent study, B and TCE and mixtures were
given to rats at two dose levels. Routine toxicological
examinations did not reveal marked hepatotoxicity.
Individual livers were analyzed using oliognucleotide
microarrays, testing the expression of around 5000
genes. Differences and similarities were observed in
expression changes by B and TCE, and for each gene,
the effect of the mixtures of B and TCE was deter-
mined. Many enyzmes responsible for the bioactivation
of B and TCE in liver were differentially expressed by the
treatments, with possible implications for the expo-
sures to mixtures. Genes important in diverse cellular
pathways were deregulated, and possible implica-
tions of combined action for toxicity are discussed.
W5 - Workshop 5
Oral Poster Presentation
OW5002
EFFECTS OF CHLORPROMAZINE WITH AND WITH-
OUT UV IRRADIATION ON GENE EXPRESSION OF
HEPG2 CELLS
RF Froetschl1, S Weickhardt1, S Staszewski1,
G Kaufmann1, K Buss2, P Kasper1
1 Federal Institute for Drugs and Medical Devices,
BONN, Germany2 Memorec Biotec GmbH, COLOGNE, Germany
Data from standard genotoxicity tests provide only
little mechanistic insight into the mode of action of
genotoxic effects and labourious additional tests are
usually needed to gain such information. Microarray
technology has the potential to produce toxicity-
related pattern of gene expression changes and may
thus be used to classify genotoxic compounds with
different mechanisms of action.
Using HepG2 cells and PIQOR cDNA microarrays rep-
resenting 1089 genes related to e.g. DNA damage and
repair, cell cycle, transcription, metabolism and other
important cell functions we are currently investigat-
ing the gene expression profiles of several known
genotoxicans with different modes of action. This
presentation focus on data with the known photo-
mutagen chlorpromazine which itself is not genotox-
ic but upon UV irradiation (with a non-genotoxic
dose in the UVA range) produces reactive free radicals
with DNA damaging properties. Genotoxicity in
HepG2 cells has been assessed concomitantly to gene
expression profiling using the Comet assay.
The available results from repeat experiments show
significantly different gene expression pattern for
the relevant sample groups. In addition, time depend-
ent changes in gene expression were observed within
these groups. Genes showing differential expression
include stress induced SQSTM1, mitogen activated P-
kinase 6, mismatch repair protein1, apoptosis
inhibitor CARD4, thyroid receptor interacting protein
NFKBIB and others. Relationship of these genes to
treatment-related mode of action of genotoxicity will
be discussed.
We are continuing efforts to complete time and con-
centration kinetics with several other genotoxicants,
assess the reproducibility of the data generated and
apply further and different approaches in analysing
the profiling data.
37
abstractboek 20-10-2004 10:28 Pagina 37
W5 - Workshop 5
Oral Poster Presentation
OW5003
IDENTIFICATION OF GENOTOXINS BY MARKER
GENE EXPRESSION BASED ON A SUPERVISED
CLASS PREDICTION APPROACH
D Bauer1, C McGinnis2, P Grass1, F Staedtler1, L Urban2,
W Suter1, L Mueller1
1 Novartis Pharma AG, BASEL, Switzerland2 Novartis Institutes (NIBRI), CAMBRIDGE,
United States of America
Testing chemical compounds for genotoxicity is often
performed using a few highly standardized in vitro
assays. Nevertheless, relevance and predictivity
regarding in vivo results esp. in humans is limited. An
assay based on existing knowledge (e.g. well-known
reference substances) and being optimized on correct
classification would be of clear advantage. Therefore,
we performed a feasibility study in order to elucidate
whether an unbiased supervised class prediction
approach based on large scale gene expression pro-
files may lead to a stable prediction model for pre-
selected classes. In addition, the number of genes
taken into account for the discriminant function
should be reduced as far as possible in order to avoid
microarray experiments in a final testing situation.
Transcription profiles were obtained from TK6 human
lymphoblastoid cells treated with three known geno-
toxic compounds (cis-Platinum, Mitomycin C, Methyl
methansulfonate) and three compounds considered
non-genotoxic (trans-Platinum, Rifampicin, Sodium
chloride). Treatment was performed at nearly equicy-
totoxic conditions (cell count and Alamar Blue deter-
mination) using six replicates. Total RNA was
hybridized to Affymetrix HG-U133A microarrays
(~22000 probe sets) and raw expression data was fur-
ther analyzed using the software packages MAS5
(Affymetrix), Genespring 6 (Silicon Genetics) and
SIMCA-P 10 (Umetrics AB). In a supervised learning
approach discriminant functions involving the
expression pattern of only six genes were identified
that are capable of separating samples treated with
genotoxic and non genotoxic compounds without
any misclassification. According to validation by
response permutations it was found that the predic-
tive power of the model is strong and far from
random. Thus, this proof-of-concept study demon-
strates the general feasibility of a marker gene based
genotoxicity test system. Particularly, supervised
approaches using class prediction methods seem to
outperform conventional cluster analysis techniques.
Nevertheless, additional efforts are required in order
to accomplish a validated and predictive assay.
W5 - Workshop 5
Oral Presentation
OW5004
USE OF GENOMICS FOR REGULATORY SCIENCE –
INDUSTRY PERSPECTIVE
S Albertini, L Suter-Dick, S Ruepp, T Weiser
F. Hoffmann-La Roche AG, BASEL, Switzerland
Because of uncertainties how (toxico-)genomics data
will be used in regulatory decision industry was and
still is reluctant in submitting such data. To help clar-
ify the regulatory needs, FDA has initiated a guidance
document: ‘Guidance for Industry: Pharmacogenomic
Data Submission’. One clear statement is, that
‘because the field of pharmacogenomics is relatively
new, most experimental results may not be well
enough established to be suitable for regulatory deci-
sion making. Pharmaceutical companies, neverthe-
less use such data for internal decision making
during the drug development process. What is the
rational behind such an approach and where do we
stand? What are the issues the pharmaceutical indus-
try is facing to improve the attrition rate and thereby
increase the success rate?
It is believed, that toxicogenomics can significantly
contribute to this endeavour. Analysis of gene expres-
sion patterns seems suitable to detect organ-toxicity.
Fingerprints are used as working hypothesis to test
compounds in order to validate this technology as a
tool for prediction of possible toxicants, especially in
man. The major issue currently facing relates to the
interpretation of the obtained data in order to trans-
form the huge amount of information into knowl-
edge. Building a high quality reference database is
one of the major cornerstones for improving predic-
tivity by toxicogenomics. Approaches we followed
will be shown and insights in the complex data eval-
uation will be given focusing on hepatotoxicity pre-
diction.
Toxicogenomics represents an exciting, new
approach and has a great potential to influence posi-
tively the predictability of preclinical safety assess-
ments.
38
abstractboek 20-10-2004 10:28 Pagina 38
W6 - Workshop 6
Oral Poster Presentation
OW6001
FRONTLOADING GENETIC TOXICOLOGY: EVALUA-
TION OF DEREK, AMESII AND GREENSCREEN AS
SCREENING TOOLS
JAJ van Gompel, D Beerens, F Woestenborghs, C Mackie
Johnson & Johnson Parmaceutical R&D, BEERSE,
Belgium
In the early Hit-to Lead phase, several assays are used
to obtain information on solubility, permeability,
metabolic stability, CYP450 interaction, cardiovascu-
lar safety pharmacology and genetic toxicology. With
5 mg of test compound, the AmesII assay gives a good
prediction towards the results in the downstream
GLP Ames assay. Due to the decision making position
of this assay, continuous monitoring of the predictive
value is a critical task. These downstream data are
normally only obtained for compounds entering the
next phase (Drug Evaluation) and whenever com-
pound quantities allow, agar based assays with TA98
and TA100 are performed. Since we lack eukaryotic
endpoints in this early phase, we are currently evalu-
ating GreenScreen, a yeast based assay (Cahill et al.
Mutagenesis 19,2, 2004). This assay detects a different
spectrum of compounds to the bacterial genotoxicity
assays and could therefore be complementary to the
AmesII bacterial screen. The bottleneck for the
assessment of this assay is however the more time-
consuming and cumbersome nature of downstream
assays (the mouse lymphoma assay and/or micronu-
cleus assay). Emerging structure-toxicity relation-
ships detected by these kind of medium throughput
assays can then be translated in new 'in-house' alerts
and fed back into in silico prediction tools like DEREK.
The combination of the ADME profiling data with
these in silico, prokaryotic and eukaryotic data pro-
vides a good hazard detection and allows the medici-
nal chemists to advance the most optimal hit series to
lead candidates.
W6 - Workshop 6
Oral Presentation
OW6002
NEW STRATEGIES IN TOXICOLOGICAL RISK ASSESS-
MENTS: TRANSPARENT AND EFFICIENT APPROACH-
ES IN DETERMINING HAZARD AND RISK
BJ Blaauboer
Utrecht University, UTRECHT, The Netherlands
The principles of risk assessment have been devel-
oped over the past 50 years and take into account the
intrinsic activity of a compound to cause a harmful
effect, the characterization of its hazard, i.e. the toxic
phenomena and the dose-effect relation and the
characteristics of exposure.
A prominent part of the data is derived from animal
models. These data ideally allow a quantification of
the dose-effect relationship, on the basis of which an
extrapolation can be made to humans, making use of
safety factor, which take into account the possible
higher sensitivity of human individuals.
These bioassays are highly standardised, laid down in
OECD guidelines. This has resulted in a system for
risk evaluation that allowed the relatively safe use of
chemicals.
However, this system meets criticism, mainly related
to its high degree of animal use and its relatively
fixed character. For instance, it is not easily allowing
adaptation of new scientific knowledge. The vast
increase in insight in the mechanisms of toxic action
should be taken into account.
In response to this criticism, proposals were made,
recently discussed in a report of the Health Council of
the Netherlands. Key is the use of schemes in which
knowledge is integrated from different domains, e.g.
structure, chemical functionalities, QSARs, mathe-
matical models for exposure and biokinetics and in
vitro data on toxicodynamics. Integrated approaches
need to be organized in the form of decision trees
with transparent criteria for expert judgments.
Examples of integrated approaches will be given and
the consequences for the hazard and risk assessment
in the fields of genotoxicity and carcinogenicity will
be discussed.
39
abstractboek 20-10-2004 10:28 Pagina 39
W6 - Workshop 6
Oral Presentation
OW6003
EVALUATION OF THE PERFORMANCE OF A SMALL
BATTERY OF IN VITRO TESTS IN DETECTING CAR-
CINOGENS
DJ Kirkland1, MJ Aardema2, LM Henderson3, L Müller4
1 Covance Laboratories Ltd., HARROGATE,
United Kingdom2 Procter & Gamble, CINCINNATI, United States of
America3 Henderson ScientificConsulting, NORTHWICH,
United Kingdom4 Novartis Pharma AG, BASEL, Switzerland
The performance of a battery of 3 in vitro genotoxici-
ty tests (Ames + MLA + in vitro MN) has been evalu-
ated for its ability to detect rodent carcinogens, from
the databases of Gold et al, NTP and IARC. Because
there are so few published data on the in vitro MN,
and the correlation between induction of MN and
chromosomal aberrations [CA] in vitro is so high, in
vitro chromosomal aberration data were also includ-
ed as relevant. The sensitivity of the 3-test battery
was high. Of the >400 chemicals for which there
were valid data, 97% gave positive results in at least 1
of the 3 tests. Only 14 carcinogens gave negative,
equivocal or inconclusive genotoxicity results in a full
3-test battery. All were either non-genotoxic carcino-
gens (liver enzyme inducers, peroxisome prolifera-
tors, hormonal carcinogens) many of which act via
mechanisms not relevant for humans, tumour pro-
moters (e.g. phenytoin), or were extremely weak
(presumed) genotoxic carcinogens (e.g. N-nitroso-
diphenylamine). The specificity of the 3-test battery
was poor. We identified about 200 chemicals that
were non-carcinogenic after testing in both male and
female rats and mice. For the approximately 50% of
non-carcinogens with relevant data, >80% gave posi-
tive results in 1 or more tests. Thus the specificity
(ability to accurately detect non-carcinogens as nega-
tive) was <20%. Some of the positive non-carcinogens
were aneugens or produced clastogenicity only at
high osmolality, but a large number of these positives
are unexplained. This performance highlights the
importance of understanding the mechanism by
which genotoxicity may be induced (whether it is rel-
evant for the whole animal or human) and using
weight of evidence approaches to assess the carcino-
genic risk from a positive genotoxicity signal.
W6 - Workshop 6
Oral Presentation
OW6004
OVERVIEW OF VALIDATION AND INTERNATIONAL
ACCEPTANCE OF TEST METHODS FOR HAZARD
ASSESSMENT IN THE OECD
AM Wagner
OECD, PARIS, France
Validation issues related to testing methods are
important to the chemical safety work of the
Organisation for Economic Co-Operation and Develop-
ment (OECD). In 1996 the OECD conducted the Solna
workshop, which resulted in a set of principles and
criteria for validation and regulatory acceptance of
new and alternative test methods that are important
for consideration during OECD Test Guideline devel-
opment. A Guidance Document has subsequently
been drafted (Guidance Document No 34 on the
'Validation and International Acceptance of New or
Updated Test Methods for Hazard Assessment'),
which provides specific guidance on the issues relat-
ed to validation of new or updated test methods, both
for in vivo or in vitro, and ecotoxicity tests. Validation
can be conducted prospectively (during the develop-
ment of a test method) or retrospectively (using exist-
ing data). The ultimate aim of GD34 is to facilitate the
development of OECD Test Guidelines and the inter-
national acceptance of data for hazard and risk
assessment. Test method validation is an evolving
area, and the finalisation of GD34 is now one of the
priority areas of the OECD. Test method validation is a
real issue for all areas in toxicology, including geno-
toxicity. Currently, a new draft Test Guideline for the
In Vitro Micronucleus Test is under consideration by
the OECD. A process of prospective validation was
undertaken for this test method, where several stud-
ies that investigated different protocol parameters
were conducted by various parties before the guide-
line was written, and the results of the validation
exercise were reviewed during an International
Meeting. In addition, a retrospective evaluation of
the validity of this in vitro genotoxicity assay, based
on available data, is currently underway and co-ordi-
nated by ECVAM (European Centre for the Validation
of Alternative Methods). Further information on the
OECD validation process will be described.
40
abstractboek 20-10-2004 10:28 Pagina 40
W7 - Workshop 7
Oral Presentation
OW7001
ROLE OF THE BLOOM’S SYNDROME HELICASE IN
MAINTENANCE OF GENOME STABILITY
ID Hickson
Institute of Molecular Medicine, OXFORD,
United Kingdom
The RecQ family of DNA helicases is highly conserved
in evolution from bacteria to man. There is a single
family member in unicellular organisms, but multi-
ple members in metazoans, with five being present in
humans. Of these, three are of particular interest
because germ-line mutations in these genes leads to
well-characterized disorders: BLM is mutated in
Bloom’s syndrome, WRN in Werner’s syndrome, and
RECQ4 in Rothmund-Thomson syndrome. These dis-
orders are associated with both cancer predisposition
(particularly evident in Bloom’s where cancers of all
types are seen) and premature aging (most notably in
Werner’s syndrome). Defects in RecQ family helicases
give rise to genomic instability in all organisms ana-
lyzed to date. This instability may take several forms,
but a consistent feature is aberrant genetic recombi-
nation – generally hyperrecombination between
homologous sequences and/or a failure to suppress
recombination between non-identical DNA sequences.
The focus of our work is the BLM gene product and its
homologue in budding yeast, Sgs1p. Mutant cells
lacking either BLM or Sgs1p show an increased fre-
quency of sister-chromatid exchanges and hypersen-
sitivity to agents that perturb DNA replication, such
as hydroxyurea. Moreover, BLM and Sgs1p act in con-
cert with a second protein, topoisomerase III, to effect
their functions. Our recent work has shown that BLM
and topoisomerase III cooperate to resolve late-stage
intermediates in the homologous recombination
repair pathway that contain Holliday junctions. Our
working model is that RecQ helicase and topoiso-
merase III act in a recombination–dependent path-
way that operates specifically at sites of damaged
replication forks.
W7 - Workshop 7
Oral Poster Presentation
OW7002
THE STUDIES ON LOCALIZATION OF HUMAN
RECQ HELICASES FUSED WITH FLUORESCENT
PROTEINS
R Rusin1, M Krzesniak1, R Vaitiekunaite1,
D Butkiewicz1, M Mrzyglodzik1, CC Harris2
1 Center of Oncology, GLIWICE, Poland2 National Cancer Institute, NIH, BETHESDA,
United States of America
The long-known, senescence-accelerating, cancer
prone diseases: Werner, Bloom and Rothmund-
Thomson syndromes are caused by mutations in
WRN, BLM and RTS genes, respectively, coding for
RecQ family helicases. In humans, there are other
members of RecQ helicases: RECQL and RECQ5. The
RecQ helicases are crucial for genomic stability and
DNA metabolism, however many of their functions
remain unknown. In order to better understand the
functioning of RecQ helicases, we generated the fluo-
rescently tagged proteins by attaching the monomer-
ic red fluorescent protein (mRFP1) cDNA to the 5’cDNA
end of WRN, BLM, and RECQL. Subsequently, we intro-
duced missense mutations in mRFP1-WRN by site-
directed mutagenesis and transfected the wild-type
and mutant genes into monkey cells (COS-7), human
lung carcinoma cells (NCI-H1299) and colon adeno-
carcinoma cells (HCT-116). Moreover, in order to per-
form the co-localization studies, we co-transfected
the helicase constructs with the plasmids producing
either fluorescently tagged PML protein, TRF1 protein
or selected proteins involved in DNA repair. We
noticed that, apart from the nucleolar and focal stain-
ing, the most remarkable feature of mRFP1-WRN
localization was its presence in donut-shaped struc-
tures of various sizes, some of them reaching 30%
diameter of cell nucleus. Neither helicase nor exonu-
clease activities are required for WRN entering nucle-
oli, 'donuts' and foci. The mutation of the key amino
acid residue in HRDC domain abolishes mRFP1-WRN
'donut' formation. Instead, mRFP1-WRN is present in
irregular, nuclear „speckles'. The mRFP1-WRN 'donuts'
resemble the PML bodies present in cells with alter-
native mechanism of telomere maintenance.
However, the cotransfection of mRFP1-WRN and
EGFP-PML constructs do not show any evidence of co-
localization in the 'donuts'. The nature and function
of these subnuclear structures remain unknown. The
mRFP1-WRN 'donuts' contain some other proteins
involved in DNA repair. We conclude that our fusion
constructs are useful tools for studying the structure-
function relationship in human RecQ helicases.
41
abstractboek 20-10-2004 10:28 Pagina 41
W7 - Workshop 7
Oral Poster Presentation
OW7003
DNA DAMAGE-REPAIR, APOPTOSIS AND NECROSIS
IN CHILDREN, ADULTS AND OLD AGE HUMANS
G Karanastasi1, N Anagnostakis1, N Messini-
Nikolaki2, S Doudounakis3, S Tsilimigaki1,
SM Piperakis1
1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 Aghia Sophia Hospital, ATHENS, Greece
Our aim using the comet assay, was:
1) To determine the amount of endogenous DNA
damage in human lymphocytes of children,
adult and old age human populations.
2) To find the sensitivity of their lymphocytes to the
exposure of increased concentrations of g-irradia-
tion and H2O2.
3) To investigate the DNA repair efficiency in these
populations, and finally,
4) To measure apoptosis and necrosis in the same
populations.
Our results indicate that:
1) The endogenous DNA damage is smaller in the
child than the other two population.
2) There is a difference in the sensitivity of the three
populations lymphocytes after been exposed to
g-irradiation or H2O2.
3) The DNA repair was more efficient in the child
than the other three populations, and finally,
4) The amount of apoptotic cells appears to be less in
the child than the other populations.
W7 - Workshop 7
Oral Poster Presentation
OW7004
DNA REPAIR FUNCTION, POLYMORPHISMS AND
GENOTOXICITY IN WORKERS EXPOSED TO LOW
DOSE IONISING RADIATION
V Aka1, RAM Mateuca1, JP Buchet2, H Thierens3,
M Kirsch-Volders1
1 Vrije Universiteit Brussels, BRUSSELS, Belgium2 Catholic University of Louvain, BRUSSELS, Belgium3 University of Ghent, GHENT, Belgium
Identification of higher risk individuals carrying
genetic polymorphisms responsible for reduced DNA
repair capacity has substantial preventive implica-
tions as these individuals could be targeted for appro-
priate cancer prevention. We have conducted a study
in 32 male seasonal cleaners of a nuclear plant and 31
control workers to assess the predictivity of the OGG1,
XRCC1 and XRCC3 genotypes and the in vitro single
strand break repair phenotype for the induction of
genotoxic effects (DNA damage and micronuclei). At
the population level, a significant contribution of the
OGG1 genotypes to the in vitro DNA strand break
repair capacity was found. Genetic polymorphisms in
XRCC1 and XRCC3 did not significantly influence
repair capacity. At an individual level, the OGG1 vari-
ants Ser/Cys and Cys/Cys genotypes showed a slower
in vitro DNA repair than the Ser/Ser OGG1genotype. A
multivariate analysis performed with genotypes, age,
cumulative dose, exposure status and smoking as
independent variables indicated that in the control
population, repair capacity is influenced by age and
polymorphisms in OGG1. In the exposed population,
DNA damage is greater in older men and in smokers.
Repair capacity is slower in individuals with Ser/Cys
or Cys/Cys OGG1 genotypes compared to those with
the Ser/Ser OGG1 genotype. Micronuclei frequencies
increased with age and the cumulative dose of
gamma rays. Analysis of the total population
revealed that genetic polymorphisms in XRCC1 result-
ed in higher residual DNA values and the Met/Met
variant of XRCC3 gave an increased frequency of
micronuclei. A combined analysis of the three geno-
types, OGG1, XRCC1 and XRCC3 polymorphisms is
advised in order to assess individual susceptibility to
ionising radiation. As an alternative or complement,
the in vitro DNA strand break repair phenotype
which integrates several repair pathways is recom-
mended.
42
abstractboek 20-10-2004 10:28 Pagina 42
W7 - Workshop 7
Oral Poster Presentation
OW7005
AGE-RELATED GENOME INSTABILITY IN DNA
REPAIR-DEFICIENT MICE
MET Dollé1, R Busuttil2, L Niedernhofer3, B van der
Horst3, J Hoeijmakers3, SWP Wijnhoven1, P Lohman1,
H van Steeg1, J Vijg4
1 Nat.Inst.of Publ. Health and Environment,
BILTHOVEN, The Netherlands2 Univ TX Health Science Center, SAN ANTONIO,
United States of America3 Erasmus University, ROTTERDAM, The Netherlands4 University Texas Health Science Centre,
SAN ANTONIO, United States of America
Genomic instability has been implicated as a major
cause of both cancer and aging. Previously we have
shown that mutations accumulate with age in an
organ-specific manner, using a transgenic mouse
model with a chromosomally integrated lacZ muta-
tional reporter gene. To examine the correlation
between age and mutation load, we are investigating
genomic instability in aging DNA repair-deficient
mice. Here we present our preliminary findings on
different mouse mutants with deficiencies in
nucleotide excision repair (NER) and DNA interstrand
crosslink (ICL) repair. NER removes a broad range of
lesions, such as UV-induced damage, other bulky
adducts and some forms of oxidative damage. Life
span studies and mutant frequency determinations
in liver and kidney were performed on male mice
with defects in global and/or transcription-coupled
repair (Xpa, Csb or Xpd(ttd)). We observed a reduced
life span for Xpa and Xpd(ttd), as compared to Csb and
wild type control animals. Conform to our initial
studies with just wild type mice, all NER-deficient
models showed an age-related increase of mutant
frequencies in liver and kidney. However, only the
Xpa-mice showed elevated mutant frequencies over
control animals in both organs. In addition, we exam-
ined mutant frequencies in liver of the very short-
lived Ercc1(*292) mouse. This mouse model lacks the
last 7 amino acids of Ercc1 and is deficient in both NER
and DNA ICL repair. We observed a two-fold increase
in mutant frequency over control mice at 23 weeks,
which is close to their maximum life span. A compar-
ison of mutation spectra in liver revealed that the
general NER-deficiency of XPA-mice resulted in point
mutations, predominantly consisting of one base pair
deletions. The increased mutant frequency in the
Ercc1(*292) mice was due to both point mutations and
translocations, the latter presumably reflecting the
additional cross-link repair deficiency compared to
Xpa-mice.
YSAL - Young Scientist Award Lectures
Oral Presentation
OYSAL1
OXIDATIVELY DAMAGED DNA: FROM GENES TO
POPULATIONS
MS Cooke
University of Leicester, LEICESTER, United Kingdom
There is an ever increasing number of physico-chem-
ical agents reported to induce oxidative damage to
DNA. Elevated levels of, and failure to repair, such
damage, has been associated with a wide variety of
pathological conditions. Nevertheless, proof of a
causative effect remains elusive. Attempts towards
obtaining a comprehensive understanding of the fac-
tors affecting the induction and repair of oxidatively
damaged DNA, have been facilitated by a diverse
range of approaches. For example, preferential
removal of lesions from transcriptionally active
genes, combined with transcribed strand-specific
repair can facilitate cell survival even when total
lesion levels would be expected to be toxic. This may,
in part, be responsible for the difficulties associating
elevated lesion levels with disease, and has prompted
the development of assays to examine damage
to individual genes. Furthermore, simultaneous
measurement of oxidative (e.g. 7,8-dihydro-8-oxo-
2'deoxyguanosine, 8-OHdG) and non-oxidative lesions
(cyclobutane thymine dimer, T<>T), has highlighted
similarities in the removal of both classes of lesion
from the genome, allowing conclusions to be drawn
regarding the processes involved. Indeed, it was the
measurement of multiple lesions, in various biologi-
cal matrices, including urine, that enabled the
hypothesis for the induction of DNA repair by vita-
min C, to be suggested. Although the proposal of a
number of confounding factors have meant urinary
8-OH-dG measurements are often considered to be no
more than reflective of whole body oxidative stress,
the potential exists for a non-invasive assay of DNA
repair.
The plethora of sources of oxidative damage to DNA
means that little precise exposure data can be gained
from studies which involve biomonitoring. However,
were these measurements able to provide informa-
tion concerning an individual's cellular defenses, and
DNA repair, for example, they would represent a far
more useful tool in human population studies.
43
abstractboek 20-10-2004 10:28 Pagina 43
YSAL - Young Scientist Award Lectures
Oral Presentation
OYSAL2
CELL-TYPE AND DNA DAMAGE-SPECIFIC RESPONSE
OF HUMAN EPIDERMAL CELLS
M D'Errico1, M Teson2, A Calcagnile1, T Nardo3,
P Jaruga4, G Zambruno2, M Stefanini3, M Dizdaroglu4,
E Dogliotti1
1 Italian National Health Institute, ROME, Italy2 I.D.I., IRCCS, ROME, Italy3 CNR, PAVIA, Italy4 NIST, GAITHERSBURG, United States of America
The epidermis is the primary target for environmen-
tal insults. In this study we present a systematic com-
parison of the response to UVB and X-rays of human
primary keratinocytes and fibroblasts. Nucleotide
excision repair (NER)-defective cells derived from XP-
C (no global genome repair, GGR) and CS-A (no tran-
scription-coupled repair, TCR) patients were used to
investigate the effects of alterations in the two NER
sub-pathways on DNA damage response.
A cell-type specific response was detected after UVB
treatment. Keratinocytes were more resistant to the
lethal effects of UVB than fibroblasts and removed
cyclobutane pyrimidine dimers (CPD) more efficient-
ly than fibroblasts. This improved repair was ascribed
to a more efficient GGR. A defect in TCR was associat-
ed with a strong apoptotic response in fibroblasts but
not in keratinocytes, whereas a defect in GGR had no
effect on the apoptotic response of either cell type.
We speculate that the persistence of CPD in the tran-
scribed sequences triggers apoptosis in fibroblasts
but not in keratinocytes where GGR operates as back-
up system to remove transcription-blocking lesions.
As observed for UVB, X-ray-induced cytotoxicity was
cell-type specific with keratinocytes more resistant to
the lethal effects of X-rays than fibroblasts. Similar
levels of DNA single-strand breaks and modified
nucleosides [8-hydroxy-2'-deoxyguanosine, 8-hydroxy-
2'-deoxyadenosine, (5'S)-8,5’-cyclo-2'-deoxyadenosine]
were detected in the two cell types after irradiation.
Normal keratinocytes repaired to completion modi-
fied DNA bases within one hour, whereas CSA ker-
atinocytes accumulated significant amounts of these
lesions. In contrast to UVB, the apoptotic response to
X-rays was not cell-type dependent. In both normal
and NER-defective skin cells apoptosis was triggered
at highly cytotoxic doses.
All together these results indicate that the response
to environmental agents is cell type- and DNA dam-
age-specific. Moreover, we provide evidence that CSA
has a cell-type specific impact in the response to UVB
and participates to repair oxidative DNA damage.
44
abstractboek 20-10-2004 10:28 Pagina 44
45
Poster presentations
PS1 - Poster Session 1
Poster Presentation
PW1001
EVALUATION OF THE MUTAGENIC PROPERTIES OF
PENTA® DRINKING WATER USING CYTOGENETIC
METHODS
AN Chebotarev, NV Kosyakova, VI Platonova
Research Center for Medical Genetics, MOSCOW,
Russia
The clastogenic properties of Penta® drinking water
has been performed using cytogenetic methods to
determine the chromosome aberration frequency
(CAF), sister chromatid exchange (SCE) number and
cell cycle duration. Penta® water samples have been
provided by Bio-Hydration Research Lab (USA).
Penta® water is manufactured using a process that
transforms cavitation acoustic energy into thermal
energy in a specially designed cavitation chamber
(US patent 6,521,248). Experiments have been con-
ducted on human lymphocytes that were cultured
using a standard protocol. The average number of cell
divisions in culture in the presence of 5BdU was
determined using the formula: (i x ni/2i-1)/( ni/2i-1),
where i = mitosis number and ni = cell number of i-
mitosis. Cell cycle duration was determined by 5BdU
presence time in cell media divided by the average
number of cell divisions. In addition to experiments
on spontaneous mutagenesis in various cell media,
induced clastogenic effects of mitomycin C (100, 200,
and 400 ng/ml) added 14 hours prior to the cell fixa-
tion have been studied. When Penta® water was used
in cell media preparation without a mutagen added,
the SCE number per cell = 3.38±0.12 and CAF = 0.92%.
These figures were lower than results seen with the
control: SCE number per cell = 4.01±0.15 and
CAF=2.50%. Cell cycle duration was the same for both
Penta' and deionized water based cell media (21.2
hours). The SCE number was the same for all concen-
trations of mutagen used in both types of waters in
cell media preparation. Regression analysis of the
mutagenic effect dependent on Mitomycin C concen-
tration has shown that the frequency of aberrant
metaphases is two times lower and the number of
chromosome breaks per cell is 1.5 times lower when
Penta® water is used compared to the control.
abstractboek 20-10-2004 10:28 Pagina 45
46
PS1 - Poster Session 1
Poster Presentation
PW1002
ARISTOLOCHIC ACID AS A PROBABLE HUMAN
CANCER HAZARD IN HERBAL REMEDIES
VM Arlt1, GM Lord2, M Hollstein3, V Alunni-Perret4,
JP Cosyns5, HH Schmeiser3
1 Institute of Cancer Research, SUTTON, SURREY, United
Kingdom2 Hammersmith Hospital NHS Trust, LONDON, United
Kingdom3 German Cancer Research Center, HEIDELBERG,
Germany4 Hopital Pasteur, NICE, France5 Cliniques St. Luc, BRUSSELS, Belgium
The old herbal drug aristolochic acid (AA), derived
from Aristolochia spp., has been associated with the
development of a novel nephropathy, designated
aristolochic acid nephropathy (AAN), that predispos-
es patients to a high risk of urothelial carcinoma.
AAN was reported for the first time in a Belgian slim-
ming clinic. To date, more than 100 patients have
been identified in Belgium. All cases could be traced
to the herbal ingestion of a preparation containing
AA, namely Aristolochia fangchi. We found that the
adenosine adduct of aristolochic acid I [7-(deoxya-
denosin-N6-yl)aristolactam I, dA-AAI] is the predom-
inant DNA adduct detectable in urothelial tissue. In
the present study we describe the first fully docu-
mented cases of AAN in the UK and France.
Histopathologic and DNA adduct analyses performed
on autopsy samples revealed that these cases did
indeed represent authentic AAN outside Belgium. It is
of interest to note that some patients had developed
a transitional cell carcinoma in the urinary tract.
Given the availability of several tissue samples from
patient's post-mortem examination, we were able to
detect the dA-AAI adduct in various tissues outside
the urinary tract (kidney, bladder, ureter, liver, lung,
stomach, small intestine, spleen, adrenal, and brain)
indicating that other factors than DNA adduct forma-
tion by AA may also be critical for the high incidence
of urothelial tumours. Interestingly, a characteristic A
to T transversion mutation was found at the first ade-
nine of codon 139 (AAG) of the p53 gene in urothelial
tumour cells of one patient. A to T transversions are
the typical mutations observed in the H-ras gene of
AA-induced tumours in rodents and correspond with
DNA adducts at adenosine residues. These data indi-
cate the probable molecular mechanism whereby AA
causes urothelial tumours. These cases highlight the
need for continuing vigilance to ensure that AA is not
present in herbal medicinal remedies.
Good Food, Good
La passion de la
abstractboek 20-10-2004 10:28 Pagina 46
47
abstractboek 20-10-2004 10:28 Pagina 47
48
PS1 - Poster Session 1
Poster Presentation
PW1003
CORRELATION BETWEEN STABILIZATION OF
CLEAVABLE COMPLEXES EVOKED BY ANTHRACY-
CLINES AND APOPTOSIS IN CELLS
BM Gruber1, EL Anuszewska1, W Priebe2, I Fokt2,
A Gozdzik1
1 National Institute of Public Health, WARSAW,
Poland2 M.D. Anderson Cancer Center, HOUSTON, United
States of America
Apoptosis is one of the possible mechanism responsi-
ble for cytotoxicity of anthracyclines. Last years
brought the new aspects of stimulating apoptotic
effects by these drugs including NFkappaB. As was
shown it can be activated by anthracyclines, leading
to apoptosis via various mechanisms. It is suggested
the role of DNA lesions generated through stabiliza-
tion of cleavable complexes DNA-topo II in activation
of NFkappaB and, indirectly, in apoptosis induction.
The aim of this study was to establish the possible
dependance between stabilization of cleavable com-
plexes by anthracyclines and intensity of apoptotic
effects. In the current work, the following cells differ-
ing in sensitivity to anthracyclines were used: human
melanoma cells(Me18); it's subline resistant to
DOX(Me18/R); human cervix carcinoma cells(HeLa);
it's subline resistant to vinblastine(KB-V1).Different
new anthracycline analogs synthetized in M.D.
Anderson Cancer Center, Houston, USA were tested.
As a result, quantitative differences in topo II protein
between the cells, in part correlated with sensitivity
to anthracyclines were observed. Also, some correla-
tion was shown between sensitivity to the tested
drugs, 'quenching' of topo II protein by these com-
pounds and the intensity of caspase dependent apop-
tosis in human cervix carcinoma cells.
PS1 - Poster Session 1
Poster Presentation
PW1004
THE EFFECT OF RESVERATROL ON THE EXPRES-
SION OF ENZYMES METABOLIZING XENOBIOTICS
IN MOUSE EPIDERMIS
W Baer-Dubowska, V Krajka-Kuzniak, H Szaefer
Poznan University of Medical Sciences, POZNAN,
Poland
Resveratrol (3,4',5-trihydroxystilbene), a polyphenol
found at high levels in a wide variety of plant species
including peanuts, mulberries and grapes, has been
reported to exhibit a wide range of beneficial biolog-
ical properties including the modulation of multi-
stage carcinogenesis. This compound inhibited 7,12-
dimethylbenz[a]anthracene (DMBA)-induced preneo-
plastic lesion formation in mouse mammary organ
culture and reduced the incidence and multiplicity of
DMBA/TPA-induced papillomas in the two-stage
mouse skin model. Antimutagenic activity of resvera-
trol was also demonstrated against the foodborne
heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyri-
do[4,3-b]indole (Trp-P-1) and 2-aminofluorene in
Salmonella bacterial tester strains. These chemical
carcinogens require metabolic activation by P450
dependent enzymes in order to exert their genotoxic
effect. However phase II enzymes, particularly GST
isoforms may modify their metabolic profile.
In the present study we examined the effect of
resveratrol on the expression of CYP1A1/1A2, CYP1B1
and GST theta in female Swiss mouse epidermis by
Western blot assays.
Resveratrol was applied topically to the shaved dorsal
skin at the doses of 8 or 16 mmoles in 0.2 ml of ace-
tone. A group treated with 5,6-benzoflavone served as
a positive control. A control group of mice received
acetone alone.
The constitutive expression of all tested enzymes was
observed, however their expression levels were differ-
ent. Topical application of resveratrol affected the
most GST theta. In both tested doses the significant
increase of this isoform protein was observed. This
compound was also a weak inducer of epidermis
CYP1B1. Treatment of mouse epidermis with resvera-
trol did not affect on the level of CYP1A1/1A2 in this
tissue.The results of our study indicate that although
moderate, the effect of resveratrol on expression of
CYP1A1/1A2, CYP1B1 and GST theta in mouse epider-
mis may play certain role in its anticarcinogenic
activity.
abstractboek 20-10-2004 10:28 Pagina 48
49
PS1 - Poster Session 1
Poster Presentation
PW1005
CHARACTERIZATION OF HUMAN DIETARY COM-
PONENTS IN HUMAN DERIVED LIVER CELLS
F Darroudi1, S Knasmueller2, V Mersch-Sundermann3,
E Lhoste4, A Bader5
1 Leiden University Medical Center, LEIDEN,
The Netherlands2 University of Vienna, VIENNA, Austria3 University of Giessen, GIESSEN, Germany4 INRA, JOUY EN JOSAS, France5 University of Leipzig, LEIPZIG, Germany
It is well documented that diet plays a crucial role in
the etiology of cancers in humans, and efforts were
also made to identify protective (anti-mutagenic and
anti-carcinogenic) substances in food. The traditional
risk assessment process applied successfully to food
additives relies on toxicology testing in vivo in ani-
mals at intake levels many times higher than is likely
in humans. The in vitro assays with metabolically
incompetent cells require addition of exogenous
enzyme homogenate (i.e. rat liver fractions) to cat-
alyze the activation of genotoxic food derived car-
cinogens. However, it became evident that certain
mode of action (i.e. detoxification process and to
detect protective / synergistic effects) are not repre-
sented adequately in the in vitro models.
Different biological assays were developed, validated
and applied using human derived liver cells (Hep G2
and hepatocytes) to detect the mutagenic potentials
of various classes of human food constituents (i.e.
polycyclic aromatic hydrocarbons, heterocyclic aro-
matic amines, nitrosamines and mycotoxins). HepG2
cells were used as the metabolic activation system as
well as target for evaluating DNA damage. Different
biological end-points, such as sister-chromatid
exchanges, micronuclei in binucleated cells, cytotoxi-
city, gene mutation (at HPRT locus) and Comet assay
were used. Furthermore, co- and anti-mutagenic
potential of human dietary food constituents (i.e.
vitamins, cruciferous vegetables, beverages and
heavy metals) was also investigated. The use of
human hepatoma (Hep G2) cell system in mutagenic-
ity testing further validated through gene expression
analysis and a comparison was made with human
hepatocytes.
The results indicate that the human hepatoma cell
systems reflect the activation / detoxification of
genotoxic carcinogens better than other indicator
cells that are currently being used and therefore have
an increased predictive value for the identification of
mutagenic, co- and anti-mutagenic constituents of
human foods.
GlaxoSmithKline is pleased to supportEEMS 2004
abstractboek 20-10-2004 10:28 Pagina 49
50
PS1 - Poster Session 1
Poster Presentation
PW1006
THEOPHYLLINE INHIBITS POLY-(ADP-RIBOSE)POLY-
MERASE-1 (PARP-1) ACTIVITY IN HUMAN PUL-
MONARY EPITHELIAL CELLS
H.J.J. Moonen, L. Geraets, A. Vaarhorst, A. Bast,
G.J. Hageman
Maastricht University, MAASTRICHT,
The Netherlands
Background: Theophylline is still widely used in the
treatment of asthma and chronic obstructive pul-
monary disease (COPD), and is believed to exert anti-
inflammatory properties. Although several possible
mechanisms underlying the beneficial effects of
theophylline have been described, inhibition of the
nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-
1), which appears to be a major player in the patho-
physiology of chronic inflammatory diseases such as
COPD, has not been investigated in this perspective
before.
Objective: In this study, we investigated the effect of
theophylline on PARP-1 activity in human pulmonary
epithelial cells (A549).
Methods: PARP-1 activity in A549 cells was deter-
mined by measuring decreasing intracellular NAD+
levels upon exposure to 300 µM of hydrogen perox-
ide, with or without pre-incubation with either 0.1,
0.2, 1 or 10 mM theophylline. Electron spin resonance
(ESR) spectroscopy was used to investigate the antiox-
idant capacity of theophylline.
Results: Intracellular NAD+ levels dramatically
decreased upon exposure to hydrogen peroxide, indi-
cating strong induction of PARP-1 activity. Pre-incuba-
tion with theophylline was found to prevent the
decrease in NAD+ in a dose dependent manner, thus
showing that theophylline inhibits the activity of
PARP-1 in these cells. Furthermore, theophylline did
not show any antioxidant capacity, thus ruling out
scavenging of oxygen radicals.
Conclusions: Theophylline inhibits PARP-1 activity in
human pulmonary epithelial cells, and does not exert
any anti-oxidant effect in these cells. These findings
provide new insights into the therapeutic effect of
theophylline.
PS1 - Poster Session 1
Poster Presentation
PW1007
GENOTOXIC EFFECTS OF OCHRATOXIN A ON THE
HUMAN KIDNEY HK-2 CELL LINE
A L C Lopez de Cerain1, S de Blaes2, O Ezpeleta1,
L Arbillaga1, A Lopez de Cerain1
1 University of Navarra, PAMPLONA, Spain2 Wageningen University, WAGENINGEN,
The Netherlands
Ochratoxin A (OTA) is a nephrotoxic mycotoxin classi-
fied by the International Agency for the Research on
Cancer (IARC) as a carcinogen category 2A. It is a
potent renal carcinogen in male rats although its
mechanism of action is not known. Few data on the
genotoxicity of OTA in human cells are available. The
aim of the present work was to investigate the muta-
genic effects of OTA using the micronucleus (MN)
assay in a human derived kidney cell line (HK-2).
A previous MTT cytototoxicity assay was carried out
with different concentrations of OTA and S9-mix in
order to select subtoxic OTA concentrations for the
mutagenicity assay. Cells were treated with OTA -
range 0.1- 100 µM- for 2, 4 and 6 h, in the presence of
rat kidney or liver S9-mix (4-10%). For the MN assay
the following conditions were decided: 10 and 40 µM,
5 hours, 4% S9-mix. The MN assay was performed
using the protocol of Fenech et al. (1999), modified for
HK-2 cells. Cytochalasin B (1 µM) was added for the
last 21-h of incubation. MN were identified in 500
binucleated cells, and scored according to the criteria
described by Bonassi et al. (2001). Data were statisti-
cally analysed with the Mann-Whitney U test (SPSS
program, version 11.0).
OTA caused an increase in the number of MN in the
absence of S9-mix (approximately 6-fold). In the pres-
ence of liver or kidney S9-mix, a 3-fold MN induction
was observed. Non significant differences were
obtained between the two concentrations tested.
