Download Genetic Susceptibility at Low Dose Exposure

“Genetic Susceptibility
at Low Dose Exposure”
Abstracts
31st Annual Meeting
of the European Environmental
Mutagen Society
September 1 – 5, 2001
Ghent, Belgium
Contents
Index
4
Symposium 1: Genetic susceptibility
14
Symposium 2: DNA repair
18
Workshop 1: Mitosis versus meiosis
23
Symposium 3: Ecogenotoxicology
28
Symposium 4: Molecular epidemiology & biomonitoring
34
Symposium 5: Low doses & thresholds
39
Workshop 2: Electromagnetic fields
44
Workshop 3: Microarray systems
49
Symposium 6: Regulations update & genotoxicity screening
54
Symposium 7: Polymorphism in risk assessment and therapy
58
Symposium 8: Genotoxicity of metals
63
Symposium 9: Chromosomal sensitivity towards genotoxic agents
68
Poster session 1
74
Poster session 2
109
Poster session 3
146
Author index
182
2
Organising Associations
European Environmental Mutagen Society
Belgian Environmental Mutagen Society
Local Organising Committee
E. Brits
M. De Boeck
J. de Gerlache
L. De Ridder
M. Kirsch-Volders
C. Laurent
D. Lison
H. Thierens
F. Van Goethem
J. Van Gompel
P. Van Hummelen
P. Vanparys
L. Verschaeve
H. Veulemans
A. Vral
(Mol: VITO)
(Brussels: VUB)
(Brussels: Solvay)
(Ghent: RUG)
(Brussels: VUB)
(Liege: ULg)
(Louvain-la-Neuve: UCL)
(Ghent: RUG)
(Beerse: Janssen Research Foundation)
(Beerse: Janssen Research Foundation)
(Leuven : VIB MicroArray Facility)
(Beerse: Janssen Research Foundation)
(Mol: VITO)
(Leuven: KUL)
(Ghent: RUG)
Scientific Committee
H. Autrup
J. de Gerlache
P. Farmer
M. Kirsch-Volders
H. Thierens
P. Vanparys
(Denmark: University of Aarhus)
(Belgium: Solvay)
(United Kingdom: University of Leicester)
(Belgium: VUB)
(Belgium: RUG)
(Belgium: Janssen Research Foundation)
Conference Chairperson
Prof. H. Thierens
Faculty of Medicine - Ghent University
Proeftuinstraat 86, B-9000 Ghent, Belgium
Phone: +32-9-264-66-43, Fax: +32-9-264-66-96
E-mail: [email protected]
3
Index
Symposium 1: Genetic susceptibility
GENETIC SUSCEPTIBILITY: AN INTRODUCTION
Angelo Abbondandolo S1/1 Page 15
CYTOGENETIC BIOMARKERS AND GENETIC POLYMORPHISMS
H. Norppa; J. Tuimala; G. Szekely; S. Gundy; A. Hirvonen S1/2 Page 16
DNA REPAIR POLYMORPHISMS: DISCOVERY, PHENOTYPE AND RISK.
Douglas A. Bell, S1/3 Page 17
Symposium 2: DNA repair
NUCLEOTIDE EXCISION REPAIR: FROM HUMAN SYNDROMES TO MOUSE MODELS AND
DYNAMICS IN LIVING CELLS
J.H.J. Hoeijmakers, D. Hoogstraten, V. van den Boom, E. Citterio, A. B. Houtsmuller,
W. Vermeulen, J. de Boer, and G.T.J. van der Horst. S2/1 Page 19
TRANSCRIPTION-COUPLED REPAIR OF 8-OXOGUANINE IN VARIOUS HUMAN CELLS.
A. Sarasin ; J. Cabral-Neto ; M. Pastoriza ; F. Le Page. S2/2 Page 20
THE ROLE OF DNA LIGASE IV IN NON-HOMOLOGOUS END JOINING AND DEFECTS IN
PATIENTS.
O’Driscoll M, Cerosaletti K, Girard P, Priestly A, Sperling K, Gennery A, Concannon P
Jeggo P. S2/3 Page 21
ROLE OF THE BREAST CANCER SUSCEPTIBILITY GENE Brca2 AND THE Rad51C GENE IN
PROVIDING GENOME STABILITY AND PROTECTION AGAINST DNA DAMAGE
M.Z. Zdzienicka S2/4 Page 22
Workshop 1: Mitosis versus Meiosis
MITOSIS : A UNIFYING MODEL FOR THE METAPHASE/ANAPHASE TRANSITION
M. Kirsch-Volders and I. Decordier W1/1 Page 24
CELL CYCLE INSIGHTS OF MOUSE PRIMARY SPERMATOCYTES
P. de Boer and O. van den Broek W1/2 Page 25
DIFFERENCES IN THE PROCESS OF MITOTIC AND MEIOTIC DIVISIONS AND THEIR
CONSEQUENCES FOR CHEMICAL EFFECTS.
Ilse-Dore Adler W1/4 Page 27
Symposium 3: Ecogenotoxicology
TOXICITY AND GENOTOXICITY OF HYDROPHOBIC CHEMICALS SAMPLED
SEMIPERMEABLE MEMBRANE DEVICES (SPMDs) IN THE AQUATIC ENVIRONMENT
D. Sabaliunas; J. Lazutka; I. Sabaliuniene S3/1 Page 29
WITH
CYTOGENETIC STUDIES ON THE EFFECT OF IONISING RADIATION BY SOLID-STAIN AND
FISH TECHNIQUES.
J.F. Barquinero; S. Cigarrán; A. Duran; M.R. Caballín, M. Ribas; L. Barrios S3/2 Page 30
ASSESSMENT OF AIR QUALITY IN SILESIA, POLAND BASED ON CHEMICAL ANALYSES AND
SALMONELLA ASSAY – NOW AND 5 YEARS AGO
D. Mielyska; E. Siwińska; A. Bubak; L. Kapka S3/3 Page 31
4
ASSESSMENT OF TOXICITY AND GENOTOXICITY OF INDUSTRIAL EFFLUENTS IN SOUTHERN
ITALY
Dell’Aquila; A. Diodati; M. Isidori; M. Lavorgna; A. Mancini S3/4 Page 32
EMOTIONAL STRESS MODIFY GENOME SENSITIVITY TO ENVIRONMENTAL MUTAGENS.