These results show that OTA treatment induces the
production of MN in a human kidney derived cell
line. As we do not know the metabolic capability of
this cell line, the importance of the metabolic activa-
tion could not be completely assessed.
abstractboek 20-10-2004 10:28 Pagina 50
51
PS1 - Poster Session 1
Poster Presentation
PW1008
DNA DAMAGE IN HUMAN CELLS TREATED WITH
DRINKING WATER DISTRIBUTED IN THREE NET-
WORKS
C Pellacani, A Buschini, M Furlini, P Poli, C Rossi
Universita di Parma, PARMA, Italy
Epidemiological studies and mutagenic short-term
bioassays have revealed the presence of genotoxic
disinfection by-products in drinking water. Few stud-
ies have been evaluated the influence of distribution
systems on mutagen formation. Mutagenic com-
pound could be leached from internal surface of
municipal water tanks and pipelines, which are fre-
quently coated with coal tar or from plastic pipelines.
The aim of this study is the evaluation of toxic and
genotoxic effects of drinking water in the networks of
three Italian towns with different water source sup-
plying systems. The drinking water have been sam-
pled in different pipelines points, after and before the
distribution systems. The water has been concentrat-
ed with semipermeable membrane devices (SPMDs),
membrane-based passive samplers, a promising tool
for the time-integrated monitoring of hydrophobic
pollutants in aquatic systems. SPMDs have been
exposed to water flow (1litre/min) during 30 days,
dialyzed in a ethyl acetate, acetone, and methanol
solution (1:1:1), and concentrated with a rotary evapo-
rator. The concentrated samples have been tested on
human leukocytes by the Comet assay for DNA dam-
age. Cell viability was checked by fluorescine diac-
etate/ethidium bromide-assay. All the water samples
were able to induce DNA damage even if with differ-
ent behaviour and efficacy. 'Town A' (groundwater):
DNA damage resulted hardly increased (≈15 times) in
cell treated with water concentrate equivalent to 12-
hour’s SPMD exposure before distribution system
with respect to the control; distribution system did
not increase water genotoxicity. 'Town B' (surface
water): before distribution-water induced a DNA
damage increase (≈3 times the control) whereas this
increase was ≈20 times for water sampled on the
pipeline farthest point. 'Town C' (surface water): DNA
damage was increased ≈15 times before the distribu-
tion and this effect become further greater (≈25
times) in tap water; in an other pipeline point it was
also observed a strong cytotoxic effect.
PS1 - Poster Session 1
Poster Presentation
PW1009
ANTIGENOTOXIC EFFECTS OF ALOE AND ITS
MODE OF ACTION
BM Lee, E Jeong Yoo
Sungkyunkwan University, SUWON, South-Korea
Antigenotoxic effects of aloe were investigated in 3 dif-
ferent tests: sets of Ames, chromosome and micronuclei
tests. Aloe reduced in a dose-dependent manner the
mutational frequencies of Salmonella strains, chromo-
some aberration, and micronuclei formation in mouse
bone marrow induced by benzo(a)pyrene. In addition,
aloe significantly increased glutathione levels in cells
used for the tests, which might be involved in possi-
ble mechanisms of aloe antigenotoxicity.
These results suggest that aloe could be a chemopre-
ventive potential by modulating cellular glutathione
levels.
abstractboek 20-10-2004 10:28 Pagina 51
52
PS1 - Poster Session 1
Poster Presentation
PW1010
CURCUMIN INTERACTS WITH INDUCTION OF
UROKINASE-TYPE PLASMINOGEN ACTIVATOR
(UPA) BY THE ALKILATING AGENT MNNG
JS Soric, J. Loniarek
Faculty of Pharmacy and Biochemistry, ZAGREB,
Croatia
We have demonstrated that the uPA gene is tran-
scriptionaly induced by MNNG, starting 24 hours post
treatment. ERK MAP kinase specific inhibitor did not
change neither constitutive neither basal uPA enzy-
matic activity, while p38 MAP kinase inhibitor could
partially abolish the uPA induction. Curcumin
appeared as most potent inhibitor of uPA induction,
decreasing basal level of uPA and abolishing the
inducible effect of MNNG. Curcumin addition and
removal from the cellular media at different time
points after MNNG treatment, showed that for sup-
pression of uPA by curcumin, the most relevant fact
was its constant presence in the growing medium.
Furthermore we observed its reversible effect, since
curcumin removal from the cell culture media abort-
ed its inhibiting ability. It indicated existence of an
persistent signal triggered by MNNG inducing uPA
expression and its inhibition by curcumin. Aditionally,
here we show that curcumin influences cellular prolif-
eration and disturbs the action of btubulin, as showed
by immunostaining. Although the precize mechanism
of curcumin action is currently unknown, the results
demonstrated, among possible other events, its addi-
tional action upon microtubular organization and
consequent induction of mitotic catastrophe (micro-
nucleation).
abstractboek 20-10-2004 10:28 Pagina 52
53
PS1 - Poster Session 1
Poster Presentation
PW1011
32P-POSTLABELLING OF ACRYLAMIDE DNA AD-
DUCTS
K Segerbäck1, N Kotova1, T Juren1, P Rydberg2
1 Karolinska Institute, HUDDINGE, Sweden2 Stockholm University, STOCKHOLM, Sweden
An important issue in the discussions on the risk con-
nected to consumption of food containing the muta-
gen acrylamide is the dose-response relationship for
formation of biological effects in cancer tests in
rodents. DNA adducts is considered to be a marker of
biological significant dose and measurements of such
products could therefore be very useful for the risk
assessment of acrylamide. Acrylamide is metabolised
to the reactive epoxide glycidamide (GA) and it is
believed that most of the genotoxic effects of acry-
lamide is mediated via GA. As an alkylating agent GA
reacts primarily with ring nitrogens in DNA bases.
Besides the major product with N-7-guanine there are
also adducts at N-1- and N-3-adenine. We have previ-
ously been using the nuclease P1 enhancement of the
32P-postlabelling assay for analysis of the N-1-ade-
nine adduct of propylene oxide. In this work we man-
age to reach a sensitivity of 1 adduct per 10exp10
normal nucleotides. This was accomplished by con-
verting the N-1-adenine adduct to the N6-adenine
adduct and separation by HPLC with on-line radioiso-
tope detection. The same method was now developed
for the N-1-adenine adduct of GA. GA was reacted
with deoxyadenosine, deoxyadenosine-3'-monophos-
phate and deoxyadenosine-5'-monophosphate. The
N-1 products were isolated and characterized by UV
and mass spectrometry. The GA-N-1-deoxyadenosine-
3'-monophosphate standard was postlabelled and
analysed by HPLC. GA-reacted DNA was enzymatical-
ly digested and analysed by the developed postla-
belling assay. The results show high sensitivity and
specificity of the assay. This method will be used for
analysis of DNA from experimental animals exposed
to low doses of acrylamide.
The support of the EU CA Childrengenonetwork
QLK4-ct-2002-02198 is acknowledged.
PS1 - Poster Session 1
Poster Presentation
PW1012
DNA-DAMAGE REDUCING EFFECTS OF FLAVONOID
SUPPLEMENTATION IN A HUMAN INTERVENTION
STUDY
LC Wilms, JCS Kleinjans
Maastricht University, MAASTRICHT,
The Netherlands
Flavonoids are natural polyphenols, which are impor-
tant constituents of fruits, vegetables, nuts, tea and
red wine. Flavonoids are claimed to be able to protect
against cardiovascular disease, certain forms of can-
cer and ageing. In this project quercetin is used as a
model for the abundant group of flavonoids.
Aim of this study is to determine whether a four-
week period of increased quercetin intake through
foodstuffs enhances the anticarcinogenic defence
mechanism in healthy volunteers.
During a four-week period eight volunteers increased
their quercetin intake through consuming one litre of
a mixture of blueberry juice (50%) and apple juice
(50%). Blood was collected before, during and after
the intervention, and dietary flavonoid intake was
determined based on the subject’s dietary records.
Quercetin levels and antioxidative capacity were
determined in plasma. Peripheral blood lymphocytes
(PBL) were isolated and exposed ex vivo to an effec-
tive dose of benz(a)pyrene (B(a)P) and hydrogen per-
oxide. As indices for DNA damage BPDE-DNA adducts
and oxidative damage were measured by 32P-post-
labelling and COMET-assay, respectively.
Results: four weeks of supplementation of quercetin
through foods led to a significant increase in plasma
quercetin content (p=0.0277). Consequently, the total
antioxidative capacity of plasma, reflected by the
TEAC (trolox equivalent antioxidative capacity) value,
showed a significant increase (p=0.0425). The inter-
vention appeared to have protective effects on ex vivo
induced DNA damage in PBL. After the intervention
the level of oxidative damage upon exposure to H2O2
was decreased (p=0.0679, n=4). BPDE-DNA adduct
levels vary enormously between subjects, but in 5 out
of 7 subjects the DNA adduct level at t=4 is lower than
that at t=0. Further, large interindividual differences
in these effects and in absolute levels of damage were
observed, possibly indicating a role of polymor-
phisms in genes encoding anticarcinogenic/antiox-
idative defence.
abstractboek 20-10-2004 10:28 Pagina 53
PS1 - Poster Session 1
Poster Presentation
PW1013
ANTIMUTAGENIC EFFECT OF PHENETHYL ISO-
THIOCYANATE AND ITS EFFECT ON THE IMMUNE
RESPONSE IN MICE
IVO Barta, P Smerak, Z Polfvkova, J Bártová, B Turek,
M Langová
Charles University, PRAGUE, Czech Republic
Phenethyl isothiocyanate (PEITC) was applied to mice
for three days in doses of 50 mg/kg, the third day a
carcinogen was also applied - either aflatoxin B1
(AFB1) 1 mg/kg, 2-amino-3-methylimidazo-4,5-f quino-
line (IQ) 20 mg/kg and N-nitroso-N-methylurea
(MNU) 20 mg/kg. PEITC demonstrably inhibits the
mutagenic activity of the three mutagenes: IQ, AFB1
and MNU in the mammalian model (micronucleus
test) in the bone marrow of laboratory mice. In the
procaryotic model (Ames test) a strong inhibition of
the mutagenic activity of IQ and AFB1 by PEITC has
been demonstrated. The protective effect of PEITC
was assessed by chemiluminiscence method and by
the blastic transformation method testing the func-
tional capacity of T-lymphocytes. The phagocytic
function of murine peritoneal granulocytes was sup-
pressed significantly by all 3 carcinogenes tested just
like blast formation from mitogen-stimulated
murine splenocytes. There was no statistically signif-
icant differences between groups of animals treated
with PEITC alone and those not treated, in chemilu-
miniscence follow-up and as regards blastic transfor-
mation. In all experiments PEITC to a significant
degree repaired the immunosuppressive effects of all
three carcinogenes tested. Results of this study indi-
cate that, PEITC may play an important role in pre-
vention of carcinogenesis by capability to modulate
immunosuppresion caused by carcinogenes.
Supported by grant IGA No. 6138 and Research Plan
No. MSM 111200001.
PS1 - Poster Session 1
Poster Presentation
PW1014
IDENTIFICATION OF PROTEINS INVOLVED IN THE
PROTECTION AGAINST COLORECTAL CANCER BY
VEGETABLES
G Breikers, SGJ van Breda, FG Bouwman, MH van
Herwijnen, JW Renes, ECM Mariman, JCS Kleinjans,
JHM van Delft
Maastricht University, MAASTRICHT,
The Netherlands
Colorectal cancer is one of the most common cancers
worldwide. There is abundant epidemiologic evi-
dence that colorectal cancer risk is decreased by con-
sumption of diets high in vegetables. Although the
evidence from animal studies is less clear, it was
found that vegetables or their components reduced
colorectal cancer risk. Vegetable components can,
directly or indirectly, influence gene expression lead-
ing to a change in transcription rate of a given gene.
Changes in gene expression will be translated into
the proteome. At present, however, the molecular tar-
gets at the genome and proteome level are largely
unknown. In our study, the effect of a vegetable rich
diet on protein expression in the colon mucosa of
healthy mice was studied by means of Proteomics.
We found that the expression of several proteins was
changed upon vegetable consumption, including
GAPDH, carbonic anhydrase I and high mobility
group protein 1 that are proteins involved in apopto-
sis or differentiation. In addition, we convincingly
showed that it is possible to link the up- or downreg-
ulation of certain proteins with their gene expres-
sion. In conclusion, these data indicate that vegetable
rich diets may exert their anti-carcinogenic effects by
changing gene- and protein expression profiles.
54
abstractboek 20-10-2004 10:28 Pagina 54
55
PS1 - Poster Session 1
Poster Presentation
PW2001
THE ROLE OF PARP-1 AND XRCC1 IN CAMP-
TOTHECIN-INDUCED HOMOLOGOUS RECOMBI-
NATION
KM Parker, T Helleday
University of Sheffield, SHEFFIELD, United Kingdom
Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear
protein responsible for detecting and signalling DNA
strand breaks and has a role in base excision repair
(BER) through interaction with XRCC1. We hypothe-
sise that the loss of PARP-1 or XRCC1 results in an
increase in homologous recombination, due to the
loss of a functional BER pathway.
In this study, we have used camptothecin (CPT), a
potent inhibitor of topoisomerase I (which creates
DNA double strand breaks (DSBs)), and derivatives of
which are used in the treatment of several cancers.
Recently it has been discovered that co-treatment
with CPT and PARP-1 inhibitors increases the cytotox-
icity of CPT. Here, we investigate the role of PARP-1
and XRCC1 in CPT toxicity, in the hope that this data
will prove useful in the future treatment of cancers.
We show that the treatment of PARP-1 -/- mouse
embryonic fibroblasts with CPT increases cell death,
and when treated with CPT for 24 hours, these cells
have an increased number of DSBs. The same effect
was seen when wildtype cells were co-treated with
CPT and PARP-1 inhibitors. We also show that the
number of RAD51 foci is spontaneously increased in
PARP-1 -/- cells and increases further after treatment
with CPT. We also observe that XRCC1 -/- cells have
increased sensitivity to CPT, have increased DSBs
after treatment with CPT, and have increased sponta-
neous and CPT-induced RAD51 foci.
We conclude that PARP-1’s role in BER protects CPT-
induced single strand breaks from causing DSBs,
through interaction with XRCC1 and allowing the BER
enzymes to repair the damage. In the absence of
either of these proteins, the single strand breaks
remain unrepaired, and DSBs are formed, leading to
homologous recombination. Therefore, co-treatment
with CPT and PARP-1 inhibitors increases cytotoxicity
through abolition of the BER pathway.
PS1 - Poster Session 1
Poster Presentation
PW2002
IN-VIVO GENOTOXICITY OF THE SYNTHETIC
PYRETHROID PESTICIDE 'CYPERMETHRIN' IN
RATE LIVER CELLS BY COMET ASSAY
N El-Khatib1, M Abd El-Aziz2, Y Badr2, N Kamal1
1 Central Agricultral Pesticides Lab., GIZA, Egypt2 National Institute of Laser Sc, GIZA, Egypt
The comet assay (single-cell gel electrophoresis,
'SCGE') is a simple method for measuring deoxyri-
bonucleic acid (DNA) strand breaks in eukaryotic
cells. The assay has applications in testing different
chemical and physical agents for genotoxicity and
monitoring environmental contamination with
genotoxins. The objective of the present study was to
evaluate the genotoxic effects of the synthetic
pyrethroid pesticide 'cypermethrin', which is widely
used in Egypt in pest-control programs in agriculture
and in public health as well. Male rats were sacrificed
1, 7 or 14 days after administration of single oral dose
1/30, 1/10 or 1/5 LD50 of commercial formulation of
cypermethrin. Single liver cell suspensions were pre-
pared and a Comet assay was performed. With the
SCGE assay, a clear induction of DNA was observed. It
is generally noticed that all pesticide treatments
yielded statistically significant (p<0.0001) DNA dam-
age. In conclusion, cypermethrin induced a clear sig-
nificant positive dose-dependent increase in DNA
damage in the rat liver cells exposed to cypermethrin
as compared with controls. But the effects in the SCGE
were generally decreased with time after treatments.
The results of the present work suggested that comet
assay might be a suitable and sensitive endpoint in
genotoxicity evaluation of pesticides, but we confirm
that various tests should be used for detecting the
mutagenic activity of pesticides.
abstractboek 20-10-2004 10:28 Pagina 55
PS1 - Poster Session 1
Poster Presentation
PW2003
DNA ADDUCTS AND MUTAGENIC SPECIFICITY OF
THE UBIQUITOUS ENVIRONMENTAL POLLUTANT
3-NITROBENZANTHRONE IN MUTA MOUSE
VM Arlt1, T Suzuki2, L Zhan2, HH Schmeiser3,
M Honma2, M Hayashi2, DH Phillips1
1 Institute of Cancer Research, SUTTON, SURREY,
United Kingdom2 Federal Institute of Health Sciences, TOKYO, Japan3 German Cancer Research Center, HEIDELBERG,
Germany
3-Nitrobenzanthrone (3-NBA) is an extremely potent
mutagen in the Ames assay and a suspected human
carcinogen identified in diesel exhaust and in ambi-
ent airborne particulate matter. To evaluate the in-
vivo mutagenicity of 3-NBA, we analysed the mutant
frequency (MF) in the cII gene of various organs in the
lambda/lacZ transgenic mouse (Muta Mouse) after
intraperitoneal treatment with 3-NBA (25 mg/kg
body weight per week for 4 weeks). Increases in MFs
were found in colon, liver and bladder, with 7.0-, 4.8-
and 4.1-fold increases above the control value, respec-
tively, whereas no increase in MF was found in lung,
kidney, spleen, and testis. The sequence alteration of
the cII gene recovered from 41 liver mutants from 3-
NBA-treated mice were compared with 32 sponta-
neous mutants from untreated mice. Base
substitution mutations predominated for both 3-
NBA-treated (80%) and untreated (81%) groups.
However, the proportion of G:C to T:A transversions in
the mutants from 3-NBA-treated mice was higher
(49% versus 6%) and the proportion of G:C to A:T tran-
sitions was lower (10% versus 66%) than in the
mutants from untreated mice. The increase in MF in
the liver was associated with strong DNA binding by
3-NBA, whereas in lung, in which there was no
increase in MF, a low level of DNA binding was
observed (268.0-282.7 versus 8.8-15.9 adducts per 10(8)
nucleotides). DNA adduct patterns with multiple
adducts spots (spots 1-6) were observed in all tissues
examined. Using HPLC co-chromatographic analysis
we confirmed that all major 3-NBA-DNA adducts pro-
duced in vivo in mice are derived from reductive
metabolites bound to purine bases (with 70-80%
deoxyguanosine and with 20-30% deoxyadenosine in
liver). These results suggest that G:C to T:A transver-
sions induced by 3-NBA are caused by misreplication
of adducted guanine residues through incorporation
of adenine opposite the adduct ('A'-rule).
PS1 - Poster Session 1
Poster Presentation
PW2004
MOUSE MICRONUCLEUS TEST ON PERIPHERAL
BLOOD RETICULOCYTES: FLOW CYTOMETRIC
ANALYSIS AFTER MULTIPLE BLOOD SAMPLING
M de Boeck, BJ van der Leede, A de Smedt,
M Steemans, F van Goethem, A Lampo, PH Vanparys
Johnson & Johnson Parmaceutical R&D, BEERSE,
Belgium
The current OECD and ICH guidelines for the mam-
malian erythrocyte micronucleus test consider flow
cytometry as an acceptable substitute for microscop-
ical evaluation, provided that the applied method is ade-
quately validated. The MicroFlow'PLUS Micronucleus
Analysis Kit (Litron Laboratories, Rochester, USA) con-
ceivably meets this requirement and determines
micronuclei in both reticulocytes and normochro-
matic erythrocytes of mouse peripheral blood using
flow cytometry. The method is based on the binding
of FITC-labelled antibodies to CD71 of immature reticu-
locytes and on parallel RNA degradation and propidi-
um iodide staining of DNA present as micronuclei.
A biological standard of mouse erythrocytes from ani-
mals infected with the malaria parasite Plasmodium
berghei is used to assure an accurate and reliable
flow cytometer set up.
The objective was to employ this method and to eval-
uate it for its utility and sensitivity following our in-
house experimental testing strategy. Time- and
dose-dependent induction of micronucleated reticu-
locytes was assessed in peripheral blood of mice
treated with nine reference compounds applying a
multiple blood sampling regimen of the same ani-
mals (i.e. before and at 48h and 72h after treatment).
Five known clastogens, two known aneugens and two
compounds previously reported to be inactive in the
mouse micronucleus test were evaluated. All known
mutagens produced a dose-dependent increase in
micronucleated reticulocytes with the highest
response at 48h after treatment, except in the case of 5-
fluorouracil which had its peak response at 72h. The
compounds previously shown to be inactive in the in
vivo micronucleus test were also negative using the
present methodology. Multiple blood sampling of the
same animal before and after treatment could be
applied without altering the sensitivity of the assay.
The flow cytometric assessment of MN-RET in mouse
peripheral blood using MicroFlow'PLUS is a sensitive
method with high analysis throughput and robust
quality control.
56
abstractboek 20-10-2004 10:28 Pagina 56
PS1 - Poster Session 1
Poster Presentation
PW2005
INDUCTION OF EXCISION REPAIRABLE DNA
LESIONS IN LYMPHOCYTES EXPOSED TO AMINOLE-
VULINIC ACID IN-VITRO
Y Duydu, A Dur
Ankara University, ANKARA, Turkey
The inhibitory effect of lead compounds on ≠ -amino-
levulinic acid dehydrogenase (ALAD) accounts for an
accumulation of aminolevulinic acid (ALA) that can
generate reactive oxygen species (ROS) leading to an
oxidative DNA damage in lead exposure. Recently
this indirect mechanism has become more popular in
evaluating the genotoxic potential of lead com-
pounds because of the fast oxidation of ALA with con-
comitant generation of ROS. In the present study lead
and ALA were compared in terms of their ability to
induce excision repairable DNA lesions in lympho-
cytes. Because of its high sensitivity and specificity in
detecting agents inducing excsion repairable DNA
lesions, cytosine arabinoside/cytokinesis block
micronucleus (ARA-C/CBMN) assay was used in order
to convert excision repairable DNA lesions to MN.
Accordingly whole blood lymphocyte cultures were
exposed to lead and ALA at the moment of culture ini-
tiation (G0 phase) at three increasing doses (5, 10, 50
µM) with and without ARA-C treatment. The degree
of synergism (DS) between ALA and ARA-C was calcu-
lated as 0.87, 1.51 (p<0.01) and 1.78 (p<0.01) at doses of
5, 10, 50 µM ALA respectively. However the calculated
DS between lead and ARA-C was 0.83, 1.41 and 1.43 at
doses of 5, 10, 50 µM lead respectively. When this
results were taken into consideration 10 and 50 µM
ALA exposure clearly increased the number of lesions
converted to MN (DS=1.51, 1.78 respectively, 'p<0.01')
wheras the DS between lead and ARA-C was not sta-
tistically significant. Finally our results could be eval-
uated as a supportive evidance for the ALA mediated
indirect mechanism for genotoxic effects of lead
exposure.
PS1 - Poster Session 1
Poster Presentation
PW2006
MUTATIONAL SPECIFICITY OF ESCHERICHIA COLI
DNAQ49 AND THE EFFECT OF UMUD(D’)C PRO-
TEINS
EG Grzesiuk, M Wrzesinski, JN Nieminuszczy,
A Nowosielska
Institute of Biochemistry and Biophysics PAS,
WARSZAWA, Poland
The high replication fidelity in Escherichia coli is
ensured by the proofreading activity of the ´ subunit
of the main replicative polymerase, pol III. Mutations
in encoding ´ - dnaQ lead to defective pol III, and are
strong mutators.
The contribution to translesion replication of pol III
and induced as a part of the SOS response umuDC-
encoded pol V has not been satisfactorily explained.
Some observations regarding E.coli dnaQ49 indicate
pol V as the only polymerase involved in replication
past cis-syn cyclobutane thymine dimers, whereas
others, indicate efficient TR past this lesion in strains
deficient in pol V and with an inactivated ´ subunit.
In the latter case it is evident that both polymerases,
pol V and pol III mutated in ´ , influence the level and
specificity of spontaneous mutations in E.coli.
The aim of this study was to analyse the mutational
specificity of dnaQ49 in umuDC+ and umuDC- back-
ground in the genetic system developed by J.H.Miller.
The system includes a set of 11 lacZ mutants and
allows to identify 6 substitutions and 5 specific
frameshifts. Using this system we established the
specificity of recessive dnaQ49 allele grown at per-
missive 28°C and non-permissive 37°C. We have found
that dnaQ49 grown at 37°C induces mainly GC:TA
transversions and +1A frameshifts; at 28°C AT:TA
transversions dramatically decreased and +1G pre-
vailed among the frameshifts. Introduction of
umuDC deletion into the dnaQ49 strain at 28°C result-
ed in noticeable increase in AT:TA transversions and
decrease in GC:AT transitions. At 37°C no such differ-
ences between dnaQ49 and dnaQ49dumuDC have
been observed. However, there were enormous differ-
ences in frameshifts between these strains. In
dnaQ49dumuDC there were only two groups of
frameshifts, -1G and +1A. At 28°C mutations resulted
mainly in -1G, whereas at 37°C both groups were
equally numerous.
57
abstractboek 20-10-2004 10:28 Pagina 57
PS1 - Poster Session 1
Poster Presentation
PW2007
CONTRIBUTION OF E.COLI ALKB PROTEIN IN THE
ALKYLATING DNA REPAIR
JN Nieminuszczy, C Janion, M Wrzesinski, EG Grzesiuk
Institute of Biochemistry and Biophysics PAS,
WARSZAWA, Poland
Potentially mutagenic and genotoxic alkylating
agents are widely spread in nature. They are present
in the living cells and are major deleterious agents in
the environment. It is widely accepted that the muta-
genic effect of the alkylanes results from alkylation of
bases in DNA. All organisms possess mechanisms
that protect cells against the harmful effects of alky-
lanes. In E. coli these defence systems include: tag and
ogt genes, expressed constitutively; ada, alkB (form-
ing one operon) and alkA and aidB that are expressed
after induction of the adaptive response.
Even though AlkB protein is known since 1983 its role
in damaged DNA repair has been established recent-
ly. AlkB is a dioxygenase that oxidatively demethy-
lates N1meA and N3meC in DNA in a reaction
involving ketoglutarate, O2, and Fe II, resulting in
recovery of the natural A and C bases. In this work we
show that AlkB protects the cells against mutagenic
activity of MMS and non-repaired N1 meA and
N3meC being a potent inducers of mutations. In E. coli
AB1157 defective in alkB the frequency of MMS-
induced argE3:Arg+ reversion is much higher than in
AB1157 alkB+ strain, and depends on the type of alkB
mutation (point or disrupted). These Arg+ reversions
are mainly due to AT:TA transversions, and are fully
dependent on umuC encoded DNA polymerase V. In E.
coli AB1157alkB umuC36 double mutant the frequency
of argE3:Arg+ reversion is dramatically reduced.
Therefore, we infer that alkB protects the cells not
only against deleterious, but also mutagenic activity
of MMS. In Miller’s strains bearing point mutations,
we have found that specificity of MMS-induced
mutations depends on growth conditions. When
MMS-treated cells were incubated in minimal medi-
um (MM) deprived of proline, mutations arise mainly
by GC:AT transitions, whereas in MM enriched with
proline they arise mainly by GC:TA transversions.
PS1 - Poster Session 1
Poster Presentation
PW2008
DAMAGE TO DNA AND ITS REPAIR IN RADIO-
IODINE TREATED PATIENTS WITH AUTONOMOUS
THYROID NODULE
EG Grzesiuk1, JN Nieminuszczy1, M Kruszewski2,
MT Plazinska3, W Grzesiuk3
1 Institute of Biochemistry and Biophysics PAS,
WARSZAWA, Poland2 Institute of Nuclear Chem. Technol, WARSZAWA,
Poland3 University Medical School, WARSZAWA, Poland
The purpose of this study is to evaluate the DNA
breakage and base damage with the use of comet assay
in radioiodine treated patients with autonomous
thyroid nodule.
In all the patients thyroid scintigram was performed
using 131-I. The thyroid scan showed a single 'hot nod-
ule' with suppressed radioactive iodine uptake in
remaining thyroid tissue. The dose of administered
131-I was from 14 to 16 mCi. Damage to DNA was esti-
mated in a tissue from 'hot nodule' by fine needle
aspiratory biopsy and in lymphocytes (PBL). Samples
were taken three times: before radioiodine treatment,
12 and 54 days after.
Preliminary results (on four patients) indicate a high
diversity in the level of DNA damage between indi-
vidual patients. Generally, in lymphocytes 12 days
after 131-I application significant level of DNA break-
age and base damage was still observed. However,
after 54 days the level of DNA damage in lymphocytes
was even lower than in the control. On the contrary,
in 'hot nodule' cells DNA damage persisted till 54th
day after 131-I treatment. Differences in the type of
damage between thyroid cells and lymphocytes were
also observed. In lymphocytes there were more base
damages while in nodule cells single strand DNA
breaks prevailed.
Our preliminary results indicate that the comet assay
can be a valuable tool for monitoring radioiodine
treated patients. It can also allowed to estimate prop-
er for each patient 131-I dose. Differences in the type
and persistence of DNA damage in lymphocytes and
thyroid nodule cells might indicate the different
mechanism of DNA damage induction and/or differ-
ences in DNA damage repair mechanisms.
58
abstractboek 20-10-2004 10:28 Pagina 58
59
abstractboek 20-10-2004 10:28 Pagina 59
PS1 - Poster Session 1
Poster Presentation
PW2009
EVALUATION OF CONDENSATE EFFECTS FROM
STANDARD CIGARETTES USING IN VITRO ASSAYS
CA Andreoli, F Flamma, A Martino, A Nunziata
Eti SpA, ROME, Italy
Cigarette smoking is a known risk factor for the
development of several pathologies. In this context,
the study of cellular response induced by condensate
smoke cigarette (CSC) can be useful in view of better
understanding its biological activity.
In this study, CSC, collected on Cambridge filter and
dissolved in DMSO, was used. All experiments were
performed on Swiss/3T3 fibroblast. Cell survival, after
24h of treatment with 25, 50, 100, 150 mg/mL of CSC,
was evaluated employing NRU, MTT test and plating
efficiency cloning (PE). The same experimental condi-
tions were adopted to study the apoptosis induction
through an in situ marker to measure caspase activa-
tion followed by staining with Hoechst. Finally DNA
lesions were evaluated with comet assay treating
cells with 50, 100, 150, 180 mg/mL of CSC for 90min.
NRU and MTT assays demonstrated that CSC induced
dose-related cytotoxicity response, with IC50 values
of 80-85 mg/mL. The results from PE assay showed
that cells lost their ability of forming colonies after
treatment with 100mg/mL and the presence of giant
cells, morphologically like senescent cells with flat-
tened cytoplasm and multinucleated, was observed
at lower doses after 168h from the end of treatment.
Apoptosis studies highlighted cells caspase-positive
after CSC treatment. To examine whether activation
of caspases correlates with changes in nuclear mor-
phology, cells were labelled with Hoechst-33258
revealing the presence of apoptotic bodies and chro-
matin aggregation dose-depending. Preliminary
results from comet assay showed that CSC was able to
induce DNA strand breaks in a dose-dependent rela-
tionship.
These results show that cytotoxicity and genotoxicity
in vitro assays can be used for studying the biological
activity of complex mixtures, such as CSC. Moreover
these assays are a suitable strategy for measuring the
effects of the reduction or removal of hazardous com-
pounds and the progresses towards the production of
safer cigarettes.
PS1 - Poster Session 1
Poster Presentation
PW2010
SYNTHESIS OF OLIGONUCLEOTIDE ANALOGUE
FOR DNA REPAIR
N Koissi, H Lönnberg
University of Turku, TURKU, Finland
The process by which normal cells become progres-
sively transformed to malignancy is now known to
require the sequential acquisition of mutations
which arise as a consequence of damage to the
genome. Mutagenic compounds act on DNA most
commonly by forming adducts; other types of dam-
age include oxidation, deamination and depurination
of the nucleosides.
DNA damages, mediated by bifunctional chemical
agents are known to form covalent cyclic adducts
with DNA bases. While cyclic adducts as such may be
classified as mutagenic DNA lesion, those bearing
additional functional groups that are able to interfere
with the base-pairing are of particular interest. Thus,
evolution has developed complex enzymatic path-
ways that recognize and repair DNA damage. The rate
of cleavage was found to differ with the lesion and
was also affected by neighbour sequences geometry
and the resulting local conformational changes,
which can be sequence-dependent.
For an understanding, the systematic study of the
biochemical and biological effects of these various
types of DNA damage, DNA in which the adduct or
other damage is placed in a structurally specific man-
ner in the DNA is needed. It has been our goal to char-
acterize adducts,1 and to develop convenient and
versatile synthetic approaches to oligodeoxynu-
cleotides containing structurally defined adducts.2
The adducted oligonucleotides have been useful not
only for establishing the biological effects of lesions,
and the fluorescent properties of the oligonucleotide,
but also for biophysical studies, in particular two-
dimensional NMR, with the goal of establishing the
structural basis for replication and other types of bio-
logical processing.
[1] Neuvonen K., Koissi N., Lönnberg H.: J. Chem. Soc.,
Perkin Trans. 2, 2002, 173
[2] Koissi N., Lönnberg H.: Nucleosides, Nucleotides &
Nucleic Acids, 2003, 22, 1135.
60
abstractboek 20-10-2004 10:28 Pagina 60
PS1 - Poster Session 1
Poster Presentation
PW2011
SYNTHESIS AND CHARACTERIZATION OF DNA
ADDUCTS FROM MUTAGENIC 3-NITROBENZAN-
THRONE PRESENT IN ATMOSPHERIC ENVIRON-
MENT
TT Takamura1, M Kawanishi2, Y Nakagawa3,
T Watanabe3, T Hirayama3, K Wakabayashi1,
Y Hisamatsu4, T Yagi2
1 National Cancer Center Reserach Institute, TOYKO,
Japan2 Osaka Prefecture University, OSAKA, Japan3 Kyoto Pharmaceutical University, KYOTO, Japan4 National Institute Public Health, TOKYO, Japan
3-Nitrobenzanthrone (3-NBA) is a powerful mutagen
present in air-borne particulate matter and is sug-
gested to be a carcinogen in rodents. 3-NBA is also
reported to be metabolically activated to form several
DNA adducts when subjected to mammalian cell
lines or rats. However, the chemical structures of
these adducts are not fully understood. In this study,
we analyzed the chemical structures of several DNA
adducts derived from 3-NBA in vitro. We have already
reported an efficient preparation method of dG-C8
adducts with several amino/nitroarenes. Using this
method, it becomes possible to prepare general types
of DNA adducts with 3-NBA; ie. N-(2'-deoxyguanosin-
8-yl)-3-aminobenzanthrone (dG-C8-ABA), 2-(2'-deoxy-
guanosin-N2-yl)-3-aminobenzanthrone (dG-N2-ABA)
and their N8-acetyl counterparts (N-Ac-dG-C8-ABA
and N-Ac-dG-N2-ABA). With authentic samples, we
found that dG-C8-ABA and dG-N2-ABA were formed
in the ratio of 10/1 in the reaction mixture of dG with
N-acetoxy-3-aminobenzanthrone, which was pre-
pared by selective O-acetylation of 3-hydroxyamino-
benzanthrone. The other putative ultimate mutagen,
N-acetoxy-N-acetyl-3- aminobenzanthrone (N-Aco-N-
Ac-ABA), was also found to give N-Ac-dG-C8 ABA and N-
Ac-dG-N2-ABA, accompanied with an unusual C-C
bonded type of DNA adduct, 2-(2'-deoxyguanosin-8-yl)-3-
acetylaminobenzanthrone, whose formation has
already been reported by us. Moreover, N-Aco-N-Ac-
ABA was shown to easily react with 2'-deoxyadeno-
sine (dA) to give a novel type of DNA adduct in which
an aziridine ring formation took place between the
double bond of the 1,11b-position of 3-acety-
laminobenzanthrone and N6 of dA (1,11b-(2'-
deoxyadenosin-N6-yl)-3-acetylaminobenzanthrone).
The efficiency of the formation of this aziridine types
of adduct was similar to that of N-Ac-dG-C8-ABA. We
are now undataking detection of in vivo formation of
these DNA adducts.
PS1 - Poster Session 1
Poster Presentation
PW2012
ROLE OF DAMAGE SPECIFIC DNA POLYMERASES
IN MUTAGENICITY OF LIPID PEROXIDATION-
INDUCED DNA ADDUCTS
B Tudek, B Rusin, B Tudek
Institute of Biochemistry and Biophysics, WARSAW,
Poland
Lipid peroxidation leads to the formation of a large
family of alkenals, among which trans-4-hydroxy-2-
nonenal (HNE) is one of the most ubiquitous. HNE
forms adducts to all four DNA bases, which are cyclic
propano- or ethenoadducts bearing a hexyl- or hep-
tyl- side chains. We were studying mutagenesis and
repair of HNE-DNA adducts in E. coli. dsM13 phage
DNA was modified in vitro with HNE, and transfected
into wild type E. coli as well as DNA repair and DNA
polymerases II, IV and V deficient mutants. The defi-
ciency of nucleotide excision repair, increased E. coli
susceptibility to HNE, as well as increased mutation
frequency in M13 lacZ gene. In uvrA strain about 44%
of mutations were recombinations between sequences
of M13 lacZ gene and lacZ gene of E. coli F’ factor, while
in the wild type the level of recombinations remained
low (8%). Additional dysfunction of DNA polymerase
IV in uvrA mutant dramatically decreased the survival,
but had no effect on mutation frequency in compari-
son to the wild type E. coli. In the strain lacking
UvrABC excinuclease and DNA polymerase V, the sur-
vival was higher than in uvrA din strain, but no HNE-
induced mutations were observed. In uvrA din polB
triple mutant, survival decreased dramatically, and
mutation frequency increased substantially in com-
parison to the wild type strain. These results suggest
that, in E. coli, HNE adducts to DNA bases are
processed by nucleotide excision repair system,
recombination and damage-specific DNA polymeras-
es. The data suggest that DNA polymerase II and
probably DNA polymerase IV are able to bypass HNE-
adducts in an error-proof mode, while DNA poly-
merase V is responsible for mutation induction on
HNE-damaged templates.
61
abstractboek 20-10-2004 10:28 Pagina 61
62
PS1 - Poster Session 1
Poster Presentation
PW2013
INHIBITION OF MYELOPEROXIDASE ACTIVITY BY
NITRITE: IMPLICATIONS FOR NEUTROPHIL-MEDI-
ATED DNA STRAND BREAKAGE
AM Knaapen1, RPF Schins2, PJA Borm2, FJ van Schooten1
1 Maastricht University, MAASTRICHT,
The Netherlands2 IUF GmbH Düsseldorf, DUSSELDORF, Germany
Hydrogen peroxide has been shown to be a crucial
factor in neutrophil-induced DNA strand breakage. In
general, H2O2 is consumed by myeloperoxidase (MPO)
to form HOCl. However, recently it was shown that
nitrite, also present at sites of inflammation, is a
potent MPO inhibitor. The consequences of this effect
for neutrophil-induced genotoxicity remain to be elu-
cidated. To this end, we co-incubated activated neu-
trophils with rat alveolar epithelial cells (RLE) in the
presence of nitrite (0-100 µM) or the specific MPO
inhibitor 4-amino benzoic acid hydrazide (4-ABAH, 0-
100 µM). DNA strand breakage in the RLE cells was
evaluated using single cell gel electrophoresis. H2O2
release and MPO activity were determined spec-
trophotometrically. Both nitrite and 4-ABAH were
found to inhibit MPO activity in a dose-dependent
manner. This was paralleled by a significant increase
in DNA damage in RLE cells, whereas no strand break-
age was induced upon incubation with nitrite or 4-
ABAH in the absence of neutrophils. To further
evaluate the underlying mechanisms, in vitro experi-
ments were performed in which H2O2 was allowed to
react with purified MPO before addition to the RLE
cells. In the presence of MPO no subsequent cellular
DNA damage could be detected. However, when
nitrite (100 µM) was added to these pre-incubations,
DNA damage in the RLE cells was comparable with
levels as induced by H2O2 alone. Inhibitory experi-
ments with catalase further confirmed that these
effects were H2O2 dependent. In conclusion, the pres-
ent data demonstrate that physiological levels of
nitrite increase the DNA damaging capacity of neu-
trophils. This effect is likely due to nitrite-induced
inhibition of MPO, which increases the availability of
H2O2. Further studies are needed to assess the impli-
cation of these observations for in vivo DNA damage
in relation to neutrophilic inflammation.
abstractboek 20-10-2004 10:28 Pagina 62
PS1 - Poster Session 1
Poster Presentation
PW2014
MECHANISMS OF PRILOCAINE AND LIDOCAINE
CYTOTOXIC EFFECTS IN HEPG2 AND V79 CELL
LINES
GR Rodrigo, B Mayerhofer, E Richter
Ludwig Maximilian Universität, MUNICH, Germany
Prilocaine as lidocaine are amide-type local anes-
thetic agents that are used for regional, nerve block
and topical anesthesia. They are eliminated almost
exclusively by hepatic biotransformation and renal
excretion. In humans, the main metabolic pathway of
lidocaine is sequential N-deethylation to monoethyl-
glycinexylidide and glycinexylidide. Both metabolites
can be hydrolyzed to 2,6-dimethylaniline, which is
further oxidized to its main urinary metabolite 4-
hydroxyxylidine. Prilocaine is marketed as a racemic
mixture and both enantiomers have similar biologi-
cal activity. However, the S-(+) enantiomer is slowly
hydrolyzed, while the R-(-) enantiomer is quickly
hydrolyzed by hepatic amidase to 2-methylaniline (o-
toluidine). O-toluidine is metabolized in vivo into a
number of compounds, some of which are active
genotoxins. It produces DNA damage (single-strand
breaks and unscheduled DNA synthesis, UDS) and
causes cell transformation. Both 2,6-dimethylaniline
and o-toluidine have been shown to be carcinogenic
in animal studies. In vitro, metabolism of lidocaine as
well as o-toluidine has been shown to be mediated by
cytochrome P450 (CYP) 3A4 or CYP1A2. N-acetyltrans-
ferase (NAT) 1 in extra-hepatic tissues or NAT 2 in liver
could have a modulating effect in toxification of
o-toluidine.
Our study models are constituted of 2 cell lines, V79
and HepG2. V79 cell line derivated from Chinese ham-
ster lung fibroblasts has no CYP activity or hepatic
amidase expression. HepG2 cell line is derived from
human hepatocellular carcinoma and has the same
genetic potential as hepatocytes.
Accordingly, this study has the following aims:
1. Characterization of the metabolic pathways
involved in the activation of prilocaine and lido-
caine, leading to cytotoxic effects in both cell lines.
2. Evaluation of the effect of various modulators of
CYP activity on the cytotoxic potential of prilo-
caine and lidocaine in HepG2 cell line.
3. Determination of the metabolites involved in
adduct formation in both cell lines.
PS1 - Poster Session 1
Poster Presentation
PW2015
GENOTOXICITY IN VITRO OF CHLORINATED
DRINKING WATERS OBTAINED FROM SURFACE
AND WATER TABLE
S Frigerio, S Radice, M Ferraris, E Chiesara, L Marabini
University of Milan, MILAN, Italy
We examined possible genotoxic effects of chlorinat-
ed drinking water concentrates in metabolically com-
petent human hepatoma cells (HepG2) using the
micronucleus assay (MN), single cell gel electrophore-
sis (comet assay) and cell cycle cytofluorometric
analysis.
The water samples were obtained from the aqueducts
of two towns with different types of water supplies:
town #1 uses water from a river, whereas town #2 is
supplied from a deep water table. In the case of town
#1 were also analysed the waters before conditioning.
Both towns use CLO2 for conditioning purpose. The
samples were concentrated by adsorption on silica
C18 cartridges and subsequently eluted.
In this study, the samples denominated RW (raw
waters) were obtained by concentrating the surface
water before conditioning, the A, R1 and R2 samples
were taken after conditioning at the point of admis-
sion into the aqueduct, and at two very distant from
each other points in distribution network respectively.
A preliminary toxicity evaluation was made using
NRU (neutral red uptake) and LDH (lactate dehydro-
genase) tests, wich revealed toxicity for concentrate
amounts starting from 0.5 Leq/ml of the A and R1
samples from town #2. No toxicity was found in all
other samples of both towns.
The short-term genotoxicity tests carried out with
waters samples at maximal non-cytotoxic doses
revealed a significant increase of tail moment (comet
assay) for point R1 of both town concentrates. For MN
test the results did not indicate statistically signifi-
cant effects for any examinated sample.
The results obtained indicate that non-cytotoxic
doses of water concentrates induce genotoxic dam-
age pointed out by comet assay and not by MN. These
evidences are not conflicting, because the two tests
have different genotoxic endpoints. Moreover these
data are in agreement with the unchanged cell cycle
distribution analysis.
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PS1 - Poster Session 1
Poster Presentation
PW2016
MITOCHONDRIAL DNA SEQUENCE ANALYSIS
AND MOLECULAR PHYLOGENY OF ORCHESTIA
CAVIMANA (CRUSTACEA) IN FRESHWATER GENO-
TOXICOLOGICAL STUDIES.
DD Davolos1, BM Pietrangeli1, N Maclean2
1 ISPESL, ROME, Italy2 University of Southampton, SOUTHAMPTON,
United Kingdom
The use of freshwater amphipods in toxicity assess-
ment is increasing since their usefulness has become
clear. However, various parameters must be consid-
ered when studying the toxicity of components to
wild life populations, taking into account the genetic
population structure and the evolutionary history of
the taxa. The talitrid amphipod Orchestia cavimana,
inhabiting beaches of freshwater lakes and rivers in
Europe and UK, is here chosen as a potential candi-
date for freshwater genotoxicology evaluations. We
initiated a molecular phylogenetic study of this semi-
terrestrial taxon based on the sequences of part of the
mitochondrial genes cytochrome oxidase subunit I
and subunit II (COI, COII). We analysed portions of the
COI and COII genes using neighbour-joining and
maximum parsimony inference using all nucleotides
plus their amino acid translations which have been
examined also by comparing the structural classes of
the COI and COII enzymes. In all the phylogenetic
trees, O. cavimana shows a basal placement with
respect to other talitrid ampihpods even of the same
genus (O. gammarellus, O. mediterranea). The low
genetic variation levels detected in samples of O. cav-
imana validate this crustacean for environmental
genotoxicity investigations at both micro- and
macrogeographic scales. Moreover this organism,
easily reared in laboratory terraria, could be used
under a variety of genotoxicological exposures. With
these considerations, molecular methods were con-
sidered for evaluating mitochondrial genotoxicity in
tissues of exposed bred organisms and populations
collected from contaminated sites.
PS1 - Poster Session 1
Poster Presentation
PW2017
BREAST CANCER PATIENTS DNA DAMAGE AND
REPAIR EFFICIENCY
N Anagnostakis1, G Karanastasi1, N Messini-Nikolaki2,
K Gourgoulianis3, K Christou3, E Athanasiou3,
S Tsilimigaki1, SM Piperakis1
1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 University of Thessaly, LARISA, Greece
In this study the effects of g-irradiation and ethanol
on lymphocytes from breast cancer patients was
examined.
Using the comet assay we estimated the DNA dam-
age and the repair efficiency on the above population
in comparison to controls.
The apoptotic and necrotic cell population was also
estimated.
Our results show a decreased DNA repair capacity in
lymphocytes from breast cancer patients. An increased
apoptosis was found if compared to healthy individuals.
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PS1 - Poster Session 1
Poster Presentation
PW2018
LUNG CANCER PATIENTS DNA DAMAGE AND
REPAIR EFFICIENCY
G Karanastasi1, N Anagnostakis1, N Messini-
Nikolaki2, K Gourgoulianis3, K Christou3,
S Tsilimigaki1, SM Piperakis1
1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 University of Thessaly, LARISA, Greece
We examined the effects of ethanol and g-irradiation
in lymphocytes from lung cancer patients in compar-
ison to healthy individuals.
With the comet assay technique we estimated the
DNA damage and the repair capacity of the above
populations.
We also estimated apoptosis and necrosis in these
populations.
Our results show that lung cancer patients have a
reduced DNA repair capacity. Apoptosis was found
to increase in lung cancer patients if compared to
healthy individuals.
PS1 - Poster Session 1
Poster Presentation
PW2019
VITAMINS' C AND E "PROTECTION" OF LYMPHO-
CYTES EXPOSED TO EXTERNAL AGENTS
K Maridaki1, G Christopoulos1, N Messini-Nikolaki2,
S Tsilimigaki1, SM Piperakis1
1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece
In the present work we examined the 'protection'
offered in human lymphocytes by vitamins C and E
from the effects of UV and g-ray and H2O2.
We also measured the number of apoptotic and
necrotic cells after staining with acridine orange and
ethidium bromide.
Our results show that vitamin E and the mixture of
E+C show to 'protects' cells from the effects of UV,
g-ray and H2O2. Vitamin C alone however does not
show such clear 'protection'.
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PS1 - Poster Session 1
Poster Presentation
PW2020
GENOTOXICITY EVALUATION OF A DI-N-OXIDE
QUINOXALINE UNDER HYPOXIC CONDITIONS BY
THE COMET ASSAY
A Azqueta1, G Pachón2, A. Lopez de Cerain1
1 University of Navarra, PAMPLONA, Spain2 University of Barcelona, BARCELONA, Spain
Bio-reductive agents have being designed in order to
take advantage of the particular metabolic character-
istics of the resistant hypoxic regions of the solid
tumours. They are activated inside the hypoxic cells
to give active species that, in the presence of oxygen,
are oxidised back to the non-toxic parent compound.
Several quinoxalines 1,4-di-N- oxides have been
described as potential bio-reductive agent, among these,
the 7-cloro-3-[[(N,N-dimethylamino) propy]amino]-2-
quinoxalinecarbonitrile 1,4-di-N-oxide (Q-85), appears
the most promising.
In the present work the genotoxic effect of Q-85 in
hypoxia was studied in Caco-2 cells with the alkaline
comet assay. Cells were treated with 0.2 and 0.4 µM of
Q-85 during 2 hours under an hypoxic atmosphere.
The number of viable cells after the treatment was
determined by the Trypan blue exclusion method
and 18x104 cells/well were seeded in six-well plates.
After incubation at 37ºC and 5% CO2 for 24, 48, 72 and
168 hours, cells were trypsinized and counted with
the Tripan blue exclusion assay. The comet assay was
carried out just after the treatment and 24 and 48
hours later. Comets were classified by the visual scor-
ing method. All this procedure was carried out 3
times.
Just after treatment with 0.2 µM and 0.4 µM, viability
percentages were 95.6±4.7 and 88.2±8.9, but 24 h later,
these percentages decreased until 35.8±19.6 and
7.2±7.2, respectively. Only some cells treated with 0.2
µM were able to proliferate after 72 hours. The DNA
was very damaged just after the treatment (total
comet score, control = 42± 11; 0.2 µM = 343±30, 0.4 µM
= 399±1). After 24 and 48 hours, viable cells showed an
important repair of the DNA, which was reflected by
a decrease in the comet score, although control values
were not reached with either dose. These results indi-
cate that Q85 has a genotoxic effect.
PS1 - Poster Session 1
Poster Presentation
PW2021
APNEA PATIENTS LYMPHOCYTES DNA DAMAGE
AND REPAIR CAPACITY
N Kontogianni1, N Messini-Nikolaki2, K Christou1,
C Pastaka1, K Gourgoulianis1, S Tsilimigaki3,
SM Piperakis3
1 University of Thessaly, LARISA, Greece2 University of Athens, ATHENS, Greece3 NCSR Demokritos, ATHENS, Greece
In this study we examined in a population of patients
with apnea, using the comet assay technique, the
DNA damage caused by g-irradiation and ethanol.
Furthermore, we studied apoptosis and necrosis.
Analysis of our results indicate that apnea patients
are more sensitive to the effects of external factors.
Apoptosis was found to be somehow higher in apnea
patients.
66
abstractboek 20-10-2004 10:28 Pagina 66
67
PS1 - Poster Session 1
Poster Presentation
PW2022
DNA DAMAGE AND REPAIR EFFICIENCY IN COM-
MON VARIABLE IMMUNODEFICIENCY PATIENTS
LYMPHOCYTES
P Kanavetas1, N Messini-Nikolaki2, M Kanariou3,
S Tsilimigaki1, SM Piperakis1
1 NCSR Demokritos, ATHENS, Greece2 University of Athens, ATHENS, Greece3 Aghia Sophia Hospital, ATHENS, Greece
In the present study we examined with the comet
assay technique in a population of patients with
common variable immunodeficiency (CVID), the DNA
damage and repair efficiency after H2O2 and g-irradi-
ation treatment.
Apoptosis and necrosis were also investigated.
Analysis of our results indicates that CVID patients
are more sensitive to the effects of external factors.
Apoptosis was also found to be somehow higher in
CVID patients.
PS1 - Poster Session 1
Poster Presentation
PW2023
PHARMACOLOGICAL BLOCKING OF ABC-TRANS-
PORTERS AND TOXICITY OF BENZO(A)PYRENE IN
MCF-7 CELLS
PK Myllynen1, T Kajosmaki1, L Vaskivuo1,
KV Vähäkangas2
1 University of Oulu, OULU, Finland2 University of Kuopio, KUOPIO, Finland
Benzo(a)pyrene, a polycyclic aromatic hydrocarbon
(PAH) present also in cigarette smoke, has a complex
metabolism in humans. The ultimate carcinogenic
metabolite, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE),
can bind to DNA. ATP-binding cassette (ABC) trans-
porters protect cells from many foreign chemicals. In
addition to specific inhibitors many widely used
drugs inhibit function of these transporters. The aim
of this study is to clarify whether pharmacological
blocking of ABC-transporters affects benzo(a)pyrene
adduct formation. Also, it has been shown that the
level of BPDE-DNA- adducts correlates with the expres-
sion of p53 tumor suppressor protein. Therefore, p53
expression was also analysed.
MCF-7 breast adenocarcinoma cells were cultured
with benzo(a)pyrene (1microM) and the ABC-trans-
porter inhibitor verapamil (0.125 microM to 100
microM) for 24 and 48 hours. DNA was isolated from
samples using a phenol extraction/ethanol precipita-
tion method and BPDE-DNA adducts were analysed by
synchronous fluorescence spectrophotometry. The
level of p53 protein was studied with immunoblotting
using DO7 antibody.
Preliminary results show that BPDE-DNA adduct for-
mation after benzo(a)pyrene treatment was increased
by verapamil both at 24 and 48 hours (approximately
1.5 and 3.8 fold, respectively). Benzo(a)pyrene treat-
ment induced p53 protein as expected. We also have
some indications that low, but not high dose of vera-
pamil caused further increase in p53 expression at 48
hours.
According to these preliminary results it seems that
blocking of the ATP- binding cassette transporters
enhances benzo(a)pyrene toxicity in MCF-7 cells.
abstractboek 20-10-2004 10:28 Pagina 67
PS1 - Poster Session 1
Poster Presentation
PW2024
SYNERGISTIC EFFECT OF CADMIUM AND ARO-
MATIC DNA ADDUCTS ON MUTAGENESIS DUR-
ING FETAL DEVELOPMENT
RWL Godschalk1, J Hogervorst1, H Albering1,
P Mercelina-Roumans2, FJ van Schooten1, J de Haan2,
JCS Kleinjans1
1 Maastricht University, MAASTRICHT,
The Netherlands2 Academic Hospital Maastricht, MAASTRICHT,
The Netherlands
The foetus is exposed to xenobiotics via the mother’s
circulation, which is possibly involved in the develop-
ment of diseases in later life. Cadmium and lipophilic
genotoxins in umbilical cord blood of newborns may
have synergistic effects on mutagenesis in the hypox-
anthine-phosphoribosyl-transferase (HPRT) reporter
gene. Concentrations of zinc (Zn), lead (Pb) and cad-
mium (Cd) were determined in cord blood of 16 non-
smoking and 9 smoking healthy mothers by atomic
absorption spectrometry (AAS). Lipophilic-DNA
adducts in lymphocytes were determined in the same
subjects by 32P-postlabeling and the HPRT-mutant
frequency was assessed by selection of 6-thioguanine
resistant cells. Although the Cd/Zn ratio was 2.5 fold
higher in blood of smoking women than in non-
smoking women (0.10±0.02 and 0.04±0.01, respec-
tively, P=0.007), this difference could not be observed
in umbilical cord blood (0.03±0.01 and 0.03±0.01,
respectively, P=0.66). Similarly, mean DNA adduct
levels were increased in lymphocytes of smoking
women when compared to non-smoking controls
(0.99±0.31 adducts per 108 nucleotides and 0.43±0.12,
respectively, P=0.009), but were only marginally
higher in newborn children of smokers than in their
non-smoking counterparts (0.57±0.29 and 0.24±0.09,
respectively, P=0.38). Since Cd is known to effectively
inhibit DNA repair, we hypothesized that concomi-
tant exposure of neonates to Cd and genotoxic com-
pounds may result in an increased fixation of DNA
damage into somatic mutations. Indeed, taking both
biomarkers into account, the number of HPRT-muta-
tions per adduct correlated positively with the Cd
concentration in cord blood (r=0.61, P=0.001). These
data suggest a molecular link between DNA damage,
inhibition of DNA repair by Cd and in vivo mutagen-
esis during fetal development. Thus, simultaneous
exposures to heavy metals and DNA damaging com-
pounds act synergistically and may result in biologi-
cally relevant genotoxic effects in neonates.
68
BIOPREDIC INTERNATIONAL14-18, rue Jean Pecker - 35000 Rennes, France Tel: +33 (0)2 99 14 36 14Fax: +33 (0)2 99 54 44 72Web : www.biopredic.comE-mail : [email protected]
Biopredic international is a provider of assay systems for invitro toxicology:* S9mix* Peripheral Blood mononuclear cells (human origin)* Bcl2 recombinant CTLL2 cells, resistant against apoptosisand very valuable to discriminate between pro-apoptotic andmutagenic properties of xenobiotics,* and numerous other systems for drug discovery and drug developpment such as human and animal hepatocytes, kera-
abstractboek 20-10-2004 10:28 Pagina 68
PS1 - Poster Session 1
Poster Presentation
PW2025
GENOTOXICITY OF FINE AND COARSE PARTICULATE
SAMPLES COLLECTED DURING CONTRASTING
POLLUTION SITUATIONS IN EUROPE (PAMCHAR)
R Duffin1, H Li1, ME Gerlofs-Nijland2, FR Cassee2,
FJ van Schooten3, RO Salonen4, PJA Borm5,
RPF Schins1
1 IUF, DUSSELDORF, Germany2 Nat.Inst.of Publ. Health and Environment,
BILTHOVEN, The Netherlands3 Maastricht University, MAASTRICHT,
The Netherlands4 KTL, KUOPIO, Finland5 IUF GmbH Düsseldorf, DUSSELDORF, Germany
Epidemiological studies have shown significant posi-
tive associations between ambient air particle (PM)
concentration and morbidity and mortality due to
cardiopulmonary diseases and lung cancer. These
adverse health effects have been consistently associ-
ated with inhalable (PM10) and fine (PM2.5) particles,
but the responsible biological mechanisms are large-
ly unknown. Coarse (PM10-2.5) and fine PM (PM2.5-
0.2) were collected using a high-volume cascade
impactor (HVCI) in six European cities: Duisburg
(autumn), Prague (winter), Amsterdam (winter),
Helsinki (spring), Barcelona (spring) and Athens
(summer). DNA strand breakage was assessed using
the comet assay and PAH-DNA adduct formation
was measured by 32-P postlabelling upon treatment
of A549 human lung epithelial cells. Considerable
sampling location/season and size fraction-specific
differences were observed in both endpoints.
Furthermore, a clear correlation with regard to the
fine PM was observed between particle PAH-content
and resulting DNA adduct formation. In ongoing
experiments, selected samples (fine and coarse PM
from Prague and fine PM from Duisburg), have been
used for intratracheal instillations in spontaneously
hypertensive rats (SHR). Subsequent genotoxicity
tests are carried out in isolated type II lung epithelial
cells from these animals. Preliminary results indicate
enhanced DNA strand breakage in animals instilled
with coarse PM. The contribution of pulmonary
inflammation to the observed DNA damage is cur-
rently under evaluation.
PS1 - Poster Session 1
Poster Presentation
PW2026
UV-INDUCED SKIN TUMORIGENESIS IN MOUSE
STRAINS IMPAIRED IN THE INK4A/ARF LOCUS
AND DNA REPAIR
A van Schanke1, HJ van Kranen2, LHF Mullenders1,
FR de Gruijl1, LHF Mullenders1
1 Leiden University Medical Center, LEIDEN,
The Netherlands2 National Institute of Public Health and the
Environment, BILTHOVEN, The Netherlands
We aimed to develop a mouse model for UV induced
malignant melanoma based on impairment of genes
involved in predisposition of humans to this disease
and environmental risk factors derived from epidemi-
ological data. Familial melanomas are often caused by
disruptions of CDKN2A (INK4a/ARF). Patients suffer-
ing the genetic disorder Xeroderma Pigmentosum are
also at high risk of developing melanoma, as a result
of dysfunctional DNA repair.
Mouse strains displaying such mutations were
exposed (neonatallly/adult) to ultraviolet (UV) light
and/or a chemical carcinogen and/or tumor promo-
tor (DMBA/TPA respectively). Ink4a/Arf ko (1), XPA ko
(2) and XPA* Ink4a/Arf double ko mice developed a
variety of skin lesions, most prominently papillomas
and nevi. Progression of benign nevi to malignant
melanomas was, however, not observed. The UV
exposure interval and dose with the highest yield of
nevi was also the most efficient inducer of
melanocyte proliferation in short-term experiments.
This regimen was used in further UV exposure exper-
iments with p16Ink4a specific mutant (3, with intact
p19Arf) and CDK4R24C mutant mice (4), both of
which developed spindle cell tumors but only few
nevi which is in contrast to previous experiments
with chemical carcinogens. A possible obstacle to the
formation of UV induced melanomas is the localiza-
tion of murine melanocytes, relatively deep in the
dermis where UVB light cannot penetrate efficiently.
Also the number of melanocytes may be low in nor-
mal murine skin. To circumvent the latter, pilot
experiments were started with K-14Slf mice (5), which
are strongly enriched in their epidermis for
melanocytes (due to K-14 driven expression of Steel
factor).
1. de Vries et al.(1995) Nature 377
2. Serrano et al.(1996) Cell 85
3. Krimpenfort et al (2001) Nature 413
4. Sotillo et al.(2001) EMBO 20
5. Kunisada et.al (1998) Development 125
(Dutch Cancer Society, UL2000-2303; European
Committee, QLK4-CT-199901084)
69
abstractboek 20-10-2004 10:28 Pagina 69
PS1 - Poster Session 1
Poster Presentation
PW2027
IN VIVO GENOTOXIC EFFECTS OF A SYNTHETIC
PYRETHROID INSECTICIDE, CYHALOTHRIN IN
FISH
QM Khan, MA Haq, H Nisar
NIBGE, FAISALABAD, Pakistan
Lambda-cyhalothrin (Kerate) is one of the largest sell-
ing insecticides in Pakistan and many other coun-
tries. Pyrethroids can enter the aquatic environment
during agricultural use or by direct spraying of water
bodies. Thus study of their possible genotoxicity is of
immediate interest. The presence of genotoxins, even
in low doses, concerns aquatic as well as non aquatic
species through the food chain and drinking water.
The comet assay, can act as a biomarker of genetic
toxicity in fish.
The comet assay was used to test genotoxicity of
cyhalothrin in the common edible fresh water fish,
Cirrhinus mrigala (Mori), found through out the
Indian subcontinent. Six fish were exposed to each
concentration of cyhalothrin (0, 0.1, 0.2. 0.3 and 0.4
ug/L) for 36 h. The test concentrations of the insecti-
cide were based on LC50 doses of cyhalothrin for this
fish. Cyclophosphamide (5mg/L), was used as positive
control. Blood was collected from the caudal vein at 0,
4, 8, 24 and 36 h following cyhalothrin exposure and
comet assays were performed. A significant increase
in comet tail length, indicating DNA damage was
observed at all the doses with cyhalothrin when com-
pared to negative control. The mean tail length
showed a dose dependent increase. The maximum
increase in mean tail length for doses was observed
at 8 h. The reduction in tail length was evident after
24 h. After 36 h tail length values were near to nega-
tive control levels for all doses. This indicates repair of
damaged DNA. The results of the study suggest that
even low levels of cyhalothrin in the aquatic environ-
ment may have a significant genotoxic effect on fish
and accumulation of this pesticide in fish tissue could
also be of concern to human health.
PS1 - Poster Session 1
Poster Presentation
PW2028
THE NOVEL UVS11 GENE OF CHLAMYDOMONAS
REINHARDTII AFFECTS CELL RESPONSE TO DNA
DAMAGE
EG Galova, A Sevcovicova, B Sviezena, M Slaninova,
D Vlcek
Comenius University FNS, BRATISLAVA, Slovak
Republic
The study of cell cycle regulation and checkpoints at
different levels has gained further insight into these
processes in different species. In comparison with
other lower eukaryotes there has been much less
progress in understanding cell cycle control in algae.
The first putative checkpoint mutant of green alga
Chlamydomonas reinhardtii have been isolated in
our lab and termed as uvs11. It has been predicted to
be UV-sensitive and repair-deficient. We assume now
that product of UVS11 gene is not required for DNA
repair itself but it is involved in cell response to DNA
damage.
In our work, we have assayed the activity of histone
H1 kinases presented in total protein extract and
those, mitosis specific ones bound to p13suc1-
Sepharose. The kinase activity was monitored in syn-
chronized cultures of untreated wild type and
mutant cells and compared with those irradiated by
UV-light (254nm) before mitosis. No substantial dif-
ferences between wild and mutant cells were found
in untreated cells. However, shortly after UV irradia-
tion the kinase activity in wild type has considerably
decreased, to recover about five hours later. The activ-
ity drop was accompanied by a prolongation of the
cell cycle due to a delay in mitoses, protoplast fis-
sions, and daughter cell release. The mutant strain
has shown no significant change in the course of
kinase activity and in other processes related to the
cell cycle after UV irradiation. Our present results
indicate that inhibition of histone H1 kinase could be
a way to arrest the initiation of mitosis in
Chlamydomonas and delay cell cycle progress in
response to DNA damage. We suggest that product of
the UVS11 gene affects cell response to DNA damage
and directly causes a decrease in kinase activity. This
implicates the role of UVS11 gene in regulation of the
cell cycle.
70
abstractboek 20-10-2004 10:28 Pagina 70
71
PS1 - Poster Session 1
Poster Presentation
PW2029
ALKYLATING AGENTS INDUCE HOMOLOGOUS
RECOMBINATION IN ABSENCE OF DETECTABLE
DOUBLE STRAND BREAKS IN MAMMALIAN CELLS
CL Lundin1, F Johansson1, E Bolderson2, D Jenssen1,
K Erixon1, T Helleday2
1 Stockholm University, STOCKHOLM, Sweden2 University of Sheffield, SHEFFIELD, United Kingdom
Alkylating agents are potent inducers of sister-chro-
matid exchanges and chromosome aberrations, sug-
gesting that the subsequent lesions can be repaired
by homologous recombination (HR). Indeed, methyl
methanesulfonate (MMS) has been suggested to
induce DNA double-strand breaks (DSBs) in yeast and
is certainly inducing HR in that system. Here, we have
investigated the role of HR in repair of lesions induced
by MMS and N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG) in mammalian cells. We show that the HR-
deficient hamster cell line irs1SF is equally sensitive
to MMS as the base excision repair (BER) defective
EM9 cell line and even more sensitive to MNNG,
showing that HR repairs alkylated DNA lesions. Both
agents induce HR at the hprt gene in the SPD8 cell
line, which we found is related to the amount of
O6-methylguanine produced by these agents.
Furthermore, inhibition of O6-methylguanine-DNA
methyltransferase increases recombination frequen-
cy following MMS or MNNG treatments, which alto-
gether suggest O6-methylguanine to be inducing HR.
In addition, and in contrast to what is believed to be
the case in yeast, we show that neither agent pro-
duces DSBs. We conclude that HR is a major pathway
in repair of O6-methylguanine, which is independent
on formation of a detectable DSB intermediate. We
speculate that HR is involved in repair of single-
strand break intermediates formed during BER.
abstractboek 20-10-2004 10:28 Pagina 71
PS1 - Poster Session 1
Poster Presentation
PW2030
BCR/ABL ONCOGENIC TYROSINE KINASE STIMU-
LATES DNA REPAIR AND RESISTANCE TO DOX-
ORUBICINE IN K562 LEUKEMIC CELLS
IM Majsterek1, J Blasiak1, A Slupianek2, D Pytel1,
T Sliwinski1
1 University of Lodz, LODZ, Poland2 Temple University, PHILADELPHIA, United States
of America
Several fusion tyrosine kinases (FTKs) have been
detected in hematopoietic malignancies, one of the
most extensively studied being BCR/ABL, considered
as the pathogenic principle of Philadelphia (Ph) chro-
mosome-positive human leukemias. The BCR/ABL
fusion generated by a t(9;22) translocation mediates
its biological effects through deregulated, constitu-
tively active tyrosine kinase activity. BCR/ABL is
reported to participate in drug resistance in leukemo-
genesis. Our recent studies revealed a novel potential
mechanism of resistance in Ph-cells underlined by
the stimulation of DNA repair. In this work we exam-
ined a role of BCR/ABL in K562 cells derived from
human chronic myeloid leukemia in response to dox-
orubicin treatment. We used cells acquired resistance
to doxorubicin during long term exposure to the drug
in culture (K562R, resistant), and wild type K562 cells
(K562S, sensitive). The drug resistance was measured
by MTT assay. We found that K562R in contrast to
wild type K562S cells, exhibited accelerated kinetic of
DNA repair after doxorubicin treatment in the range
0.01–1 µM evaluated by the alkaline comet assay. Pre-
treatment of the K562R with a selective FTKs
inhibitor, imatinib (STI751) at 1 µM, reverted drug
resistance and slowed down DNA repair. Western blot
analysis with CRKL antibodies followed by cells cul-
ture in the presence of STI571 revealed inhibition of
the phosphorylation of the CRKL adaptor protein spe-
cific for Ph-leukemias confirming BCR/ABL inactiva-
tion by STI571. The results obtained suggest that
BCR/ABL in K562 cells may stimulate the repair of
DNA damages in response to the drug treatment and
be relevant to the resistance of the leukemic cells to
anti-cancer therapy.
Financial support by grant KBN 3P04 A03225.
PS1 - Poster Session 1
Poster Presentation
PW2031
NEW APPROACHES TO IMPROVE HOMOLOGOUS
RECOMBINATION IN CHLAMYDOMONAS REIN-
HARDTII
M Slaninova1, L Fritsche2, M Pacanovska1, M Fusekova1,
G Treuner2, D Vlcek1, W Mages2
1 Comenius University FNS, BRATISLAVA, Slovak
Republic2 University Regensburg, REGENSBURG, Germany
The unicellular biflagellate green alga Chlamydomonas
reinhardtii is a popular model system used to study
photosynthesis, gametogenesis and mating, flagellar
assembly and cell division. The Chlamydomonas
genome which has recently been completed is cur-
rently being annotated and in the coming years func-
tional gene analysis in this organism will be in the
focus of interest. To this end, homologous recombina-
tion would be a highly desirable method, but unfor-
tunately Chlamydomonas (like higher organisms)
shows a very low rate of homologous recombination
by nature. Using two new dominant selectable
bacterial resistance markers directed against the
aminoglycoside antibiotics hygromycinB and paro-
momycin, respectively, we have constructed a trans-
genic Chlamydomonas strain as part of an in vivo
transformational readout system for homologous
recombination. In theory, this system allows positive
selection of homologous recombination events.
Currently we are trying to achieve this recombination
and in a second step we want to improve the rate of
recombination by pre- or post-treatment of trans-
formed cells with different physical or chemical stim-
uli.
In a parallel approach, we are trying to express a key
protein of bacterial recombination, the E. coli RecA
protein, in C. reinhardtii. For this purpose, we have
constructed a cassette in which the recA gene is
expressed from a (previously-published) strong C.
reinhardtii hybrid promoter consisting of constitu-
tive rbcs2 and inducible HSP70A promoter elements.
At the 3' end, the construct is terminated by the C. rein-
hardtii rbcs2 gene 3`region. This cassette is being used to
transform C. reinhardtii cells mutant in argininosucci-
nate lyase (arg2/arg7-8). Strains expressing the heterol-
ogous RecA protein will then be transformed again,
this time with truncated double stranded DNA
fragments of the argininosuccinate wild type gene
overlapping the arg2 mutation. Homologous recom-
bination should result in complementation of the
arg2 mutation in this latter strategy. Results will be
reported.
72
abstractboek 20-10-2004 10:28 Pagina 72
PS1 - Poster Session 1
Poster Presentation
PW2032
REPLICATION BYPASS OF UV AND BPDE INDUCED
LESIONS IN REPAIR DEFICIENT CHINESE HAM-
STER CELL LINES
FJ Johansson, A Lagerqvist, K Erixon, D Jenssen
Stockholm University, STOCKHOLM, Sweden
The aim of the present study was to investigate the
kinetics of replication bypass of UV lesions and
adducts induced by benzo[a]pyrene-diol-epoxide
(BPDE) measured as replication fork progression in
Chinese hamster ovarian (CHO) cells. The fork pro-
gression was here measured by one of us developed
method based on the Alkaline DNA unwinding (ADU)
technique. The ADU technique is a method to meas-
ure single strand interruptions in cellular DNA, which
is based on the principle that strand separation in
alkali requires strand breaks. The fraction of single
stranded DNA will be a measurement of the number
of strand breaks. This method has been applied to
study replication fork progression, where each repli-
cation fork is expected to provide two single strand
breaks. Initially pulse labeled DNA is found in the sin-
gle stranded fraction, but with chase time it succes-
sively turns into double stranded DNA, due to fork
progression and replicon merging. Inhibition of fork
progression by DNA lesions will delay this process.
Using cells lines deficient in nucleotide excision
repair (NER) and homologous recombination repair
(HRR), the repair kinetics of UV- and BPDE-lesions
may reveal the type of bypass mechanisms involved
in restoration of forks. The results demonstrated that,
replication forks stalled by UV or BPDE induced
lesions were delayed in restoration and most pro-
nounced in NER-deficient cells. It was found that this
process was further blocked by post-treatment with
caffeine. Caffeine has been suggested to bind to sin-
gle stranded DNA at replication forks and thereby
stalling the post replication repair/translesion syn-
thesis. These results awoke the question whether also
deficiency in replication bypass in addition to repair
of damaged DNA could explain the sensitivity for UV
and BPDE, which will be further discussed in this
presentation.
PS1 - Poster Session 1
Poster Presentation
PW2033
DNA DAMAGE, MUTAGEN SENSITIVITY AND DNA
REPAIR EFFICACY IN HELICOBACTER PYLORI-
INFECTED GASTRIC MUCOSA CELLS
M Arabski1, J Blasiak1, J Chojnacki2, J Drzewoski2,
P Kazmierczak2, G Klupinska2, M Kasprzak1,
M Wisniewska-Jarosinska2
1 University of Lodz, LODZ, Poland2 Medical University, LODZ, Poland
Helicobacter pylori is a common human pathogen
and its infection is believed to contribute to gastric
cancer. The malignant transformation of gastric
mucosa cells may be fueled up by impaired DNA
repair due to the accumulation of spontaneous muta-
tions in target genes and increasing susceptibility to
exogenous carcinogens. Moreover, the effectiveness
of DNA repair may contribute to failure of chemo-
therapy and resistance of gastric cancer cells to drugs.
To evaluate the role of the infection of H. pylori on
DNA repair we determined: 1) the kinetics of removal
of DNA damage induced by hydrogen peroxide and
the antibiotic amoxycillin, and 2) the level of basal,
oxidative and alkylative DNA damage in the H. pylori-
infected and non-infected gastric mucosa cells. The
level of DNA damage and the kinetics of DNA repair
were evaluated by alkaline single cell gel elec-
trophoresis (comet assay). Oxidative and alkylative
DNA damage were assayed with the use of DNA
repair enzymes formamidopyrimidine-DNA glycosy-
lase (Fpg) recognizing oxidized DNA bases and
3-methyladenine-DNA glycosylase II (AlkA) recogniz-
ing alkylated bases. We observed slower kinetics of
DNA repair after treatment with hydrogen peroxide
and amoxicillin in H. pylori-infected than non-infect-
ed gastric mucosa cells. The level of basal, oxidative
and alkylative DNA damage was higher in the infect-
ed compared to non-infected cells. Our results indi-
cate that H. pylori-infected gastric mucosa cells have
more damaged DNA and display decreased DNA
repair efficacy. Therefore, these features can be con-
sidered as risk markers for gastric cancer associated
with H. pylori infection and the comet assay may be
applied to evaluate these markers.
73
abstractboek 20-10-2004 10:28 Pagina 73
PS1 - Poster Session 1
Poster Presentation
PW2034
CELL ADHESION MOLECULES ICAM-1 (CD54) IN
CANCER INVASION
MZ Milicevic1, B Bajic2, JC Ciric3
1 Vinca Institute of Nuclear Sciences, BELGRADE,
Yugoslavia2 Galenika Pharmaceuticals, BELGRADE, Yugoslavia3 Institute of Endocrinology, BELGRADE, Yugoslavia
Several families of adhesion molecules have been
identified over the past several years and their syn-
thesis and expression on the cell membrane studied
in relation to the invasive and metastatic behaviour
of cancers. These adhesion molecules may be deleted
selectively or exhibit specific patterns of expression.
They may also be differentially expressed as a metas-
tasis–associated phenomenon. Intercellular and cell-
matrix interactions are also a major element of
cancer cell invasion and dissemination. The expres-
sion of CD-54 in thyroid specimens from 10 cases of
papillary adenocarcinoma, 10 cases of follicular ade-
noma and 10 normal thyroid specimens was exam-
ined and quantified by an immunohystochemical
method, using frozen sections and a standard avidin-
biotin–peroxidase complex method. Thyroid epithe-
lial cells from all cases of papillary adenocarcinoma
expressed the CD54. The ICAM-positive staining was
detected predominantly on the apical site of malig-
nant thyroid epithelial cells, but not in the cytoplasm
of those cells. Intense expression of the ICAM-1 mole-
cule was detected on the lesion were malignant cells
were extended. Normal thyroid tissues showed weak
expression of ICAM-1 on capillary and post –capillary
endothelial cells but not on thyroid epithelial cells.
We found that carcinomas expressing CD54 had a sig-
nificantly higher incidence of lymph node metastasis
than tumours not expressing CD- 54. Those results
suggest that ICAM expression is up-regulated in thy-
roid adenocarcinoma cells and that ICAM-1 is
involved in malignant processes in thyroid gland.
Staining for this molecule might be useful adjunct for
distinguishing of benign and malignant thyroid dis-
eases. Increased expression of CD-54 correlated with
invasiveness of human thyroid papillary adenocarci-
noma. The study of CD-54 cell adhesion molecules has
added to our understanding of how alterations in
specific molecules may contribute to the neoplastic
phenotype. CD 54 may have much more specialized
roles in controlling cellular processes.
PS1 - Poster Session 1
Poster Presentation
PW2035
LIFESPAN OF HUMAN LYMPHOCYTES EXAMINED
AFTER CHEMO- OR RADIOTHERAPY
S Gundy, M Baki, I Bodrogi, O Esik
National Institute of Oncology, BUDAPEST, Hungary
Estimates of the mean lifetimes of lymphocytes
based on the frequencies of unstable chromosomal
aberrations (CAs) seem to be ranged from 110 days to
10 years. Great differences in the estimations might
be accounted for since many circumstances influence
the aberration rate. The main objectives of our follow-
up study were to investigate the lifespan of human
lymphocytes in patients who had received either
radiotherapy in different volumes and locations of
the body, or chemotherapeutical treatment including
a combination of chemicals with a radiomimetic
drug causing the same variety of DNA damage as
radiation does. Cytogenetic effects of treatments
were examined prior to, during, immediately and up
to 7 years after the termination of different treat-
ments. The yields of unstable CAs showed a 3-fold
individual variability within the groups of patients
treated either with the same doses of chemicals or
radiotherapy. It was also found that not only the
extents of the irradiated volumes, but also the loca-
tions of lymph nodes play a role in both the yields
and the disappearance of unstable CAs. The average
half-life of the lymphocytes having suffered irradia-
tion is much shorter than reported so far. Our new
finding is that the disappearance of chemotherapy-
induced aberrations shows 2 peaks: the first one
occurs 3 years, and the second one occurs 6 years after
the end of treatment. It seems that the lifespan of
peripheral blood lymphocytes largely depends on the
nature of exposures. The amount of irreparable DNA
damage in precursor cells, which leads later to aber-
ration-formation is bigger when patients are chemo-,
rather than radiotherapy-treated. This phenomenon
must be taken into consideration when CAs are used
as biomarkers for the estimation of cancer risk.
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PS1 - Poster Session 1
Poster Presentation
PW2036
REACTIONS OF N,N-BIS(2-CHLOROETHYL)-P-AMINO-
PHENYLBUTYRIC ACID (CHLORAMBUCIL) WITH
2’-DEOXYCYTIDINE, 2’-DEOXY-5-METHYLCYTIDINE,
AND THYMIDINE
D Florea-Wang, J Hovinen
University of Turku, TURKU, Finland
N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid
(chlorambucil), alone or in combination with pred-
nisone, is routinely used in the chemotherapy of
chronic lymphocytic leukemia. Chlorambucil is also
used clinically for Hodgkin's lymphoma, non-Hodgkin's
lymphoma, Waldenström's macroglobulineamia, ovar-
ian and breast cancer, some other tumors, and certain
autoimmune diseases. Chlorambucil is an alkylating
agent that binds covalently to many types of cellular
molecules, such as DNA, RNA, and proteins. Because
the alkylating agents bind at sites of DNA that are
actively transcribed, they are more toxic to cancer
cells than to normal cells. However, like other alkylat-
ing agents, chlorambucil is potentially mutagenic,
teratogenic, and carcinogenic, and an increased inci-
dence of acute leukemias and other secondary malig-
nancies has been reported in patients who have
received this drug.
Chlorambucil was allowed to react with 2'-deoxycyti-
dine, 2’-deoxy-5-methylcytidine and thymidine at
physiological pH, and the reactions were followed by
HPLC-MS and HPLC-MS/MS techniques.1 Although
the predominant reaction observed was chlorambucil
hydrolysis, chlorambucil reacted with various het-
eroatoms of the nucleoside. The principal site of alky-
lation with all pyrimidines nucleosides was N3. Also,
several other adducts were detected. The N3, O2, 5’-O,
deglycosylated and deaminated derivatives were
characterized by means of MS/MS, UV, and 1H NMR in
the case of 2’-deoxycytidine and 2’-deoxy-5-methyl-
cytidine. C5-Adduct was also detected in the case of
2’-deoxycytidine. Thimidine was the least reactive
pyrimidine nucleoside studied, and in addition of the
N3 derivative, it reacted only at the carbohydrate
moiety.1 Diana Florea-Wang, Elina Haapala, Jorma Mattinen, Kristo
Hakala, Juhani Vilpo, and Jari Hovinen, Chemical Research
in Toxicology, Vol. 17, No. 3, pages 383-403, (2004)
PS1 - Poster Session 1
Poster Presentation
PW2037
GENOTOXICITY AND NEPHROTOXICITY BY A
COMMON REACTIVE INTERMEDIATE OF ULTRA-
SHORT ACTING NON-DEPOLARIZING TROPINYL
DIESTER NEUROMUSCULAR BLOCKING AGENTS
FAA van Acker, DJ van den Dobbelsteen,
GJMJ Horbach
N.V. Organon, OSS, The Netherlands
Muscle relaxation is achieved during clinical anaes-
thesia by the intravenous administration of neuro-
muscular blocking agents (NMBAs). These agents
produce muscle paralysis, primarily by occupying
nicotinic acetylcholine receptors located in the motor
end-plate region of striated muscle. In general, recov-
ery of neuromuscular block is due to the rapid metab-
olism, enzymatic/chemical degradation or biliary or
renal clearance yielding pharmacologically inactive
metabolites.
From a series of di- and tri-substituted benzyl bisqua-
ternary ammonium derivatives of a bis (tropan-3a-0l)
diester, two compounds were selected for toxicological
evaluation prior to human testing. During these stud-
ies it was revealed that for Org A a reactive intermedi-
ate was formed, which is believed to be responsible
for the observed genotoxic and nephrotoxic proper-
ties. In non-rodent toxicity studies clinical chemistry
and histopathological investigations revealed that
kidney was the primary target organ even upon sin-
gle dosing. In addition, the in vitro chromosomal
aberration test showed clastogenic activity both in
the presence and absence of a metabolizing system.
Subsequently, the structural analogue Org B was eval-
uated for its genotoxic properties and consistent with
the genotoxicity of Org A, this derivative was also
found to be clastogenic.
The proposed mechanism of toxic action is the for-
mation of a quinone-methide product from the
chemical and enzymatic degradation of the phenolic
acetate function into a transient phenol which
undergoes Hofmann-like elimination.
Quinone-methides have been shown to engage in
electrophilic reactions resulting in toxicity by react-
ing with cellular macromolecules such as proteins
and DNA. Therefore, the quinone-methide metabo-
lites are believed to result in the clastogenic effect of
the NMBAs. Upon excretion in the urine and concen-
tration of the primary urine the proximal tubular
cells are targetted by this common reactive metabo-
lite.
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PS1 - Poster Session 1
Poster Presentation
PW2038
POLYNUCLEOTIDE KINASE PARTICIPATES IN NON-
HOMOLOGOUS END JOINING IN RESPONSE TO
IONIZING RADIATION
AR Rasouli-nia, F Karimi-Busheri, M Weinfeld
University of Alberta, EDMONTON, Canada
Human polynucleotide kinase (hPNK) catalyzes phos-
phorylation of 5'-DNA termini and dephosphoryla-
tion of 3'-DNA termini. The protein participates in
processing and rejoining of single- and double-
strand-break termini, and has been shown to interact
with XRCC1, DNA polymerase beta, DNA ligase III and
probably XRCC4. Down regulation of hPNK expres-
sion by a double stranded small-interfering RNA mol-
ecule (siRNA) in a human lung adenocarcinoma cell
line resulted in hypersensitivity to a wide variety of
genotoxic agents and an elevation in spontaneous
mutation frequency. Since double-strand breaks are
considered to contribute significantly towards
genomic instability and cell lethality induced by ion-
izing radiation, we sought further evidence for direct
involvement of hPNK in the non-homologous end
joining (NHEJ) pathway by comparing the influence
of hPNK down-regulation on the radiation response
of NHEJ-proficient and deficient cells. We observed
that while reduced expression of hPNK in the NHEJ-
proficient cell lines increased cellular sensitivity to
ionizing radiation, it had no effect on the response of
the NHEJ-deficient cell line. Together with an exami-
nation of the repair response of the cells, these data
provide evidence for hPNK participation in NHEJ.
PS1 - Poster Session 1
Poster Presentation
PW2039
DAMAGE, REPAIR, DNA EVOLUTION
G de Buendia
Universidad Antonio Narinio, BOGOTA, Colombia
As many biological functions, DNA repair mecha-
nisms have also been evolving, so it is possible to
identify among the repair mechanisms those which
have appeared in the earlier stages of evolution.
Above all, evolution is a process which goes from sim-
plicity and unspecificity to complexity and specifici-
ty. From this point of view, translesion synthesis (TS),
non homologous recombination (NonHR) and the
addition and capture of telomeric sequences (ACTS)
can be considered as primeval DNA repair mecha-
nisms, and nucleotide excision repair (NER) and base
excision repair (BER) can be considered as evolved
DNA repair mechanisms.
TS, NonHR and ACTS are very simple mechanisms
because they consist of only one protein in the major-
ity of cases. They are general mechanisms because
they operate the same way in response to any lesion.