PARALLELS IN RESULTS ON MICE AND HUMAN INVESTIGATIONS
F. Ingel S3/5 Page 33
Symposium 4: Molecular epidemiology and biomonitoring
DNA ADDUCTS IN PAH EXPOSED PERSONS MODULATED BY GENETIC POLYMORPHISMS IN
CARCINOGEN METABOLIZING ENZYMES
F.J. Van Schooten S4/1 Page 35
TOBACCO SMOKE-INDUCED GENOTOXICITY IN LARYNGEAL CANCER SUBJECTS
K.Szyfter, P.Jałoszyński, J.Banaszewski, M.Pabiszczak, L.Möller, W.Szyfter S4/2 Page36
MICRONUTRIENTS, GENE-DIET INTERACTION AND GENOMIC STABILITY
Michael Fenech S4/3 Page 37
EFFECT OF METABOLIC GENOTYPES ON THE FORMATION OF BUTADIENE-HEMOGLOBIN
ADDUCTS
M. Warholm; P. Begemann; A. Colombi; S. Fustinoni; H.G. Neumann; A. Rannug; L. Soleo; J.
Swenberg; L. Vimercati S4/4 Page 38
Symposium 5: Low doses and thresholds
THE RISK ASSESSMENT OF GENOTOXIC CHEMICALS
James M. Parry S5/1 Page 40
LOW DOSES AND THRESHOLDS IN HEPATOCARCINOGENESIS
G. Williams, M.D. S5/2 Page 41
SPONTANEOUS MUTATION RATE • THE APPARENT THRESHOLD
R.C. von Borstel S5/3 Page 42
FISH-DETECTED ASYNCHRONOUS REPLICATION IN HUMAN CELLS EXPOSED TO
GENOTOXIC AGENTS
C.Z. Cotrim; A. Brás; I.Vasconcelos ;M. Sá da Costa;J.Rueff S5/4 Page 43
Workshop 2: Electomagnetic fields
ARE EXTREME LOW AND RADIOFREQUENCY FIELDS HAZARDOUS TO HUMANS? AN
INTRODUCTION.
L. Verschaeve W2/1 Page 45
ELECTROMAGNETIC FIELDS AND CARCINOGENESIS: INTERACTION OF GENOTOXIC AND
NON-GENOTOXIC FACTORS
J. Juutilainen W2/2 Page 46
GENOTOXIC EFFECTS OF BOTH ELF AND RADIOFREQUENCY ALONE AND IN COMBINATION
WITH EXPOSURE TO CHEMICALS
MR. Scarfì W2/3 Page 47
STATIC AND EXTREMELY LOW FREQUENCY ELECTROMAGNETIC FIELDS: EVALUATION OF
CANCER HAZARDS
R. A. Baan W2/4 Page 48
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Workshop 3: Microarray systems
TOXICOGENETICS AND TOXICOGENOMICS: PROMISES AND REALIZATIONS.
J.H.M. van Delft W3/1 Page 50
IDENTIFICATION OF GENE EXPRESSION PROFILES IN DIFFERENT MOUSE TISSUES USING
CDNA MICROARRAY.
P. Van Hummelen; J. Mathys; K. Marchal; P. Glenisson; Y. Moreau. W3/2 Page 51
NORMALIZATION AND ERROR ANALYSIS OF DNA MICROARRAY DATA: AFFECT ON
INTERPRETATION OF BIOLOGICAL RESULTS
Roger E. Bumgarner W3/3 Page 52
POTENTIAL APPLICATION OF DNA-CHIP TECHNOLOGY (MICROARRAY) IN TOXICOLOGY
Silvio Albertini and Laura Suter-Dick W3/4 Page 53
Symposium 6: Regulations update and genotoxicity screening.
GENOTOXICITY ASSESSMENT IN DRUG DISCOVERY AND EARLY DEVELOPMENT
L. Müller S6/1 Page 55
UK COM GUIDANCE ON A STRATEGY FOR TESTING CHEMICALS FOR MUTAGENICITY
David J Tweats S6/2 Page 56
PROGRESS TOWARDS HARMONISATION IN GENOTOXICITY TESTING THROUGH THE
INTERNATIONAL WORKSHOPS (IWGT)
D. Kirkland S6/3 Page 57
Symposium 7: Polymorphisms in risk assessment and therapy.
POLYMORPHISMS OF DRUG METABOLISING ENZYMES AND XENOBIOTIC TOXICITY
Magnus Ingelman-Sundberg S7/1 Page 59
GENETIC POLYMORPHISM
XENOBIOTICA
H.Autrup S7/2 Page 60
IN GLUTATHIONE S-TRANSFERASE
AND RESPONSE TO
INTERINDIVIDUAL VARIABILITY IN RESPONSE TO RADIATION EXPOSURE: ANALYSIS OF
DNA REPAIR IN CANCER PATIENTS.
C. Alapetite S7/3 Page 61
IMPACT OF POLYMORPHISMS IN PHASE I OR PHASE II ENZYMES ON LYMPHOCYTE DNA
ADDUCTS IN POPULATIONS SUFFERING MEDIUM TO LOW EXPOSURE TO PAH.
P. Georgiadis ; J. Topinka, M. Stoikidou; S. Kaila; M. Gioka, K. Katsouyianni, R. Sram; H. Autrup; A.
Kyrtopoulos S7/4 Page 62
Symposium 8: Genotoxicology of metals
METAL- AND REDOX- REGULATION OF P53 PROTEIN FUNCTIONS.
P.Hainaut S8/1 Page 64
DETERMINANTS OF THE GENOTOXICITY OF METALLIC COMPOUNDS.
D. Lison S8/2 Page 65
CARCINOGENIC METAL COMPOUNDS: INTERFERENCE WITH DNA REPAIR PROCESSES AND
CELL CYCLE CONTROL
A.Hartwig, M.Asmuss, A. Buerkle, I. Ehleben, D. Kostelac, A. Pelzer, T. Schwerdtle S8/3 Page 66
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ELUCIDATION OF THE BIOCHEMICAL PATHWAYS INVOLVED IN THE MUTAGENICITY,
ASSOCIATED WITH ISOLATED PARTICULATE DEBRIS FROM THE PERI-PROSTHETIC TISSUE
OF FAILED PROSTHESIS.
S.Clerkin, C.P.Case. S8/4 Page 67
Symposium 9: Chromosomal sensitivity towards genotoxic agents.
CHROMOSOMAL ABNORMALITIES IN MITOMYCIN C-SENSITIVE CHINESE HAMSTER CELLS
DEFECTIVE IN THE Brca2 AND Rad51C GENES
P.P.W. van Buul; A. van Duijn-Goedhart; M. Kraakman-van der Zwet; B.C. Godthelp; M.Z. Zdzienicka
S9/2 Page 70
HOW RELIABLE ARE CHROMOSOMAL ABERRATION ASSAYS AS BIOMARKERS OF
INDIVIDUAL SENSITIVITY TOWARDS IONISING RADIATION?
A. Vral, H. Thierens, A. Baeyens and L. De Ridder S9/3 Page 71
DISAPPEARENCE OF CHROMOSOMAL ABERRATIONS FROM THE BLOOD CIRCULATION
DEPENDS ON THE LOCATION OF IRRADIATED LYMPH NODES
S. Gundy, G. Székely, Zs. Kelecsényi, O. Ésik S9/4 Page 72
ARA A ENHANCEMENT OF CHROMATID BREAKS IN IRRADIATED CHO CELLS: LACK OF
CORRELATION WITH DSB REJOINING
P. E. Bryant and C. Finnegan, S9/5 Page 73
Poster Session 1: Genetic susceptibility, DNA repair, ecogenotoxicology.
DIFFERENCES IN MECHANISMS OF APOPTOSIS IN THE HL60 CELL LINE AND SYRIAN
HAMSTER EMBRYO (SHE) CELLS.
Alexandre S., Rast C. and Vasseur P. P1/1 Page 75
DEVELOPMENTAL ABNORMALITIES INDUCED BY X-IRRADIATION IN P53 DEFICIENT OR
HETEROZYGOUS MICE.
S. Baatout; P. Jacquet; A. Michaux; J. Buset; W. Schoonjans; J. Yan; A. Benotmane; L. de SaintGeorges; C. Desaintes; M. Mergeay P1/2 Page 76
THE P53 CODON 72 SNP AND LUNG CANCER
E. Biros; I. Biros; A. Kohut; I. Kalina; E. Bogyiova; J. Stubna P1/4 Page 78
ASSESSMENT OF CHEMOTHERAPY-INDUCED DNA DAMAGE IN PERIPHERAL BLOOD
LEUKOCYTES OF CANCER PATIENTS USING THE ALKALINE COMET ASSAY
N. Kopjar; V. Garaj-Vrhovac; I. Milas P1/5 Page 79
GENETIC SUSCEPTIBILITY TO BLADDER CANCER IN SLOVAK-CAUCASIANS: ROLE OF NAT2
POLYMORPHISM
V. Habalová; L. Klimčáková; J. Šalagovič; I. Kalina; M. Hrivňák; H. Schneider P1/6 Page 80
POLYMORPHISM OF THE GSTM1 GENE ASSOCIATED WITH SUSCEPTIBILITY TO LUNG
CANCER IN RELATION TO THE DURATION OF SMOKING IN SLOVAK POPULATION
J. Šalagovič ; J. Štubňa* ; I. Kalina; L. Klimčáková; V. Habalová P1/7 Page 81
A YEAST GENOTOXICITY AND CYTOTOXICITY ASSAY FOR HIGH-THROUGHPUT SCREENING
N. Billinton ; P. Cahill ; A. W. Knight ; R. M. Walmsley P1/9 Page 83
POLYMORPHISMS IN DNA REPAIR GENES, XPB, XPC AND hHR23B, IN POLISH POPULATION
– A PRELIMINARY STUDY.
D. Butkiewicz; M. Rusin; M. Pawlas; M. Chorazy P1/10 Page 84
INFLUENCE OF VHL EXPRESSION ON DNA REPAIR
C. Flohr ; E. Weidt ; J. Decker ; B. Epe P1/11 Page 85
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DNA REPAIR CAPACITY AND EXPRESSION PROFILES OF DNA REPAIR GENES IN RESTING
AND PHA-STIMULATED HUMAN PERIPHERAL BLOOD LYMPHOCYTES
C. Mayer, O. Zelezny, M.C. von Brevern, A. Bach, H. Bartsch and P. Schmezer P1/12 Page 96
PARP INHIBITOR 3-AMINOBENZAMIDE DOES NOT INCREASE THE YIELDS OF
CHROMOSOMAL ABERRANT CELLS INDUCED BY BORON NEUTRON CAPTURE REACTION IN
V79 CHINESE HAMSTER CELLS
N.G. Oliveira; M. Castro; A.S. Rodrigues; I.C. Gonçalves; R. Cassapo; A.P. Fernandes; T. Chaveca;
J.M. Toscano-Rico and J. Rueff P1/14 Page 88
INFLUENCE OF NITRIC OXIDE ON DNA REPAIR
N. Phoa , B. Epe P1/15 Page 89
DNA DAMAGE AND REPAIR EFFICIENCY IN SCHIZOPHRENIC PATIENTS
D. Psimadas , N. Messini-Nikolaki , A. Fortos , S. Tsilimigaki
and S. Piperakis P1/16 Page 90
SEARCHING FOR REPAIR COMPETENCE OF CELLS OF LARYNX CANCER PATIENTS –
GENETIC INSTABILITY AND ALLELIC LOSSES IN GENES CONTROLLING CELL CYCLE AND
DNA REPAIR.
Stembalska – Kozowska A. , R. Smigiel , T. Krcicki , M. Blin , F. Mirghomizadeh , K. Bartusiak, M.
Sasiadek . P1/17 Page 91
COMPARISON OF DIETHYL SULFATE MUTAGENICITY ON FEMALE AND MALE GERM CELLS
OF DROSOPHILA MELANOGASTER UNDER DIFFERENT REPAIR CONDITIONS.
J. Hernando; M. A. Comendador; L. M. Sierra. P1/18 Page 92
EXCISION OF PYRIMIDINE RING-RUPTURED 1,N6-ETHENOADENINE BY THYMINE GLYCOLDNA GLYCOSYLASE
M. Bajek, J.M. Ciela, B. Tudek P1/19 Page 93
UNSCHEDULED DNA SYNTHESIS: MEASUREMENT OF DNA REPAIR IN A HUMAN HEPATOMA
CELL LINE (HEPG2 CELLS)
I. Valentin; Y. Lossouarn; V. Thybaud; J-C. Lhuguenot and M-C. Chagnon P1/20 Page 94
GENOTOXICITY DETECTED WITH COMET ASSAY AND MICRONUCLEUS TEST IN CYPRINUS
CARPIO SPECIMENS EXPOSED IN SITU TO TRASIMENO LAKE WATERS TREATED WITH
DISINFECTANTS FOR POTABILIZATION.
Buschini A., Martino A., Gustavino B., Monfrinotti M., Poli P., Rossi C., Santoro M. & Rizzoni M.
P1/23 Page 97
EFFECTS OF BRUSSELS SPROUTS EXTRACTS AND THE ACTIVE CONSTITUENTS ON
OXIDATIVE DNA DAMAGE AND THE ACTIVITY OF NAD(P)H: QUINONE REDUCTASE IN HEPA
1c1c7 CELLS
C.Y. Zhu; S. Loft P1/24 Page 98
THE USE OF IN VITRO ASSAYS TO TEST SOUTH AFRICAN MEDICINAL PLANT EXTRACTS
FOR MUTAGENIC ACTIVITY
E.E. Elgorashi; J.L.S. Taylor; L. Regniers; L. Verschaeve; A. Maes; N. De Kimpe; J. van Staden; A.