In contrast, NER and to a greater extent BER are very
specific for lesions and they recognize them before
the synthesis phase. In addition, they are complex
mechanisms because they need the operation of a
high number of proteins. In relation to complexity of
DNA sequences they can maintain, NER and BER
restitute the high variability of genetic information
with high fidelity, whereas TS, NonHR and ACST
reconstruct DNA chain continuity patching this with
monotonous sequences, as mutation spectra studies
as shown. The studies of DNA sequence analysis of
DNA repair genes in several organisms are also in
agreement with this model. It was found that the
genes of DNA polymerases which operate in TS in the
three superkingdoms (archaea, prokaryotes and
eukaryotes) are very similar, whereas genes of
methyl glycosylases which belong to BER vary from
one organism to another. This could be due to the fact
that they appeared recently in the evolution of the
analyzed organisms and so they do not have a com-
mon ancestor.
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PS1 - Poster Session 1
Poster Presentation
PW2040
ANALYSIS OF MICE WITH A SERINE TO ALANINE
SUBSTITUTION AT AA389 OF THE P53 GENE
W Bruins1, E Zwart1, E Hoogervorst1, LD Attardi2,
J van den Berg1, CTHM van Oostrom1, RB Beems1,
GJ van den Aardweg3, G. Lozano4, T Jacks5,
A de Vries1, H van Steeg1
1 National Institute of Public Health and the
Environment, BILTHOVEN, The Netherlands2 Stanford University Center, STANFORD,
United States of America3 Josephine Nefkens Institute, ROTTERDAM,
The Netherlands4 The University of Texas M.D., HOUSTON,
The Netherlands5 Center for Cancer Research,MIT, CAMBRIDGE,
United States of America
It is well known that p53 plays an important role in
suppression of human cancer. Mice with a complete
loss of p53 or with an overexpression of mutant p53
have been extensively studied, but these models do
not fully recapitulate the human situation. To mimic
the human situation, we have generated a subtle
germline mutation in the p53 gene, changing Serine
389 (392 human) into Alanine. Presumably, phospho-
rylation of S389 activates p53 and enables the protein
to exert its functions in cell cycle arrest and/or apop-
tosis. S389 is specifically phosphorylated after UV-
light, whereas after gamma irradiation S389 shows
no phosphorylation.
Site-directed mutagenesis of relevant p53 exon
sequences, homologous recombination in ES cells and
cre-mediated excision of the selectable marker gene
have resulted in p53.S389A cells and mice. It is expect-
ed that this approach will only disturb specific
subsets of p53 protein functions rather than all. Aging
experiments showed no increased incidence of
tumors and no reduced lifespan up to the age of 2.5
years in p53.S389A mice. However, carcinogen-
induced tumorigenesis experiments showed an
increased incidence of 2-AAF induced bladder neo-
plastic lesions, and a decreased latency time of UV-
induced skin tumors in p53.S389A mice.
MEFs were treated with UV-light, and a reduction in
apoptosis, which was even more evident in MEFs iso-
lated from crosses between p53.S389A and DNA
repair deficient XPA-/- mice, was observed. Also,
p53 protein levels were reduced after UV. After
gamma-irradiation however, G1-arrest was compara-
ble to that in wild type cells. Thus far, our results indi-
cate that blocking phosphorylation at codon 389 does
not have an effect on p53 responses after DNA double
strand breaks, but does seem to affect responses after
UV-induced DNA damage. This work was supported
by the Dutch Cancer Society and the NIEHS
(Comparative Mouse Genomics Centers Consortium).
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PS1 - Poster Session 1
Poster Presentation
PW2041
CARCINOGEN-INDUCED TUMOR DEVELOPMENT
IN MICE WITH AN R270H MUTATION IN P53
SWP Wijnhoven1, D Tuveson2, E Zwart1, N Willis2,
K Olive2, E Speksnijder1, M Schaap1, CTHM van
Oostrom1, T Jacks2, A de Vries1, H van Steeg1
1 National Institute of Public Health and the
Environment, BILTHOVEN, The Netherlands2 MIT, BOSTON, United States of America
In the p53.R270H 'knock-in' mouse model, an
Arginine to Histidine mutation was introduced in
codon 270 of the mouse p53 gene. This mutation,
equivalent to human R273H, is a 'hot-spot' mutation
in several human and mouse tumor types (lung,
breast, colon) and is frequently found as a germ-line
mutation in the human Li-Fraumeni syndrome. In
addition, a transcriptional stop-cassette flanked by
loxP sites was introduced in intron 1 of the p53 gene.
As a consequence, the expression of the mutant p53
protein can be controlled by crossing the mice with
(tissue-specific) Cre-transgenic mice.
In order to study the effects of the R270H mutation on
DMBA-induced (mammary) tumorigenesis, p53.
R270H mice were bred to mammary gland-specific
WAP-Cre mice. DMBA-treated heterozygous p53.
R270H mice developed mammary tumors with a high
incidence and short latency time, whereas equally
treated wild type mice did not respond. This indicates
that the R270H point mutation is an important trig-
ger for mammary tumor development and can act in
a dominant-negative manner.
Another indication for a potential dominant-negative
phenotype of the R270H mutation was observed after
chronic exposure of skin-specific p53.R270H/ K14-Cre
mice to a subtoxic dose of UVB-irradiation. The laten-
cy time for UV-induced skin tumor development was
strongly reduced in p53.R270H mutant mice com-
pared to wild type mice, and moreover, the tumor
multiplicity seems to be increased in heterozygous
point mutant mice.
Finally, 2-AAF exposure in p53.R270H/ TgN-balancer
Cre mice with a ubiquitous but mosaic expression in
all tissues tested, only leads to minor differences in
tumor development between p53.R270H mutant and
wild type mice.
In conclusion, the p53.R270H point mutation seems to
explore a dominant-negative tumor phenotype after
carcinogen exposure in a highly tissue-specific man-
ner.
This work was supported in part by the Dutch Cancer
Society and the NIEHS (Comparative Mouse Genomics
Centers Consortium).
PS1 - Poster Session 1
Poster Presentation
PW2042
USE OF METABOLIC COMPETENT CELLS TO EVALU-
ATE THE GENOTOXICITY OF TOBACCO CONDEN-
SATES
S Simi1, M Casella1, D Zampieri1, C Sorrentino2,
A di Muro2, L del Piano2, J Doehmer3, M Abet2, S Simi1
1 Institute of Clinical Physiology CNR, PISA, Italy2 Ist. Sperim. per il Tabacco, SCAFATI (SA), Italy3 GenPharmTox, MARTINSRIED/PLANEGG, Germany
Genetically engineered cells expressing cytochrome
P450 (CYP 450), a key enzyme for xenobiotic biotrans-
formation, have provided a new tool to investigate
both the genotoxicity and the P450 mediated meta-
bolic activation of a wide variety of chemicals includ-
ing drugs, environmental pollutants, mutagens and
carcinogens.
To study the genotoxicity of tobacco smoke, the major
cause of premature deaths in developed countries,
two Chinese hamster cell lines transfected with rat
cytochromes CYP 1A1 and CYP 1A2, known to be
induced by tobacco smoke, were exposed to two differ-
ent tobacco condensates.
Plating efficiency, sister chromatid exchanges and
chromosome aberrations were used as biomarkers of
genetic damage.
Preliminary results seem to show that all these end-
points are differently induced, suggesting also that
the two cytochromes operate in different way.
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abstractboek 20-10-2004 10:28 Pagina 78
PS1 - Poster Session 1
Poster Presentation
PW2043
DNA INSTABILITY IN DM1 MUSCLE CELLS
S Simi, M Casella, P Beffy, R del Carratore, S Simi,
M Simili
Institute of Clinical Physiology CNR, PISA, Italy
Myotonic Dystrophy type 1 (DM1) is an inherited
human disease whose molecular defect is the expan-
sion of a trinucleotide DNA sequence in the 3’UTR of
the DMPK gene. The triplet expansion is present in up
to 2,000 copies in DM patients while normal individ-
uals have 10–30 copies.
Since DM1 shares several features with fragile X syn-
drome, another 'unstable triplet syndrome' particularly
characterised by chromosome instability, cytogenetic
analyses (micronucleus assay, FISH assay) were per-
formed on human lymphocytes to verify if also DM1 is
prone to chromosome instability. A significant correla-
tion was found between the presence of the disease
and MN frequency increase, mainly due to MN bear-
ing centromere positive signals, thus indicating a
high incidence of chromosome loss.
Since it is known that the triplet expansion in muscle
cells is much higher than in lymphocytes, DM muscle
cells (DM-HFM, DM1 human foetal skeletal muscle
cells, 2800 CTG repeats) and control cells (C-HFM,
<200 CTG repeats) were kindly provided by J.
Puymirat (Quebec, Canada) and cultured to deter-
mine whether the chromosome instability occurred
also in muscle.
Cytogenetic analysis show that MN frequency in DM-
HFM is significantly higher than in C-HFM, strongly
suggesting a genomic instability phenomenon.
A possible mechanism of MN formation could involve
nuclear budding, a process which has been hypothe-
sised as the way by which amplified DNA selectively
localised to specific sites at the periphery of the
nucleus could be eliminated.
Our results of nuclear buds scoring show that the
buds frequency in DM-HFMC is significantly higher
than in C- HFM.
Experiments will be carried out to verify the presence
of amplified triplets in buds.
PS1 - Poster Session 1
Poster Presentation
PW2044
DNA PROTECTIVE PROPERTIES OF A 1,4-DIHY-
DROPYRIDINE DERIVATIVE IN HUMAN CELLS IN
VITRO
NI Ryabokon1, NV Nikitchenko1, J Rzeszowska-
Wolny2, GJ Duburs3, RI Goncharova1
1 Institute of Genetics&Cytology, MINSK, Republic
of Belarus2 Center of Oncology, GLIWICE, Poland3 Latvian Institute of Organic, RIGA, Latvia
Beta-carbonyl-1,4-dihydropyridines (1,4-DHPs) are
synthetic analogs of dihydronicotinamide, i.e. the
hydrogen and electron transferring part of redox
coenzymes NADH and NAD(P)H, which are involved
in many reactions, including energy transduction,
signaling pathways, DNA repair and, thus, are crucial
for living cells. 1,4-DHPs are widely known and used
for their important chemical, biochemical and phar-
macological properties. They also have high efficiency
as antimutagenic compounds. So, among 12 1,4-DHPs
screened in the Antimutagenesis Laboratory, Institute
of Genetics and Cytology, Minsk, 6 showed antimuta-
genic activity, significantly reducing spontaneous
and alkylation-induced genetic damage in Drosophila
[Goncharova et al., 1980; Kuzhir, 1999], as well as radia-
tion-induced injuries in pond fish [Goncharova, 2000].
In this study, we revealed that AV-153, one of the most
effective 1,4-DHPs in experiments with animals, is
nontoxic in a wide range of concentrations for lym-
phocytes of healthy donors in vitro and culture lines
of human cells. It significantly reduces cell death fre-
quency and different types of DNA damage induced
spontaneously or by alkylating and oxidizing agents,
as well as by gamma- and X-rays. The reduction fac-
tors reach 70%. The analysis of DNA repair kinetics
showed that AV-153 increases the effectiveness of
repair process in human cells during the first 15–60
min after treatment. These data demonstrate that AV-
153 could be studied and used as a DNA protective
and, thus, a cancer preventive compound. The role of
AV-153 in DNA repair, as well as the mechanisms of its
action, are studied and will be discussed.
The work has been carried out in the framework of
collaborative research between the Institute of
Genetics and Cytology (Minsk) and the Center of
Oncology (Gliwice) (2003–2005) and partially sup-
ported by fellowship grants from the Association for
Supporting Cancer Research (Poland), UNESCO (Polish
brunch) and National Cancer Institute (Bethesda,
USA).
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PS1 - Poster Session 1
Poster Presentation
PW2045
ROLE FOR MISMATCH REPAIR IN THE CELLULAR
RESPONSE TO UV-C IRRADIATION
V Borgdorff, S van Hees-Stuivenberg, N de Wind
Leiden University Medical Center, LEIDEN,
The Netherlands
Mismatch repair is the cellular process responsible
for the correction of replication errors such as mis-
matches and insertion/deletion loops. This process is
essential for the maintenance of the cell’s genomic
integrity. To investigate whether mismatch repair, in
addition to removing misincorporations, plays a role
in the processing of lesions induced by genotoxic
agents, we studied the cellular response of Msh2-defi-
cient mouse embryonic stem (ES) cells to UV-C irradi-
ation. UV-C treatment induced fivefold more
mutations in Msh2-deficient ES cells than in wild-
type ES cells. This increased UV-induced mutagenesis
is dependent on the Msh2/Msh6 mismatch recogniz-
ing heterodimer, since it is also observed in Msh6-
deficient cells, whereas cells deficient for the
Msh2/Msh3 heterodimer show a similar mutation
induction as wild-type cells by UV exposure. No sig-
nificant differences in the UV-induced mutation spec-
trum were observed between Msh2-deficient- and
wildtype ES cells. However, we did observe UV-
induced mutation hotspots at unique sites in the
Msh2-deficient cells. In addition, Msh2-deficient cells
displayed a decreased S/G2 arrest accompanied by a
slightly reduced level of UV-C-induced apoptosis.
Taken together, our data demonstrate an important
role for mismatch repair in counteracting UV-induced
mutagenesis. We hypothesize that mismatch repair
mediates sequence context-dependent removal of
nucleotides misincorporated opposite to UV-induced
pyrimidine dimers (compound lesions). Alternatively,
the mismatch repair-dependent cell cycle arrest may
allow nucleotide excision repair to remove damage in
a DNA sequence context-dependent way, resulting in
reduced mutagenesis and an altered distribution of
mutations.
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PS1 - Poster Session 1
Poster Presentation
PW2046
BIOLUMINESCENT SALMONELLA REVERSE MUTA-
TION ASSAY: A HIGH THROUGHPUT SCREEN FOR
DETECTING MUTAGENICITY
J Aubrecht, JJ Osowski, WW Ku, JR Cheung,
JI Ackerman, SM Hoogendoorn, JF Blake, P Persaud
Pfizer, GROTON, CT, United States of America
Here, we describe the development and evaluation of
a novel bioluminescent high throughput Salmonella
reverse mutation assay applicable to the screening of
large numbers of small molecules including combi-
natorial libraries. The bioluminescent Salmonella assay
utilizes genetically engineered standard Salmonella
tester strains TA98 and TA100 expressing the luxCDABE
operon from Xenorhabdus luminescence. In princi-
ple, the assay employs bioluminescence as a sensor of
metabolic activities in living cells. The assay provides
highly concordant data with the outcome in the
standard Salmonella plate incorporation reverse
mutation assay. Since the results of the standard
Salmonella assay are required by various regulatory
agencies for approval of new drugs, the biolumines-
cent Salmonella assay can be effectively used for pri-
oritization of compounds in drug discovery. Because
of its high throughput attributes, the assay permits
effective, fast and economical screening of a large
series of structural analogs enabling the investiga-
tion of structure activity relationships. In general, the
application of bioluminescent sensors for detection
of metabolically active cells is expected to have
broader application in the design of other high
throughput clonogenic assays in various cell systems
PS1 - Poster Session 1
Poster Presentation
PW2047
TP53 MUTATIONS IN EXPOSED WORKERS
KV Vähäkangas1, P Hainaut2
1 University of Kuopio, KUOPIO, Finland2 Intl. Agency for Research on Cancer, LYON, France
Kirsi Vähäkangas, Department of Pharmacology and
Toxicology, University of Kuopio, POB 1627, FIN-70211
Kuopio, Finland
Pierre Hainaut, International Agency for Research on
Cancer, Lyon, France
In some cases, evidence exists that carcinogenic
exposures contribute to the mutation spectrum of the
TP53 gene in human cancers. Most of this data comes
from dietary and environmental exposures, and very
few examples are available from occupational expo-
sures. In populations exposed to dietary Aflatoxin B1
with liver cancer (AFB1) and ultraviolet (UV)-radiation
with skin cancer, a single specific-looking TP53 muta-
tion has been described in some of the tumours.
Whether these fingerprints in the TP53 gene can be
used to reveal occupational etiology remains to be
shown. Uranium miners have probably been studied
enough to say that radon does not induce a specific
p53 mutation in lung cancer. In other cases, although
differences in the TP53 mutation spectrum exist, they
are more diffuse and difficult to interpret at this
point. Implications for a putatively specific p53 muta-
tion can be found in the literature for vinyl chloride,
mustard gas, metal industry and petrochemical
industry, and await further studies for confirmation.
Cigarette smoking seems to induce long-lasting
molecular footprints in TP53, but their use to rule out
other occupational exposures as etiological factors in
occupational cancers is still very questionable, espe-
cially due to the putatively synergistic effects of ciga-
rette smoke with other carcinogens. In IARC p53
mutation database (www.iarc.fr/p53) very few data
about occupations exist. Thus, although interesting
implications of possibly typical mutation spectra
among cancers with other occupational etiologies
exist, the data is scanty and awaits further develop-
ment of p53 mutation database.
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PS1 - Poster Session 1
Poster Presentation
PW2048
THE NOVEL UVS11 GENE OF CHLAMYDOMONAS
REINHARDTII AFFECTS CELL RESPONSE TO DNA
DAMAGE
M Slaninova, EG Galova, A Sevcovicova, B Sviezena,
D Vlcek
Comenius University FNS, BRATISLAVA,
Slovak Republic
The study of cell cycle regulation and checkpoints at
different levels has gained further insight into these
processes in different species. In comparison with
other lower eukaryotes there has been much less
progress in understanding cell cycle control in algae.
The first putative checkpoint mutant of green alga
Chlamydomonas reinhardtii have been isolated in
our lab and termed as uvs11. It has been predicted to
be UV-sensitive and repair-deficient. We assume now
that product of UVS11 gene is not required for DNA
repair itself but it is involved in cell response to DNA
damage.
In our work, we have assayed the activity of histone
H1 kinases presented in total protein extract and
those, mitosis specific ones bound to p13suc1-
Sepharose. The kinase activity was monitored in syn-
chronized cultures of untreated wild type and
mutant cells and compared with those irradiated by
UV-light (254nm) before mitosis. No substantial dif-
ferences between wild and mutant cells were found
in untreated cells. However, shortly after UV irradia-
tion the kinase activity in wild type has considerably
decreased, to recover about five hours later. The activ-
ity drop was accompanied by a prolongation of the
cell cycle due to a delay in mitoses, protoplast fis-
sions, and daughter cell release. The mutant strain
has shown no significant change in the course of
kinase activity and in other processes related to the
cell cycle after UV irradiation. Our present results
indicate that inhibition of histone H1 kinase could
be a way to arrest the initiation of mitosis in
Chlamydomonas and delay cell cycle progress in
response to DNA damage. We suggest that product of
the UVS11 gene affects cell response to DNA damage
and directly causes a decrease in kinase activity. This
implicates the role of UVS11 gene in regulation of the
cell cycle.
PS1 - Poster Session 1
Poster Presentation
PW2049
DUAL EFFECT OF HTLV-1 TAX PROTEIN ON
NUCLEOTIDE EXCISION REPAIRIN HUMAN T-
CELLS
Y Schavinsky-Khrapunsky, E Priel, M Aboud
Ben Gurion University of the Negev, BEER SHEVA,
Israel
HTLV-I is the etiological agent of adult T-cell
leukemia. Its Tax oncoprotein is regarded as a key ele-
ment in initiating the viral-associated leukemogenic
process. Tax oncogenic potential is partially ascribed
to its interference with various modes of DNA repair,
which leads to genetic instability. In this study we
were particularly interested in elucidating Tax effect
on nucleotide excision repair (NER) in human T-cells,
since NER is one of the major repair pathways used by
eukaryotic cells to maintain their genomic integrity
and because T-cells are the main targets of HTLV-I in
human infection. We show here that at low doses Tax
rather stimulates NER, implying that the low Tax
level in the infected T-cells of latent HTLV-I carriers
might protect these cells from mutagenesis and con-
tribute, thereby, to the long lasting clinical latency of
HTLV-I infection. However at high doses Tax inhibits
this repair, suggesting that Tax must be elevated in
the carriers’ infected T-cells for initiating their
leukemogenic progression. This dual Tax effect proved,
in this study, to be mediated by NF-kappaB. Furthermore
we demonstrate that NER inhibition by Tax is exerted
through NF-kappaB-dependent interference with p53-
transcriptional activity and consequently with its role in
NER.
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PS1 - Poster Session 1
Poster Presentation
PW2050
NIEHS - COMPARATIVE MOUSE GENOMICS CENTERS
CONSORTIUM: UNDERSTANDING THE BIOLOGICAL
SIGNIFICANCE OF HUMAN POLYMORPHISMS
JP Packenham
NIEHS/NIH, RESEARCH TRIANGLE PARK,
United States of America
The National Institute of Environmental Health
Sciences (NIEHS) Comparative Mouse Genomic
Centers Consortium (CMGCC) is a cross-disciplinary,
multi-Institutional, program that falls under the aus-
pices of the NIEHS Environmental Genome Project
(http://www.niehs.nih.gov/cmgcc). This program
was initiated with the goal of developing transgenic
and knockout mouse models based on human DNA
sequence variants in the environmentally responsive
genes discovered under phase I of the EGP. Ultimately,
these models will be used as tools to improve our
understanding of the biological significance of
human DNA polymorphisms and the role of such
variation in environmentally related diseases.
Initially, this program has focused on DNA polymor-
phisms that occur in DNA repair and cell cycle envi-
ronmentally responsive genes. The Consortium is
accomplishing its goals through, mouse model devel-
opment, technology development, and resources
developed by the Consortium. To date the Consortium
has over 32 mouse models under development and
has developed through its bioinformatics team a
Mouse Federated database, containing three major
components: mouse phenotypic assessment that
allows for functional prediction of gene variants to
model, collection and analysis of phenotypic data
including gene expression data, and mouse model
dissemination. Through this database, SNPs are ana-
lyzed for their functional impact using SNP location
and Haplotype frequency data, structural analysis
and prediction, literature mining and potential com-
binatorial effects through pathway data. Models are
generated and analyzed for pathology and other phe-
notypic endpoints under various environmental con-
ditions. This combined knowledge is then integrated
to generate knowledge of the human gene in the per-
turbed mouse background and map new information
onto existing knowledge and finally, the experimen-
tal data is used to refine and adapt models in order to
validate causality pathways. Validated mouse models
and resources developed within the CMGCC are avail-
able to the general scientific community without
charge.
PS1 - Poster Session 1
Poster Presentation
PW2051
PRE-EXPOSURE: MODULATION OF FREQUENCIES
AND REPAIR OF DNA DAMAGE?
P Cramers1, AA van Zeeland2, LHF Mullenders2,
JCS Kleinjans1
1 Maastricht University, MAASTRICHT,
The Netherlands2 Leiden University Medical Center, LEIDEN,
The Netherlands
Ionizing radiation (IR) interacts with DNA either by
direct hit, or by the production of reactive oxygen
inducing strand breaks and base modifications.
Alleviation of biological effects after pre-exposure to
low doses of IR has been described. Low dose expo-
sure might reduce damage induction from the high
dose, or might induce repair mechanisms.
The aim of this study was to test whether low radia-
tion doses can cause protection against high dose
effects by modulation of initial damage and repair.
By using confluent (G1 phase) human primary fibro-
blasts, we ruled out possible effects of the condition-
ing dose on the cell cycle.
Comet assay measurements showed that pre-expo-
sure of cells with 0.1 Gy prior to a challenge dose (0-8
Gy) resulted in a protective effect that was most pro-
nounced at higher challenge doses. No difference was
observed in repair kinetics between conditioned and
unconditioned cells.
DNA DSB were detected with gamma H2AX fluores-
cence. A linear dose response relationship was found
in the dose range 0-3 Gy. Damage induction experi-
ments have not yet been completed. No difference in
repair kinetics between conditioned and uncondi-
tioned cells was observed.
Taken together, our results indicate that in confluent
cells, pre-exposure gives a small modulation of DNA
damage induction and no enhancement of repair.
Another part of our project focuses on pre-exposure
to X-rays and the effects on UV-induced repair.
Recently several authors showed upregulation of
repair proteins after X-rays, including the NER pro-
tein XPC. It is therefore interesting to investigate
whether X-rays can trigger adaptation against UV
and vice versa. Repair replication experiments are
currently under way to study the inducibility of exci-
sion repair after low doses of ionizing radiation and
adaptation after both X-rays and UV conditioning
and challenging (cross-reactivity). Preliminary
results suggest an increased UV-repair in X-ray
pre-treated cells.
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PS1 - Poster Session 1
Poster Presentation
PW2052
THE ROLE OF LYS63-LINKED POLYUBIQUITIN
CHAINS IN REPAIR AND MUTAGENICITY OF
BENZO[A]PYRENE-DIOL-EPOXIDE (BPDE) DNA
ADDUCTS
SAS Langie, AM Knaapen, RK Chiu, RWL Godschalk,
CHMA Ramaekers, BG Wouters, FJ van Schooten
Maastricht University, MAASTRICHT,
The Netherlands
Humans are continuously exposed to environmental
carcinogens including polycyclic aromatic hydrocar-
bons like benzo[a]pyrene (B[a]P) that can induce DNA
adducts. Since DNA adducts can not always be suc-
cessfully repaired prior to cell-division, cells have
evolved tolerance mechanisms to replicate lesion-
containing DNA. Template switching and lesion
bypass are two mechanisms that ensure this DNA
damage tolerance, and there is evidence for the
involvement of Lys63-linked multi-ubiquitination in
these pathways. We hypothesised that the absence
of Lys63-linked multi-ubiquitination will cause
increased mutagenicity upon exposure to benzo[a]
pyrene-diol-epoxide (BPDE). Therefore, A549 cells
(human epithelial lung carcinoma cells) overexpress-
ing ubiquitin in its Wt-form (Ub+) or as a K63R
mutant configuration, were applied to further study
the role of Lys63-linked multi-ubiquitination mediat-
ed DNA damage tolerance mechanisms in the muta-
genicity of B[a]P. 32Postlabeling results showed no
significant differences in decline of the BPDE-DNA
adduct levels between K63R and Ub+-cells over a peri-
od of 24 hours (P>0.5), indicating that Lys63-ubiquiti-
nation is not directly involved in DNA adduct
removal. Furthermore, we demonstrated that Ub+-
and K63R-cells, either exponentially growing or
arrested in G0/G1, show no differences in survival
upon exposure to low doses (( 1 µM) of BPDE, whereas
K63R-cells were more sensitive at concentrations
>1µM. G0/G1-arrested cells seemed to be more resist-
ant towards the toxic effects of (±)anti-BPDE. Finally,
we demonstrated that BPDE-exposure (0.5 or 1.0 µM)
causes higher mutation frequencies in the HPRT-gene
of K63R-cells, as compared to Ub+-cells. In conclusion,
our data show that absence of Lys63-multi-ubiquiti-
nation (K63R) causes enhanced BPDE-induced muta-
genicity. Thus, our data suggest that inhibition of
Lys63-linked multi-ubiquitination prevents error-free
template switching and shifts DNA damage tolerance
to error-prone lesion bypass.
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PS1 - Poster Session 1
Poster Presentation
PW3001
THE ROLE OF ALLELIC POLYMORPHISM IN COLO-
RECTAL CANCER RISK
CSA Csejtei1, A Tibold2, ZS Faluhelyi3, I Kiss2, I Ember2
1 Markusovszky Teaching Hospital, SZOMBATHELY,
Hungary2 Public Health Inst. Uni. PTcs, PTCS, Hungary3 Baranya County Hosp., PTCS, Hungary
Allelic polymorphisms of metabolyzing enzymes and
other genes (e.g.onco/supressor genes) might be able
to have an influence on colon cancer susceptibility. In
our present study we tested the effect of glutathione-
S-transferase M1 (GTSM1), and p53 polymorphisms on
the risk of colorectal cancer in the Hungarian popula-
tion. DNA was isolated from deparaffinized sections
of colorectal cancer samples, genotyped for 0 and +
allels (GSTM1 and T1) and Arg/Pro alleles (p53), and
compared to the data of healthy controls. The allelic
polymorphisms were determined by a simultaneous
polymerase chain reaction (GSTM1 and T1) in the pres-
ence of a positive control, and by allelic-specific poly-
merase chain reaction (p53). The allelic distributions
were compared, odds rations (OR) were calculated to
show the difference between the occurrence of „low-
risk” and „high-risk” alleles among cases and con-
trols. Our results indicated a statistically significant
connection between the occurrence of colorectal
tumours and GTSM1 0-allele, and p53 Pro allele.
Combination of the „high-risk” alleles further increased
the risk.
Certain alleles of metabolyzing enzymes and
onco/suppressor genes were proved to occure more
frequently in colon cancer patients than in healthy
controls. Therefore, genetic polymorphisms can be
used to gain useful data on the individual suscepti-
bility to colon cancer.
PS1 - Poster Session 1
Poster Presentation
PW3002
CYP1B1 LEU432VAL POLYMORPHISM, SMOKING
HABITS AND THE RISK OF BREAST CANCER
PJ Sillanpää1, K Mitrunen1, V Kataja2, M Eskelinen2,
VM Kosma3, M Uusitupa3, H Vainio1, A Hirvonen1
1 Finnish Institute of Occupational Health,
HELSINKI, Finland2 Kuopio University Hospital, KUOPIO, Finland3 University of Kuopio, KUOPIO, Finland
Cytochrome P450 (CYP) 1B1 enzyme is involved in the
conversion of oestradiol and a number of PAHs to
mutagenic intermediates, capable of causing poten-
tial DNA damage. Several polymorphisms have been
described in the CYP1B1 gene.
Several previous studies have examined the associa-
tion between inherited differences in the CYP1B1 gene
and breast cancer risk with contrasting results. We
examined this issue further in a Finnish Caucasian
study population consisting of 483 breast cancer
patients and 482 healthy population controls. The
genotypes were determined by PCR-based allele-spe-
cific fluorogenic probes (Applied Biosystems). Odds
ratios (ORs) and 95% confidence limits (95% CIs) were
calculated by unconditional logistic regression analy-
ses adjusting for known or suspected risk factors for
breast cancer.
Overall, the frequencies of CYP1B1*1/*3 and *3/*3 geno-
types were not significantly different between cases
and controls (OR 1.19, 95% CI 0.89-1.59 and OR 1.17, 95 %
CI 0.77-1.78, respectively). However, when the data
was stratified according to smoking habits, an
increased risk for breast cancer was seen between
CYP1B1*3 alleles and number of cigarettes/day (p for
interaction 0.02). Women who carried at least one
CYP1B1*3 allele and smoked 1-9 cigarettes/day were at
3-fold increased risk of developing breast cancer (OR
3.06, 95% CI 1.32-7.12) compared to women who
smoked the same amount and had two CYP1B1*1 alle-
les. Moreover, a significant trend (p for trend 0.02) of
increasing risk with increasing number of CYP1B1*3
alleles was seen among women smoking 1-9 ciga-
rettes/day; ORs were 2.63 (95 % CI 1.07-6.46) and 5.09
(95% CI 1.30-19.9), for women with one or two copies
of the CYP1B1*3 variant alleles, respectively.
Our preliminary results suggest that the CYP1B1*3
allele may pose increased breast cancer risk among
light smoking Finnish women.
85
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PS1 - Poster Session 1
Poster Presentation
PW3003
GENETIC SUSCEPTIBILITY TO COPD AND LUNG
CANCER: COMBINED ANALYSIS OF GSTM1 AND MEH
POLYMORPHISMS
JS Salagovic, R Tkacova, V Habalova, J Zidzik,
B Fleischer, I Kalina
P. J. Safarik University, KOSICE, Slovak Republic
Objective: Cigarette smoking is the major risk factor
for the development of both chronic obstructive pul-
monary disease (COPD) and bronchogenic carcinoma,
but only 10-20% of heavy smokers develop the dis-
eases which suggests the presence of genetic suscep-
tibilty. The genetic susceptibility to lung cancer and
COPD might depend on variation in antioxidative
enzyme activities that detoxify cigarette smoke prod-
ucts such as microsomal epoxide hydrolase (mEH)
and glutathione S-transferase M1 (GSTM1).
Methods: The polymorphisms in GSTM1 gene and
mEH gene (EPHX1) were examined in 156 lung cancer
patients, 148 patients with COPD and 150 healthy con-
trol subjects using PCR and PCR-RFLP based methods.
The frequencies of polymorphic genotypes of mEH
and GSTM1 genes were compared both individually
and in combination in patients and healthy controls.
Results: Our findings suggest that the presence of at
least one mEH „slow allele” significantly increases
lung cancer risk (OR=1.71; 95% CI 1.02-2.89), especially
among smokers (OR=1.83; 95% CI 1.0-3.4). Moreover,
carriers of both slow alleles (homozygotes) with very
low mEH activity are at increased lung cancer risk
(OR=2.14; 95% CI 0.86-5.61), but significantly only
among nonsmokers. The odds ratio of individuals
with the both potentially risk genotypes: homozy-
gotes for slow allele in mEH gene and for null allele in
GSTM1 gene versus that of other genotypes combined
was 3.53 (95% CI 0.98-16.5; p=0.02, Fisher´s exact test).
The risk of development of COPD in lung cancer
patients was associated only with GSTM1 0/0 geno-
type (OR=2.98; 0.73-1.19).
Conclusions: Our study suggests that lung cancer risk
is associated with the presence of slow mEH allele
and GSTM1 0/0 genotype and that in patients with
lung cancer the presence of at least one active allele
in GSTM1 gene has a protective effect against the
development of COPD.
PS1 - Poster Session 1
Poster Presentation
PW3004
THE INFLUENCE OF MPO, MTHFR, XRCC1 POLY-
MORPHISMS ON AROMATIC DNA ADDUCT LEVELS
IN BRONCHUS
E Gyorffy1, L Anna1, J Segesdi2, Z Gyori2, I Soltesz3,
S Kostic3, A Csekeo3, J Minarovits2, B Schoket1
1 National Center for Public Health, BUDAPEST,
Hungary2 National Center for Epidemiology, BUDAPEST,
Hungary3 National Institute of Pulmonology, BUDAPEST,
Hungary
There is a large database in the literature on the
impact of major metabolic polymorphisms on lung
cancer risk and on biomarkers of environmental
genotoxic exposures. However, there is little knowl-
edge and contradictory reports on the risk of more
recently introduced genetic polymorphisms, such as
MPO, MTHFR and XRCC1. The aim of our study was to
investigate the effect of these three polymorphisms
on smoking-related aromatic DNA adduct level in
bronchial tissue of lung patients. The study popula-
tion consisted of 215 Hungarian patients who under-
went lung resection. Smoking status was self-reported
in a questionnaire. Current smokers and those who
had given up smoking within one year before surgery
were considered as smokers (n=143). Life-time non-
smokers and those who had given up smoking in
more than one year were categorised as non-smokers
(n=72). Levels of aromatic DNA adducts in macroscop-
ically normal bronchial tissue were determined by
the 32P-postlabelling method with nuclease P1
enrichment. MPO G463A, MTHFR Ala222Val and
XRCC1 Arg399Gln genotypes were detected by PCR-
based methods. For MPO, there were no clear differ-
ences in DNA adduct levels in relation to its
polymorphism. For MTHFR, adduct levels were 12 to
33% lower in individuals with Ala/Val or Val/Val vari-
ant genotypes compared to the wild- type genotype
(P=0.078 in smokers and P=0.026 in non-smokers).
XRCC1 repair polymorphism did not affect the adduct
levels. However, in combination with MTHFR, adduct
levels were 30% lower in Gln/Gln subjects compared
to those with Arg/Arg (P=0.025) and Arg/Gln
(P=0.028), considering the sub-population of MTHFR
wild-type carrier and smoking individuals only. The
underlying molecular mechanisms need further
investigations.
The research project has been supported by the
Hungarian OTKA T 034616 research grant.
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PS1 - Poster Session 1
Poster Presentation
PW3005
DELTA-AMINOLEVULINIC ACID DEHYDRATASE
POLYMORPHISM AND SUSCEPTIBILITY TO LEAD
EXPOSURE IN OCCUPATIONALLY LEAD EXPOSED
WORKERS
Y Duydu, HS Suzen
Ankara University, ANKARA, Turkey
In this study, the cytogenetic response to lead expo-
sure in storage battery manufacturing workers
carrying different alleles of ≠ -aminolevulinic acid
dehydratase (ALAD 1 and ALAD 2) was evaluated. The
cytogenetic response was measured by analysis of
the frequency of sister chromatid exchange (SCE) and
the number of high-frequency cells (HFCs) in periph-
eral blood lymphocytes from workers occupationally
exposed to lead. A total of 71 voluntary male workers
were enrolled in the study. According to our genotype
analysis, 50 workers had the ALAD 1-1 genotype and 21
workers had the ALAD 1-2 genotype. When HFCs were
compared between ALAD 1-1 and ALAD 1-2 workers,
the percentage of HFC was statistically (T2-test,
P < 0.05) higher in ALAD 1-1 workers. However the dif-
ference in mean values of SCE/cell between ALAD 1-1
and ALAD 1-2 workers was statistically not signifi-
cant. On the basis of this result we suggest that ALAD
1-1 subjects might be more susceptible to cytogenetic
effects of lead exposure than ALAD 1-2 subjects.
This study was supported by Research Found of
Ankara University, Project no. 98.03.00.01.
PS1 - Poster Session 1
Poster Presentation
PW3006
POLYMORPHISM IN DNA REPAIR GENE XPD AND
BREAST CANCER RISK IN SMOKING WOMEN
M Mitrunen1, V Kataja2, M Eskelinen2, VM Kosma3,
M Uusitupa3, A Hirvonen1
1 Finnish Institute of Occupational Health, HELSINKI,
Finland2 Kuopio University Hospital, KUOPIO, Finland3 University of Kuopio, KUOPIO, Finland
DNA repair is essential for protecting cells from the
genotoxic effects of carcinogenic exposures. The XPD
protein takes part in the nucleotide excision repair
pathway, which recognizes and repairs wide variety
of structurally unrelated lesions. Mutations in the
XPD gene can diminish the activity of the repair and
therefore lead to increased DNA damage and ulti-
mately to carcinogenesis. Four polymorphisms result-
ing in amino acid changes have been reported in the
XPD gene, of which the Lys751Gln change in exon 23
has been anticipated to have the most profound
effect on the activity of the enzyme. We examined if
this polymorphism was associated with breast cancer
risk in Finnish Caucasian population. A PCR-based
RFLP-analysis was used for the genotype Odds ratios
(OR) and 95% confidence intervals were calculated by
unconditional logistic regression. Our preliminary
statistical evaluations revealed no significant overall
association between the Lys751Gln polymorphism
and breast cancer risk; OR for subjects with two Gln
alleles was 1.13 (95% CI 0.76-1.66). Neither was any sig-
nificant difference seen by menopausal status,
whereas a statistically significant interaction was
seen with smoking (p for interaction 0.019); women
who reported ever smoking and had two Gln alleles
were at significantly increased risk of breast cancer
(OR 2.42, 95% CI 1.22 - 4.78) compared to women who
carried one or two Lys alleles. The risk was confined to
women who had smoked over 10 cigarettes/day.
These preliminary data suggests that the XPD
Lys751Gln polymorphism may be an important modi-
fier of breast cancer risk in smoking Finnish women.
This work was supported by the Academy of Finland
and by EVO funds from Kuopio University Hospital.
87
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PS1 - Poster Session 1
Poster Presentation
PW3007
THE FUNCTIONAL IMPACT OF POLYMORPHISMS
IN DNA REPAIR GENES: XPA, XPB AND RECQL1.
M Krzesniak1, D Butkiewicz1, M Mrzyglodzik1,
R Vaitiekunaite1, CC Harris2, M Rusin1
1 Center of Oncology, GLIWICE, Poland2 National Cancer Institute, NIH, BETHESDA,
United States of America
Reduced capacity to repair DNA damage can lead to
cancer formation, which is examplified by many can-
cer-prone diseases caused by mutations in DNA
repair genes. The inter-individual variation in DNA
repair capacity and cancer risk are associated with
polymorphisms of DNA repair genes. Previously, we
and others have found sequence alterations within
the coding and non-coding regions of genes involved
in DNA metabolism. It is not known whether and
how these particular sequence alterations change the
functioning of the encoded proteins. We focused on
two genes involved in nucleotide excision repair sys-
tem (XPA, XPB) and on RECQL1, which encodes the
smallest known human helicase from the RecQ fami-
ly. This group of helicases contains at least four other
genes, three of which (WRN, BLM and RTS) are associ-
ated with cancer-prone genetic diseases. For our func-
tional analyses we have selected the common
polymorphism of XPA (-4G>A) located four residues
upstream the start codon, which in previous molecu-
lar epidemiological analyses showed significant
association with modulation of lung cancer risk. Two
relatively infrequent XPB polymorphisms (117:Lys>Arg;
402:Gly>Cys) were also selected due to their localiza-
tion within the evolutionary conserved regions of the
protein. Finally, we studied the functional impact of
three sequence alterations of RECQL1 helicase gene: 248:
Ala>Pro, 487:Lys>Thr (polymorphic allele), 566:Thr>Ala
(sequence alteration found in HeLa cells) arranged as 8
different sequence combinations. This helicase is not
well studied and its high expression in lung suggests
that its sequence alterations may potentially influ-
ence the lung cancer risk. Using the luciferase
reporter assay, host cell reactivation assay, in vivo
protein labeling by the fluorescent tags and Western
blotting we assessed the influence of the sequence
alterations on the expression level, activity and cellu-
lar localization of the studied DNA repair proteins.
The detailed results of the analyses will be presented.
88
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PS1 - Poster Session 1
Poster Presentation
PW3008
EPHX1 GENE POLYMORPHISMS AND INDIVIDUAL
SUSCEPTIBILITY TO LUNG CANCER
AV Voho1, K Mitrunen1, S Anttila1, O Impivaara2,
J Jarvisalo3, H Vainio1, K Husgafvel-Pursiainen1,
A Hirvonen1
1 Finnish Institute of Occupational Health, HELSINKI,
Finland2 National Public Health Institute, TURKU, Finland3 Social Insurance Institution, TURKU, Finland
Microsomal epoxide hydrolase (EPHX1) is a widely
expressed enzyme involved in the first pass metabo-
lism of polyaromatic hydrocarbons (PAHs), especially
benzo(a)pyrene (BaP) from tobacco smoke. At least
two functional polymorphisms in the EPHX1 gene are
common in Caucasians. We conducted a case-control
study in a Finnish study population to investigate the
role of the EPHX1 Tyr113His and His139Arg polymor-
phism in lung cancer susceptibility, both alone and in
combination with glutathione S-transferase M1 (GSTM1)
genotypes. Subjects with homozygous Tyr113/Tyr113
genotype were at increased risk of lung cancer (OR,
1.47; 95% CI, 1.06-2.04) compared with those having
His113 allele-containing genotypes. Similarly, the
homozygous His139/His139 genotype posed an
increased risk for this malignancy (OR, 1.58; 95% CI,
1.08-2.30) compared with the Arg139 allele-contain-
ing genotypes. However, lung cancer risk was not
related to the EPHX1 phenotypes predicted from the
exon 3 and exon 4 genotype data. When combined
genotype effects were examined, the OR for EPHX1
Tyr113/Tyr113 genotype in combination with GSTM1
null genotype was 1.73 (95%CI, 1.09-2.74). Subjects who
had this genotype combination and a smoking histo-
ry of over 40 pack-years (PYs) showed a 4-fold risk of
lung cancer (OR, 4.05; 95%CI, 1.27-12.92). The combination
of GSTM1 null and EPHX1 His139/His139 genotypes was
also associated with significantly increased lung cancer
risk (OR, 1.79; 95%CI, 1.06-3.02). Moreover, subjects
with this genotype combination and over 40 PYs had
a 4.5-fold risk of lung cancer (95%CI, 1.28-15.8). Our
results support the hypothesis that combinations of
even modest risk genotypes may lead up to a remark-
ably increased risk of lung cancer among smokers.