Fossey P1/25 Page 99
GENOTOXICITY EVALUTATION OF TRITERPENES FROM SAMBUCUS NIGRA
T. Cangiano, M. Della Greca, A. Fiorentino, A. Gentili, M. Isidori P1/26 Page 100
INDUCTION OF ANEUPLOIDY IN HUMAN CELL LINES BY HORMONES
Mahmood A. Kayani, James, M. Parry P1/28 Page 102
INDUCTION OF MICRONUCLEI AND CHROMOSOME NON-DISJUNCTION AFTER SHORT-TERM
EXPOSURE TO CARBENDAZIM IN CULTURED HUMAN LYMPHOCYTES
8
Mahmood. R and Parry. J. M P1/29 Page 103
IN SITU MONITORING
WITH TRADESCANTIA- MICRONUCLEUS ASSAY ON
GENOTOXICITY OF URBAN AIR IN SOUTHERN ITALY
F. Cundari ; M. Isidori ; A. Nardelli ; A. Parrella ; O. Pepe P1/30 Page 104
THE
THE MICRONUCLEUS ASSAY IN HAEMOCYTES OF Dreissena polymorpha FOR THE
DETECTION OF GENOTOXICITY IN FRESHWATER ENVIRONMENTS
M. Pavlica; G.I.V. Klobuar; R. Erben; D. Papeš P1/31 Page 105
EVALUATION OF GENOTOXIC ACTIVITY OF OXIDIZING TREATMENTS TO REMOVE SIMAZINE
FROM WATER
Sueiro R.A.; Suárez S.; Rubio A., Araujo M., Garrido M. J. P1/32 Page 106
UTILIZATION OF THE VITOTOX GENOTOXICITY TEST AND ACUTE AND CHRONIC
MICROBIOTESTS TO ASSESS THE ENVIRONMENTAL RISKS OF SOLID INDUSTRIAL WASTES.
A. Van Cauwenberge, P. Bouviez and E. Noël P1/33 Page 107
COMPARATIVE ASSESSMENT OF WEAK GENOTOXIC PESTICIDE EFFECTS IN PLANTS AND
HUMAN CELL CULTURES
M. Wilder; B. Volkmer; E. A. Sanders; R. Greinert; E.W. Breitbart; D. Pollet
P1/34 Page 108
Poster Session 2: Molecular epidemiology and biomonitoring, low doses and
thresholds, genotoxicity of metals.
POTENTIAL GENOTOXIC RISK FOR HUMANS BY THE ENVIRONMENTAL CONTAMINANT 3NITROBENZANTHRONE
V.M. Arlt; C.A. Bieler; M. Wiessler; D.H. Phillips ; H.H. Schmeiser
P2/1 Page 110
CHINESE HERBS NEPHROPATHY AND UROTHELIAL CARCINOMA: AN OUT-BREAK IN
BELGIUM
V.M. Arlt; J.L. Nortier; J.-L. Vanherweghem; H.H. Schmeiser P2/2 Page 111
THE CHANGES OF SPONTANEOUS FREQUENCY OF CHROMOSOMAL ABERRATIONS IN THE
20 YEARS PERIOD IN THE CZECH REPUBLIC POPULATION GROUPS
H. Bavorova; D. Ocadlikova; P. Rössner; R. J. Sram P2/3 Page 112
MICRONUCLEI IN UNCULTURED T-LYMPHOCYTES OF RAILROAD WORKERS EXPOSED TO
TRANSIT CHEMICALS
G. Falck; H. Järventaus; T. Kallas ; J. Catalán; L. Pitkämäk; and H. Norppa P2/4 Page 113
DIFFERENCES IN SMOKING-RELATED DNA ADDUCT LEVELS IN TUMOROUS AND NONTUMOROUS TISSUES FROM LUNG CANCER PATIENTS
E. Gyrffy ; Z. Gyri; I. Soltész; S. Kostič; A. Cseke; J. Minárovits ,; B.Schoket P2/5 Page 114
BIOLOGICAL SAMPLE COLLECTION AND PROCESSING IN AN ON SITE LABORATORY IN
ESTONIAN SHALE OIL MINE - BIOMODEM STUDY
L.E. Knudsen; A. Jensen; J. Kusova; J. Kubackova; V. Muzyka; R. Anzion; P. Scheepers P2/6 Page
115
URINARY MUCONIC ACID AND PHENYL MERCAPTURIC ACID EXCRETION IN ESTONIAN
SHALE OIL MINE WORKERS DEPEND ON GST - GENOTYPES
L.E. Knudsen; A. Jensen; S.Loft; H.Autrup; J.Poole P2/7 Page 116
LEUKOCYTE DNA DAMAGE IN FIBERGLASS-REINFORCED PLASTIC WORKERS MEASURED
BY THE COMET ASSAY
B. Laffon; E. Pásaro; J. Méndez P2/8 Page 117
ANALYSING MUTATION SPECTRA
P. D. Lewis and J. M. Parry P2/9 Page 118
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MICRONUCLEI ANALYSIS IN PERIPHERAL LYMPHOCYTES OF HOSPITAL WORKERS
OCCUPATIONALLY EXPOSED TO IONIZING RADIATIONS.
F. Maffei, S. Angelini, G. Cantelli Forti, V. Lodi, F.S. Violante, S. Mattioli, P. Hrelia P2/10 Page 119
INFLUENCE OF PHASE II ENZYMES ON BIOMARKERS OF EXPOSURE AND EFFECT IN
SILESIAN CHILDREN EXPOSED TO POLYCYCLIC AROMATIC HYDROCARBONS
D. Mielyska, K. Szyfter, E. Siwińska, L. Kapka, R. Jaskua –Sztul P2/11 Page 120
BIOMONITORING STUDY OF A GROUP OF WORKERS OCCUPATIONALLY EXPOSED TO
PAINTS AND SOLVENTS.
Migliore L., R. Bibbiani, Z. Ricevuto, M. Vicentini, N. Serretti P2/12 Page 121
CYTOGENETIC STUDIES IN SOMATIC AND GERM CELLS OF MALE INDIVIDUALS EXPOSED
TO STYRENE IN THE WORKPLACE.
Naccarati A.; A. Zanello; R. Scarpato; L. Lastrucci; L. Migliore P2/13 Page 122
A BIOMARKER APPROACH TO DETECT EARLY DNA DAMAGE AND GENOTOXIC RISK OF
COMMON ENVIRONMENTAL POLLUTANTS IN ADOLESCENTS
T Nawrot; E Den Hond; HA Roels; L Verschaeve; G Koppen, JA Staessen P2/14 Page 123
INFLUENCE OF CYP1A2 AND NAT2 PHENOTYPES ON URINARY MUTAGENICITY IN
CIGARETTE SMOKERS.
S.Pavanello, P. Simioli, S. Lupi, P. Gregorio, and E. Clonfero P2/15 Page 124
A STUDY ON THE EFFECTS OF SEASONAL SOLAR RADIATION ON EXPOSED POPULATIONS
S. Tsilimigaki , N. Messini-Nikolaki , M. Kanariou , and S. Piperakis P2/16 Page 125
DNA DAMAGE-REPAIR IN A POPULATION WITH CHRONIC PSYCHOGENIC STRESS
E. Dimitroglou , M. Zafiropoulou , N. Messini-Nikolaki , S. Ntountounakis , S. Tsilimigaki and S.
Piperakis. P2/17 Page 126
BIOLOGICAL DOSIMETRY IN A GROUP OF NUCLEAR POWER PLANT WORKERS IN BELGIUM
BY MEANS OF CHROMOSOME PAINTING.