PS1 - Poster Session 1
Poster Presentation
PW3009
PREDICTING MULTIPLE GENE-ENVIRONMENT
INTERACTIONS USING HIGH THROUGHPUT SIN-
GLE NUCLEOTIDE POLYMORPHISM GENOTYPING
HB Ketelslegers, RWH Gottschalk, RWL Godschalk,
AM Knaapen, FJ van Schooten, JCS Kleinjans,
JHM van Delft
Maastricht University, MAASTRICHT, The Netherlands
The majority of cancers in humans are caused by cig-
arette smoking. Cigarette smoke contains over 3500
chemicals, including carcinogenic polycyclic aromat-
ic hydrocarbons, n-nitrosamines and aromatic amines.
These compounds are metabolized in a complex path-
way of absorption, distribution, activation and detoxi-
fication leading to reactive compounds that can
interact with DNA, so called DNA adducts. Previous
studies have shown that there is a relationship
between cigarette smoke constituents, carcinogenic
DNA adducts formation and cancer. Moreover, there
is a positive relation between dose (number of ciga-
rettes per day) and effect (DNA adducts). However,
within this relationship there is a wide inter-individ-
ual variation in DNA adduct levels in individuals with
similar exposures. This variation can at least partly be
explained by polymorphisms in genes that code for
enzymes involved in metabolic pathways of the spe-
cific carcinogens.The majority of studies on these
biomarkers of susceptibility focused on single poly-
morphisms. But taking in account that cigarette smoke
is a complex mixture of a large number of carcinogens,
simultaneous assessment of multiple genotypes
seems to be necessary.
The current pilot study was performed to determine
whether multiple genotyping can indeed explain
part of the inter-individual variation. The susceptibil-
ity of smoking individuals towards the formation of
DNA adducts was investigated. 24 Single Nucleotide
Polymorphisms in 18 genes, involved in DNA repair,
oxidative stress and biotransformation were geno-
typed in 65 smoking individuals using the Snapshot
method. A significant relationship was found between
the sum of all 'bad' polymorphisms and the adduct
level adjusted for cigarettes per day. From this it was
concluded that the sum of putatively bad polymor-
phisms determines the efficiency of adduct forma-
tion at a certain dose.
89
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PS1 - Poster Session 1
Poster Presentation
PW3010
ASSOCIATION BETWEEN XRCC3 POLYMORPHISMS
AND BREAST CANCER RISK IN FINNISH POPULA-
TION
L Heikinheimo1, P Siivola1, V Kataja2, M Eskelinen2,
VM Kosma3, M Uusitupa3, K Mitrunen1, A Hirvonen1
1 Finnish Institute of Occupational Health,
HELSINKI, Finland2 Kuopio University Hospital, KUOPIO, Finland3 University of Kuopio, KUOPIO, Finland
Genetically determined variability in DNA repair
capacity has been suggested to contribute to inter-
individual differences in susceptibility to breast can-
cer. We explored this issue by evaluating the potential
association between the XRCC3 Thr241Met and intron
5 A>G polymorphisms and breast cancer risk in our
Finnish Caucasian study population consisting of 483
cancer patients and 482 healthy controls. PCR-RFLP and
Taqman analysis were used to determine the geno-
types. Odds ratios (ORs) and 95 % confidence intervals
(CIs) were estimated by unconditional logistic regres-
sion. No significant overall effect was seen between
these XRCC3 polymorphisms and breast cancer risk.
However, when stratified by the use of alcohol,
women with at least one Met allele were found to be
at 1.5-fold risk for this malignancy (OR 1.54, 95% CI
1.05–2.26) if they reported ever using alcohol. This
association was most evident among women who
reported using alcohol daily or weekly (OR 2.05, 95%
CI 1.03–4.09). A statistically significant interaction
was also found between the intron 5 polymorphism
and body mass index (BMI) (p for interaction = 0.046);
women who had BMI less than 25.4 and carried at
least one variant G allele were found to be at some-
what increased risk for breast cancer (OR 1.46, 95% CI
0.97–2.20). Similarly, women who had ever used HRT
and carried at least one variant G allele had a tenden-
cy of increased risk for breast cancer (OR 1.63, 95% CI
0.93–2.84). No association was found when stratified
by the use of alcohol or tobacco. Neither were com-
bined effects found for intron 5 and Thr241Met poly-
morphisms. The results suggest that XRCC3 Thr241Met
and intron 5 A>G polymorphisms may be modest mod-
ifiers of individual breast cancer risk in Finnish
women. This work was supported by the Academy of
Finland and EVO funds from Kuopio University
Hospital.
PS1 - Poster Session 1
Poster Presentation
PW3011
EFFECT OF POLYMORPHISMS OF DNA REPAIR AND
FOLATE METABOLISM ON CHROMOSOMAL ABER-
RATIONS
I Heilimo, P Siivola, H Maunu, H Jarventaus, K Mitrunen,
A Hirvonen, H Norppa
Finnish Institute of Occupational Health, HELSINKI,
Finland
The frequency of chromosomal aberrations (CAs) in
cultured peripheral blood lymphocytes has for many
years been applied as a biomarker of early effects of
genotoxic carcinogens. The relevance of CA level as an
indicator of cancer predisposition was further sup-
ported by epidemiological studies suggesting that a
high frequency of CAs is predictive of an increased
risk of cancer. The cancer risk predictivity of CAs was
not explained by tobacco smoking or occupational
carcinogen exposure and did not depend on the time
between CA analysis and cancer detection, indicating
that the association also concerns 'unexposed' sub-
jects but does not reflect undetected cancer. The find-
ings suggest a role for individual susceptibility to
chromosome damage. In particular, variation in DNA
repair capacity, possibly related to polymorphisms of
DNA repair proteins, might provide an explanation.
Several of such polymorphisms have been proposed
to be associated with an increased cancer risk.
However, very little is known about their effects on
cytogenetic biomarkers in humans. Our previous
findings have suggested that polymorphisms of
XRCC1 and XPD may affect the level of CAs in human
lymphocytes. In the present study, we have examined
a population of 170 occupationally unexposed
Finnish subjects. We have genotyped major genetic
polymorphisms in DNA repair genes XRCC1 (codons
194, 280 and 399), XRCC3 (codon 241), XPD (exon 23),
and hOGG1 (codon 326) using methods based on PCR-
RFLP. Additionally, two polymorphisms in folate
metabolism enzymes, MTHFR (C677T) and MS
(A2756G) were examined. A preliminary analysis sug-
gested that polymorphisms in XRCC1 codons 194 and
280 affect the frequency of chromosome-type aberra-
tions. Polymorphisms in XPD exon 23 and MTHFR
C677T appeared to influence the level of chromatid-
type aberrations. [Supported by QLK4-CT-2000-
00628].
90
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PS1 - Poster Session 1
Poster Presentation
PW3012
GENETIC POLYMORPHISMS IN XENOBIOTIC-
METABOLISING ENZYMES AND THEIR POSSIBLE
LINKS WITH CHROMOSOMAL ABERRATIONS
P Siivola, I Heilimo, K Mitrunen, H Jarventaus, H Maunu,
A Hirvonen, H Norppa
Finnish Institute of Occupational Health, HELSINKI,
Finland
Genetic polymorphisms of various xenobiotic-
metabolising enzymes (XMEs) have been associated
with cancer risk. This association is assumed to
reflect the role of XMEs in the metabolic activation
and detoxification of environmental carcinogens. The
effect of genetic polymorphisms on susceptibility to
specific genotoxic exposures can be studied by exam-
ining biomarkers, such as chromosomal aberrations
(CAs), in exposed and unexposed subjects. For
instance, the GSTM1 null genotype has been associat-
ed with an increased CA level in smokers. Besides
affecting the frequency of CAs induced by genotoxic
exposures, genetic polymorphism may also modulate
the background level of CAs. This kind of an effect has
earlier been described for NAT2 slow and GSTT1 null
genotypes. In the present study, we examined a pop-
ulation of 170 occupationally unexposed subjects. We
analysed the major genetic polymorphisms in several
genes responsible for carcinogen metabolism (i.e.
GSTM1, GSTM3, GSTP1, GSTT1, NAT2, EPHX exon 3 and
4) using PCR-RFLP and fluorogenic allele-specific
probes –based genotyping methods. According to our
preliminary evaluation, EPHX genotype expected to
result in low enzyme activity was associated with an
elevated frequency of CAs, especially of the chro-
matid-type. [Supported by QLK4-CT-2000-00628].
PS1 - Poster Session 1
Poster Presentation
PW3013
MICRONUCLEUS TEST AS AN INDEX OF INDIVID-
UAL CANCER SUSCEPTIBILITY: THE PLEURAL
MALIGNANT MESOTHELIOMA
C Bolognesi, E Perrone, E Giordano, P Roggieri,
R Filiberti, M Neri, R Andreatta, R Puntoni
National Cancer Research Institute, GENOA, Italy
Inherited or acquired genome instability character-
izes the cancer susceptibility in humans and increas-
es the sensitivity of DNA to various physical and
chemical carcinogenic agents. Malignant pleural
mesothelioma (MM) is a rare highly aggressive neo-
plasm. Although it is well established that asbestos is
the major risk for MM, accounting for about 80% of
cases, the molecular steps in the carcinogenic process
is unknown and other ethiological factors have been
suggested such as SV40, ionising radiation and genet-
ics. The evidence of a complex heterogeneity of struc-
tural chromosomal aberrations in MM seems reflect
an intrinsic predisposition of the cells to accumulate
genomic damage. A number of biomarkers have been
widely applied in molecular epidemiology for identi-
fying subjects at risk of developing cancer. They
should allow to evaluate environmental and occupa-
tional exposure and further give information on the
status of susceptibility. One of the most used method-
ologies for assessing genomic damage in human lym-
phocytes is the micronucleus assay (MN). This assay
seems to be an useful method for monitoring indi-
viduals with genetic instability and as a screening
test for carriers of specific mutations in evaluating
cancer susceptibility.
A biomonitoring study was carried out to evaluate
the MN frequency in PBLs of patients with MM and of
their first-degree relatives with respect to lung cancer
patients and two groups of controls. The study includ-
ed 47 patients with MM, 10 first-degree relatives, 48
patients with lung cancer, 53 risk controls and 88
healthy controls. Preliminary results revealed a signif-
icant increased MN frequency in patients with MM
and in their relatives: the mean MN is double in com-
parison with all the other groups. No association was
found between MN and asbestos exposure. The analy-
sis is ongoing: the final results will be presented.
91
abstractboek 20-10-2004 10:28 Pagina 91
PS1 - Poster Session 1
Poster Presentation
PW3014
DNA REPAIR GENE POLYMORPHISMS AND GENO-
TOXICITY BIOMARKERS IN WORKERS EXPOSED
TO COBALT CONTAINING DUST
RAM Mateuca1, V Aka1, M de Boeck1,
M Kirsch-Volders1, D Lison2
1 Vrije Universiteit Brussels, BRUSSELS, Belgium2 Catholic University of Louvain, BRUSSELS, Belgium
The aim of this biomonitoring study was to identify
the link between relevant polymorphisms in DNA
repair genes (hOGG1, XRCC1 and XRCC3) and biomark-
ers of genotoxicity in workers exposed to cobalt metal
alone or in combination with tungsten carbide at a
mean level corresponding to the current TLV-TWA
(20µg Co/m3). The study comprised 21 workers exposed
to metallic Co alone, 26 workers exposed to a WC-Co
mixture and 26 matched controls. The genotoxicity
biomarkers represented both initial DNA damage
(8-hydroxydeoxyguanosine [8-OHdG] in urine and
comet assay on lymphocytes) and definitive chromo-
some breakage/loss (micronuclei in lymphocytes).
The predictivity of DNA repair genotypes was analysed
by direct comparisons between groups (Mann-Whitney
U test) and by multiple regression analysis taking into
account, age, exposure type, smoking, and interac-
tions between genotypes, smoking and exposure.
Multiple regression analysis indicated that in both
the exposed and total populations, MNMC frequen-
cies were higher in workers with variant hOGG1326
and XRCC3241 genotypes.
In the Co population, smokers with variant XRCC3241
genotypes had high frequencies of MNCB. In the WC-
Co exposed population, hOGG1 was predictive for
MNCB. Multiple regression analysis revealed that
workers with variant hOGG1326 and XRCC3241 geno-
types and smokers with variant hOGG1326 genotype
show a higher MNMC frequency. Direct comparison
between WC-Co exposed workers with Ser/Ser and
variant hOGG1326 genotypes showed a higher MNMC
frequency in the latter ones. Polymorphisms in
XRCC1280 resulted in an increased baseline DNA dam-
age in the total and exposed populations. There was
no influence of genetic polymorphisms on the uri-
nary levels of 8-OHdG.
It was concluded that for a better individual follow-
up of workers exposed to Co containing dust, and in
particular to hard metal dust, smoking should be
avoided and if unexpectedly high frequencies of MN
are observed at individual level, genotyping for
hOGG1326 and XRCC3241 should be advised.
92
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PS1 - Poster Session 1
Poster Presentation
PW3015
POLYMORPHISMS OF TNF-A, IL-10 AND IL1RN
AND RISK OF NON-SMALL CELL LUNG CANCER
HL Lind, S Zienolddiny, AAGE Haugen
National Institute of Occupational Health, OSLO, Norway
Studies have indicated that inflammation and reac-
tive oxygen species induced by smoking, may play a
role in lung carcinogenesis. We have previously
shown that certain variants of interleukin-1 beta
(IL1B) and cyclooxygenase-2 (COX2) genes are associ-
ated with increased risk of non-small cell lung cancer.
We have now extended our genotyping studies to
include several other genes involved in the inflam-
matory pathway. Here, we report results from analy-
sis of SNPs in genes for tumor necrosis factor alpha
(TNF-a -308 G/A and +488 G/A), interleukin-10 (IL-10 -
1082 A/G and –592 C/A) and interleukin-1 receptor
antagonist (IL1RN 86 bp VNTR intron 2). The SNPs in
TNFa and IL-10 were determined using the TaqMan
methodolgy, while the 86 bp VNTR in intron 2 of the
IL1RN gene was determined using PCR and gel elec-
trophoresis. Both the cases and controls were of
Norwegian origin and were frequency matched on
the basis of age and smoking status. In summary, this
is the first study investigating polymorphisms in
TNF-a, IL-10 and IL1RN in relation to lung cancer.
PS1 - Poster Session 1
Poster Presentation
PW3016
POLYMORPHISMS IN ARACHIDONIC ACID PATH-
WAY GENES AND FISH CONSUMPTION ARE MOD-
IFYING COLORECTAL ADENOMA RISK
HJ van Kranen1, CLE Siezen1, AIM van Leeuwen2,
NR Kram1, MEM Luken1, E Kampman2
1 National Institute of Publ. Health and Environment,
BILTHOVEN, The Netherlands2 WUR, WAGENINGEN, The Netherlands
The association between polymorphisms in genes
involved in lipid metabolism, particularly the arachi-
donic acid (AA) pathway, and colorectal adenomas
was investigated in a Dutch case control study includ-
ing 384 cases and 403 polyp-free controls. Twenty-one
polymorphisms in seven candidate genes were stud-
ied, and a potential modifying effect of fish con-
sumption was considered.
Inverse associations were found between colorectal
adenomas and the CT genotype of SNP H477H in
PPARg and the GC genotype of SNP V102V in COX-2
(OR, 0.63; 95%CI, 0.45-0.89 and OR, 0.65; 95%CI, 0.46-
0.92 respectively) compared to their homozygote
major genotypes. A positive association was observed
for the TC genotype of SNP c.2242T>C in COX-2 (OR,
1.47; 95%CI, 1.07-2.00) compared to the TT genotype.
Extended analysis with estimated haplotypes not
only confirmed these associations but also revealed
three additional associations between haplotypes in
COX-2, sPLA2 and 15LOX and colorectal adenomas.
Interactions between fish consumption and the
genotypes of COX-2 and PPAR d were observed. Firstly
for SNP c.-789C>T in PPAR d (P=0.01), an inverse asso-
ciation was observed for individuals with the CC
genotype and highest tertile (T3) of fish consumption
as compared to the lowest tertile (T1) of fish con-
sumption (OR, 0.65; 95%CI, 0.41-1.02). However, for
those with the CT or TT genotype, fish consumption
increased risk (OR T3 versus T1 and CC genotype, 2.22;
95%CI, 0.78-6.36). Secondly, inverse associations were
observed for SNPs V102V and c.2242T>C in COX-2. The
interaction between fish consumption and c.2242T>C
was statistically significant (p=0.01), with an OR for
the TT genotype and high fish consumption of 0.52
(95%CI, 0.27-1.01) as compared to low fish intake.
These results indicate that SNPs in various genes
involved in the AA-pathway are associated with
colorectal adenoma risk. Part of these associations is
modified by fish consumption.
93
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PS1 - Poster Session 1
Poster Presentation
PW3017
GSTM1, NUCLEOTIDE EXCISION REPAIR AND
APOPTOTIC CAPACITY POLYMORPHISMS ON LYM-
PHO-MONOCYTIC ANTI-BPDE-DNA ADDUCTS IN
COKE-OVEN WORKERS
SP Pavanello1, A Pulliero1, E Siwinska2, D Mielzynska2,
E Clonfero1
1 University of Padova, PADOVA, Italy2 Institute Occupational Health, SOSNOWIEC, Poland
Polymorphisms of nucleotide excision repair (NER)
genes (XPC-PAT +/-, XPA 5’ non-coding region -A23G,
XPD-exon 23 A35931C Lys751Gln, XPD-exon 10 G23591A
Asp312Asn), glutathione S-transferase µ 1 (GSTM1-active
or -null) and Fas-A670G (also know as Apo1/CD95 one of
the most important genes involved in the apoptotic
pathway) on anti-benzo[a]pyrenediolepoxide (BPDE)-
DNA adduct levels from the lympho-monocytes (LMF)
of highly PAH-exposed coke oven workers were eval-
uated. The sample cohort included 67 male Polish
coke-oven workers (67% current smokers), all belong-
ing to the high exposure group (individual urinary
post-shift 1-pyrenol >2.28 µmol/mol creatinine, the
proposed BEI). The bulky anti-BPDE-DNA adduct
levels were detected by HPLC/fluorescence analysis
and genotypes by PCR-RFLP.
The low DNA repair capacity of XPC-PAT +/+ and XPA-
A23A genotypes significantly (p<0.05) double up the
mean anti-BPDE-DNA adduct levels. As already
reported in our previous studies DNA adducts are also
strongly influenced by GSTM1 genotype (p<0.05). The
higher frequencies of workers with unfavourable XPC-
PAT +/+ and XPA- A23A NER genotypes, alone or com-
bined with GSTM1-null belonged to the tertile with the
highest adduct level (> 4.11 adducts/ 108 nucleotides,
p<0.01). The few subjects with the unfavourable geno-
typic combination, XPC- PAT +/+ or XPA- A23A jointly to
GSTM1-null and Fas A670A, all belonged to the tertile
with highest adduct level. No association of dietary
habits and tobacco smoking with anti-BPDE-DNA
adducts in the LMF of coke oven workers was found.
The modulation of anti-BPDE-DNA adducts in the
LMF, by GSTM1-null and some low-capacity geno-
types of NER, may be considered as genetic suscepti-
bility factors capable of modifying the individual
response to the high PAH(BaP) genotoxic exposure
and the consequent cancer risk in coke oven workers.
PS1 - Poster Session 1
Poster Presentation
PW3018
GENETIC POLYMORPHISM OF XENOBIOTIC META-
BOLISING ENZYMES AND GENOTOXICITY OF
STYRENE
M Migliore1, A Naccarati1, F Mercati1, G de Palma2,
P Manini2, E Scotti3, R Andreoli3, A Mutti2
1 University of Pisa, PISA, Italy2 University of Parma, PARMA, Italy3 ISPESL, PARMA, Italy
We studied the modulating role of the genotypes
involving polymorphisms of xenobiotic metabolising
enzymes (XME) in the genotoxic responses associated
with occupational exposure to styrene.
A cross-sectional study was performed on 46 styrene-
exposed workers (26 males); 98 styrene unexposed
healthy subjects were also enrolled as control group.
Either the main [mandelic and phenylglyoxylic acids
(MA and PGA)] and the minor [4-vinylphenol (4-VP)]
styrene metabolites were determined by LC/MS/MS
on urine samples. The induction of MN on peripheral
blood lymphocytes was evaluated by the citokinesis-
block micronucleus assay. FISH analysis with a pan-
centromeric probe was used to assess the frequency
of centromere-positive MN (C+MN). Genetic polymor-
phism of GSTA1, GSTM1, GSTT1, GSTP1, EPHX1 and
NQO1 were characterised by PCR based methods on
workers’ blood samples only.
Workers expressed higher MN (but not C+MN) values
than controls. Among female workers, MN was
dependent from 4-VP values and NQO1 status, where-
as among males the only significant predictor was
age. The percentage of C+MN cells was independent
from exposure and modulated by age and GSTA sta-
tus in males and by alcohol, GSTM1, GSTT1, GSTP1 and
NQO1 in females.
In the present study, styrene exposure was associated
with clastogenic effects. Such effects were sex-
dependent, being related to urinary 4-VP and modu-
lated by the NQO1 polymorphism in female, but not
in male workers. Aneuploidogenic changes were
independent from styrene exposure and modulated
in the two sexes by different combinations of poly-
morphic XME. The limited sample size does not allow
firm conclusions on defined patterns of XME respon-
sible for such alterations.
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PS1 - Poster Session 1
Poster Presentation
PW3019
MUTAGEN SENSITIVITY OF PATIENTS WITH HEAD
AND NECK CANCER OF DIFFERENT ANATOMICAL
REGIONS
G Szekely, E Remenar, M Kasler, S Gundy
National Institute of Oncology, BUDAPEST, Hungary
The bleomycin (BLM) sensitivity assay measures the
degree of cancer susceptibility on a genetic basis. The
elevated number of BLM-induced chromatid breaks
per cell (b/c) shows that patients with head and neck
squamous-cell carcinoma (HNSCC) are more sensitive
to the mutagen than the healthy controls. However,
there might be a heterogeneity of neoplastic process-
es in HNSCC of different anatomical regions, because
the areas have different duration of contacts with muta-
gens. The aim of this study was to examine whether the
mutagen sensitivity of patients is different with various
localisations of HNSCC. Conventional chromosome
analysis and BLM assay were carried out in HNSCC
patients, healthy non-smokers and non-drinkers, and
smokers but non-drinkers. The aberrant cell frequen-
cy in HNSCC patients (2.78%) and healthy smokers
(2.63%) was similar, and higher than that in non-
smoker controls (2.25%). These results indicate that
the spontaneous rate of aberrations and cancer risk
are clearly associated with mutagenic effect of tobac-
co use. Significant differences were also found in b/c
values between patients (1.11 ± 0.39) and controls (0.97
± 0.34) when overall BLM-sensitivity was studied, and
this phenomenon also demonstrated an increased
cancer risk (OR=1.83). Furthermore, patients with
HNSCC of different anatomical regions showed dif-
ferent mutagen sensitivities: b/c values of those with
oral cavity, oropharynx and hypopharynx cancers dif-
fered significantly from those of controls, but those of
patients with larynx tumour did not. Patients with
tumours of oral cavity (without lip) had the highest
cancer risk (OR=2.05) when compared to those with
other anatomical sites. These data suggest that the
mode of action of mutagens is more complex in upper
aerodigestive tract than it was previously thought.
Moreover, the concentration and the synergism of the
mutagens (alcohol, tobacco and dietary factors) is
probably more definitive in this location than in the
lower parts of the aerodigestive tract.
PS1 - Poster Session 1
Poster Presentation
PW3020
ROLE OF GST AND NAT2 POLYMORPHISMS IN
THYROID CANCER PATIENTS
R Marcos1, A Hernandez1, N Xamena1, J Surralles1,
A Creus1, P Galofre2, R Marcos1
1 Universitat Autonoma Barcelona, CERDANYOLA
DEL VALLES, Spain2 Hosp. Unive. Vall d'Hebron, BARCELONA, Spain
The existence of different genetic polymorphisms in
human populations has proven to be a susceptibility
factor for different tumours. To link thyroid cancer
incidence to genetic susceptibility biomarkers, poly-
morphisms at the GSTM1, GSTT1, GSTP1 and NAT2
genes have been determined for 176 thyroid cancer
patients and 167 controls. The results indicate that,
according to the calculated odd-ratios, the frequencies
of the different GST genotypes found in the group of
cancer patients do not significantly differ from those
values obtained in the controls. Nevertheless, for the
NAT2 polymorphisms, the allele NAT2*5 is significant-
ly linked to cancer incidence with a high risk in
NAT2*5 +/+ individuals.
Since thyroid patients are therapeutically treated
with radioiodine, it was analysed whether the sensi-
tivity to radiation was modulated by genotype. In 30
patients, the MN frequency before and after exposure
was determined. The results indicate that none of the
polymorphisms studied show any kind of association
with the basal level of micronuclei. When the same
patients were followed after radioiodine exposure, a
significant increase in the frequency of MN was
observed in all of them, indicating genotoxic activity
of the ionising radiation exposure. The increase in the
MN frequency was not associated with any of the GST
polymorphisms evaluated. Nevertheless, the pres-
ence of slow acetylator genotypes and, in particular,
the presence of the NAT2*7 allele were significantly
associated with a lower increase of the MN frequency
after radioiodine treatment.
95
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PS1 - Poster Session 1
Poster Presentation
PW3021
DNA DAMAGE AND DNA REPAIR POLYMORPHISMS
IN HEALTHY INDIVIDUALS
A Zijno1, F Marcon1, P Leopardi1, S Rossi1, CA Andreoli2,
L Conti1, R Galati3, A Verdina3, R Crebelli1
1 Istituto Superiore di Sanita, ROME, Italy2 Eti SpA, ROME, Italy3 Istituto Regina Elena, ROME, Italy
The high variability observed among individuals in
DNA repair capacity (DRC) may result in different
response to DNA damage and thus different suscepti-
bility to environmental and occupational carcino-
gens. Apart from subjects affected by pathological
syndromes, healthy individuals also differ in intrinsic
DNA repair capacity that has been shown to be mod-
ulated by single nucleotide polymorphisms. These
polymorphisms are generally associated with only
moderate structural modifications of repair enzymes
but are prevalent in the population and may con-
tribute to the overall population risk of cancer. In the
present study, the relationships among polymor-
phisms in DNA repair genes and biomarkers of geno-
toxicity, such as SCE, micronuclei, single strand
breaks have been analysed in a healthy population.
The population (169 subjects) was genotyped for rele-
vant genes involved in different DNA repair path-
ways: APE1 (Asp148Glu) and XRCC1 (Arg399Gln and
Arg280His), key enzymes in base excision repair
(BER); XRCC3 (Thr241Met), involved in homologous
recombinational pathway for repairing double-
strand breaks; XPD (Lys751Gln) that participates in
nucleotide-excision repair (NER).
Results: Allelic frequencies were in the range
described for Caucasian population. All genotype
distribution was compatible with Hardy-Weinberg
equilibrium. Among the studied polymorphisms,
non-parametric test indicated that only the XPD poly-
morphism may modulate DNA damage. SCE frequen-
cies in double variant, heterozygous and wild type
were 5.63, 5.45 and 5.13, respectively (p=0.061). SCEs in
double variant were significantly higher than in wild
type (p=0.033) as well as in variant carriers (variant
homozygous plus heterozygous) respect to wild type
(p=0.32). A slight not significant increase of SCE in
relation to XPD polymorphism was observed in vari-
ant carriers respect to wild type in smoker group
(6.08 and 5.57, respectively, N=56, p=0.089). Overall
evaluation of these data indicated a slight role of the
studied DNA repair polymorphisms on basal and
smoking related DNA damage in the investigated
population.
PS1 - Poster Session 1
Poster Presentation
PW3022
GERMLINE MUTATIONS OF HMSH2, HMLH1 AND
HMSH6 IN 48 CHINESE HEREDITARY NONPOLY-
POSIS COLORECTAL CANCER FAMILIES
H Yan, J HeiYing, S ShuHan, H Yan
Second Military Medical University, SHANGHAI,
China
Hereditary nonpolyposis colorectal cancer (HNPCC)
(MIM # 114500), also know as Lynch syndrome, is the
most frequent autosomal dominant predisposition to
the development of colorectal cancer. It is caused by
germline mutations in human homologues of the
bacterial mismatch repair (MMR) genes MutL and
MutS: hMSH2, hMLH1, hMSH6, hPMS1, hPMS2. Up to
date, more than 300 different predisposing muta-
tions have been identified; more than 90% of these
mutations were in hMLH1, hMSH2 and hMSH6. In
present study, we have screened 48 Chinese HNPCC
families fulfilling the Amsterdam criteria for muta-
tions in the DNA-mismatch repair genes hMLH1,
hMSH2 and hMSH6, using dHPLC and direct DNA
sequencing. The families were selected on the basis of a
family history of HNPCC-related tumors. Furthermore,
we required that tumor tissue from at least one indi-
vidual in the family had to display microsatellite
instability. We identified 10 novel germ-line muta-
tions in 10 families. Four families had mutations in
hMLH1, three mutations in hMLH2 and three muta-
tions in hMSH6. Among these mutations, 2 are splic-
ing site mutations, 4 are nonsence mutation and 4 are
frameshift mutations, the pathogenic effects of these
mutations in the process of carcinogenesis were also
analyzed.
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abstractboek 20-10-2004 10:28 Pagina 96
PS1 - Poster Session 1
Poster Presentation
PW3023
RELATIONSHIP BETWEEN SMOKING-RELATED
BULKY DNA ADDUCTS AND P53 ARG72PRO GENE-
TIC POLYMORPHISM IN LUNG CANCER PATIENTS
L Anna
National Center for Public Health, BUDAPEST,
Hungary
P53 tumour suppressor gene has a regulatory role in
various cellular mechanisms including DNA repair.
We hypothesize that its genetic polymorphism is a
potential modulator of the level of DNA damage
induced by smoking in pulmonary tissues. The aim of
the study was to investigate the association between
p53 Arg72Pro genetic polymorphism and smoking-
related bulky DNA adducts, and its modulation by
XRCC1 Arg399Gln DNA repair genotype in a study
population of lung cancer patients who underwent
lung resection (n=148). Smoking status was self-
reported by the patients. p53 Arg72Pro and XRCC1
Arg399Gln genotype was determined by PCR-RFLP
methods. Levels of bulky DNA adducts were deter-
mined by 32P-postlabelling in macroscopically nor-
mal bronchial tissues. Among current smokers and
short-term former smokers who stopped smoking
less than a year before surgery, those individuals who
were heterozygote or homozygote with the variant
Pro allele had elevated level of DNA adducts com-
pared to those who were of Arg/Arg genotype
(p=0.04). There was a statistically significant dose-
related increase of DNA adduct level up to 20 ciga-
rettes/day among current smokers with the variant
Pro allele (p=0.0004), whereas bronchial DNA adduct
level was not apparently influenced by cigarette
smoking in the subgroup of Arg/Arg homozygous
individuals. XRCC1 Arg399Gln polymorphism did not
apparently influence the impact of p53 genotypes on
bronchial DNA adduct levels. The results suggest that
p53 72Pro allele is a risk factor for increased primary
DNA damage in the bronchial tissue by smoking.
Further analyses are in progress to confirm the obser-
vations in a larger study population.
Supported by Hungarian OTKA T034616 and HU-FIN
S&T Research Fund SF-02/01.
PS1 - Poster Session 1
Poster Presentation
PW4001
HYPERSENSITIVITY TO MITOMYCIN C-INDUCED
SISTER CHROMATID EXCHANGES (SCE) IN LYM-
PHOCYTES FROM HYPERBARIC OXYGEN EXPOSED
PATIENTS
Y Duydu1, A Aydin2, A Dur1, A Eken2, K Dundar2,
G Uzun2
1 Ankara University, ANKARA, Turkey2 Gata, Centre of HBOT, ANKARA, Turkey
Hyperbaric oxygen therapy (HBOT) has been success-
fully used for the treatment of a various clinical con-
ditions such as wound healing, carbon monoxide
poisoning, soft tissue infections, and radiation necro-
sis. However, as well known from the previously pub-
lished studies, HBO also leads to an increase in the
reactive oxygen species in the blood. This situation
resulted in an induction of DNA damage in leuco-
cytes as reported in many earlier studies. However,
studies on possible interactions between HBO and
other genotoxic chemicals are not available so far. In
this study, we investigated the SCE induction in
Mitomycin C (MMC) exposed and unexposed blood
samples from patients undergoing HBOT. Twelve vol-
untary patients (wound healing) were enrolled in
this study and exposed to 10 consecutive HBO treat-
ments (1 session/day). The patients inhaled 100% oxy-
gen under a pressure of 2.5 ATA. The mean SCE/Cell
level of patients were 5.92±1.37 before undergoing
HBOT. After the first session of HBOT the mean
SCE/Cell level reached the maximum (16.4±2.19). After
fifth (14.34±2.09) and last session (12.24±2.27) the
mean SCE/Cell levels were statistically lower (Mann-
Whitney U-test, p<0.05) then the first session of
HBOT. Nevertheless, the mean SCE/Cell level observed
after the last session of HBOT was still statistically
higher (Mann-Whitney U-test, p<0.05) than the
beginning of the HBOT. The same blood samples were
also exposed to MMC in-vitro at two doses (20, 40
ng/ml). MMC exposed samples (at both doses)
reached the maximum SCE/Cell levels after the first
session of HBOT as were observed in MMC unexposed
samples. However, there was no statistically signifi-
cant decrease in mean SCE/Cell levels of MMC
exposed samples after the fifth and even after the last
session of the HBOT. Such a result might be evaluated
as a hypersensitivity to MMC-induced SCE in HBOT.
97
abstractboek 20-10-2004 10:28 Pagina 97
PS1 - Poster Session 1
Poster Presentation
PW4002
CYTO-AND GENOTOXIC POTENTIAL OF BETA-
CAROTENE CLEAVAGE PRODUCTS
PM Eckl1, A Alija1, N Bresgen1, O Sommerburg2,
W Siems3
1 University of Salzburg, SALZBURG, Austria2 University of Ulm, ULM, Germany3 Herzog-Julius Hospital, BAD HARZBURG, Germany
Free radical attack on b-carotene results in the forma-
tion of high amounts of cleavage products with
prooxidant activities. Since this finding may give an
explanation for the increased risk of lung cancer in
smokers observed in the Alpha-Tocopherol Beta-
carotene-Cancer prevention (ATBC) study and the
beta-Carotene and RETinol Efficacy (CARET) Trial a b-
carotene cleavage products mixture (CP), apo8'-
carotenal (apo8') and b-carotene were tested in the
highly sensitive primary rat hepatocyte genotoxicity
assay. The endpoints tested were: the mitotic index,
the percentage of necrotic and apoptotic cells,
micronucleated cells, chromosomal aberrations and
SCE (sister chromatid exchanges). Our results indicate
a genotoxic potential of both CP and apo8' already in
the concentration range of 100 nM and 1 µM, i.e. at
pathophysiologically relevant levels of b-carotene
and b-carotene breakdown products. A 3h treatment
with CP induced statistically significant levels of
micronuclei at concentrations of 0.1, 1 and 10µM, and
chromosomal aberrations at concentrations of 1, 5 and
10µM. Apo8' induced statistically significant levels of
micronuclei at concentrations of 0.1, 1 and 5µM, and
chromosomal aberrations at concentrations of 0.1, 1
and 10µM. In contrast, no significant cytotoxic effects
of these substances were observed. Since b-carotene
induced neither significant cytotoxic nor genotoxic
effects at concentrations ranging from 0.01 up to 10
µM, it can be concluded that most likely b-carotene
breakdown products are responsible for the occur-
rence of carcinogenic effects found for b-carotene in
clinical efficacy and cancer prevention trials.
PS1 - Poster Session 1
Poster Presentation
PW4003
INFLUENCE OF TCDD ON 8-HYDROXY-2'-DEOXY-
GUANOSINE FORMATION IN HUMAN LIVER CELL
LINES
MO Eom, KP Byun, MS Park, OH Kim, HK Jung,
HI Kang
Korea Food and Drug Administration, SEOUL,
South-Korea
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known
to induce a wide spectrum of biological responses,
including changes in gene expression, metabolism,
cell growth and differentiation. However, little is
known concerning the mechanisms that TCDD exerts
its effect at the cellular or molecular level. To enhance
our understanding of toxicity by TCDD at cellular
level, we investigated TCDD-induced responses in
two human liver cell lines, hepatoma HepG2 and non-
tumorigenic fetal hepatic WRL68 cells. We have
found that growth of HepG2 cell was decreased over
50% after treatment with over 1nM TCDD, but TCDD
does not have any effect on proliferation in WRL68
cell. Also, the levels of 8-hydroxy-2'-deoxyguanosine
(8-OHdG) was increased in a time dependent manner
after exposure to 0.1nM TCDD in HepG2 cell. The gene
expressions of 8-oxo-dGTPase (hMTH1) and adenine
DNA glycosylase (hMYH) were increased at 6h follow-
ing exposure to TCDD and then recovered at 24h. The
8-oxoguanine DNA glycosylase (hOGG1) gene expres-
sion was decreased at 72h after treatment with TCDD.
In WRL68 cells, the level of 8-OHdG increased 2 hours
after treatment with 0.1nM TCDD and then returned
to control level at 6 hours. Similarly, increase of the
repair enzymes hOGG1, hMTH and SOD expression
were observed at 6 hours and decreased at 12 hours in
WRL68 cells. These results suggest that the different
levels of 8-OHdG may be attributable to toxicity by
TCDD and enzymes related to oxidative stress may be
responsible to repair of 8-OHdG in our experimental
systems.
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PS1 - Poster Session 1
Poster Presentation
PW4004
ANTIMUTAGENIC PROPERTIES OF NATURAL
ANTIOXIDANTS IN THE ESCHERICHIA COLI K12
REVERSION ASSAY
B Nikolic, J Knezevic-Vukcevic, B Vukovic-Gacic,
D Mitic-Culafic, T Beric-Bjedov, J Stanojevic,
S Stankovic, D Simic
Faculty of Biology, BELGRADE, Yugoslavia
Natural antioxidants, widely distributed in plant
extracts, might protect genomic DNA from lesions
induced by reactive oxygen species, a significant
source of endogenous mutagenesis. The study of their
antimutagenic potential can promote the prevention
of carcinogenesis, immunodeficiency, neurodegener-
ation and aging.
The potential of vitamin E (alpha-tocopherol), essen-
tial oil (EO) of basil (Ocimum basilicum L.) and its
major constituent linalool to reduce the oxidative
mutagenesis, was studied in the E.coli K12 (arg- in
Arg+) reversion assay. Test A is created to evaluate the
antimutagenic potential against t-butyl hydroperox-
ide (t-BOOH)-induced oxidative mutagenesis and is
performed on repair proficient strain. Test B is per-
formed on mutator strains mutS (deficient in mis-
match repair) and mutT (deficient in removing
8-oxo-G); it is designed to determine the effect of
antioxidants on spontaneous mutagenesis and to
determine the molecular mechanism of its action.
The protective effect of model antioxidant vitamin E
against oxidative mutagenesis was detected in both
tests, but the strongest inhibition was detected in
mutS strain (76%). EO of basil, as well as linalool,
exhibited moderate inhibitory potential in Test A
(about 35%), while powerful inhibition was detected
with both test substances in mutS strain (over 60%).
EO of basil showed no effect on mutagenesis mediat-
ed by 8-oxo-G lesions, while investigation of linalool
in mutT strain is under way.
PS1 - Poster Session 1
Poster Presentation
PW4005
ANTIMUTAGENIC PROPERTIES OF NATURAL ANTI-
OXIDANTS IN THE WP2 ANTIMUTAGENICITY TEST
B Vukovic-Gacic, J Stanojevic, J Knezevic-Vukcevic,
D Mitic-Culafic, T Beric-Bjedov, B Nikolic, S Stankovic,
D Simic
Faculty of Biology, BELGRADE, Yugoslavia
Oxidative damage to DNA by reactive oxygen species
is very important in mutagenic, carcinogenic, and
aging processes. The study of active substances from
medicinal and aromatic plants has revealed that
many of them possess antioxidative activity and may
be potent inhibitors of mutagenesis and carcinogen-
esis.
We tested essential oil (EO) of basil (Ocimum
basilicum L.) and its major monoterpene – linalool for
antimutagenesis due to their antioxidative proper-
ties. Vitamin E (alpha-tocopherol), potent scavenger
of reactive oxygen species, was used as the model
antioxidant.
The potential of antioxidant to reduce spontaneous
and t-butyl hydroperoxide (t-BOOH)-induced oxida-
tive mutagenesis were screened using strains E. coli
IC185 (WP2 lamB+) and IC202 (WP2 oxyRlamB+/pKM101).
Antimutagenic potential was observed by monitoring
trpE65 in Trp+ reversions. Strain IC202, highly sensi-
tive to oxidative damage due to its deficiency in the
OxyR function, shows increased mutability with
respect to the OxyR+ strain.
Vitamin E inhibited induced mutagenesis in both
strains (64% in IC202 and 43% in IC185). Slight inhibi-
tion of spontaneous mutagenesis was detected in
oxyR strain (17%). After validation with vitamin E, we
used highly sensitive oxyR strain for further investi-
gation.
A significant reduction of spontaneous mutagenesis
was obtained with both EO (63%) as well as linalool
(62%). The stronger antimutagenic potential against
induced mutagenesis was detected with EO (77%) in
respect to linalool (68%).
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PS1 - Poster Session 1
Poster Presentation
PW4006
EVALUATION OF OXIDATIVE DNA DAMAGE IN
ROME AIRPORT PERSONNEL
CLU Ursini1, D Cavallo1, C Chianese1, A di Francesco1,
M Gismondi2, S Iavicoli1
1 ISPESL, MONTEPORZIO CATONE, ROME, Italy2 Occup.Medic.Rome airport, ROME, Italy
Airport personnel is occupationally exposed on flight
lines and during aircraft routine maintenance proce-
dures to several polycyclic aromatic hydrocarbons
(PAH) produced by jet fuel, diesel fuel or kerosene
combustion. We evaluated, by enzyme Fpg-modified
comet test, direct and oxidative DNA damage on exfo-
liated buccal cells and lymphocytes of airport workers
(n=15) occupationally exposed to complex mixtures of
PAH and in a selected control group (n=7). For each
subject we calculated tail moments (product of relative
tail intensity and length) of 50 comets from fpg-
enzyme treated cells (TMenz) and from untreated
cells (TM), which indicate oxidative and direct DNA
damage respectively. For exfoliated cells, both TM and
TMenz mean values were higher in the exposed than
in the controls (TM p=0,12; TMenz p=0,05). For lym-
phocytes no significant differences were shown in
the exposed respect to control (TM p=0,4; TMenz
p=0,14). The presence of oxidative DNA damage was
evaluated in each subject considering TMenz/TM
ratio. When this ratio was higher than 1,5 the subject
was estimated to have oxidative damage. We found
the presence of oxidative DNA damage in 47% and in
13% respectively on lymphocytes and on exfoliated
cells of exposed in respect to the absence of oxidative
damage of controls. These results demonstrate a
great sensitivity of exfoliated cell to point out direct
DNA damage probably induced by substances con-
tained in the PAH mixture able to directly damage
DNA because they are the first target of interaction
with such substances. While the high presence of
subjects with oxidative DNA damage shown on lym-
phocytes could be related to effects of diol-epoxides
which are oxidant metabolites of PAH. The perform-
ing of enzyme-modified comet assay on both exfoli-
ated buccal cells and lymphocytes seems to be a
useful approach to study populations chronically
exposed to PAH.
PS1 - Poster Session 1
Poster Presentation
PW4007
EXERCISE-INDUCED OXIDATIVE STRESS AND
PARP-1 ACTIVATION IN COPD PATIENTS BEFORE
AND AFTER REHABILITATION
GJ Hageman, EM Mercken, AMWJ Schols,
GJJM Haenen, EFM Wouters, A Bast
Maastricht University, MAASTRICHT, The Netherlands
Previously, exercise-induced oxidative stress has
been shown after streneous exercise in patients with
COPD (chronic obstructive pulmonary disease). We
compared the exercise-induced systemic and local
oxidative stress response between a maximal and sub-
maximal cycle ergometry test and studied the influ-
ence of pulmonary rehabilitation. 11 (6M/5F) stable
COPD patients (FEV1=40 13%) were included. All sub-
jects performed an incremental cycle ergometry test
until exhaustion and a continuous work rate test at
60% of peak work load before and after 8 weeks pul-
monary rehabilitation. The standard Comet assay
and immunecytochemical staining for the presence
of poly(ADP-ribose) polymerase (PARP-1) polymers
were used to evaluate reactive oxygen species (ROS)-
induced DNA damage in peripheral blood lympho-
cytes. Breath condensate hydrogen peroxide was
measured as marker of pulmonary oxidative stress.