Roncancio C.L.; Laurent C.; Thierens H. & Lambert V P2/18 Page 127
THE INFLUENCE OF OCCUPATIONAL EXPOSURE TO PAHs ON THE EXPRESSION OF p53
AND p21/WAF1 PROTEINS
P. Rössner Jr.; B. Binková; R. J. Šrám P2/19 Page 128
SPERM CHROMATIN STRUCTURE IN WORKERS EXPOSED TO LOW LEVELS OF INORGANIC
LEAD
M. Spano, F. Caruso, G. Leter, E. Cordelli, M. Joffe, P. Apostoli, S. Porru, P. Kiss, M. Vanhoorne, F.
Comhaire, A. Giwercman, L. Bisanti, W. Zschiesche, J.P. Bonde
P2/20 Page 129
BIOMONITORING OF HUMAN POPULATION EXPOSED TO SIMAZINE IN TAP WATER
Suárez S., Rubio A., Sueiro R.A., Garrido M. J P2/21 Page 130
SISTER CHROMATID EXCHANGE AND PROLIFERATIV RATE INDEX IN A CROATIAN
POPULATION OCCUPATIONALY EXPOSED TO PESTICIDES
D. Željei and V. Garaj-Vrhovac P2/22 Page 131
COMPARISON OF Saccharomyces cerevisiae TEST AND COMET ASSAY ON HUMAN
LEUKOCYTES IN THE LOWEST EFFECTIVE DOSE OF CHLORINE DISINFECTANTS
Annamaria Buschini, Paola Poli, Luca Pasini, Chiara Alessandrini, Carlo Rossi P2/23 Page 132
DEVELOPMENT OF AN IN SITU METHOD FOR MICRONUCLEUS ASSAY WITH HEP G2 AND
CHO CELLS.
Fessard V.; Valentin I.; Mourot A. ; Chagnon M.C.; Poul J.M.; Lhuguenot J.C. P2/24 Page 133
10
GRISEOFULVIN : DOSE-RESPONSE STUDIES IN HEPATOCARCINOGENESIS MEDIUM-TERM
ASSAY AND IN IN VITRO ANEUPLOIDY INDUCTION IN SPLEEN LYMPHOCYTES IN THE RAT
K. Labay ; M. Ould Elhkim ; M. Poul ; G. Jarry ; S. Marteau ; J.M. Poul ; P. Sanders
P2/25
Page 134
LOW DOSES OF GAMMA RAYS: MOLECULAR ALTERATION INDUCED IN HUMAN TUMOR
CELLS
M. Osmak; A. Brozovi P2/26 Page 135
AN INVESTIGATION OF THE MUTAGENIC DAMAGE THAT IS CAUSED IN HUMAN CELLS BY
DEBRIS FROM WORN ORTHOPAEDIC JOINT REPLACEMENTS
W. Niedzwiedz, C.P. Case P2/28 Page 137
APPLICATION OF THE MICRONUCLEUS ASSAY IN PATIENTS TREATED WITH RADIOUCLIDE
THERAPIES: RESULTS AND LIMITATIONS.
M. Monsieurs, A. Vral, B. Brans, L. De Ridder, RA Dierckx and H. Thierens
P2/30 Page 139
IN VITRO APOPTOSIS TESTING COMPARED TO THE CLINICAL CHARACTERISTICS
J. Philippé, A. Janssens, F. Offner, H. Thierens P2/31 Page 140
MODULATION OF MUTAGENIC ACTIVITY IN MEAT SAMPLES DEEP-FRIED UNDER DIFFERENT
CONDITIONS
C. Perez; A. Lopez de Cerain ; J. Bello P2/32 Page 141
INDUCTION OF MICRONUCLEI BY COMBINED TREATMENTS OF HETEROCYCLIC AMINES
(HAs) IN V79 CELLS
C. Perez; A. Lopez de Cerain ; L. Alvarez; J. Bello P2/33 Page 142
DEVELOPMENT OF AN IN VITRO ASSAY TO DETECT MUTATION INDUCTION IN THE LACZ
TRANSGENE OF MUTATMMOUSE CULTURED SPLENOCYTES
M. Ballantyne; R. Marshall; A. Wolfreys; G. Ellis P2/34 Page 143
IN VITRO DNA ADDUCT FORMATION BY HETEROCYCLIC AMINES, ACTIVATED VIA
DIFFERENT METABOLIC PATHWAYS
H.J.J. Moonen ; T.M.C.M. de Kok ; J.C.S. Kleinjans P 2/35 Page 144
HISTORY OF THE CYTOGENETIC ANALYSIS IN THE CZECH REPUBLIC
Z. Smerhovsky; P. Rossner; R.J. Sram, and K. Landa, G. Loprieno P2/36 Page 145
Poster Session 3: Chromosomal sensitivity towards genotoxic agents, mitosis
versus meiosis, electromagnetic fields.
A COMPARISON OF THE CYTOGENETIC RESPONSE TO IRRADIATION OF RESTING
PERIPHERAL BLOOD LYMPHOCYTES AND EPSTEIN-BARR VIRUS TRANSFORMED
LYMPHOBLASTOID CELLS.
A.Baeyens, A. Vral, H. Thierens and L. De Ridder P3/1 Page 147
GENOTOXIC EFFCTS OF TWO PESTICIDES AND THEIR MIXTURES:
IN-VIVO CHROMOSOMAL ABERRATIONS AND MICRONUCLEUS ASSAY
E.N. El-Khatib; and H.A. Rokaya P3/3 Page 149
THE PROTECTIVE EFFECT OF VITAMIN C AGAINST CYFLUTHRIN INDUCED CLASTOGENICITY
IN RAT BONE MARROW CELLS
H. N. EL-KHATI0B P3/4 Page 150
CYTOGENETIC BIOMONITORING OF EGYPTIAN WORKERS EXPOSED TO PESTICIDES:
MICRONUCLEI ANALYSIS IN PERIPHERAL BLOOD LYMPHOCYTES
H. N. EL-KHATIB and F. M. Hammam P3/5 Page 151
11
PHENOLIC COMPOUNDS FROM RED WINE ARE PROTECTIVE AGAINST THE DNA DAMAGING
EFFECT OF IONISING RADIATION EX VIVO
W. Greenrod; C. Stockley; M. Abbey; M. Fenech P3/6 Page 152
INDIVIDUAL VARIABILITY IN THE YIELD OF CHROMOSOMAL ABERRATIONS AFTER LOW
DOSE GAMMA-RAY IRRADIATION
A.Kiuru ; C. Lindholm ; A. Koivistoinen ; R. Mustonen P3/7 Page 153
INFLUENCE OF AGE ON VINBLASTINE-INDUCED CHROMOSOME MALSEGREGATION IN
PERIPHERAL LYMPHOCYTES OF FEMALE DONORS.