Measurements were done before, immediately after
and after 4h, and 24h.
Before rehabilitation: ROS-induced DNA strand
breaks were detected with the Comet assay immedi-
ately after both exercise tests (p<0.05), while breath
H(2)O(2) increased only immediately after and 4 h
after maximal exercise (p<0.05). The percentage of
PARP-polymer positive cells was significantly
increased only immediately after the submaximal
work load.
After rehabilitation: A significant improvement was
seen in peak work load (+24%, p<0.001) and in sub-
maximal exercise duration (+73%, p<0.002).
Remarkably, for the maximal exercise test a similar-
ly increased oxidative stress response was found with
the Comet assay, while no increase in ROS-induced
DNA damage or PARP-activation was detected after
the submaximal exercise test. These data indicate
that pulmonary rehabilitation positively influences
exercise-induced oxidative stress. Additional analy-
ses will possibly elucidate whether this positive effect
is caused by an improved antioxidant status.
Sponsored by a University Hospital Grant.
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abstractboek 20-10-2004 10:28 Pagina 100
PS1 - Poster Session 1
Poster Presentation
PW4008
SIGNIFICANT EFFECT OF ASCORBIC ACID ON DNA
CONFORMATION IN RELATION TO ITS ANTIOXI-
DANT ACTIVITY
YY Yoshikawa
Nagoya Bunri College, NAGOYA, Japan
Ascorbic acid is often regarded as an antioxidant in
vivo, where it protects against cancer by scavenging
DNA-damaging reactive oxygen species. In the pres-
ent paper, we will report another potent scenario on
the protective effect of ascorbic acid through signifi-
cant change in the higher order structure of DNA.
Recently, we examined the effect of ascorbic acid on
the higher-order structure of DNA through single
DNA observation with fluorescence microscopy, and
found that ascorbic acid generates a pearling struc-
ture in single giant DNA molecules, where elongated
and compact parts coexist along a molecular chain.[1]
The results of observations with electron microscopy
and atomic force microscopy indicate that the com-
pact parts assume a loosely packed conformation.
As the extension, here we study the protective effect
against double strand breakage by reactive oxygen at
different concentrations of ascorbic acid, in relation
to the change of the higher order structure of giant
DNA molecules. We have performed the real time
observation on the double strand breakage reaction
on individual giant DNA molecules by use of fluores-
cence microscopy. We have found that the double
strand break is markedly protected when coexisting
ascorbic acid is over millimolar concentrations. It is
found that such a protective effect of ascorbic acid
corresponds well to the above mentioned change on
the higher order structure of giant DNA molecules.
It has been reported that human circulating immune
cells, such as neutrophils, monocytes and lympho-
cytes, accumulate ascorbic acid in millimolar concen-
trations. Therefore, it is suggested that the ascorbic
acid concentration that induced the large conforma-
tional change in DNA in this study may be of physio-
logical significance.
Ref. [1] Y. Yoshikawa, et a., Eur.J.Biochem., 270,
3101(2003).
PS1 - Poster Session 1
Poster Presentation
PW4009
THE MODULATING EFFECTS OF THYME AND ITS
MAJOR INGREDIENTS ON OXIDATIVE DNA DAM-
AGE
A Basaran, S Aydin
Hacettepe University Faculty of Pharmacy, ANKARA,
Turkey
Thyme has been commonly used in foods mainly for
its flavour, aroma and preservation and also in folk
medicine for years. Thyme essential oil and its ingre-
dients have been shown to exhibit a range of biologi-
cal activities. Antibacterial, antifungal, antitussive,
antispasmodic, antiplatelet aggregation and also
antioxidant activities were reported mostly with thy-
mol and carvacrol. On the other hand very few stud-
ies have been performed on the mutagenic and/or
antimutagenic effects of thyme and its ingredients.
In the present study the mutagenic potential of major
compound of thyme oil, i.e. thymol, carvacrol, and g-
terpinene and the methanolic extracts of thyme,
were investigated by single cell gel electrophoresis in
human lymphocytes and the effects of these sub-
stances on H2O2 (0.1 mM) induced oxidative DNA
damage were evaluated. No additional DNA strand
breakage was observed at thymol and g-terpinene
concentrations below 0.1 mM but at the higher con-
centrations of 0.2 mM significant increases were seen
in DNA damage. Carvacrol, which is an isomer of thy-
mol, seemed to induce DNA damage also in a dose-
manner and below concentrations of 0.1 mM no
damage in lymphocytes were seen whereas at the
higher concentrations carvacrol induced DNA dam-
age. The n-hexane and ethyl acetate fractions pre-
pared from the concentrated aqueous methanolic
extract of thyme showed no significant DNA damage
within the concentration range of 0.0075-7.5 mg/L,
and 10-50 mg/L respectively. Thymol and carvacrol at
the concentrations below 0.1 mM and 0.05 mM
respectively protected lymphocytes against H2O2
induced DNA damage but no protective effect was
observed with g-terpinene. N-Hexane and ethyl
acetate fractions of thyme containing phenolic, ter-
penic substances were also found to prevent the
oxidative DNA damage in human lymphocytes and it
can be concluded that phenolic compounds such as
thymol and carvacrol were responsible for the antiox-
idant effects of thyme.
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PS1 - Poster Session 1
Poster Presentation
PW4010
MODULATION OF OXIDATIVE CELL DAMAGE BY
POLYPHENOLIC APPLE JUICE COMPOUNDS/
EXTRACTS IN HUMAN COLON CELLS
CJ Janzowski1, S Schaefer1, M Baum1, F Will2,
H Dietrich2, G Eisenbrand1
1 University of Kaiserslautern, KAISERSLAUTERN,
Germany2 Institute of Enology and Beverages, GEISENHEIM,
Germany
Diets rich in fruits and vegetables are associated with
a lower risk of tumor induction in the intestine and
other sites. Many food ingredients have been claimed
to exhibit anticarcinogenic potential. We hypothe-
sized that apple juice with especially high amounts of
antioxidative polyphenols might be able to protect
the intestine and colon against oxidative cell damage.
We used the human colon cancer cell lines Caco-2 and
HT29. Cells were preincubated with selected polyphe-
nolic constituents/extracts for 24h. (Oxidative) DNA
damage was subsequently induced by treatment
with menadione (1h) and was detected by single cell
gel electrophoresis with/without additional treat-
ment with the repair enzyme formamidopyrimidine-
DNA-glycosylase (FPG). Lipid peroxidation was
induced by H2O2/ Fe2+ and monitored by HPLC/fluo-
rescence of the thiobarbituric acid derivative of mal-
ondialdehyde (MDA). Stability of parent compounds
during incubation was studied by HPLC/UV.
When incubating cells with menadione alone (1h,
37°C), higher concentrations were needed for HT29
(20µM) than for Caco-2 (3µM) cells to generate a simi-
lar extent of DNA damage. Preincubations with
quercetin and phloretin induced concentration
dependent modulation of DNA damage, HT29 being
more sensitive than Caco-2 cells. The respective glyco-
sides rutin and phloridzin (1-100µM) were not effec-
tive, epicatechin, chlorogenic acid and caffeic acid
were weakly effective. Preincubation with quercetin
increased the repair of oxidative DNA damage, the
extent of induced MDA formation was decreased.
During incubation of HT29, quercetin was rapidly
eliminated from the medium DMEM (t1/2: 117min);
rutin was eliminated much slower (t1/2>24h). Apple
juice extract efficiently diminished (oxidative) DNA
damage in HT29 and Caco-2 cells at 50µg/mL and
100µg/mL, respectively.
In conclusion, apple juice extract as well as quercetin
and phloretin are effective antioxidants, reducing
oxidative cell damage in human colon cells in a con-
centration dependent manner. (Support: BMBF grant
no. 01EA 0101).
PS1 - Poster Session 1
Poster Presentation
PW4011
DNA DAMAGE ASSESSMENT IN TRANSGENIC
TOBACCO LINES AFTER EXPOSURE TO OXIDATIVE
STRESS
C Pellacani, A Mancini, FM Restivo, A Buschini, P Poli,
C Rossi
Universita di Parma, PARMA, Italy
Every year, environmental stress causes considerable
losses in crop quality and productivity. One of the
important mechanisms by which plants are damaged
during adverse environmental conditions is the
excessive production of ROS. Plants have evolved non-
enzymic and enzymic protection mechanisms that
efficiently scavenge ROS. Major ROS-scavenging
mechanisms of plants include superoxide dismutase
(SOD) and catalase (CAT). The aim of this work was to
verify the leaves cells sensitivity to oxidative stress in
terms of DNA damage in SR1 Nicotiana tabacum wt
line and in transgenic derivative lines (kindly provid-
ed by Dr Frank van Breusegem, Ghent University,
Belgium) CAT1, SOD+ and SOD-. CAT1 is an antisense
line for the N.plumbaginifolia catalase isoform 1 and
contains approximately 10% of normal catalase activ-
ity respect to SR1. SOD+ is an over-expressing line for
the N. plumbaginifolia mitochondrial SOD Mn-iso-
form, with enzyme activity ≈2 fold higher than in
untransformed plant. SOD- is a new transgenic line
obtained with antisense technology. After treatment
with oxygen peroxide for 2, 3 and 4hs at 25°C, the
leaves of 70 day-old plants were sliced to directly col-
lect nuclei for the Comet assay. DNA migration, as
DNA damage parameter, and cells with complete
DNA migration (GC), as necrosis/apoptosis parame-
ter, were recorded. Oxygen peroxide treatment
increased both the parameters in all the tobacco
lines. SOD+ showed behaviour similar to the wt line
SR1 both for DNA damage and GC induction; the oxi-
dant treatment produced GC more than DNA damage
in CAT1, whereas both the effects were evident in
SOD-. A time-dependent increase of DNA migration
values is observed still after 3h-exposure with subse-
quent decrease after 4h-exposure and simultaneous
increase of GC percentage. In conclusion, DNA dam-
age: CAT1 significantly less sensitive (P<0.05) than the
other lines; GC: SOD- more sensitive than SR1 and
SOD+ (P<0.01), and CAT1 than SR1 (P<0.05).
102
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PS1 - Poster Session 1
Poster Presentation
PW4012
GREEN TEA POLYPHENOLS, FLAVONOIDS, VITA-
MINS AND LACTOSERUM: PERSPECTIVES FOR COM-
BINATION CHEMOPREVENTION
TMCM de Kok, J de Jonge, MH van Herwijnen,
JJ Briedé, LC Wilms, LM Maas, P Kempers,
JCS Kleinjans
Maastricht University, MAASTRICHT, The Netherlands
Many naturally occurring dietary compounds protect
against oxidative and alkylating DNA damage. These
chemopreventive effects can be induced either by the
inhibition of radical formation, direct radical scav-
enging, regeneration of antioxidants or modulation
of bioactivating or detoxifying enzymes. To achieve
maximal chemopreventive effects, a balanced mix-
ture of different compounds, combining several pro-
tective mechanisms, may give better results than the
individual factors. In this respect, Rivella green, a pop-
ular Swiss soft drink based on a combination of a
green tea, lactoserum, vitamin C and herbal and fruit
extracts, has in potential a promising chemopreven-
tive formula. Therefore, we investigated the effect of
Rivella green and its individual ingredients on in vitro
radical generation, induction of oxidative DNA dam-
age (8-oxodG) and DNA alkylation by benzo[a]pyrene
(BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine (PhIP). The effects were compared to those
of two model green tea polyphenols, EGCG and ECG,
and quercetin as model flavonoid.
The green tea extract, lactoserum, sugar free base
(containing the herbal extract) and the combination
as present in the soft drink itself showed strong
inhibiting effects on superoxide radical formation
and induction of 8-oxodG by a superoxide generating
system. Even though lactoserum showed stimulation
of hydroxyl radical formation by hydrogen peroxide
(probably caused by stimulation of the Fenton reac-
tion by traces of transition metals in lactoserum), lev-
els of 8-oxodG were reduced; this indicates the
presence of other antioxidative agents. Rivella green,
the green tea extract, lactoserum and the sugar free
base showed furthermore inhibition of DNA alkyla-
tion by both BaP and PhIP. Based on these findings
and taking into account that green tea polyphenols
and flavonoids may also protect against DNA alkyla-
tion in vivo by modulation of phase I and II detoxify-
ing enzymes, a human dietary intervention study
appears relevant to evaluate the full potential of this
specific combination of chemopreventive com-
pounds.
PS1 - Poster Session 1
Poster Presentation
PW4013
OXYGEN RADICAL SCAVENGING ACTIVITY AND
PREVENTION OF DNA STRAND BREAKAGE BY
QUERCETIN IN HUMAN LYMPHOCYTES
JJ Briedé, LC Wilms, S Peeters, EJC Moonen,
JCS Kleinjans
Maastricht University, MAASTRICHT, The Netherlands
Quercetin is a dietary flavonoid present in plant-
derived nutrients, and reported to possess many bio-
logical activities including pro- and anti-oxidative
properties. Although the compound has been exten-
sively studied for its pro- and anti-oxidative proper-
ties in vitro, cellular (anti-)oxidative activity of
quercetin needs further investigation. Therefore, we
treated human peripheral blood lymphocytes with
quercetin and followed cellular uptake kinetics by
HPLC-UV detection. Subsequently, the oxygen free rad-
ical scavenging capacity and prevention of oxygen free
radical-induced DNA strand breakage of quercetin in
lymphocytes was determined. Incubations were per-
formed at concentrations ranging from physiological
relevant blood plasma levels up to its maximal solu-
bility in watery solvents (1 - 100 µM) and were fol-
lowed by several washing procedures. It is shown that
2% of extracellular quercetin is taken up by lympho-
cytes. Subsequently, quercetin pre-incubated lym-
phocytes were exposed to increased oxidative stress
by producing either hydroxyl or superoxide anion
radicals via respectively H2O2/Fe2+ or phenazine
methosulfate/NADH containing systems. Electron
spin resonance (ESR) spectrometry in combination
with spin trappings technique revealed that these
quercetin-treated cells have an increased capability
to scavenge extracellular produced hydroxyl- or
superoxide anion free radicals. Moreover, quercetin
pre-incubated lymphocytes challenged with these
oxygen free radical producing systems showed a
decrease in DNA strand breaks using the alkaline sin-
gle cell electrophoresis assay (COMET). Altogether,
these experiments show that the uptake of quercetin
by human blood lymphocytes results in improved cel-
lular anti-oxidant capacity and DNA protective prop-
erties in circumstances of increased oxidative stress.
103
abstractboek 20-10-2004 10:28 Pagina 103
PS1 - Poster Session 1
Poster Presentation
PW4015
DNA DAMAGE IN COLON, LIVER AND LUNG AFTER
ORAL EXPOSURE TO DIESEL PARTICLES IN RATS
M Dybdahl1, AK Müller1, E Olatunde Farombi2,
P Møller3, H Wallin4, U Vogel4, L Risom3, H Autrup5,
LO Dragsted1, S Loft3, ML Binderup1
1 Danish Institute for Food and Vet. Research,
SØBORG, Denmark2 University of Nigeria, IBADAN, Nigeria3 University of Copenhagen, COPENHAGEN,
Denmark4 National Institute of Occupational Health,
COPENHAGEN, Denmark5 University of Aarhus, AARHUS, Denmark
Several chemical mutagens and carcinogens, includ-
ing polycyclic aromatic hydrocarbons (PAHs) and
nitrated PAHs, are adsorbed to the surface of diesel
exhaust particles (DEP). DEP can induce formation of
reactive oxygen species and cause oxidative DNA
damage as well as bulky DNA adducts. Lung tissue is
a target organ for DEP induced cancer following
inhalation, but recent studies have provided evidence
that the lung is also a target organ for DNA damage
and cancer after oral exposure to other complex mix-
tures of PAHs. The aim of the present study was to
investigate the genotoxic effect of DEP after oral
administration in terms of markers of DNA damage,
mutations and repair in Big Blue' rats fed a diet with
0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days.
DEP increased the formation of DNA adducts and
DNA strand breaks in colon, liver and lung. The num-
ber of oxidised DNA bases, measured as endoIII and
FPG sensitive sites, was increased in lung tissue, but
not in colon and liver. The lack of effect on DNA base
oxidation could be related to the increased expression
of the repair gene OGG1 observed in colon and liver,
but not in the lung. There was a significant increase
in ERCC1 mRNA in liver at the highest dose level, sug-
gesting a repair response to bulky DNA adducts.
There was no detectable increase in mutation fre-
quency in any of the investigated tissues.
It was shown by the present study that gastrointesti-
nal exposure to DEP at relatively low levels can induce
DNA adducts and oxidative stress resulting in DNA
strand breaks and increased expression of DNA repair
genes locally in colon and systemically in lung and
liver. The data indicate that consideration of oral
exposure should be included in risk assessment of
DEP.
PS2 - Poster Session 2
Poster Presentation
PW5001
DIFFERENTIAL GENE EXPRESSION AFTER TREAT-
MENT WITH 2,3,7,8-TETRACHLORODIBENZO-P-
DIOXIN IN HAIRLESS MOUSE SKIN
TK Ryeom, HI Kang, MK Kang, MO Eom, MS Park,
SW Jee, KS Kwon, MH Kim, OH Kim
Korea Food and Drug Administration, SEOUL,
South-Korea
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) displays
high toxicity in animals and has been implicated in
human carcinogenesis. Despite extensive research,
the mechanisms of TCDD-induced carcinogenesis are
poorly understood. To enhance our understanding of
toxicity mediated through the pathway by which
TCDD stimulates gene expression, we have used two
different microarray hybridization to detect the tran-
scriptional signature in papillomas of hairless mouse
skin induced by two stage carcinogenesis protocol.
We found that 49 and 42 genes among 5,592 genes
(Digital genomics cDNA chip) associated with protein
synthesis, cell organization, lipid transport and oxida-
tive stress in tumor and surrounding regions were
up- or down- regulated two fold or more, respectively.
In addition, 53 genes among 96 genes (pathway-spe-
cific cDNA arrays, SupperArray) involved in cell cycle,
signal transduction, apoptosis, adhesion molecule,
angiogenesis, and invasion were up-regulated in the
tumor surrounding region. In the tumor region, only
a few genes related with cell cycle and oncogenes
were up-regulated but several genes encoding inte-
grin, Akt, Raf were down-regulated. Our data suggest
that TCDD promote to development of skin tumor in
hairless mouse by changes of multiple integrated
network of signaling pathway.
104
abstractboek 20-10-2004 10:28 Pagina 104
PS2 - Poster Session 2
Poster Presentation
PW5002
HEPATOTOXICITY OF COUMARIN IN SANDWICH
CULTURED HEPATOCYTES INVESTIGATED BY
GENE EXPRESSION PROFILING
AS Kienhuis1, HM Wortelboer2, JCS Kleinjans1,
RH Stierum2, JHM van Delft1
1 Maastricht University, ZEIST, The Netherlands2 TNO Nutrition and Food Research, ZEIST,
The Netherlands
Primary hepatocyte cultures are often used to study
the hepatotoxicity of compounds. When primary
hepatocytes are cultured in sandwich configuration,
the viability and morphology of the cultures are
greatly enhanced compared to conventional mono-
layers, making the system more comparable to the in
vivo situation. Still, no ideal in vitro liver system exists.
Toxicogenomics can be used as a tool to analyse toxi-
cology at the molecular level without preconceived
notion on the molecular mechanisms involved in the
mode of action. This will make it possible to compare
the toxin-specific expression profiles in sandwich
cultures in vitro and rat livers in vivo and hence
determine the relevance of these in vitro models in
terms of their predictiveness of molecular alterations
associated with toxicity as they occur in vivo. To
investigate the in vitro - in vivo extrapolation para-
digm in toxicogenomics, coumarin is used as an
example.
In the present study, the in vitro hepatotoxicity of
coumarin was tested in rat hepatocytes cultured in
sandwich configuration. The hepatotoxic effects of
coumarin in rats are mediated by a cytochrome P450
(CYP) dependent conversion of coumarin to coumarin
epoxide and o-HPA. A major problem of in vitro hepa-
tocyte cultures is the rapid decline in CYP activity.
When hepatocytes were cultured in standard medi-
um, no cytotoxicity of coumarin up to 1.8 mM was
observed. Addition of an inducer mixture containing
low concentrations of model inducers phenobarbital,
b-naphthoflavone, and dexamthasone, significantly
increased CYP 2B and CYP 3A enzyme activities com-
pared to maintenance in standard medium. Besides,
the cytotoxicity of coumarin increased significantly
when cells were cultured in inducer-supplemented
medium as determined by MTT reduction and LDH
leakage assays. Future toxicogenomics studies will be
combined with this model to compare the gene
expression profiles of cells cultured in either standard
medium or inducer-supplemented medium.
PS2 - Poster Session 2
Poster Presentation
PW5003
INFLUENCE OF SIX PAHS ON GENE-EXPRESSION
IN HEPG2 CELLS; RELATION TO DNA-ADDUCT
FORMATION AND CARCINOGENICITY
YCM Staal, MH van Herwijnen, FJ van Schooten,
JHM van Delft
Maastricht University, MAASTRICHT, The Netherlands
Polycyclic aromatic hydrocarbons (PAHs) present in
the air originate from incomplete combustion and
some are known to cause cancer. To improve risk
assessment for PAHs, we investigated modulation of
gene expression in human cells after exposure to six
ambient PAHs. HepG2 cells were exposed to 3, 10 or
30µM of benzo[a]pyrene (B[a]P), benzo[b]fluoranthene
(B[b]F), fluoranthene (FA), dibenzo[a,h]anthracene
(DB[a,h]A), 1-methylphenanthrene (1-MPA), dibenzo[a,l]-
pyrene (DB[a,l]P) or solvent control during 6 hours in
duplicate experiments. RNA was isolated, indirectly
labelled with fluorophores and hybridised on a
Phase-I cDNA array containing 600 toxicologically
relevant genes. Samples were hybridised on the
microarray twice for each duplicate experiment, using
four fluorophores (Alexa 488, Alexa 594, Cyanine3 or
Cyanine5). Data were analysed with ImaGene and
GeneSight to identify changes in gene expression. Also,
DNA adducts were measured by 32P post-labelling.
All compounds, except 1-MPA, altered the gene
expression. In general, CYP1A1 (not 1-MPA) and CYP1A2
(not 1-MPA, FA) were up regulated. Insulin-like
growth factor binding protein 1, IgE binding protein
and Likely Ortholog of Rat Vacuole Membrane Protein
1 (VMP1) were up regulated by B[a]P, B[b]F and
DB[a,h]A (not VMP1). Histone 2A (B[a]P, B[b]F) and
Histone deacetylase (B[a]P, B[b]F, FA) were down regu-
lated. The high to low DNA-adduct forming capacity
for the PAHs was B[a]P≥DB[a,l]P≥B[b]F≥DB[a,h]A≥1-
MPA≥FA. This order is generally similar for the num-
ber of gene expression differences.
DNA adducts (2log) were correlated with (2log) gene-
expression differences for the significantly modulat-
ed genes. Significant correlations were found for 22 of
43 genes with a correlation up to 0.64 (CYP1A1). When
correlating the Potency Equivalency Factor (PEF) for
carcinogenicity (2log) values for B[a]P, B[b]F, DB[a,h]A
and DB[a,l]P with (2log) gene-expression differences,
significances were found for 20 of 43 genes with a
correlation up to 0.72 (Insulin-like growth factor I).
Different genes correlate with DNA adduct formation
or with PEF.
105
abstractboek 20-10-2004 10:28 Pagina 105
PS2 - Poster Session 2
Poster Presentation
PW5004
DIFFERENTIAL GENE EXPRESSION IN HUMAN
PBMC INDUCED BY CIGARETTE SMOKE AND ITS
CONSTITUENTS
DM van Leeuwen, JCS Kleinjans, JHM van Delft
Maastricht University, MAASTRICHT, The Netherlands
Background: Toxicogenomics is the application of
new technologies like microarrays in toxicology in
order to understand the molecular basis of the rela-
tionship between xenobiotic exposure (to e.g. car-
cinogenic agents) and early human response at
genome level, i.e. altered expression of large number of
genes that are analysed simultaneously. This project
focuses on this early response induced by tobacco
smoke related agents cigarette smoke condensate
(CSC), BaP, NNK, 4-ABP and H2O2. The aim of the study
was to research differential gene expression due to
exposure to carcinogenic compounds in human sur-
rogate cells from blood. In order to shed light on the
effects that may occur in vivo, we first conducted in
vitro studies in human PBMC.
Methods: Quiescent PBMC were exposed for 18 h to
several concentrations of the agents. Total RNA was
isolated and purified following Trizol and RNeasy
protocols. Derived cDNAs were indirectly labelled
with Cy3 or Cy5 fluorophores. Control and test sam-
ples were competitively hybridised to Phase-1
microarrays, containing 4 x 600 toxicologically rele-
vant genes. Data were analysed with the software
programs ImaGene and GeneSight to identify modu-
lated genes.
Results: Data on CSC, BaP, NNK, 4-ABP and H2O2 have
been generated. Significant modulations and >2 fold
up and down regulation of genes were clearly
detectable. Data are reported on total number of
genes modulated and genes that will be considered
as candidate biomarkers, i.e. senstively and conse-
quently responding genes. The inflammatory process
was the main pathway that was induced due to the
exposures.
Conclusions: This in vitro study showed clear gene
expression modulations after in vitro exposure of
PBMC to chemical carcinogens. Regarding
numbers of differentially expressed genes, the study
provides a basis for the selection of candidate genes
for the biomarker. However, in vivo data will be need-
ed to substantiate these results.
PS2 - Poster Session 2
Poster Presentation
PW5005
ANALYSIS OF THE ILSI GENOTOXICITY WORKING
GROUP'S AFFYMETRIX MGU74V2 ARRAY DATA
USING ROSETTA RESOLVER
JS Harvey, J Kenny, R Rees, AM Lynch
GlaxoSmithKline, HERTFORDSHIRE, United Kingdom
The ILSI Genomics subcommittee was established to
evaluate the role of genomics in mechanism based
risk assessment. Here we present an analysis of the
Affymetrix MgU74Av2 gene expression data generat-
ed by members of the ILSI Genotoxicity working
group (Proctor and Gamble, Pfizer, Aventis, Glaxo
SmithKline). Mouse lymphoma cells were treated
with reference genotoxins (Etoposide, Hydroxyurea,
Taxol, Methyl Methanesulfonate, Cisplatin and
Mitomycin C), which induced robust genotoxicity and
cytotoxicity. Differential gene expression (DGE)
changes were measured 4 hrs and 24 hrs after treat-
ment using standard methodologies. Analysis of the
MgU74Av2 arrays (~12000 sequences) from replicate
studies for each compound was performed using
Rosetta Resolver software (Merck & Co, USA).
Supervised ratio analysis identified a number of DGE
changes related to compound treatment, concentra-
tion and time. There was, however, only a low num-
ber (generally < 50 ) of concentration dependent DGE
changes (>1.5 fold) for any particular treatment.
Furthermore there was limited reproducibility in the
DGE changes for any compound tested in any two
laboratories (generally <10 maximum observed ~100).
Unsupervised 2D-cluster analysis and Principal
Component Analysis failed to identify reproducible
or meaningful relationships between control and
treated DGE array data, either between laboratories
or more surprisingly, within a laboratory. Moreover,
numerous statistical methods were applied to the
data but failed to identify any DGE changes that
could be related to a compounds distinct mechanism
of action.
In conclusion, Rosetta Resolver analysis of the
Affymetrix MgU74Av2 data resulted in the identifica-
tion of a limited list of DGE changes in mouse lym-
phoma cells following treatment with various
genotoxins. Gene pathway analysis was non-inform-
ative for the majority of the genes identified.
Statistical analysis of the data (using numerous
methods), however, was unable to identify any DGE
changes that could be specifically related to a com-
pounds mechanism of action ie. direct or indirect
genotoxicity.
106
abstractboek 20-10-2004 10:28 Pagina 106
PS2 - Poster Session 2
Poster Presentation
PW5006
IMPACT OF GENETIC POLYMORPHISMS ON BITH
WEIGHT
RJ Sram, J Dejmek, B Binkova, I Chvatalova,
A Milcova, M Dostal, P Rössner, I Solansky
Institute of Experimental Medicine AS CR, PRAGUE,
Czech Republic
All studies from Poland and the Czech Republic indi-
cate the relationship between ambient air pollution
and an increase of DNA adducts in maternal and cord
blood and/or in placentas, as well as the relationship
of these biomarkers to the development of newborns.
Therefore we studied the impact of genetic polymor-
phisms on pregnancy outcome as a part of CHIL-
DRENGENONETWORK project. DNA was isolated
from placenta samples collected in polluted regions
(Teplice and Prague) and control region (Prachatice).
Polymorphisms of metabolic genotypes (GSTM1, GSTP1,
GSTT1, EPHX3, EPHX4, CYP1A1 Ile/Val and CYP1A1-MspI)
were determined in 1014 placenta samples. The conse-
quence of genotypes to birth weight as well as catego-
ry LBW+prematurity (low birth weight < 2500 g,
prematurity < 37 weeks) was determined. Using mul-
tiple regression analysis, CYP1A1-MspI mutation
decreased the newborn birth weight of smoking
mothers in Teplice ( -315 g, P<0.05). GSTM1 nulll geno-
type significantly increased LBW+prematurity in all
three groups (AOR = 1.33, P<0.05, especially at Teplice
AOR = 1.54, P=0.01 and Teplice-nonsmokers AOR = 1.53,
P<0.05), CYP1A1 Ile/Val polymorphism in smoking
mothers in the first trimester (AOR = 2.89, P<0.05) and
smoking mothers in the first trimester at Teplice
(AOR = 2.91, P<0.05). Other significant predictors were
exposure to carc-PAHs, maternal smoking, passive
smoking. Combination of different polymorphisms is
further analyzed. Our results indicate that genetic
polymorphisms may affect birth weight in newborn
children, if combined with air pollution and/or
lifestyle (smoking).
Supported by the Czech Ministry of Environment
VaV/740/5/03 and by the EC QLK4-CT-2002-02198.
PS2 - Poster Session 2
Poster Presentation
PW5007
BIOINFORMATICS FOR GENETIC TOXICOLOGY
PD Lewis1, S Abeysinghe2, I Khan3
1 Cardiff University, LLANELLI, United Kingdom2 UW College of Medicine, CARDIFF,
United Kingdom3 Biostat & Bioinformatics Unit, CARDIFF,
United Kingdom
Over the last decade Bioinformatics, as a discipline in
its own right, has developed in parallel with the
'omic' revolution, particularly genomics and pro-
teomics. In particular, huge efforts have been made to
develop novel algorithms and software tools to
analyse sequence and microarray data. Thus, many
bioinformaticists/bioinformaticians have focused
their research and skills within these areas. In con-
trast, little attention has been paid to developing and
implementing tools for analysis of mutation datasets
from dedicated databases such as the Human Gene
Mutation Database and the Mammalian Gene
Mutation Database. Such databases house mutation
data that is either inherited and disease-linked or
induced by known chemical/physical mutagens in
vitro or in transgenic animals. Such data is extremely
useful for trying to predict potential cause of genetic
disease although the tools to extract useful informa-
tion and make predictions from mutation data are
currently limited. We are currently developing algo-
rithms and software tools for mutation data retrieval,
statistical analysis, pattern discovery and prediction
of mutable DNA regions which will be made publicly
available. The software will be accessed using a web
site with a user friendly interface and state of the art
2D and 3D visualisation tools. Initial studies using
such tools have already allowed us to make predic-
tions as to a potential cause of lung cancer in smok-
ers.
107
abstractboek 20-10-2004 10:28 Pagina 107
PS2 - Poster Session 2
Poster Presentation
PW5008
PROGRESS WITHIN THE CHILDRENGENONET-
WORK, A FP5 CONCERTED ACTION
LE Knudsen
Institute of Public Health, COPENHAGEN, Denmark
This Concerted Action (CA) form a European network
providing input and data for risk assessment at the
national and European level. The Children Genotoxicity
Network focus on gene-environment interactions dur-
ing the foetal, neonatal and infancy developmental
periods, concentrating on genotoxic exposures and
environmental factors with focus on air pollution (traf-
fic and tobacco). The aim of the Concerted Action is to
collect, validate and review the available, though few,
European and other studies (WP1) and study materi-
als on children’s exposure and susceptibility to geno-
toxicants (WP2), with focus on air pollution. A further
aim is to prepare protocols to conduct feasibility stud-
ies with perfused placenta (WP3) and with mothers
and their children (two siblings) (WP4), where
research will be financed by national or other
sources. Special attention is given to ethical, legal and
social issues related to environmental studies of chil-
dren (WP6). Progress witih the workpackages will be
described and the associations to the work on bio-
monitoring of children within Europe as initiated by
SCALE will be presented.
PS2 - Poster Session 2
Poster Presentation
PW6001
CHEMICAL INDUCTION OF PREMATURE CEN-
TROMERE SEPARATION: A NEW PARAMETER OF
GENOTOXICITY?
B Bajic1, SP Biljana2, D Ninoslav3, L Zivkovic2
1 Galenika pharmaceuticals, BELGRADE, Yugoslavia2 School of Pharmacy, BELGRADE, Yugoslavia3 School of Veterinary Medicine, BELGRADE,
Yugoslavia
Premature Centromere Separation (PCS) is an uncom-
mon cytogenetic abnormality which is characterized
with chromosome chromatides distinctively separat-
ed before usual time. PCS can be viewed as a primary,
yet unspecific manifestation of chromosome insta-
bility that has been associated with various malig-
nancies and chromosome-breakage syndromes.
Recent investigations have found that PCS yields are
significantly higher in population exposed to mixed
chemicals, pesticides, crude oils and cytostatic drugs.
In order to evaluate PCS as a parameter of genotoxici-
ty we have used various cytostatics, such as an
inhibitor of protein synthesis, Cycloheximide (dose, 5
µg/ml; 10 µg/ml and 25 µg/ml), a cAMP analogue, 8-Cl
cAMP (1 µM; 5 µM and 15 µM), inductor of microtubu-
lar stabilization Taxol (0,01 µM; 0,05 µM and 0,2
µM)and alkylating agent Mitomycin C (dose 0,05 µM;
0,15 µM and 0,6 µM). All investigated cytostatics can
induce PCD when added into a culture of human
peripheral blood lymphocytes. In order to evaluate
these findings we have investigated the distribution
patterns of PCS involving less than two, more than
two and PCS involving all chromosomes compared to
increasing doses of our investigated substances. Also,
by comparing distribution patterns of PCS induced by
our investigating agents to there clastogenic and/or
aneugenic potential we tried to evaluate if PCS con-
tributes to there clastogenic potential. We have found
no correlation between means of total chromosome
aberration and micronucleus frequencies in human
peripheral blood lymphocytes. Our results suggest
that PCS induced by novel investigated substances
should be included as a parameter of genotoxicity in
pre clinical and clinical risk assessment analysis.
108
abstractboek 20-10-2004 10:28 Pagina 108
PS2 - Poster Session 2
Poster Presentation
PW6002
GENOME DAMAGE CAUSED BY 5-NITROFURAN-
TOIN IN SUCKLING AND ADULT MICE DETECTED
BY MICRONUCLEUS ASSAY
A Fucic1, D Markovic2, Z Ferencic2, B Mildner2, AM
Jazbec3, J Bubic Spoljar2
1 Institute for Medical Research and Occupational
Health, ZAGREB, Croatia2 Pliva Research Institute Ltd, ZAGREB, Croatia3 Faculty for Forestry, ZAGREB, Croatia
Antibiotic 5-nitrofurantoin has widely been used in
paediatric patients to treat urosepsis for the last 20
years. Recent papers suggest the need for re-evalua-
tion of its safety due its potential genotoxic effects.
Taking into account possible differences in its metab-
olism and clearance between suckling and adult ani-
mals, we conducted a genotoxicological study to
reveal possible age-related genome damage caused
by this substance. In vivo micronucleus assay (MN)
was performed on 3 and 8- week- old mice. Each
group included 5 males and 5 females. Single doses of
5, 10 and 50 mg/kg of 5-nitrofurantoin were injected
intraperitonealy. Blood samples from tail vein were
taken before injection and 48h, 96h, 168h and 2 weeks
after the application. In each animal 1000 reticulo-
cytes were analysed.
5-nitrofurantoin caused a significantly increased MN
frequency in both suckling (mean group value 5,5 %)
and adult animals (mean group value 3,5%) than in
control animals. Mean MN frequency in suckling ani-
mals was higher than in adult animals for each dose
and sampling time. Micronucleus frequency remained
significantly higher in suckling animals even two
weeks after exposure to 5-nitrofurantoin. Our study
confirmed the genotoxic potency of 5-nitrofurantoin
in suckling and adult animals. We proved higher sen-
sitivity of suckling animals to this compound and a
significantly longer effect than in adults. This effect is
probably due to underdeveloped mechanisms of
detoxification and renal elimination of xenobiotics.
Our study confirmed the need for further analysis of
this compound and its cost-benefit ratio.
PS2 - Poster Session 2
Poster Presentation
PW6003
ESTIMATED EXPOSURE TO PHTHALATES IN COS-
METICS AND RISK ASSESSMENT
BM Lee, HJ Koo
Sungkyunkwan University, SUWON, South-Korea
Some phthalates such as DEHP and DBP, and their
metabolites are suspected of having teratogenic and
endocrine disrupting effects. To predict possible
human exposure to phthalates in cosmetics, the lev-
els of di(2-ethylhexyl) phthalate (DEHP), di-ethyl
phthalate (DEP), di-butyl phthalate (DBP), and butyl-
benzyl phthalate (BBP) were determined by HPLC in
102 branded hair sprays, perfumes, deodorants and
nail polishes. DBP was detected in 19 of the 21 nail pol-
ishes, in 11 of the 42 perfumes, and DEP in 24 of the 42
perfumes and 2 of the 8 deodorants. Exposure levels
to phthalates in cosmetics by dermal absorption were
estimated to be 6.0x10-4 µg/kg bw/day for DEHP, 0.6
µg/kg bw/day for DEP, and 0.103 µg/kg bw/day for
DBP. However, if 100% absorption was assumed
through inhalation, the daily exposure levels to
phthalates in cosmetics were estimated to be 0.026
µg/kg bw/day for DEHP, 81.471 µg/kg bw/day for DEP,
and 22.917 µg/kg bw/day for DBP, which are far lower
than the regulation levels set by the Scientific
Committee on Toxicity, Ecotoxicity and the Environ-
ment (CSTEE) (37µg/kg bw/day, DEHP), Agency for
Toxic Substances and Disease Registry (ATSDR) (7000
µg/kg bw/day, DEP), and International Programme on
Chemical Safety (IPCS) (66 µg/kg bw/day, DBP),
respectively. Based on these data, hazard indices (HI,
daily exposure level / regulation level) were calculat-
ed to be 7.0x10-4 for DEHP, 0.012 for DEP, and 0.347 for
DBP, respectively. These data suggest that estimated
exposure to phthalates in the above mentioned cos-
metics are small. However, total exposure levels may
be greater and require further investigation.
109
abstractboek 20-10-2004 10:28 Pagina 109
PS2 - Poster Session 2
Poster Presentation
PW6004
SETTING SAFE UPPER INTAKE LEVELS FOR
NUTRACEUTICALS
BM Lee, SC Kang
Sungkyunkwan University, SUWON, South-Korea
A nutraceutical is any food or food ingredient consid-
ered to provide medical or health benefits, including
the prevention and treatment of disease. Nutraceuticals
may be taken as the form of foods or of dietary supple-
ments. The scientific literature has shown that
nutraceuticals have not only beneficial properties, but
also adverse health effects. Nutraceuticals such as phy-
tosterols for cholesterol control, may be intended to be
consumed over a long period of time. However, for
many nutraceuticals including phytosterol, the safe
upper intake levels (SUIL) are not provided for con-
sumers and public concern on the safety issue has
been raised. The SUIL is the highest level of daily
nutrient intake that is likely to pose no risks of
adverse health effects to almost all individuals in the
general population. As intake of nutraceticals
increases above the SUIL, the risk of adverse effects
increases. The development of SUIL depends on the
establishment of an adequate uncertainty factor
between expected intake level and identified poten-
tial hazards. After careful review and analysis of pre-
vious reports, scientific judgement was employed to
determine how to establish SUIL. In this paper, a
methodology how to set safe upper intake levels for
nutraceuticals was proposed, based on a through
review of the scientific literature, the hazard identifi-
cation, dose-response assessment and uncertainty
factors.
(This work was supported by a grant from the
Ministry of Health & Welfare 03-PJ1-PG1-CH10-0002).
PS2 - Poster Session 2
Poster Presentation
PW6005
GENOTOXIC STUDY OF SOME MEDICINAL PLANTS
USING THE WHITE/WHITE+ SMART ASSAY IN
DROSOPHILA
N Mezzoug1, M Idaomar1, J Abrini1, A Muñoz Serrano2,
A Alonso-Moraga2
1 Abdelmalek Essaadi University, TETOUAN, Maroc2 Universidad de Córdoba, CÓRDOBA, Spain
Since the plants often synthesize plus their major
components, the chemical substances which are
potentially genotoxic and/or carcinogenic; the evalu-
ation of the genotoxicity of the plants used in the tra-
ditional medicine can present a great importance.
The present study is carried out to assess the geno-
toxicity activity of Licorice (Glycyrrizha glabra),
Nutmeg (Myristica fragrans), Black cumin (Nigella
sativa), Oregano (Oreganum compactum), Rosemary
(Rosmarinus officinales) and Fenugreek (Trigonella
foenum graecum) using the w/w+ somatic assay in
Drosophila melanogaster. Oregon K-yellow females
were crossed to Oregon k-white males. The flies were
permitted to lay eggs in bottles for 3 days on food sup-
plemented with the aqueous extract of the plant at
different concentrations. Newly hatched flies were
counted and the females were transferred to fresh
medium; 2-6 days later their eyes were inspected for
the presence of white spots. The loss of heterozygosi-
ty of recessive marker used (white) can lead to the
formation of mutant clones of larval cells, which are
then expressed as spots on the eyes of the adult flies.
Negative solvent controls were included in all experi-
ments. For the evaluation of the genotoxic effect of
plants, the frequencies of spots per eyes of a treated
series are compared to its concurrent control using
the T2- test. Our results do not show any significant
genotoxic effect of the Black cumin, Oregano and
Fenugreek. But It was found that the Nutmeg,
Rosemary and Liquorice increase the rate of sponta-
neous mutations which remains no significant
(p>0.05) with Liquorice at 2%, significant (p<0.05)
with rosemary at 5% and highly significant (p<0.001)
when the Nutmeg is administered at 3%.
110
abstractboek 20-10-2004 10:28 Pagina 110
PS2 - Poster Session 2
Poster Presentation
PW6006
GREENSCREEN EM - A YEAST BASED GENOTOXICITY
AND CYTOTOXICITY ASSAY FOR ENVIRONMEN-
TAL MONITORING
W Knight, PO Keenan, RM Walmsley
Umist, MANCHESTER, United Kingdom
GreenScreen, a new assay capable of simultaneously
measuring both genotoxicity and more general cyto-
toxicity in aqueous environmental samples, will be
described. There is currently a need for simple meth-
ods for the screening and monitoring of a wide range
of toxic contaminants in the aquatic environment.
For example, in assessing the effectiveness of waste
treatment, to control the toxicity of industrial efflu-
ent discharges, to enable compliance with pollution
prevention and control legislation, to protect water
treatment plants from toxic influents and for hazard
assessment where the deliberate release of chemical
armaments is suspected. GreenScreen allows rapid
determination of whole sample genotoxicity with
minimum sample pre-treatment.
The assay uses eukaryotic (yeast) cells, genetically
modified to express a green fluorescent protein when-
ever DNA damage, as a result of exposure to genotoxic
agents, is repaired. A measure of the reduction in cell
proliferation is used to characterise general toxicity
producing familiar EC50 and LOEC data. The assay pro-
tocol has been developed for use in the field by non-
skilled personnel, employing dedicated, portable
instrumentation. A range of environmentally relevant
substances has been evaluated using the assay, includ-
ing solutions of metal ions, solvents and pesticides.
Preliminary data from a year long direct toxicity
assessment validation programme will be presented,
comparing the assay’s response to that of standard
Daphnia and algal tests, in the analysis of 38 varied
industrial effluents. In addition, results from a project
funded by the European Commission 5th Framework
'Energy, Environment and Sustainable Development'
programme will also be discussed. In this work, a
novel at-line genotoxicity monitor has been devel-
oped and integrated with an effluent toxicity treat-
ment system. The sensitivity to a wide range of
substances and effluents suggests GreenScreen is a
useful tool for environmental toxicity monitoring
and hazard assessment.
PS2 - Poster Session 2
Poster Presentation
PW6007
HUMAN STRESS EXPRESSION IN CONTEXT OF
RISK ASSESSMENT
FI Ingel
A.N.Sysin Research Institute of Human Ec, MOSCOW,
Russia
The fact, well known to oncologists, that strong emo-
tional trauma presents in each anamneses of cancer,
unfortunately, till now did not attract an attention of
specialists in risk assessment.
Here will be present the substantiation for new
approach for human risk assessment grounded on
evaluation of biomarkers of effect.
In 2 independent researches (60 adult men and 78
young children) we measured emotional stress
expression in parallel with evaluation of spectrum of
divided cells, level of micronuclei and apoptosis in
blood cells cultures (cytokinetic block) with and with-
out 1,0 Gy of gamma-irradiation in vitro as well as
adaptive response to irradiation.
Results demonstrated similar effects both for adults
and for children (P<0,05): positive correlation between
human stress expression and frequency of accelerately
divided cells; negative correlation between level of
apoptosis and stress expression; positive correlation
between stress expression and radiosensitivity as well
as type of adaptive response.
Because: stress is the first reaction of an organism to
any influence and therefore it cannot be avoided (Selye,
1936; Khaitsev et al, 1997); human stress expression
directly correlates with toxic and genotoxic exposure
(Ingel et al, 2001); abnormal stress expression connect-
ed with an increased level of chromosomal aberra-
tion and elevated individual genome susceptibility
(Ingel 1997-2002), we may conclude that human
stress expression is important indicator of risk of
oncological and other diseases connected with
appearance of genetic damage.
Study of mammalian and human genetical effects of
emotional stress demonstrated that measurement of
stress expression in complex with individual genome
susceptibility is one of key approach for creation of
the new system of risk assessment.
The algorithm of this approach will be present.
The study was partly supported with INTAS grant
1005.
111
abstractboek 20-10-2004 10:28 Pagina 111
PS2 - Poster Session 2
Poster Presentation
PW6008
MEASUREMENT OF REACTIVE METABOLITES AS A
BASIS FOR CANCER RISK ASSESSMENT: APPLICA-
TION TO 1,3-BUTADIENE
CF Fred1, M Törnqvist1, A Kautiainen2, F Granath3
1 Stockholm University, STOCKHOLM, Sweden2 Preclinical R&D, Biovitrum AB, STOCKHOLM,
Sweden3 Karolinska Institute, STOCKHOLM, Sweden
1,3-Butadiene is a general air pollutant associated
with combustion of organic matter and is also an
extensively used monomer in polymer production.
The cancer risk estimation of 1,3-butadiene is encum-
bered with large uncertainties. Extrapolation from
tumour frequencies in long-term animal tests has led
to a relatively high figure for the risk associated with
1,3-butadiene exposure. This is mainly based on obser-
vations of very high tumour incidences in butadiene-
exposed mice, which in this respect are about 100
times more sensitive than rats. It has been hypothe-
sized that a high cancer risk from 1,3-butadiene could
be associated with its metabolism to the bifunctional
1,2:3,4-diepoxybutane (DEB) which, in comparison
with monofunctional epoxides, 1,2-epoxy-3-butene
(EB) and 1,2-epoxy-3,4-butanediol (EBdiol), is a highly
effective mutagen, i.e. cancer initiator. Measurement
of in vivo doses of DEB is therefore essential for the
risk assessment of 1,3-butadiene. Reaction products
with hemoglobin offer a possibility of measuring
reactive metabolites in vivo. Hemoglobin adducts
from EBdiol have in this study been measured with
available methods, which are, however, not applica-
ble to the bifunctional DEB.
This work presents a procedure for measurement of a
specific, ring-closed adduct, Pyr-Val, formed from the
reaction of DEB with N-terminal valines in hemoglo-
bin. It is based on LC-ESI-MS/MS analysis of the Pyr-
modified N-terminal peptides enriched after trypsin
digestion of globin. Mouse and rat could be compared
regarding the metabolism of EB, DEB and EBdiol.
From the data it was concluded that, in 1,3-butadiene
exposure, higher levels of DEB are formed in mice
compared to rats. Estimates of in vivo doses in pub-
lished cancer tests showed that carcinogenesis in
mice is mainly due to DEB, whereas in rat, and possi-
bly man, the monofunctional EBdiol is a larger risk
contributor. Preliminarily, the estimated cancer risk is
compatible with the epidemiology-based risk esti-
mate of US EPA.
PS2 - Poster Session 2
Poster Presentation
PW6009
THE APPROACH OF REGIONAL MONITORING OF
CYTOGENETIC EFFECTS IN HUMAN DUE TO ENVI-
RONMENTAL POLLUTION CONSEQUENCES
VG Druzhinin, NV Mokrushina, VI Minina,
AN Volkov, TA Golovina
Kemerovo State University, KEMEROVO, Russia
The main task while planning and carrying out cyto-
genetic monitoring is to choose the strategy of study-
ing of clastogenic effects in human populations,
when genotoxic effects are either known or supposed
to be. First of all, the object of monitoring must be any
territory characterized by both specific climatic geo-
graphic conditions of the environment and a variety
of ecological situations. It can be accounted for by the
fact that the investigation of mutation processes in
local populations (a particular plant, city or village
dwellers) does not allow one to completely and cor-
rectly estimate the degree and orientation of geno-
toxic processes. On the other hand, monitoring of too
large territories is either difficult to be carried out or
it is practically impossible by one research team. It is
the approach of regional monitoring that allows one
to estimate the degree of genotoxic parameters of the
environment, which finally must promote the devel-
opment of ecogenetical approaches for a complex of
administrative and medical preventive measures to
improve the ecological situation and hence to protect
one's health.Within the framework of the regional
approach to monitoring we proposed the method of
cytogenetic cartography whose realization is based
on several basic principles such as the unification of
investigations; sanitary - hygienic examination pre-
ceding to monitoring; selectivity and priority; a com-
plete coverage of territories; the organization of
control points; the juxtaposition of maps (Druzhinin
et al., 2003). The experimental data of 15- year moni-
toring of chromosomal aberrations in cohorts of the
inhabitants of the large industrial region of Western
Siberia, i.e. the Kemerovskaya oblast' are submitted in
our paper. The potential of the method of cytogenetic
cartography in genetics monitoring is also discussed.
112
abstractboek 20-10-2004 10:28 Pagina 112
PS2 - Poster Session 2
Poster Presentation
PW6010
BIOMARKERS OF AIR POLLUTION EXPOSURE -
FOLLOW -UP STUDY OF POLICEMEN IN PRAGUE
RJ Sram1, B Binkova1, O Beskid2, I Chvatalova1,
A Milcova1, P Rossner2, O Sevastyanova1,
Z Smerhovsky1, Z Stavkova1, J Topinka1
1 Institute of Experimental Medicine AS CR,
PRAGUE, Czech Republic2 Intitute of Experimental Medicine, PRAGUE, Czech
Republic
The effect of exposure to PAHs adsorbed onto res-
pirable air particles (<2.5 mm) on DNA adducts and
chromosomal aberrations was studied in a group of
policemen (males, aged 22-50 years) working in the
downtown area of Prague and spending >8 h out-
doors (EXP, N=53). The matched healthy volunteers
spending >90% daily time indoors were chosen as
Controls (CON, N=52). Ambient air particles (PM10,
PM2.5) and carcinogenic polycyclic aromatic hydro-
carbons (carc-PAHs) were monitored using VAPS sam-
pler, personal exposure was evaluated using personal
samplers during working shift. DNA adducts were
analyzed in lymphocytes by 32P-postlabeling assay,
chromosomal aberrations by conventional cytogenetic
analysis and fluorescent in situ hybridization (FISH).
Further biomarkers include cotinine in urine to con-
trol for exposure to tobacco smoke, plasma levels of
vitamins A, E and C, folic acid, cholesterol and trigly-
cerids. Polymorphisms of metabolic genotypes
(GSTM1, GSTP1, GSTT1, EPHX, CYP1A1-MspI) and DNA
repair genotypes (XRCC1 and XPD) were determined.
The level of 'like' B[a]P-derived DNA adduct was high-
er in exposed group (0.122±0.036 vs. 0.099±0.035
adducts/108 nucleotides, P=0.003). DNA adduct levels
were modified by XPD repair gene in exon 23 and
XRCC1 and GSTM1 genotypes. Using FISH technique
and probes for chromosomes 1 and 4 the genomic fre-
quency of translocations calculated as FG/100 was
1.72 and 1.24 for EXP and CON (P<0.05), respectively. To
see the dynamics of the observed changes reported
here, the prospective cohort study is going on. All the
biomarkers of exposure and effect are analyzed
repeatedly during the period of one year in three-
months intervals (4-times) to cover periods with high
(winter) and low (summer) levels of air pollution.
Supported by the Czech Ministry of Environment
VaV/340/2/00 and VaV/740/5/03 and by the EC IC
QLK4-2000-00091.
PS2 - Poster Session 2
Poster Presentation
PW6011
CYTOGENETIC MONITORING OF OCCUPATIONAL
EXPOSURE TO ANTINEOPLASTIC AGENTS: INFLU-
ENCE OF GENETIC POLYMORPHISMS
R Cozzi1, A Testa2, M Giachelia3, F Festa1, G Tranfo4,
G Spagnoli4, D Tirindelli3, L Baccelliere5, C Sciutto5,
N la Rosa5
1 University, ROME, Italy2 ENEA CR Casaccia, ROME, Italy3 Casaccia, ENEA, ROME, Italy4 ISPESL, ROME, Italy5 S.Martino Hospital, GENOA, Italy
A multidisciplinary monitoring study was carried out
on hospital personnel exposed to antineoplastic drugs
to investigate the risk of occupational exposure to these
substances. In this project, a cytogenetic investigation
(comet assay, chromosomal aberrations and micronu-
clei analyses) was carried out on a group of 25 female
nurses employed in oncology units and on a control
group adequately selected. All subjects (mean age = 39
years) were asked to fill in the personal healthy ques-
tionnaire proposed by the International Commission
Protection against Environmental Mutagens and
Carcinogens. Workers were also selected using a ques-
tionnaire concerning the individual occupational expo-
sure.
Furthermore, as humans vary in their response to
chemicals, considering their specific genetic constitu-
tion, we characterised the subjects for the polymor-
phisms of four genes (metabolic genes: GSTM1, GSTT1;
DNA repair genes: XRCC1, XRCC3) in order to verify the
relationship between DNA damage and genetic vari-
ants.
Regarding to the cytogenetic assessment, the exposed
group showed a significantly higher frequency of
genetic damage where compared to the control group:
chromosome and chromatid aberrations frequencies
in workers (chromosome aberration 5.44 %, chromatid
aberration 5.48 %) appeared significantly higher
(p<0.001, p<0.01 respectively) than in controls (chromo-
some aberration 1.46 %, chromatid aberration 3.07 %).
Similarly, micronucleus frequencies appeared signifi-
cantly higher (p<0.001) in workers (16.1 ‰) than in con-
trols (7.95 ‰). Concerning the evaluation of DNA
primary damage in these workers, the results indicat-
ed significantly higher tail moment values in exposed
group than in controls (p=0.001).
Finally, regression multivariate analysis showed a rela-
tion between GSTT1 null genotype and chromatid aber-
rations. This research was supported by Ministry of
Public Health (Ricerca Finalizzata, bando 2001 : 'La valu-
tazione dei rischi nella manipolazione dei chemioter-
apici antiblastici in ambiente sanitario') and partially
by MIUR Project 'Biological basis on human suscepti-
bility'. 113
abstractboek 20-10-2004 10:28 Pagina 113
PS2 - Poster Session 2
Poster Presentation
PW6012
THE AMES II™ MUTAGENICITY ASSAY: AN INTER-
NATIONAL VALIDATION STUDY PERFORMED
WITH NINETEEN CODED COMPOUNDS
SF Flueckiger-Isler1, K Braun2, G Engelhardt3,
M Baumeister4, H-G Wunderlich5, JAJ van Gompel6,
N Hasler-Nguyen7, R Reimann8, V Gervais9
1 Xenometrix by Endotell GmbH, ALLSCHWIL,
Switzerland2 Aventis Pharma Deutschland, HATTERSHEIM,
Germany3 BASF AG, LUDWIGSHAFEN, Germany4 Boehringer Ingelheim, BIBERACH, Germany5 Federal Environmental Agency, BAD ELSTER,
Germany6 Johnson & Johnson Parmaceutical R&D, BEERSE,
Belgium7 Novartis Consumer Health, NYON, Switzerland8 Schering AG, BERLIN, Germany9 Servier Group, ORLTANS-GIDY, France
An international collaborative validation study was
performed in nine laboratories to compare the Ames
II™ Mutagenicity Assay with the standard Salmonella
plate incorporation test. Nineteen coded chemicals
were tested for their mutagenic activity in the strains
TA98 and TAMix (TA7001-7006). The test compounds
were selected from a published study with a large
data set from the traditional Ames test. The results of
both assay systems were compared, and the inter-lab-
oratory consistency of the Ames II test was assessed.
Of the eight mutagens selected, six were correctly
identified with the Ames II assay by all laboratories,
one compound was judged positive by five of six
investigators and one by four of six laboratories. All
seven non-mutagenic samples were consistently neg-
ative in the Ames II assay. Of the four chemicals that
gave inconsistent results in the traditional Ames test,
three were uniformly classified as either positive or
negative in the present study, whereas one com-
pound gave equivocal results. A comparison of the
test outcome of the different investigators resulted in
an inter-laboratory consistency of 89.5%.
Owing to the high concordance between the two test
systems, and the low inter-laboratory variability in
the Ames II assay results, the Ames II is an effective
screening alternative to the standard Ames test for
screening new substances for their mutagenic poten-
tial.
Compared to the standard Ames test, the Ames II assay
offers higher speed format, the possibility of automa-
tion, the recording of all possible base-pair substitu-
tions in one culture, substantially lower amount of test
chemical, easy scoring being a colorimetric assay, easy
handling and ready to use reagents. These criteria are
essential to meet the constantly increasing number of
chemicals flowing through early phase development
and the increasing demand for early indications of
possible Mutagenicity.
114
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PS2 - Poster Session 2
Poster Presentation
PW6013
ABSENCE OF GENOTOXIC EFFECTS OF RADIO-
FREQUENCY ON HUMAN BLOOD BY USING
DIFFERENT CYTOGENETIC TESTS
A Testa, M Appolloni, E Cordelli, AM Fresegna,
C Marino, L Stronati, P Villani
ENEA CR Casaccia, ROME, Italy
The possible health hazard of environmental expo-
sure to radiofrequency (RF) signals became an issue
of considerable public concern. Despite the majority
of evidences suggest that radiofrequency has not
genotoxic potential, some studies report positive
results.
The aim of this study was to evaluate the potential
genotoxic effect of radiofrequency alone or in combi-
nation with X-rays (1 Gy) on human blood cells. Three
different conventional and molecular cytogenetic
tests: chromosome aberrations (CA), micronuclei
(MN) and alkaline comet assay were applied.
Whole blood samples from 6 healthy donors (35-45
years) were exposed to 935 MHz, 1 W/kg for 24 hours.
The exposure setup was based on two rectangular R9
waveguides (for sham and exposed cells radiated
simultaneously in blind way) in which only the fun-
damental TE01 mode can propagate for the frequency
bands of interest. In order to guarantee the same
environmental conditions (temperature, humidity,
CO2) for the exposed and sham cells, both waveguides
were placed inside the same incubator (exposure
system is provided by Foundation for Research
on Information Technologies in Society (IT’IS)
and described in J. Schuderer et al, In Vitro Exposure
Systems for RF Exposures at 900 MHz, submitted)
Results did not show any significant difference
between radiofrequency exposed and sham samples
for each cytogenetic endpoint analyzed. Similarly, the
combined exposure failed to indicate the presence of
any synergistic effect between radiofrequency and
X-rays.
This Project (PERFORM B) is partially supported by
Elettra 2000, MMF and GSM association.
PS2 - Poster Session 2
Poster Presentation
PW6014
THE EFFECTS OF ACRYLONITRILE ON LEVEL OF
CHROMOSOMAL ABERRATIONS
O Beskid1, Z Dusek2, I Chvatalova2, M Dostal2,
Z Stavkova2, P Rössner2, RJ Sram2
1 Institute of Experimental Medicine, PRAGUE,
Czech Republic2 Institute of Experimental Medicine AS CR,
PRAGUE, Czech Republic
Acrylonitrile (ACN) is classified as possibly carcino-
genic to humans (group 2B) according to IARC. It is
used in production of plastics, synthetic fibers and
rubbers. We investigated the influence of ACN on the
level of unstable and stable aberrations measured by
conventional cytogenetic analysis and FISH (whole
chromosomal painting for chromosomes 1 and 4),
respectively. The investigation was carried out on a
group of 60 chemical plant workers and a control
group of 55 healthy volunteers from the same region.
The subjects were genotyped for glutatione-S-trans-
ferases (GST) M1, P1 and T1, EPHX, p53, XPD, XRCC1,
hOGG1, MTHFR, and MS to evaluate possible differ-
ences in individual susceptibility to xenobiotics due
to genetic polymorphism of metabolizing enzymes.
Further we analysed the levels of vitamin A, C, E in
plasma for determination of antioxidative status, and
the level of cotinine in urine to objectify smoking sta-
tus. The relationship between occupational exposure
and cytogenetic endpoints was tested by Kruskal-
Wallis test.
Occupational exposure to ACN significantly affected
the frequency of chromosomal aberrations detected
by conventional method. We observed an increase of
AB.C. (aberrant cells) in the exposed group (3.27±1.91%
AB.C.) in comparison with controls (2.05±1.53% AB.C.,
P<0.01). However, there was no significant difference
between these two groups in genomic frequency of
translocations (Fg/100) measured by FISH (1.88±1.52 in
the exposed subjects vs. 1.63±1.30 in controls). No rela-
tionship between smoking status and the frequency
of aberrant cells was found neither for stable nor
unstable aberrations. The frequency of AB.C evaluat-
ed by conventional method correlated with the level
of vitamins A and C, and p53 MspI polymorphism,
while Fg/100 revealed association to age, EPHX, XPD-
6 polymorphisms, and vitamin E level.
This study was supported by EC QLK4-CT-2000-02381.
115
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PS2 - Poster Session 2
Poster Presentation
PW6015
THE IMPACT OF OCCUPATIONAL EXPOSURE TO
IRRADIATION IN CZECH NUCLEAR POWER PLANT
WORKERS
Z Dusek1, O Beskid1, I Chvatalova1, J Schmuczerova1,
Z Stavkova1, A Milcova1, P Rossner1, J Rubes2,
Z Smerhovsky1, RJ Sram1
1 Institute of Experimental Medicine AS CR,
PRAGUE, Czech Republic2 Veterinary Research Institute, BRNO,
Czech Republic
The aim of our study was to identify occupational risk
of irradiation exposure or exposure to clastogenic
chemical compounds in the Czech nuclear plant
workers. The level of chromosomal aberrations is
known as a biomarker of early biological effects and a
predictor of cancer risk. Therefore, we applied classi-
cal cytogenetic analysis and FISH (whole chromo-
some painting for chromosomes 1 and 4, combined
with pancentromeric probe) in three groups: 123 sub-
jects in the Temelin plant, 114 subjects in the
Dukovany plant (15 years in use), and 53 matched con-
trols from Ceske Budejovice. Nuclear plant workers
were divided into two groups: subjects with admit-
tance into the controlled zone, and others. Further
factors were analysed: p53 expression, XPD, XRCC1,
hOGG1, p53, MTHFR, and MS gene polymorphisms,
levels of vitamin A, C, E in plasma, and level of coti-
nine in urine. Long-term exposure to ionizing radia-
tion in the controlled zone was 0.46±1.49 mSv in
Temelin and 5.68±9.51 mSv in Dukovany. Using con-
ventional cytogenetic analysis, we observed
1.90±0.95 and 1.82±1.19 %AB.C. in Temelin, and
2.39±1.01 and 2.33±1.04 %AB.C. in Dukovany, for con-
trolled zone workers and others, respectively. In the
control group, we found 2.25±0.82 %AB.C. Genomic
frequency of translocations Fg/100 measured by FISH
was 1.89±1.40 and 2.01±1.68 in Temelin, and 2.41±1.88
and 2.12±1.61 in Dukovany for controlled zone workers
and others, respectively. In the control group, Fg/100
was 1.83±1.19. As potential confounders were identi-
fied following factors: age, XPD-6, GSTP1, and GSTT1
genotypes, long-term medication, alcohol consump-
tion and smoking status. No association between
dose of irradiation and level of chromosomal aberra-
tions in any nuclear power plant was obtained nei-
ther by conventional cytogenetic analysis nor by
FISH.
This study was supported by the Czech Government.
PS2 - Poster Session 2
Poster Presentation
PW6016
FLOW CYTOMETRIC ANALYSIS AND MANUAL
SCORING OF PERIPHERAL BLOOD MICRONUCLEUS
FREQUENCIES IN CYCLOPHOSPHAMIDE TREATED
MICE
BM van der Leede, M de Boeck, A de Smedt,
M Steemans, F van Goethem, A Lampo, PH Vanparys
Johnson & Johnson Parmaceutical R&D, BEERSE,
Belgium
The in vivo rodent erythrocyte micronucleus test is
widely used to evaluate the clastogenic and/or aneu-
genic potential of chemicals and pharmaceuticals.
Micronucleus frequencies are traditionally determined
manually by microscopy, making the micronucleus test
relatively labour intensive and time consuming.
Investigators at Litron Laboratories (Rochester, NY)
developed a flow cytometric method to measure
micronuclei in both reticulocytes and normochro-
matic erythocytes of mouse peripheral blood. This
method has many advantages over manual scoring,
including high speed data acquisition and objective
analysis. In an international multi-laboratory study
co-ordinated by Litron Laboratories the flow cytomet-
ric method is currently being validated in nine partici-
pating laboratories. Phase I validation results have
been published (Torous et al., Environ. Mol. Mut. 38:59-
68, 2001) and demonstrated high transferability of the
flow cytometric method among the participating lab-
oratories. Phase II of the validation has focused on
comparing micronucleus frequencies obtained by
microscopy with those generated by flow cytometry.
Each of the participating laboratories was assigned
one compound for evaluation. Here we present the
data obtained on mice treated with cyclophos-
phamide (0, 2.5, 5, 10 or 20 mg/kg/day by oral gavage)
for 3 consecutive days as part of our contribution to
Phase II validation study. Wright stained blood smears
were used for the microscopic analysis. In addition,
results are presented of an in-house study with
cyclophosphamide (0 or 40 mg/kg by oral gavage)
using a single dose regime and acridine orange stain-
ing of blood smears. Cyclophosphamide induced a
dose-dependent increase in micronucleated reticulo-
cytes by both flow cytometric and microscopic analy-
sis. The results indicate a high correlation between
flow cytometry and microscopy, especially using acri-
dine orange staining and also between flow cytomet-
ric analysis of the same samples at two different
laboratories.
116
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PS2 - Poster Session 2
Poster Presentation
PW6017
THE GENOTOXICITY OF IPRONIDAZOLE (GASTRO-
GAL 10) ON CULTURED HUMAN LYMPHOCYTES
B Markovic, Z Stanimirovic, N Djelic, V Bajic
Faculty of Veterinary Medicine, BELGRADE,
Yugoslavia
The genotoxic effects of antimicrobial preparation
Gastrogal 10 (ICN Galenica, Belgrade, Pharmaceutical
Company, Serbia) has been investigated in vitro on
human peripheral blood lymphocytes by following
the capability of Gastrogal 10 to induce numerical
and structural chromosome changes. To test a possi-
ble effect of the investigated substance on DNA we
used the sister chromatid exchange (SCE) test in vitro.
The investigated substance Gastrogal 10 was tested
through three experimental concentrations: 25 µM;
50µM and 100µM. Negative control groups were
treated with physiological saline. Our results show
that all experimental doses of the investigated sub-
stance Gastrogal 10 shows the potential for transfor-
mation of the karyotype of human lymphocytes by
inducing numerical aberrations type aneuploidy and
polyploidy and structural aberrations type gaps and
lesions. The overall cytogenetic changes induced by
Gastrogal 10 in all three doses clearly shows a statisti-
cal high significant increase in respect to the control
group. We have also estimated a correlation between
an increase of dosage and cytogenetic changes.
Cytogentic changes and a dose-effect dependency
clearly show a genotoxic potential of Gastrogal 10 on
lymphocytes of human peripheral blood. All three
doses of Gastrogal 10 induced a highly statistically
increase in the frequency of SCE in respect to the
untreated control groups. High SCE frequencies
shows a possibility of the investigated substance to
generate changes in the DNA structure. Our results
classify Gastrogal 10 a clasogenic agent.
PS2 - Poster Session 2
Poster Presentation
PW6018
AN ASSESSMENT OF CUMENE HYDROPEROXIDE
IN THE IN VIVO COMET ASSAY IN MOUSE SKIN
P Clay1, A Wolfreys2, B Elliott1, E Jones1
1 Syngenta CTL, MACCLESFIELD, United Kingdom2 SEAC, Unilever Colworth, SHARNBROOK,
United Kingdom
The Comet assay has been proposed as being an
appropriate assay for the assessment of the genotoxic
potential of chemicals that may be active at the site of
contact. One such class of chemicals is strong oxidis-
ing agents acting through reactive oxygen, many of
which are genotoxic in vitro. However, these chemi-
cals act via one of the mechanisms of mutagenicity
considered subject to a practical threshold and thus
there should be an effective 'non-mutagenic' dose of
this material.
The genotoxicity of cumene hydroperoxide, a strong
oxidising agent, has been assessed in an in vivo
mouse skin Comet assay following a single dermal
application. Small, but dose related, increases in the
group mean values for tail moment were observed in
all the treated groups. These increases reached statis-
tical significance (P<0.05) for the high dose group but
did not exceed twice the concurrent control value.
Although a clear positive response was not observed,
the reproducibility and dose relationship of the data
suggest that cumene hydroperoxide induces DNA
damage, as indicated by comet formation in the
mouse skin following treatment at both 2.5 and 5.0
mg/animal. This indicates that the threshold dose
lies below 2.5 mg/animal.
The maximum dose level tested was limited by
micropathological changes to the skin. The severity
of these effects was not reflected in clinical or macro-
scopic observations and underlines the importance of
micropathological assessment of the tissue of inter-
est when performing in vivo comet assays.
117
abstractboek 20-10-2004 10:28 Pagina 117
PS2 - Poster Session 2
Poster Presentation
PW6019
THE GENOTOXICITY OF IPRONIDAZOLE (GASTRO-
GAL 10) ON CULTURED HUMAN LYMPHOCYTES
B Markovic1, Z Stanimirovic1, N Djelic1, B Bajic2
1 Faculty of Veterinary Medicine, BELGRADE,
Yugoslavia2 Galenika Pharmaceuticals, BELGRADE, Yugoslavia
The genotoxic effects of antimicrobial preparation
Gastrogal 10 (ICN Galenica, Belgrade, Pharmaceutical
Company, Serbia) has been investigated in vitro on
human peripheral blood lymphocytes by following
the capability of Gastrogal 10 to induce numerical
and structural chromosome changes. To test a possi-
ble effect of the investigated substance on DNA we
used the sister chromatid exchange (SCE) test in vitro.
The investigated substance Gastrogal 10 was tested
through three experimental concentrations: 25 µM,
50µM and 100µM. Negative control groups were
treated with physiological saline. Our results show
that all experimental doses of the investigated sub-
stance Gastrogal 10 shows the potential for transfor-
mation of the karyotype of human lymphocytes by
inducing numerical aberrations type aneuploidy and
polyploidy and structural aberrations type gaps and
lesions. The overall cytogenetic changes induced by
Gastrogal 10 in all three doses clearly shows a statisti-
cal high significant increase in respect to the control
group. We have also estimated a correlation between
an increase of dosage and cytogenetic changes.
Cytogentic changes and a dose-effect dependency
clearly show a genotoxic potential of Gastrogal 10 on
lymphocytes of human peripheral blood. All three
doses of Gastrogal 10 induced a highly statistically
increase in the frequency of SCE in respect to the
untreated control groups. High SCE frequencies
shows a possibility of the investigated substance to
generate changes in the DNA structure. Our results
classify Gastrogal 10 a clastogenic agent.
PS2 - Poster Session 2
Poster Presentation
PW6020
PHOTOGENOTOXICITY TESTING; IN VIVO MICRONU-
CLEUS ASSAY IN RAT EPIDERMAL SKIN CELLS
CAM Krul, M-JST Steenwinkel, N de Vogel,
RNC van Meeuwen
TNO Nutrition and Food Research, ZEIST,
The Netherlands
Photogenotoxicity testing is recommended for cos-
metic ingredients (e.g. sunscreens) and pharmaceuti-
cals (e.g. antimicrobal agents), that absorbs UV light
and reach the skin or eyes either by dermal or oral
administration. In recent years the demand for in
vitro photogenotoxicity assessment is remarkably
increased. Most photogenotoxicity assays are adopt-
ed from standard guideline assays, such as the Ames-
and chromosomal aberration test.
In the regular tiered approach for genotoxicity test-
ing, whenever an inconclusive response is observed
in vitro, an in vivo assay is performed to establish the
genotoxic potential in vivo. For the evaluation of the
photogenotoxic potential no follow-up assay in vivo
is available (except for a photocarcinogenicity study).
Particularly photo-clastogenic responses are observed
in photogenotoxicity assays in vitro. Therefore, a feasi-
bility study was performed to demonstrate the induc-
tion of micronuclei (MN) in rat epidermal skin cells.
Fisher 344 rats were shaved and dorsal skin (12 cm2)
was treated with 8-MOP (0.9 µg/cm2) 30 minutes
prior to UV-irradiation (1 MED) simulating sunlight.
Animals were sacrificed 48 and 72h after treatment,
epidermal skin cells were isolated and slides were
prepared. The incidence of micronucleated skin cells
(MNSC) was measured in 2000 skin cells (SC). The
induction of MNSC was highest at 72h and about
5-fold increased compared to the control (non-
exposed ventral side skin). Additionally, rats were
exposed to UV (2-3 MED) in the absence of a pho-
togenotox compound. The numbers of MNSC were
significantly increased compared to the controls. This
new developed photo-MN assay can be used to exam-
ine whether pharmaceutical compounds have the
ability to cause UV-induced MN in vivo.
The in vivo photo-MN assay in rats might be very
helpful bridging the gap between in vitro photogeno-
toxicity and in vivo photocarcinogenicity testing.
118
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PS2 - Poster Session 2
Poster Presentation
PW6021
CHROMOSOMAL ABERRATIONS AND RISK OF
CANCER IN A HUNGARIAN COHORT
A Kelecsenyi, S Gundy
National Institute of Oncology, BUDAPEST, Hungary
The frequency of chromosomal aberrations (CAs) in
human peripheral blood lymphocytes (PBLs) has rou-
tinely been used to monitor occupational and envi-
ronmental exposures to genotoxic agents for over 3
decades. In 1990s a clear association was detected
between high CA frequencies and increased cancer
risk in a large collaborative project of Nordic and
Italian laboratories. In late 1990s another important
conclusion was drawn from the results that a high
level of CAs generally predicts an increased cancer
risk regardless of the reason for the elevated CAs. The
assay was introduced in Hungary in 70s. Under the
extension of Nordic-Italian 'Cancer Risk Biomarkers'
(CRB) program we also contribute in the project. The
results from other European cohorts will also be com-
bined with our ones and those already included into
the CRB project. The associations between total can-
cer incidence/mortality and the frequency of high,
medium, and low CAs, gender, age and time since
test, and exposures will be modelled by Cox's regres-
sion.
Between 1978 and 2003 we carried out more than
2000 CA analyses on cancer-free persons, but the data
of only 831 persons were fully available for the crite-
ria of the accepted protocol. CA data were tri-
chotomized into low (0-1 CA;) medium (2 CA), and
high (>2 CA). Out of 831 persons 52 cancer cases (6.3%)
occurred. The total of cancers observed in the low
(4.7%), medium (5.7%) and high (9.3%) CA groups dif-
fered significantly (log-rank test: p=0.0225). In conclu-
sion we may say that our findings also support the
hypothesis that an elevated CA frequency in PBLs pre-
dicts an increased future cancer risk, and the extent
of CAs in lymphocytes may reflect the same events,
which take place in other cells of target tissues of the
organism.
This study was supported by grant: QLK- CT-200-
00628.
PS2 - Poster Session 2
Poster Presentation
PW6022
ANEUPLOIDY STUDIES IN MOUSE GERM CELLS
WITH BISPHENOL A
MS abdo Attia1, ID Adler1, U Eichenlaub-Ritter2,
R Ranaldi3, F Pacchierotti3
1 GSF, NEUHERBERG, Germany2 University of Bielefeld, BIELEFELD, Germany3 ENEA CR Casaccia, ROME, Italy
Bisphenol A (BP-A), widely used in polycarbonate-
plastics production, has estrogenic properties and is
aneugenic in vitro (Parry et al., 2002, Mutagenesis
17:509). Recently, Hunt et al. (2003, Cur. Biol. 13:546)
have associated spindle aberrations and chromosome
congression failure during meiosis of female mice
to BP-A leakage from damaged cages and/or water
bottles. Therefore, a series of coordinated experiments
were performed within the EU-Project 'Protection of the
European Population from Aneugenic Chemicals' (PEP-
FAC) to ascertain aneugenic effects of BP-A in mouse
germ cells. Meiotic delay and aneuploidy induction in
spermatocytes (Attia et al., 2002, Mutat. Res. 520:1) of
(102/ElxC3H/El)F1 males were studied after 6 daily
oral doses of 0.002-0.2 mg/kg BP-A in corn oil.
Aneuploidy induction was studied in a) in vitro
maturing oocytes of MF1-mice exposed to 50 to 400
ng/ml BP-A in medium, b) in vitro matured oocytes
(Eichenlaub-Ritter & Betzendahl, 1995, Mutagenesis
10:477) obtained from prepubertal (C57BlxCBA/Ca)F1
females exposed to 7 daily oral doses of 20 or 40 mg
/kg BP-A (Hunt et al., 2003), and c) ovulated oocytes
(Tiveron et al., 1992, Mutat. Res. 266:181) from C57Bl
females orally treated with single doses of 0.2 or 20
mg/kg or 7 daily doses of 0.04 mg/kg BP-A. No
increases in hyperhaploid or diploid sperm frequen-
cies were found. In vitro, BP-A did not increase the
hyperhaploidy rates but significantly increased
'diploid' metaphase II oocytes at 200 ng/ml BP-A. Ex
vivo, BP-A affected nuclear maturation to metaphase
II and spindle formation in oocytes but preliminary
data of the ex vivo and in vivo oocytes studies showed
no increase in hyperhaploidy rates. Thus, the aneu-
genic effects of BP-A forecasted by Hunt et al. could
not be demonstrated so far. Further studies are under
way.
Supported by EU-Contract No. QLK-CT 2000-00058
(PEPFAC).
119
abstractboek 20-10-2004 10:28 Pagina 119
PS2 - Poster Session 2
Poster Presentation
PW6023
INTEGRATED CYTOTOXICITY ASSESSMENT IN A
DOWNSCALED IN VITRO MICRONUCLEUS TEST
FVG van Goethem, M de Boeck, B-J van der Leede,
A de Smedt, M Steemans, A Lampo, PH Vanparys
Johnson & Johnson Parmaceutical R&D, BEERSE,
Belgium
Micronucleus formation is primarily considered as a
hallmark of genotoxicity. The in vitro micronucleus
test (MNT) is a well-established assay for the assess-
ment of the mutagenic properties of new chemical
entities during the early phases of drug development.
Its capacity to simultaneously detect clastogens and
aneugens and their potential effects on cell prolifera-
tion, the simplicity of scoring, the rapid performance
and the possibility for downscaling, have made it an
attractive method. It is well known that excessive
cytotoxicity of a test compound can indirectly cause
DNA strand breaks which renders the identification
of true genotoxins difficult. Recently, the expert group
from the International Workshop on Genotoxicity
Testing stated that assessment of toxicity should be
performed by determining cell proliferation (increased
cell counts, population doubling) and by determining
other markers of cytotoxicity (confluency, apoptosis,
necrosis) which can provide additional information
(Kirsch-Volders et al., Mutat. Res., 2003). Here we pres-
ent data of the concurrent assessment, in the same
cells, of genotoxicity and cytotoxicity using a down-
scaled version of the MNT in 96-well microplates and
Alamar Blue conversion. L5178Y cells were treated
with a number of genotoxic and non-genotoxic refer-
ence compounds. The proposed experimental set-up
allowed an increased screening throughput since the
cytotoxicity profile could be determined within min-
utes where after relevant concentrations were select-
ed for further processing in the MNT. A comparison
between the Alamar Blue fluorometric procedure and
relative cell counts to determine cytotoxicity for
selected compounds, learned that there was a good
correlation between the two approaches. In addition,
results showed that the present set-up was able to
identify false positive results due to cytotoxicity
interfering with the final outcome of the genotoxici-
ty testing. Moreover, the current protocol offers the
advantage that only 5 mg of test compound is
required.
PS2 - Poster Session 2
Poster Presentation
PW6024
MICRNUCLEUS STUDIES IN MOUSE SOMATIC
CELLS WITH BISPHENOL-A (BP-A) AND NOCODA-
ZOLE (NOC)
MS abdo Attia1, A. Vanhauwaert2, M Kirsch-Volders2,
ID Adler1
1 GSF, NEUHERBERG, Germany2 Vrije Universiteit Brussels, BRUSSELS, Belgium
BP-A is widely used in polycarbonate-plastics produc-
tion, has estrogenic properties, and is aneugenic in
vitro (Parry et al., 2002, Mutagenesis 17:509). NOC is
used as an anti-tumor agent. It inhibited micro-
tubules function in vitro (De Brabander et al., 1976,
Cancer Res. 36:905) and induced chromosome loss,
non-disjunction and apoptosis human lymphocytes
(Elhajouji et al., 1995, Environ. Mol. Mutagenesis
26:292). Therefore, coordinated experiments were
performed within the EU-Project 'Protection of the
European Population from Aneugenic Chemicals’
(PEPFAC) to determine micronucleus (MN) induction
by these two aneugens in somatic cells in vivo.
Laboratory 1 used (102/ElxC3H/El)F1 males treated
with two daily oral doses of BP-A (0.2, 0.02, 0.002
mg/kg) or two daily ip doses of NOC (12.5, 25, 50
mg/kg), and performed the in vivo bone marrow MN-
test 24h after the last dosing. Laboratory 2 used (CD1)
SPF Swiss albino males treated with single oral doses
of BP-A (500, 1000, 2000 mg/kg) or single ip doses of
NOC (10, 40, 160 mg/kg), and performed the in vivo
bone marrow MN-test (only for BP-A) and the ex
vivo/in vitro MN-test with lymphocytes 24 and 48h
after exposure.
With BP-A, neither of the laboratories observed an
induction of MN in bone marrow. With NOC,
Laboratory 1 observed no induction of MN in bone
marrow but significant cytotoxicity with the highest
dose. Laboratory 2 found no MN induction with BP-A
or NOC in the ex vivo/in vitro test. Laboratory 2 will
have results from the in vivo gut MN-test performed
on the same animals soon.
The present data with NOC are in contrast to the
results reported by Tinwell and Ashby (1991;
Mutagenesis 6:193), which may be due to differences
in animals strains and/or dosing.
Supported by EU-Contract No. QLK-CT 2000-00058
(PEPFAC).
120
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PS2 - Poster Session 2
Poster Presentation
PW6025
EXPOSURE OF BIG BLUE MICE TO HC RED NO.3 DID
NOT INDUCE GENE MUTATIONS
S Pfuhler1, K Milne2, P Blackshear2, M Streicker2
1 Wella AG, DARMSTADT, Germany2 ILS, RESEARCH TRIANGLE PARK, United States of
America
HC Red No.3 is an aromatic amine which is used as
direct hair dye. It is known to trigger gene mutations
in the Ames test. In a 2-year bioassay in mice the dye
showed equivocal evidence with regard to hepatocel-
lular neoplasms in male animals. Therefore, the Big
Blue lacI/cII assay was used to clarify its potential to
induce gene mutations in cells of the liver and uri-
nary bladder.
HC Red No.3 and the vehicle control (1% CMC in deion-
ized water) were administered by gavage once daily
over a period of 8 weeks. The doses used for the
administration of the dye to six male mice per group
were 500 and 1000 mg/kg bw per day. The positive
control (50 mg/kg bw N'-N'-ethyl nitrosourea (ENU))
was applied as intraperitoneal injection for the first 5
days only. At the time of necropsy, samples of the
liver and urinary bladder were obtained for analysis
of mutations at the cII target genes of the shuttle vec-
tor. About one million plaque forming units were
evaluated per dose group. No significant increases in
cII transgene mutation frequency (MF) in the liver or
bladder compared with controls could be observed.
The MF for the bladder was 36.5 x 10-6 for the negative
control, 39.3 x 10-6 for the low dose group and 30.2
x -6 for the high dose group. For the liver the respec-
tive values were 46.8 x 10-6 , 37.0 x 10-6 and 34.5 x 10-6.
The positive control lead to a significant increase in
the MF (bladder:400 x 10-6; liver: 234 x 1010-6), con-
firming the validity of this study.
The result shows that HC Red No.3 does not cause
gene mutations in vivo and thus represents an impor-
tant additional element to assure the absence of a
carcinogenic hazard for humans.
PS2 - Poster Session 2
Poster Presentation
PW6026
PREDICTIVITY OF THE MICRONUCLEUS (MN)
ASSAY FOR THE REGULATORY CHROMOSOME
ABERRATION TEST IN HUMAN LYMPHOCYTES
A Elhajouji, W Frieauff, W Suter
Novartis Pharma AG, BASEL, Switzerland
In the early phases of drug development a variety of
screening procedures are developed to better predict
the outcome of the regulatory assays required prior to
clinical phases and registration. As part of the geno-
toxicity package future drugs are assessed for their
potential to induce chromosome aberrations in mam-
malian cells. In our laboratories micronucleus test in
mammalian cell lines is used as a medium through-
put screening tool for the prediction of the time con-
suming chromosome aberration assay. Due to the low
throughput (manual analysis of the slides) of the pri-
mary human lymphocyte cytokinesis-blocked (CB)
MN assay, this test is used more for chemical class
effect clarification and further mechanistic evalua-
tion when combined with fluorescence in situ
hybridization. We have accumulated data from differ-
ent cell systems i.e. V79 Chinese hamster cells, L5178Y
mouse lymphoma cells, human lymphoblastoid TK6
cells and the primary human lymphocytes. For a com-
parative evaluation of the different test systems three
criteria were used: sensitivity, specificity and predic-
tion of the respective cell systems. TK6 cells showed
an improved prediction of the chromosome aberra-
tion assay when compared to the other two cell lines
V79 and L5178Y with 83% in sensitivity, 81% in speci-
ficity and 81% in predicitivity. However with the
human lymphocyte CB assay all tested compounds
that were also evaluated in the chromosome aberra-
tion assay showed the same genotoxicity profile.