P. Leopardi, R. Crebelli, F. Marcon, A. Zijno, G. Dobrowolny P3/8 Page 154
MUTAGENICITY OF AMOSITE FIBRES IN THE LUNG OF lacI TRANSGENIC RATS (A REPORT
FROM THE FIBRETOX PROJECT)
P. Loli; J. Topinka; M.Hurbankova; P. Georgiadis; T. Wolff; S. A. Kyrtopoulos
P3/9 Page 155
LYMPHOCYTES FROM IODINE-131 TREATED THYROID CANCER PATIENTS UNDERGO A
TRANSIENT ADAPTATION TOWARDS MITOMYCIN C GENOTOXICITY
O. Monteiro Gil; N.G. Oliveira; A.S. Rodrigues; A. Laires; T.C. Ferreira; E. Limbert; J. Rueff
P3/10 Page 156
CYTOGENETIC BIOMONITORING OF EXTERNAL WORKERS IN THE NUCLEAR INDUSTRY:
STUDY OF EXPOSURE EFFECTS AND SUSCEPTIBILITY
H. Thierens ; A. Vral ; M. Barbé; A. Baeyens; L. De Ridder P3/11 Page 157
INTERFERING WITH HISTONE DEACETYLATION WILL CAUSE ANEUPLOIDY IN MAMMALIAN
CELLS
D. Cimini, D. Fioravanti, F. Degrassi P3/12 Page 158
APPLICATION OF THE ALKALINE SINGLE CELL GEL ELECTROPHORESIS (SCGE) ASSAY IN
ASSESSMENT OF DNA DAMAGE IN PERIPHERAL BLOOD LEUKOCYTES OF RADAR-FACILITY
WORKERS
V. Garaj-Vrhovac, N. Kopjar, D. Želježić P3/13 Page 159
CYTOGENETIC EFFECTS OF HIGH FREQUENCY ELECTROMAGNETIC FIELDS ON HUMAN
LYMPHOCYTES IN VITRO
I.-L. Hansteen ; E. H Kure; P3/14 Page 160
EFFECTS OF LOW-FREQUENCY ELECTROMAGNETIC FIELDS IN HUMAN LYMPHOCYTES
J. Delimaris , S. Tsilimigaki , N. Messini-Nikolaki , G. Ziros and S.M. Piperakis P3/15 Page 161
ABSENCE OF COOPERATIVE EFFECTS IN L929 CELLS FOLLOWING COMBINED EXPOSURE
TO MX AND A 50 Hz SINUSOIDAL MAGNETIC FIELD
O. Zeni, A. Perrotta, P. Pisani, M.R. Scarfì P3/16. Page 162
GENOTOXIC EFFECTS OF 50Hz MAGNETIC FIELDS ON HUMAN BLOOD CELLS
A. Testa, L. Stronati, D.Conti, P. Villani, , A. M. Fresegna, F. Russo, G. Lovisolo, C. Marino and E.
Cordelli P3/17 Page 163
NO INTERACTION OF ELF MAGNETIC FIELDS WITH A CHEMICAL
MICRONUCLEUS INDUCTION IN HUMAN LYMPHOCYTES.
G.R. Verheyen; G. Pauwels; L. Verschaeve; G. Schoeters P3/18 Page 164
ANEUGEN ON
NEW MOLECULAR TOOLS FOR PREDICTIVE TOXICOLOGY: THE ROLE OF MOLECULAR
DATABASES AND COMPREHENSIVE MICROARRAYS
P. Alen, K. Schmeiser, W. Whitford, S. Hicken and G. Farris P3/19 Page 165
DNA ADDUCTS, MUTANT FREQUENCY AND GENE EXPRESSION PROFILES IN BPDEEXPOSED TK6 CELLS
12
S.M. Morris, O.E. Domon,L.J. McGarrity, S.J. Culp, L. Blankenship, J.T. MacGregor, B. Rosenzweig
and F.D. Sistare P3/20 Page 166
DIFFERENTIAL GENE EXPRESSION IN RATS AFTER INJECTION WITH KIDNEY TOXICANTS
K. Schmeiser, P.Alen, G. Farris and L. Kier P3/21 Page 167
IDENTIFICATION OF MECHANISMS AT THE GENOME LEVEL FOR PROTECTION AGAINST
COLON CANCER BY VEGETABLES
S.G.J. van Breda; J.H.M. van Delft; L.G.J.B. Engels; J.C.S. Kleinjans P3/22 Page 168
CHROMOSOME ABERRATIONS, GENOTOXICITY
SELECTED CHEMICALS
P. Arni and M. Kiffe P3/23 Page 169
AND
CYTOTOXICITY
INDUCED
BY
HIGH THROUGHPUT SINGLE CELL QUANTIFICATION OF DNA DAMAGE BASED ON
CONFOCAL IMAGING OF VERTICAL COMETS
Ph. Baert; P. Van Oostveldt P3/24 Page 170
GENOTOXICITY OF ORGANIC EXTRACTS DERIVED FROM AIRBORNE PARTICULATE MATTER
IN FLANDERS, BELGIUM
E. Brits, G. Schoeters, L. Verschaeve, E. Roekens, E. Muylle P3/25 Page 171
INDUCTION OF ATHEROSCLEROSIS IN APOE-KNOCKOUT MICE
BENZO[A]PYRENE
D.M.J. Curfs; E. Lutgens; M.J.A.P. Daemen; F.J. van Schooten P3/26 Page 172
EXPOSED
TO
APOPTOSIS INDUCTION IN HUMAN LYMPHOCYTES AFTER IN VITRO EXPOSURE TO
COBALT/HARD METAL COMPOUNDS
M. De Boeck, I. Decordier, N. Lombaert, E. Cundari, D. Lison and M. Kirsch-Volders .P3/27 Page 173
RELATION BETWEEN THE INDUCTION OF APOPTOSIS AND THE INDUCTION
MICRONUCLEI AFTER IN VITRO EXPOSURE TO THE ANEUGEN NOCODAZOLE.
I. Decordier, M. Kirsch-Volders and E. Cundari. P3/28 Page 174
OF
THE USE OF IN-SILICO STRUCTURE ACTIVITY-BASED PREDICTION SYSTEMS FOR
GENOTOXICITY AT NOVARTIS
S. Glowienke ; HJ. Martus ; G. Bold, L. Mueller P3/29 Page 175
THE STUDIES OF FLUAZIFOP FROM FENOXY ACID DERIVATIVES AS PEROXISOME
PROLIFERATOR IN RAT LIVER
G. Kostka; J.K. Ludwicki; D. Palut; K. Lembowicz; B. Wiadrowska P3/30 Page 176
ANEUPLOIDY AND TUMOUR PROGRESSION IN BARRETT’S OESOPHAGUS
Elizabeth M Parry, Jeanette Croft and Shareen Doak P3/31 Page 177
TESTING MELANOIDIN FRACTIONS FOR MUTAGENICITY - THE USE OF IN VITRO TESTS
J.L.S. Taylor ; L. Regniers; L. Verschaeve; A. Maes; C. Arribas Olave; K. Abbaspour-Tehrani; E.
Elgorashi; N. De Kimpe; J. van Staden; A. Fossey P3/32 Page 178
SIMULTANEOUS ASSESSMENT OF GENOTOXICITY AND CYTOTOXICITY IN A DOWNSCALED
IN VITRO MICRONUCLEUS TEST
F. Van Goethem, V. Van Hoof, E. Hansen, K. Cools and P. Vanparys P3/34 Page 180
DEVELOPMENT OF A DISSOCIATION METHOD TO OBTAIN SINGLE COLUMNAR EPITHELIAL
COLON CELLS TO BE USED IN THE COMET ASSAY
A. Vanhauwaert P3/35 Page 181
13
Symposium 1
Genetic susceptibility
S1/1 - S1/3
14
S1/1
GENETIC SUSCEPTIBILITY: AN INTRODUCTION
Angelo Abbondandolo
National Cancer Research Institute-Genova
Genova, Italy
A basic principle of genetics is that the phenotype is rarely determined by the genotype alone
and is, much more commonly, the result of the interaction between genotype and environment. This
old principle has received a clear confirmation in the individual susceptibility to chronic diseases, and
to cancer in particular. Cancer, as it has been said many times, is a genetic disease. This means, of
course, that there are genes that can cause cancer. However, against a handful of genes that really
cause cancer, many more genes appear now to exist that rather influence the probability that cancer,
and environmental cancer in particular, will arise. Many of these genes are polymorphic, and particular
allele combinations will increase or decrease the susceptibility to environmental cancer. The early
reports of associations between polymorphic genes and environmental cancer concentrated on genes
that control the metabolism of chemical carcinogens. The attention, however, was soon shifted to
other categories of genes that may be equally or even more relevant. One of such categories is that of
DNA repair genes, for which polymorphisms are being discovered at an increasing pace. The great
hope behind these studies is that the knowledge of genetic polymorphisms will make possible in a
near future to identify individuals with an increased cancer susceptibility. An element of scepticism in
this hope comes, in my opinion, from the terrific dimension of this task. Nobody knows exactly how
many susceptibility genes exist, but their number may well be in the order of thousands or tens of
thousands. To complicate the issue, we know right from the beginning of these studies that a
genotype which increases the risk for a type of tumour may well protect from another tumour type. To
compute all possible effects of all existing polymorphisms influencing the response to all different
chemical carcinogens appears as an impossible task. Finding practical ways to use thevaluable
information that is being produced by researchers in this field for the assessment of individual
susceptibility is the challenge.
15
S1/2
CYTOGENETIC BIOMARKERS AND GENETIC POLYMORPHISMS
H. Norppa1; J. Tuimala1; G. Szekely2; S. Gundy2; A. Hirvonen1
1Department
Finland;
of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki,
2National
Institute of Oncology, Budapest, Hungary
Cytogenetic biomarkers are among the most widely used tools in biomonitoring of genotoxic effects in
humans. Yet, relatively little is known about factors affecting the individual level of chromosome
damage, besides some well-explored variables such as age, sex, and tobacco smoking. High frequency of chromosomal aberrations (CAs) was observed to be predictive of an increased cancer risk.
This association was also observed in non-smokers and in subjects with no identifiable occupational
exposure to carcinogens, which suggested that individual susceptibility plays an important role. Recent
studies indicated that common genetic polymorphisms of xenobiotic-metabolizing enzymes (XMEs)
could influence the frequencies of CAs and sister chromatid exchanges (SCEs) in peripheral
lymphocytes, interacting with genotoxic exposures or apparently modulating the spontaneous level of
these biomarkers. The homozygous deletion (null genotype) of glutathione S-transferase M1 gene
(GSTM1), resulting in total lack of GSTM1-mediated detoxification, was associated with increased
chromosome damage in lymphocytes of smokers and some occupationally exposed groups - in
agreement with the effect of the GSTM1 polymorphism on the level of DNA adducts. The GSTM1 null
genotype was also observed to increase the in vitro genotoxicity of some epoxides and genotoxins
found in tobacco smoke. The homozygous deletion of another glutathione S-transferase gene,
GSTT1, led to an elevated baseline frequency of SCEs, regardless of known exposures. In vitro
sensitivity to diepoxybutane and some other epoxides was observed to be primarily due to the GSTT1
null genotype. N-acetyltransferase 2 (NAT2) genotypes associated with slow acetylator status resulted
in a higher level of baseline CAs. Such genotype effects on the baseline level of chromosome damage
could reflect interaction with unidentified genotoxic exposures (e.g., from diet or inborn metabolism) or
the involvement of the enzyme in other metabolic routes affecting chromosome integrity. In addition to
XME genotypes, polymorphisms of proteins involved in DNA repair and enzymes functioning in folate
metabolism are expected to influence the level of chromosome damage. Their phenotypic
consequences are presently poorly known. Polymorphism of the XRCC1 gene (X-ray repair crosscomplementing group 1) was associated with increased SCEs in smokers, elevated baseline level of
CAs, and in vitro sensitivity to bleomycin and a tobacco-specific nitrosamine. The determination of
selected genotypes in genetic toxicology studies is expected to help in (i) controlling individual
variability, (ii) detection of exposure effects, and (iii) identification of sensitive subgroups.
16
S1/3
DNA REPAIR POLYMORPHISMS: DISCOVERY, PHENOTYPE AND RISK.
Douglas A. Bell,
National Institute of Environmental Health Science Research Triangle Park, NC.
Common genetic variation modulates human disease risk. Government and commercial laboratories
are funding large scale efforts to discover polymorphisms in genes that may be related to human
disease, response to drugs or response to environmental agents. Because of the importance of DNA
repair in preserving the integrity of the genome and in modulating the therapeutic effects of antitumor
agents, DNA repair genes are lead candidates for polymorphism discovery projects. Approximately
3000 polymorphisms have been reported in ~80 human DNA repair genes and more extensive data
will be available soon. Challenges with regard to evaluating functional effects of polymorphisms will be
discussed. Experiments testing the relationship between genotype and phenotype for selected genes
(XRCC1 , XRCC3 , XPD, XPF and others) will be presented. Data on the association between DNA
repair polymorphisms and cancer risk at several sites will be presented and critiqued. Results will be
discussed in relation to susceptibility at low dose exposures.
17
Symposium 2
DNA repair
S2/1 – S2/4
18
S2/1
NUCLEOTIDE EXCISION REPAIR: FROM HUMAN SYNDROMES TO MOUSE
MODELS AND DYNAMICS IN LIVING CELLS
J.H.J. Hoeijmakers1, D. Hoogstraten1, V. van den Boom1, E. Citterio1, A. B. Houtsmuller2,
W. Vermeulen1, J. de Boer1, and G.T.J. van der Horst1.
1MGC,
2Dept.