Based on the data from the different screening assays
the primary human lymphocytes showed the highest
prediction for the chromosome aberration assay. An
improvement of the throughput of the human lym-
phocytes CB assay using image analysis would lead
to the most adequate in vitro cytogenetics screening
tool.
121
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PS2 - Poster Session 2
Poster Presentation
PW6027
EVALUATION OF THE MUTAGENIC EFFECTS OF
URBAN AND INDUSTRIAL WASTE WATER IN THE
CANARY ISLANDS
E de la Pena1, O Herrero1, S Aguayo2, A de la Torre2,
M Carballo2, MJ Mu-oz2
1 Centro de Ciencias Medioambientales CSIC,
MADRID, Spain2 CISA - INIA, MADRID, Spain
It is shown the importance of mutagenic studies of
wastewater due to the presence of polluting agents,
which are mainly organic micropollutants, that affect
the recipient water quality and which are not includ-
ed in the actual safety regulations. Eighteen waste
water effluents were studied: 5 urban, 5 industrial
and 8 mixed. Their physical and chemical characteri-
zation was realized and the acute toxicity of the
whole effluent was valued by means of different
assays recommended by Whole Effluent Toxicity. The
method based on Toxicological Identification
Assessment was used with certain effluents. As a part
of the toxicological assessment the organic extract
was evaluated with acute toxicity, mutagenic, terato-
genic and estrogenic assays. The mutagenic effects
are studied with the Salmonella typhimurium assay,
using the TA100 and TA98 strains analysis. The esti-
mation of Toxicity Units was done with the existing
information of the identified compounds. We show
no mutagenic effect in the preliminary data of the
analysed effluent samples. However, estrogenic and
teratogenic effects were detected in 4 mixed and 3
industrial effluents respectively. The organic part of
the samples could be the main responsible of this tox-
icity. The analysis of the organic compounds proves
the presence of different chemical remainders in
ng/L concentrations: surfactants (nonylphenol and
octylphenol), plastic softeners (phthalates, bisphenol
A), steroid hormones (estradiol) and synthetic ones
(ethinilestradiol), PHAs, fatty acids, insecticides
(diazinon), etc. We wish to emphasize the absence of
toxicological and environmental data for almost the
50% of the identified compounds. Project REN2002-
04162-C02-02
PS2 - Poster Session 2
Poster Presentation
PW6028
BIOMONITORING OF CHILDREN - RESULTS FROM
THE WORK OF A SCALE SUBGROUP
LE Knudsen1, L Casteleyn2, C Sala3
1 Institute of Public Health, COPENHAGEN, Denmark2 Ministry of the Flemmish Community, BRUSSELS,
Belgium3 ARPA, LOMBARDIA, Italy
Monitoring of the quality of the environmental
media, air, water and soil, has a long tradition in
European countries and there is ample legislation
dealing with it. A step that is leading closer to evalu-
ating human health effects is human biological mon-
itoring, where the concentration of a pollutant - or its
metabolite(s) - is determined in a biological sample,
generally blood or urine. In a similar way, other types
of biomarkers can help assess the reaction of the
human body to environmental pollutants.
The various biomonitoring studies that are run in the
different European countries are generally not carried
out using the same methodological approach. More
harmonised biomonitoring survey programmes are
recommended. To study the cause-effect framework
and to document the level of scientific evidence of a
cause-effect link properly defined research studies
are needed. Therefore research projects should be
'grafted' on the surveillance framework where possi-
ble. Less invasive methods are to be considered
together with new techniques within genetic expres-
sion profiles.
An overview of the European research in children’s
health has identified some 100 studies including at
least 400,000 children participating in existing bio-
monitoring and or research activities conducted in
the member States and acceding Countries. Forty-
four studies dealt with exposure to heavy metals, 15
with dioxins/PCB, and 5 with exposure to endocrine
disruptors. Twenty-seven studies included the deter-
mination of biomarkers of asthma and allergy and
only a limited number investigated cytogenetic bio-
markers in relation to environmental pollution.
The ethical issues of doing good and no harm to the
participants and the information and dissemination
of project results are very important.
References to the 2 reports made by the group are
http://www.brussels-conference.org/Download/
baseline_report/BR_Biomonitoring_final.pdf
http://europa.eu.int/comm/environment/health/
finalreports_en.htm.
122
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PS2 - Poster Session 2
Poster Presentation
PW7001
AGEING AND THE SEQUENCE OF CENTROMERE
SEPARATION
B Bajic1, SP Biljana2, D Ninoslav3, L Zivkovic2,
MZ Milicevic4
1 Galenika Pharmaceuticals, BELGRADE, Yugoslavia2 School of Pharmacy, BELGRADE, Yugoslavia3 School of Veterinary Medicine, BELGRADE,
Yugoslavia4 Vinca Institute of Nuclear Sciences, BELGRADE,
Yugoslavia
Loss of control of the sequence of centromere separa-
tion or out-of-phase of centromere division is found
in ageing cells, Alzheimer’s disease patient’s (AD),
various chromosome instability syndromes and can-
cers. In ageing cells the main affected chromosome is
the X chromosome. X chromosome instability mani-
fests as premature separation of the centromere or
out-of-phase centromere division. Out-of-phase cen-
tromere division has been observed to correlate with
non-disjunction leading to aneuploidy of the X chro-
mosome in elderly human females and less in elderly
human males. Human sequence of centromere sepa-
ration is well known. In humans the earliest separat-
ing centromeres are from chromosome 18,17,2,10 and
12, respectively. Those separating last belong to chro-
mosomes 21,22,13,14 and 15. Other chromosomes and
the X chromosome separates between the two
extremes. Knowing that altered sequence of cen-
tromere separation in elderly people show that
X-chromosome separates before chromosome 18, we
used fluorescent in situ hybridization (FISH) for the a-
centromeric region of the X chromosome in order to
estimate more precisely the time when PCD arises in
the cell cycle. FISH analysis has revealed that X chro-
mosome expresses out-of-phase division in elderly
females 7.46% in metaphase of mitosis and 9.35% in
interphase nucleuses and in elderly males group
2.84% in metaphases and 5.54% in interphase nucle-
uses. Using FISH, our results show that out-of-phase
can occur much earlier than metaphase of mitosis, i.e.
in interphase of the cell cycle, immediately after
replication. Maintenance of sequential separation of
centromeres is genetically highly controlled thus sug-
gesting that out-of-phase of the X chromosome can
be viewed as a non-specific manifestation of chromo-
some instability affected by the ageing process in eld-
erly humans.
PS2 - Poster Session 2
Poster Presentation
PW7002
TRIMETHYLTIN CHLORIDE INDUCES INTRACEL-
LULAR CALCIUM ELEVATION AND APOPTOSIS IN
HELA-S3 CELLS
AM Florea, D Büsselberg, AW Rettenmeier, ED Dopp
University Hospital Essen, ESSEN, Germany
Trimethyltin chloride (TMT) is found in the environ-
ment, and cases of human intoxication have been
reported. We have previously shown that TMT induces
elevated Ca2+ transients in HeLa cells (Florea et al.,
Europ. J. Physiol., 447, 2004, S127). Calcium-dependent
mechanisms are involved in the initiation of gene
expression, in cell proliferation, cell cycling, and
apoptosis. In this study, we further investigated if
TMT-induced [Ca2+]i rise is associated with the induc-
tion of apoptosis. Laser Scanning Microscopy (LSM)
and Fluorescence Activated Cell Sorting (FACS) were
used for the measurements. Intracellular calcium
changes were determined by using Fluo 4. Assessment
of apoptosis was proceeded using propidium iodide
and annexin V/FITC staining. The results show that
TMT perturbed calcium homeostasis in HeLa-S3 at
concentrations as low as 100 nM and that this pertur-
bation correlates with the induction of apoptosis. The
[Ca2+]i elevation is mainly due to a release from inter-
nal stores. Moreover, a [Ca2+]i rise in the nuclear
region of HeLa-S3 cells was also observed. Significant
apoptotic death was induced by TMT after 12 h at 2
µM and after 48 h at 0.5 and 2 µM. Parallel staining of
Fluo 4 and annexin V showed significant increases of
[Ca2+]i, however, the exteriorisation of phosphatidyl
serine was not significant. We propose that calcium
elevation is very important in TMT-induced cytotoxi-
city in HeLa-S3 cells. Moreover, the calcium rise in the
nuclei might be related to DNA damage and might
trigger apoptosis. The data suggest that TMT exerts
its effects at least at two different sites: at the cell
membrane and inside the cell resulting finally in cell
death.
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PS2 - Poster Session 2
Poster Presentation
PW7003
ELECTROPORATION INCREASES CELLULAR UPTAKE
AND GENOTOXIC EFFECTS OF ORGANOARSENIC
COMPOUNDS IN CHO CELLS
ED Dopp1, AM Florea1, LM Hartmann2, B Shokouhi1,
AV Hirner2, AW Rettenmeier1
1 University Hospital Essen, ESSEN, Germany2 University of Duisburg-Essen, ESSEN, Germany
We have shown in previous studies that the induction
of genotoxic effects caused by methylated arsenic
species primarily depend upon their ability to pene-
trate cell membranes (Dopp et al., Occup Medic, Social
Medic, Environm Medic 39 (2004) 4, 217). In the pres-
ent investigations cellular uptake of arsenic com-
pounds was enforced by electroporation to find out
whether an increased uptake leads to enhanced cyto-
toxic and genotoxic effects in CHO cells. We also
wanted to clarify if the arsenic compounds are intra-
cellularly bound to membranes, or if they are present
in the cytosol either bound to proteins or not.
Cytotoxicity was determined the by Trypan blue
extrusion test and genotoxicity by the micronucleus
assay. Measurements of the intracellular arsenic con-
centrations were carried out by ICP-MS in (1) whole
cell extracts, (2) membrane-free cell extracts and (3)
deproteinised cell extracts. The results show that, in
the concentration range of 0.1 µM-500 µM (exposure
period: 24 h), trivalent arsenic compounds (AsNaO2,
MeAs(OH)2, Me2AsOH) exert higher cyto- and geno-
toxicity compared to that of the pentavalent arsenic
species (AsHNa2O4·7 H2O, MeAsO(OH)2, Me2AsOOH,
Me3AsO). Three compounds with a low membrane
permeability (MeAsO(OH)2 = 0.05 %, Me2AsOOH =
0.02 %, Me3AsO = 0.13 %) were used in the electropo-
ration experiments. After an exposure period of 30
min and electroporation at 290 V for 40 µs, CHO cells
were loaded with 0.25 % MeAsO(OH)2, 0.33 %
Me2AsOOH, and 0.52 % Me3AsO, respectively, present
in the cell culture medium. Micronucleus formation
was significantly increased in arsenic-exposed CHO
cells after electroporation. The ICP-MS measurement
of the arsenic derivatives revealed that these com-
pounds were present in the cytosol and not bound to
proteins or intracellular membranes.
PS2 - Poster Session 2
Poster Presentation
PW7004
GENOTOXIC POTENTIAL OF RESPIRATORY BEN-
TONITE PARTICLES WITH DIFFERENT QUARTZ
CONTENTS AND SURFACE MODIFICATIONS
S Geh1, D Höhr2, T Shi2, ED Dopp1, PJA Borm2,
AW Rettenmeier1
1 University Hospital Essen, ESSEN, Germany2 IUF GmbH Düsseldorf, DUSSELDORF, Germany
Crystalline silica has been classified as a human car-
cinogen, but there is still considerable controversy on
its fibrogenic and carcinogenic potential. In the pres-
ent study, we investigated the genotoxic potential of
bentonite particles (Ø 3 – 7 µm) with an a–quartz con-
tent of up to 6 % and different surface activation
(alkaline, acidic, organic). Human lung fibroblasts
(IMR-90) were incubated for 24 h, 48 h or 72 h with
bentonite in concentrations ranging from 1 to 100
µg/cm2. Cytotoxicity was studied by determination
of viable cells using flow cytometry and UV-
spectroscopy (Alamar blue test). Cellular uptake of
particles was estimated by transmission electron
microscopy (TEM). Genotoxicity was assessed using
the micronucleus (MN) assay and kinetochore analy-
sis. The generation of reactive oxygen species (ROS)
caused by bentonite particles was measured by the
electron spin resonance technique (ESR). Our results
show that surface- modified bentonite particles with
a quartz content of 5 % - 6 % are more cytotoxic than
untreated bentonites or bentonites with a quartz
content up to 5 %. The cellular uptake of surface-mod-
ified bentonites was significantly higher compared to
that of untreated (native) bentonite particles. The for-
mation of MN was only slightly increased (p<0.05)
after exposure of IMR-90 cells for 72 h to bentonite
samples with a higher quartz content (concentration:
15 µg/cm2). The surface modification of particles did
not influence MN formation. Clastogenic and/or
aneugenic effects were dependent upon the type of
bentonite samples applied. Generation of ROS corre-
lated with the observed genotoxic effects. We con-
clude, that the genotoxic potential of bentonite
particles depends upon the content of quartz and
available transition metals, in contrast to the cytotox-
ic potential which depends upon the chemical modi-
fication of particles and the surface structure.
124
abstractboek 20-10-2004 10:28 Pagina 124
PS2 - Poster Session 2
Poster Presentation
PW7005
MUTS-DEPENDENT GASP PHENOMENON IN
MIXED CULTURES OF ESCHERICHIA COLI AND SAL-
MONELLA ENTERICA
V Bacun-Druzina1, K Gjuracic2, J Pesut1, J Franekioli1
1 Fac.of Food Technology and Biotechnology, ZAGREB,
Croatia2 Pliva, Research Division, ZAGREB, Croatia
The major mechanisms of microbial genome ecologi-
cal specialization are the loss of preexisting genes or
gene activities during evolution. The ability of
Escherichia coli to grow during carbon starvation in
stationary phase has been termed the Growth
Advantage in Stationary Phase (GASP) phenotype.
The GASP phenomenon has been observed in range
of microorganisms, including clinical isolates of E.
coli and S. enterica. The occurrence of mutators
among isolates of E. coli and S. enterica is up to 10%.
The resulting mutants with increased fitness express
GASP phenotype enabling them to grow and displace
the parent as the majority population. The amount of
MutS, homodimer which binds to mismatched bases,
is strongly down regulated as E. coli cells enter sta-
tionary phase. During the prolonged stationary phase
decreased amounts of MutS contributed to the
increased mutagenesis resulting in mutator pheno-
type.
The aim of our investigation was to analyze the
mixed populations of E. coli K12 and S. enterica
serovar Typhimurium during the prolonged period of
carbon starvation due to the evaluation of the GASP
phenomenon and its dependents of mutS gene. The
mixture of one-day-old culture of S. enterica and ten-
day-old cells of E. coli as minority showed the GASP
phenomenon when they are resistant to a growth
inhibitor (nalidixic acid or streptomycin). The same
results were obtained when the mixture consisted of
S. enterica as a minority cells. On the other hand, in
mixed populaton of mutS mutants of both bacteria, in
various ratios and age, only abortive GASP phenotype
was observed. In addition, in the cells with GASP phe-
notype the chromosomal rearrangements were
determined by PFGE.
PS2 - Poster Session 2
Poster Presentation
PW7006
DIFFERENT NUCLEOTIDE EXCISION REPAIR-
DEFICIENT MICE SHOW DIVERGENT AGING
PHENOTYPES
SWP Wijnhoven1, RB Beems1, MET Dollé1, J Vijg2,
P Lohman1, J Hoeijmakers3, G van der Horst4,
H van Steeg1
1 National Institute of Public Health and the
Environment, BILTHOVEN, The Netherlands2 University of Texas, TEXAS, United States of America3 Erasmus University, ROTTERDAM, The Netherlands4 Erasmus Medical Center, ROTTERDAM,
The Netherlands
The accumulation of somatic DNA damage is consid-
ered to be a major cause of the aging process in vari-
ous species including mice and humans. Among the
sources of DNA damage, reactive oxygen species (ROS)
are often thought to be the ultimate cause of aging.
However, the mechanisms involved remain obscure.
To counteract the effects of DNA damage, an intricate
network of DNA repair pathways has evolved. One
major pathway is nucleotide excision repair (NER),
which removes a broad range of bulky lesions includ-
ing some forms of oxidative damage. Patients with a
defect in NER proteins like CSB and XPD, both
involved in repair as well as transcription of DNA,
appeared to have a decreased life span.
In order to investigate whether defects in genome
maintenance are correlated to accelerated aging, we
have successfully conducted several longevity and
cross sectional studies with mice having defect in
DNA repair and/or RNA transcription (i.e. Xpa-, Csb-,
Xpd(Ttd)- deficient mice as well as C57Bl/6 wild type
controls). The mean survival of female Xpd(Ttd) as
well as Xpa mice appeared to be much shorter (appr.
90 weeks) than those found for Csb and wild type lit-
termate controls (104-110 weeks). Full histopathology
has been performed in aged female mice, and gross
examination at autopsy revealed small posture,
kyphosis, large spleen, small thymus and abnormal
skin and hair especially in Xpd(Ttd) mice. The termi-
nal body weights in Xpd(Ttd) and Csb females were
decreased, with an increase in the relative weights of
several organs of Xpd(Ttd) mice, especially in the kid-
ney, spleen and the heart. Furthermore, lipofuscin
pigmentation (an aging feature, correlated to oxida-
tive damage) was found to be accumulated in the
liver of Xpd(Ttd) mice as compared to the other phe-
notypes.
This work was supported by NIH/NIA (1 PO1-AG17242).
125
^
abstractboek 20-10-2004 10:28 Pagina 125
PS2 - Poster Session 2
Poster Presentation
PW7007
PREMATURE AGING IN XPC/CSB AND XPA/CSB
DOUBLE KNOCKOUT MICE
I van der Pluijm1, J de Wit1, RB Beems2, CTHM van
Oostrom2, A Maas1, H van Steeg2, J Hoeijmakers3,
G van der Horst1
1 Erasmus Medical Center, ROTTERDAM,
The Netherlands2 National Institute of Public Health and the
Environment, BILTHOVEN, The Netherlands3 Erasmus University, ROTTERDAM, The Netherlands
The Global Genome Repair subpathway of Nucleotide
Excision Repair (GG-NER) removes helix-distorting
DNA damage in the entire genome, whereas Trans-
cription Coupled Nucleotide Excision Repair (TC-NER)
removes transcription-blocking DNA lesions. Defects
in these pathways can have deleterious effects; XP-A
patients are totally devoid of NER activity, XP-C
patients only lack GG-NER. Both show cutaneous
abnormalities and increased skin cancer predisposi-
tion, often accompanied by neurological degenera-
tion. CS patients have a defect in TC-NER and show
postnatal growth failure and severe neurodysfunc-
tion. CS is a progeroid syndrome, revealing a strong
link between DNA repair deficiency and premature
aging.
Mouse models for XP-A, XP-C and CS-B mimic many
features of the human syndromes. XPA and XPC mice
are photosensitive and display skin-cancers after UV-
exposure. Aging features of CSB mice include retinal
degeneration and weight loss. These findings led us
to hypothesize that the aging features of CS are dis-
tinct from NER function. To test this we generated
double mutant mice and (XPC/CSB, XPA/CSB and
XPA/XPC). Surprisingly, the phenotype of CSB mice is
dramatically enhanced in an XPC or XPA deficient
background. Although embryos appear to develop
normally, XPC/CSB and XPA/CSB mice soon after birth
become severely growth retarded, ataxic and kyphot-
ic. The maximum age reached is 21 days. These mouse
models demonstrate that loss of CSB promotes an
aging phenotype, which is accelerated by a total NER-
deficiency, suggesting that the aging features are
caused by accumulation of endogenous DNA damage
that is not repaired in the absence of NER. Here we
present the generation and characterization of a con-
ditional XPA knockout mouse, allowing time- and tis-
sue dependent inactivation of the XPA gene in
XPA/CSB animals in order to further investigate the
onset of the XPA/CSB phenotype.
126
abstractboek 20-10-2004 10:28 Pagina 126
Author Index
Familyname Initials Prefix Abstractcodes
Aardema MJ OW6003
Aardweg GJ van den PW2040
Aarts JMMJG OW1004
Abd El-Aziz M PW2002
Abet M PW2042
Abeysinghe S PW5007
Aboud M PW2049
Abrini J PW6005
Acker FAA van PW2037
Ackerman JI PW2046
Adler ID PW6022, PW6024
Agana L OW1003
Agen E van OS2001, OW1001
Aguayo S PW6027
Ahr J OS2002
Aka V OW7004, PW3014
Albering H PW2024
Albertini S OW5004
Albrecht C OW4001
Alija A PW4002
Alink GM OS5001
Alonso-Moraga A PW6005
Alunni-Perret V PW1002
Anagnostakis N PW2017, PW2018,
OW7003
Andreatta R PW3013
Andreoli CA PW2009, PW3021
Andreoli R PW3018
Anna L PW3023, PW3004
Anttila S PW3008
Anuszewska EL PW1003
Appolloni M PW6013
Arabski M PW2033
Arbillaga L PW1007
Arlt VM PW2003, PW1002
Athanasiou E PW2017
Attardi LD PW2040
Attia MS abdo PW6022, PW6024
Aubrecht J OS2003, PW2046
Autrup H OW3001, OW3002,
PW4015
Aydin A PW4001
Aydin S PW4009
Azqueta A PW2020
Baccelliere L PW6011
Backendorf CMP OW2003
Bacun-Druzina V PW7005
Bader A PW1005
Badr Y PW2002
Baer-Dubowska W OW1002, PW1004
Bagnasco M OW4003
Bajic B PW6001, PW7001,
PW2034, PW6019
Bajic V PW6017
Bak H OW3002
Baki M PW2035
Barale R OW3003
Bart M OW5001
Barta IVO PW1013
Bártová J PW1013
Basaran A PW4009
Bast A PW4007
Bauer D OW5003
Baum M PW4010
Baumeister M PW6012
Beems RB PW7006, PW7007,
PW2040
Beerens D OW6001
Beffy P PW2043
Berg J van den PW2040
Berggren P OW3004
Beric-Bjedov T PW4004, PW4005
Beskid O PW6014, PW6010,
PW6015
Biljana SP PW6001, PW7001
Binderup ML PW4015
Binkova B PW6010, PW5006
Blaauboer BJ OW6002
Blackshear P PW6025
Blaes S de PW1007
Blake JF PW2046
Blasco MAB OS3003
Blasiak J PW2033, PW2030
Bodrogi I PW2035
Boeck M de PW2004, PW6016,
PW6023, PW3014
Boffetta P OS4003, OW3003
Bolderson E PW2029
Bolognesi C PW3013
Bonassi S OW3003
Borgdorff V PW2045, OW2007
Borm PJA OW4001, PW2013,
PW7004, PW2025
Bouwman FG PW1014
Bracci PM OW1003
Braun K PW6012
Breda SGJ van OW1001, OS2001,
PW1014
Breikers G PW1014
Brennan P OS4003
Bresgen N PW4002
Briedé JJ PW4013, PW4012
Bruins W PW2040
Bubic Spoljar J PW6002
Buchet JP OW7004
Buendia G de PW2039
Bürkle A OW4004
127
abstractboek 20-10-2004 10:28 Pagina 127
Buschini A PW1008, PW4011
Buss K OW5002
Busuttil R OW7005
Butkiewicz D PW3007, OW7002
Büsselberg D PW7002
Byun KP PW4003
Calcagnile A OYSAL2
Camoirano A OW4003
Campisi J OS3004
Canova S OW2004
Canzian F OS4003
Carballo M PW6027
Carratore R del PW2043
Cartiglia C OW4003
Casella M PW2042, PW2043
Cassee FR PW2025
Casteleyn L PW6028
Cavallo D PW4006
Cebulska-
Wasilewska A OW3003
Celotti L OW2004
Chebotarev AN PW1001
Chen X OW2005
Chen Y OW2005
Cheung JR PW2046
Chianese C PW4006
Chiesara E PW2015
Chiu RK PW2052
Chojnacki J PW2033
Christopoulos G PW2019
Christou K PW2017, PW2018,
PW2021
Chuang EY OW2005
Chvatalova I PW5006, PW6010,
PW6015, PW6014
Ciric JC PW2034
Clay P PW6018
Clonfero E PW3017
Coleman CN OW2005
Colognato R OW4002
Conti L PW3021
Cooke MS OYSAL1
Coppedè F OW1003, OW4002
Cordelli E PW6013
Cosyns JP PW1002
Cozzi R PW6011
Cramers P PW2051
Crebelli R PW3021
Creus A PW3020
Csejtei CSA PW3001
Csekeo A PW3004
D'Errico M OYSAL2
Dahm-Daphi X OW2002
Darroudi F PW1005
Davolos DD PW2016
Dejmek J PW5006
Delft JHM van OS2001, PW1014,
OW1001, PW5003,
PW5002, PW5004,
PW3009
Demopoulos N OW3001
Dietrich H PW4010
Dizdaroglu M OYSAL2
Djelic N PW6019, PW6017
Dobbelsteen DJ van den PW2037
Doehmer J PW2042
Dogliotti E OYSAL2
Dollé MET OW7005, PW7006
Dopp ED PW7003, PW7002,
PW7004
Dostal M PW6014, PW5006
Doudounakis S OW7003
Dragsted LO PW4015
Druzhinin VG PW6009
Drzewoski J PW2033
Duburs GJ PW2044
Duffin R PW2025, OW4001
Dundar K PW4001
Dur A PW4001, PW2005
Dusek Z PW6015, PW6014
Duydu Y PW4001, PW2005,
PW3005
Dybdahl M PW4015
Eckl PM PW4002
Eichenlaub-
Ritter U PW6022
Eicker J OW4001
Eisenbrand G PW4010
Eken A PW4001
El-Awady RA OW2002
El-Khatib N PW2002
Elhajouji A PW6026
Ellinger-
Ziegelbauer H OS2002
Elliott B PW6018
Ember I PW3001
Engelhardt G PW6012
Engels LGJB OW1001
Eom MO PW4003, PW5001
Epe B OW4004
Erixon K PW2029, PW2032
Esik O PW2035
Eskelinen M PW3006, PW3010,
PW3002
Ezpeleta O PW1007
Fabiánová E OW3003
Faluhelyi ZS PW3001
Fenech M OW3003
Ferencic Z PW6002
Ferraris M PW2015
Festa F PW6011
Filiberti R PW3013
128
abstractboek 20-10-2004 10:28 Pagina 128
Flamma F PW2009
Fleischer B PW3003
Flohr CF OW4004
Flora S de OW4003
Florea-Wang D PW2036
Florea AM PW7002, PW7003
Flueckiger-Isler SF PW6012
Fokt I PW1003
Fontana I OW4002
Forrest MS OW1003
Francesco A di PW4006
Franekioli J PW7005
Fred CF PW6008
Freidig AP OW5001
Fresegna AM PW6013
Frieauff W PW6026
Frigerio S PW2015
Fritsche L PW2031
Froetschl RF OW5002
Fronza G OW2006
Fucic A PW6002, OW3003
Furlini M PW1008
Fusekova M PW2031
Gadisetti CVR OW2005
Gajecka M OS4002
Galati R PW3021
Galofre P PW3020
Galova EG PW2028, PW2048
Gant TW OS2004
Geh S PW7004
Gemignani F OS4003
Georgiadis P OW3001
Gerlofs-Nijland ME PW2025
Gervais V PW6012
Giachelia M PW6011
Giordano E PW3013
Gismondi M PW4006
Gjuracic K PW7005
Godschalk RWL PW2024, PW3009,
PW2052
Goethem FVG van PW2004, PW6016,
PW6023,
Gold LS OS5004
Golovina TA PW6009
Gompel JAJ van OW6001, PW6012
Goncharova RI PW2044
Gottschalk RWH PW3009
Gourgoulianis K PW2017, PW2018,
PW2021
Gozdzik A PW1003
Granath F PW6008
Grass P OW5003
Grifalconi M OW2004
Groten JP OW5001
Gruber BM PW1003
Gruijl FR de PW2026,OW2003
Grzesiuk EG PW2006, PW2008,
PW2007
Grzesiuk W PW2008
Gundy S PW2035, PW3019,
PW6021, OW3003
Gyorffy E PW3004
Gyori Z PW3004
Haan J de PW2024
Habalova V PW3003
Haenen GJJM PW4007
Hageman GJ PW4007
Hagmar L OW3003
Hainaut P PW2047
Hall J OS4003
Hansteen I-L OW3003
Haq MA PW2027
Harris CC OW7002, PW3007
Hartmann LM PW7003
Harvey JS PW5005
Hasler-Nguyen N PW6012
Hastings H OS6002
Haugen AAGE PW3015
Hayashi M PW2003
Hees-
Stuivenberg S van PW2045
Heijne WHM OW5001
Heikinheimo L PW3010
Heilimo I PW3011, PW3012
HeiYing JIN PW3022
Helleday T PW2029, PW2001
Hemminki KH OS4001
Henderson LM OW6003
Hernandez A PW3020
Herrero O PW6027
Herwijnen MH van OS2001, PW1014,
PW4012, PW5003
Hickson ID OW7001
Hirayama T PW2011
Hirner AV PW7003
Hirvonen A PW3008, PW3010,
PW3011, PW3012,
OW3005, PW3006,
PW3002
Hisamatsu Y PW2011
Hoeijmakers J OW7005, PW7006,
PW7007
Hogervorst J PW2024
Höhr D PW7004, OW4001
Hollstein M PW1002
Holly EA OW1003
Honma M PW2003
Hoogenboom LAP OW1004
Hoogendoorn SM PW2046
Hoogervorst E PW2040
Horbach GJMJ PW2037
129
abstractboek 20-10-2004 10:28 Pagina 129
Horst B van der OW7005
Horst G van der PW7007, PW7006
Hou S OW3004
Hovinen J PW2036
Hubbe P OW2002
Hung RJ OS4003
Husgafvel-
Pursiainen K PW3008
Iavicoli S PW4006
Idaomar M PW6005
Impivaara O PW3008
Ingel FI PW6007
Izzotti AI OW4003
Jacks T PW2040, PW2041
Jagerstad I OS5003
Janion C PW2007
Jansen JG OW2007
Janzowski CJ PW4010
Jaruga P OYSAL2
Jarventaus H PW3011, PW3012
Jarvisalo J PW3008
Jazbec AM PW6002
Jee SW PW5001
Jenssen D PW2029,PW2032
Johansson F PW2029
Johansson FJ PW2032
Jones E PW6018
Jonge J de PW4012
Jonker D OW5001
Jung HK PW4003
Juren T PW1011
Kajosmaki T PW2023
Kalina I PW3003
Kamal N PW2002
Kampman E PW3016
Kanaar R OS1002
Kanariou M PW2022
Kanavetas P PW2022
Kang HI PW5001, PW4003
Kang MK PW5001
Kang SC PW6004
Kannouche PL OS1004
Karanastasi G PW2017, PW2018,
OW7003
Karimi-Busheri F PW2038
Kasler M PW3019
Kasper P OW5002
Kasprzak M PW2033
Kataja V PW3006, PW3010,
PW3002
Katsouyianni K OW3001
Kaufmann G OW5002
Kautiainen A PW6008
Kawanishi M PW2011
Kazmierczak P PW2033
Keenan PO PW6006
Kelecsenyi A PW6021
Kempers P PW4012
Kenny J PW5005
Ketelslegers HB PW3009
Khan I PW5007
Khan QM PW2027
Kienhuis AS PW5002
Kim MH PW5001
Kim OH PW4003, PW5001
Kirkland DJ OW6003
Kirkwood TBL OS3001
Kirsch-Volders M PW6024, PW3014,
OW3003, OW7004
Kiss I PW3001
Kleinjans JCS PW1014, PW5002,
PW5004, PW1012,
OS2001, PW3009,
PW2024, PW2051,
PW4012, PW4013
Klupinska G PW2033
Knaapen AM PW2013, PW3009,
PW2052
Knasmueller S PW1005
Knezevic-
Vukcevic J PW4004, PW4005
Knight W PW6006
Knudsen LE OW3003, PW5008,
PW6028
Koissi N PW2010
Kok TH de OW1004
Kok TMCM de PW4012
Kontogianni N PW2021
Koo HJ PW6003, PW6003
Kosma VM PW3006, PW3010,
PW3002
Kostic S PW3004
Kosyakova NV PW1001
Köteles GJ OW3003
Kotova N PW1011
Krajka-Kuzniak V OW1002, PW1004
Kram NR PW3016
Kranen HJ van PW2026, PW3016
Krul CAM PW6020
Kruszewski M PW2008
Krzesniak M PW3007, OW7002
Ku WW PW2046
Kumar R OW3004
Kwon KS PW5001
Kyrtopoulos SA dr. OW3001
Lagerqvist A PW2032
Lambert B OW3004
Lampo A PW2004, PW6016,
PW6023
Landi S OS4003
Langie SAS PW2052
Langová M PW1013
130
abstractboek 20-10-2004 10:28 Pagina 130
Larsson P OW3004
Lazutka J OW3003
Lee BM PW6003, PW6004,
PW1009
Leede BJ van der PW2004, PW6023
Leede BM van der PW6016
Leeuwen AIM van PW3016
Leeuwen DM van PW5004, OS2001
Lehmann AR OS1004
Leopardi P PW3021
Levi F OS6001
Lewis PD PW5007
Lhoste E PW1005
Li H PW2025, OW4001
Liber HL OW2005
Liberti SE OW2001
Lind HL PW3015
Lindholm C OW3003
Lison D PW3014
Loft S PW4015
Lohman P OW7005, PW7006
Longobardi M. OW4003
Loniarek J. PW1010
Lönnberg HARRI PW2010
Lopez de Cerain A PW1007, PW2020
Lord GM PW1002
Lozano G. PW2040
Luken MEM PW3016
Lundin CL PW2029
Lützen A OW2001
Lynch AM PW5005, OW4005
Maas A PW7007
Maas LM PW4012
Mackie C OW6001
Maclean N PW2016
Mages W PW2031
Magrini R OW2006
Majsterek IM PW2030
Mancini A PW4011
Manini P PW3018
Marabini L PW2015
Marcon F PW3021
Marcos R PW3020
Maridaki K PW2019
Mariman ECM PW1014
Marino C PW6013
Markovic B PW6019, PW6017
Markovic D PW6002
Martino A PW2009
Mateuca RAM PW3014, OW7004
Maunu H PW3011, PW3012
Mayerhofer B PW2014
Maywood E OS6002
McGinnis C OW5003
Meeuwen RNC van PW6020
Menichini T OW2006
Mercati F PW3018
Mercelina-
Roumans P PW2024
Mercken EM PW4007
Merello A OW4003
Mersch-
Sundermann V PW1005
Messini-
Nikolaki N PW2017, PW2018,
PW2019, PW2021,
PW2022, OW7003
Mezzoug N PW6005
Mielzynska D PW3017
Migliore M PW3018, OW4002
Milcova A PW6010, PW6015,
PW5006
Mildner B PW6002
Milicevic MZ PW2034, PW7001
Milne K PW6025
Minarovits J PW3004
Minina VI PW6009
Mirkova E OW3003
Mitchell JB OW2005
Mitic-Culafic D PW4004, PW4005
Mitrunen K PW3008, PW3010,
PW3011, PW3012,
PW3002
Mitrunen M PW3006
Mognato M OW2004
Mokrushina NV PW6009
Müller P PW4015
Moonen HJJ PW1006
Moonen EJC PW4013
Moonen JC OW1001
Mrzyglodzik M PW3007, OW7002
Mu-oz MJ PW6027
Mueller L OW5003
Mullenders LHF PW2026, PW2026,
PW2051, OW2003
Müller AK PW4015
Müller L OW6003
Muñoz serrano A PW6005
Muro A di PW2042
Mutti A PW3018
Müller L OW6003
Myllynen PK PW2023
Naccarati A PW3018
Nakagawa Y PW2011
Nardo T OYSAL2
Neri M PW3013
Nexo BA OW3002
Niedernhofer L OW7005
Nieminuszczy JN PW2007, PW2006,
PW2008
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Nikitchenko NV PW2044
Nikolic B PW4004, PW4005
Ninoslav D PW6001, PW7001
Nisar H PW2027
Norppa H PW3011, PW3012,
OW3003
Nowosielska A PW2006
Nunziata A PW2009
Olatunde
Farombi E PW4015
Olive K PW2041
Ommen B van OW1005, OW5001
Oosten M van OW2003
Oostrom CTHM van PW2040, PW7007,
PW2041
Osowski JJ PW2046
Overvad K OW3002
Pacanovska M PW2031
Pacchierotti F PW6022
Pachón G PW2020
Packenham JP PW2050
Palma G de PW3018
Park MS PW5001
Park MS PW4003
Parker KM PW2001
Pastaka C PW2021
Pavanello SP PW3017
Peeters S PW4013
Peijnenburg A OW1004
Pellacani C PW1008, PW4011
Pena E de la PW6027
Perrone E PW3013
Persaud P PW2046
Peöut J PW7005
Pfuhler S PW6025
Phillips DH PW2003
Piano L del PW2042
Pietrangeli BM PW2016
Piperakis SM PW2017, PW2018,
PW2019, PW2021,
PW2022, OW7003
Platonova VI PW1001
Plazinska MT PW2008
Pluijm I van der PW7007
Polfvkova Z PW1013
Poli P PW1008, PW4011
Powell SN OW2002
Priebe W PW1003
Priel E PW2049
Pulliero A PW3017
Puntoni R PW3013
Pytel D PW2030
Raaschou-
Nielsen O OW3002
Radice S PW2015
Radicella JP OW4004
Ramaekers CHMA PW2052
Ranaldi R PW6022
Rasmussen LJR OW2001
Rasouli-nia AR PW2038
Reddy A OS6002
Rees R PW5005
Reimann R PW6012
Remenar E PW3019
Renes JW PW1014
Restivo FM PW4011
Rettenmeier AW PW7002, PW7003,
PW7004
Richter E PW2014
Rietjens IMCM OS5001
Risom L PW4015
Rodrigo GR PW2014
Roggieri P PW3013
Rosa N la PW6011
Rossi C PW1008, PW4011
Rossi S PW3021
Rössner P OW3003, PW5006,
PW6010, PW6014,
PW6015
Rubes J PW6015
Ruepp S OW5004
Rusin B PW2012
Rusin M PW3007
Rusin R OW7002
Russo D OW2006
Ryabokon NI PW2044
Rydberg P PW1011
Rydzanicz M OS4002
Ryeom TK PW5001
Ryk CM OW3004
Rzeszowska-
Wolny J PW2044
Sala C PW6028
Salagovic JS PW3003
Salonen RO PW2025
Sarasin AS OS3FS1
Schaap M PW2041
Schaefer S PW4010
Schanke A van PW2026
Schavinsky-
Khrapunsky Y PW2049
Schins RPF OW4001, PW2013,
PW2025
Schmeiser HH PW2003, PW1002
Schmuczerova J PW6015
Schoket B PW3004
Schols AMWJ PW4007
Schooten FJ van OW1004, PW5003,
PW3009, PW2052,
PW2013, PW2024,
PW2025
Schulz I OW4004
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Sciutto C PW6011
Scotti E PW3018
Segerbäck K PW1011
Segesdi J PW3004
Sevastyanova O PW6010
Sevcovicova A PW2028, PW2048
Shi T OW4001, PW7004
Shiloh Y OS1001
Shokouhi B PW7003
Shtylik A OW2007
ShuHan SUN PW3022
Siciliano G OW4002
Siems W PW4002
Siezen CLE PW3016
Siivola P PW3010, PW3011,
PW3012
Sillanpää PJ PW3002
Simi S PW2042, PW2043,
PW2042, PW2043
Simic D PW4004, PW4005
Simili M PW2043
Siwinska E PW3017
Skibola CF OW1003
Slaninova M PW2031, PW2048,
PW2028
Sliwinski T PW2030
Slupianek A PW2030
Smedt A de PW2004, PW6016,
PW6023
Smerak P PW1013
Smerhovsky Z PW6010, PW6015
Smith MT OW1003
Solansky I PW5006
Soltesz I PW3004
Sommerburg O PW4002
Sorensen M OW3002
Soric JS PW1010
Sorrentino C PW2042
Spagnoli G PW6011
Speksnijder E PW2041
Sram RJ PW6010, PW5006,
OW3003, PW6015,
OW3001, PW6014
Staal YCM PW5003, OS2001
Staedtler F OW5003
Stanimirovic Z PW6019, PW6017
Stankovic S PW4004, PW4005
Stanojevic J PW4004, PW4005
Staszewski S OW5002
Stavkova Z PW6015, PW6010,
PW6014
Steeg H van PW7007, PW7006,
PW2040, OW7005,
PW2041
Steemans M PW2004, PW6016,
PW6023
Steenwinkel M-JST PW6020
Stefanini M OYSAL2
Steineck G OW3004
Stephanou G OW3001
Stierum RH PW5002, OW5001
Stoikidou M OW3001
Stopper H OS5002
Stout GJ OW2003
Streicker M PW6025
Stronati L PW6013
Surralles J PW3020
Suter-Dick L OW5004
Suter W PW6026, OW5003
Suzen HS PW3005
Suzuki T PW2003
Sviezena B PW2028, PW2048
Szaefer H OW1002, PW1004
Szekely G PW3019
Szyfter K OS4002
Szymoniak K OS4002
Takamura TT PW2011
Tamagno P OW2006
Tampa E OW4003
Teson M OYSAL2
Testa A PW6013, PW6011
Thierens H OW7004
Tibold A PW3001
Tirindelli D PW6011
Tjønneland A OW3002
Tkacova R PW3003
Tognoni G OW4002
Topinka J OW3001, PW6010
Törnqvist M PW6008
Torre A de la PW6027
Tranfo G PW6011
Treuner G PW2031
Tsai M-H OW2005
Tsilimigaki S PW2017, PW2018,
PW2019, PW2021,
PW2022, OW7003
Tudek B PW2012, PW2012,
PW1013
Tuveson D PW2041
Urban L OW5003
Ursini CLU PW4006
Uusitupa M PW3006, PW3010,
PW3002
Uzun G PW4001
Vähäkangas KV PW2047, PW2023
Vainio H PW3008, PW3002
Vaitiekunaite R OW7002, PW3007
Vanhauwaert A. PW6024
Vanparys PH PW2004, PW6016,
PW6023
Vaskivuo L PW2023
Verdina A PW3021
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Vijg J OS3002, OW7005,
PW7006
Villani P PW6013
Vineis P OS4004
Vlachodimi-
tropoulos D OW3001
Vlcek D PW2031, PW2048,
PW2028
Vogel N de PW6020
Vogel U OW3002, PW4015
Voho AV PW3008
Volkov AN PW6009
Vries A de OS1003, PW2040,
PW2041
Vukovic-Gacic B PW4004, PW4005
Waard WJ de OW1004
Wagner AM OW6004
Wakabayashi K PW2011
Wallin H PW4015, OW3002
Walmsley RM PW6006
Watanabe T PW2011
Weickhardt S OW5002
Weinfeld M PW2038
Weiser T OW5004
Wierzbicka M OS4002
Wijnhoven SWP OW7005, PW2041,
PW7006
Will F PW4010
Willers H OW2002
Willis N PW2041
Wilms LC PW1012, PW4012,
PW4013
Wind N de OW2007, OW2003,
PW2045
Wisniewska-
Jarosinska M PW2033
Wit J de PW7007
Woestenborghs F OW6001
Wolfreys A PW6018
Wortelboer HM PW5002
Wouters BG PW2052
Wouters EFM PW4007
Wrzesinski M PW2006, PW2007
Wunderlich H-G PW6012
Xamena N PW3020
Yagi T PW2011
Yan H OW2005
Yan HE PW3022
Yan HONG PW3022
Yoo E jeong PW1009
Yoshikawa YY PW4008
Zambruno G OYSAL2
Zampieri D PW2042
Zeeland AA van PW2051
Zhan L PW2003
Zhang SD OS2004
Zhao S OW2005
Zidzik J PW3003
Zienolddiny S PW3015
Zijno A PW3021
Zivkovic L PW6001, PW7001
Zwart E PW2040, PW2041
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