CBG, Dept. of Cell Biology and Genetics,
of Pathology, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Gene cloning and in vitro assays have resolved the ‘cut and patch’ core mechanism of nucleotide
excision repair (NER) involving 25-30 proteins. However, little is known about the dynamics and
organisation of NER in vivo and its functioning amidst other processes. Confocal analysis and
photobleaching in living cells revealed that NER factors tagged with green fluorescent protein are
highly mobile, have a unique intranuclear distribution and diffusion rate, depending on their MW
arguing against a preassembled NER ‘holocomplex’. After induction of damage a fraction of NER
proteins becomes transiently immobile in a dose-dependent fashion, corresponding with one repair
event taking ~4 minutes. These findings favour a model involving consecutive assembly of NER
factors on the site of a lesion. NER proteins involved in NER and basal transcription, such as CSB and
subunits of TFIIH, exhibited dramatic changes in intranuclear distribution and mobility in response to
damage and after treatment with inhibitors of transcription. These results provide insight into the
nuclear dynamics of inducible processes in living cells.
Defects in NER are associated with 3 UV-sensitive, genetically heterogeneous syndromes: xeroderma
pigmentosum, Cockayne syndrome and trichothiodystrophy. Mouse models for each of these
conditions have provided insight into the complex genotype-phenotype relationship and revealed
cancer predisposition and prominent ageing features. We propose that DNA damage compromises the
transcriptional capacity inducing cell malfunctioning and apoptosis, leading to ageing. These findings
link ageing with the condition of our genes and have important implications for the molecular basis of
ageing.
19
S2/2
TRANSCRIPTION-COUPLED REPAIR OF 8-OXOGUANINE IN VARIOUS HUMAN
CELLS.
A. Sarasin ; J. Cabral-Neto ; M. Pastoriza ; F. Le Page.
UPR 2169 CNRS, Cancer Research Institute, Villejuif, France.
Various irradiation processes as well as the aerobic metabolism are accompanied by the formation of
reactive free radicals that can damage cellular components and produce DNA lesions, such as the 8oxoguanine. The possible mispairing between 8-oxoguanine and adenine induces G to T transversion
in the absence of efficient repair. Our results show that replication of a unique modified base in a
double-stranded plasmid in repair-proficient human cells and repair-deficient cells from classical
xeroderma pigmentosum (XP) patients induces a similar mutation frequency of about 1-3% (G to T
transversions in 95% of mutants). Cells isolated from Cockayne's syndrome patients or XP patients
exhibiting also Cockayne symptoms are unable to repair the 8-oxoguanine leading therefore to at least
10 times more G to T transversions than in normal cells. This low repair level is associated with
mutations on the XPB, XPD, XPG or CSB gene. Full complementation with the expression of the wild
type gene indicates that this high mutation frequency is only due the genetic defects of the repair gene
(1). This high mutagenic potency of 8-oxoguanine is only observed when this lesion is located on the
transcribed strand (in such case the transcription machinery is blocked) while normal repair is
observed when this lesion is located on the non-transcribed strand. Human cells which are deficient in
the susceptibility genes for breast and ovarian cancer, BRCA1 or BRCA2, are also fully defective in
the TCR of 8-oxoguanine. Expression of wt BRCA1 gene from a recombinant adenovirus, fully
complements the repair defect in BRCA1-deficient cells (2). These results imply that the recognition
and the repair of 8-oxoguanine and probably other oxidative lesions, need the cooperation of protein
complexes involved in DNA repair, cell cycle regulation and tumour suppressor activity. Preliminary
results indicate that some mismatch DNA repair proteins are necessary for the TCR of 8-oxoguanine.
This pathway may be important for the recognition of the lesion and eventually for blocking the RNA
polymerase II at the lesion site.
(1) Le Page, F, Kwoh, EE, Avrutskaya, A, Gentil, A, Leadon, SA, Sarasin, A and Cooper PK., Cell,
2000, 101, 159-171.
(2) Le Page, F, Randrianarison, V, Marot, D, Cabannes, J, Perricaudet, M, Feunteun, J and Sarasin
A., Cancer Research, 2000, 60, 5548-5552.
20
S2/3
THE ROLE OF DNA LIGASE IV IN NON-HOMOLOGOUS END JOINING AND
DEFECTS IN PATIENTS.
O’Driscoll M1, Cerosaletti K2, Girard P1, Priestly A1, Sperling K4, Gennery A3, Concannon P2,Jeggo P1.
1
MRC Cell Mutation Unit, University of Sussex, Brighton, UK.
2
Molecular Genetics Program, Virginia
Mason Research, Centre Seattle WA, USA. 3 Newcastle General Hospital, Newcastle, UK.
4
Institute
of Human Genetics, Humbolt University, Berlin, Germany.
DNA double strand breaks (DSBs) are the most biologically significant lesion induced by ionising
radiation but can also arise endogenously at replication forks, during meiosis and V(D)J
recombination, and possibly during other developmental or metabolic processes. The presence of
DSBs in cells elicits a number of distinct response mechanisms that include processes of DNA repair,
cell cycle checkpoint arrest and/or the onset of apoptosis. Together these processes serve to maintain
genetic stability and limit the proliferation of damaged cells. Defects in some of the proteins involved in
these processes have been shown to contribute to hereditary disorders as well as to clinical
radiosensitivity. The most renowned example is Ataxia telangiectasia (A-T) a syndrome arising from
defects in ATM. A distinct but overlapping disorder is Nijmegen Breakage syndrome (NBS), which has
been shown to be due to defects in Nbs1. Like A-T, NBS patients have immunodeficiency and cancer
predisposition but they do not display either ataxia or telangiectasia. In contrast to A-T they frequently
show growth and developmental delay including microcephaly. Recently, hypomorphic mutations in
hMre11 have been detected in ATLD (A-T like disorder), a condition manifest as clinically mild A-T. In
mammalian cells, the principle mechanism for the repair of DSBs induced in the G1 phase of the cell
cycle is non-homologous end joining (NHEJ). Five proteins have been shown to function in NHEJ.
Three of these constitute the DNA dependent protein kinase (DNA-PK) complex, the two subunits
(Ku70 and Ku80) of the Ku protein and the DNA-PK catalytic subunit, termed DNA-PKcs. In addition,
Xrcc4 and DNA ligase IV, two proteins that co-associate strongly, are required for NHEJ. The current
model is that Ku binds to DNA double strand ends, recruits DNA-PKcs and activates its kinase activity,
as well as recruiting the Xrcc4/DNA ligase IV complex that carries out the final ligation step. A hallmark
of cells defective in NHEJ is marked radiosensitivity and impaired ability to rejoin DSBs as monitored
by pulse field gel electrophoresis, consistent with the notion that NHEJ represent a major DSB repair
mechanism in mammalian cells. To evaluate the contribution of possible defective NHEJ to human
health, one approach adopted was to investigate cell lines from clinically normal patients who
dramatically over-responded to radiotherapy. A second approach used was to examine cells lines
derived from patients with overlapping clinical features to those already observed in A-T and NBS that
do not harbour mutations in the known genes. A third strategy based on the prediction that patients
defective in NHEJ would be impaired in V(D)J recombination was to examine patients displaying
uncharacterised immunodeficiency. Here, we describe how combining these approaches have
revealed patients with defects in DNA ligase IV. We describe the cellular and clinical features
associated with this deficiency, which we have called “Ligase IV Syndrome”, and identify it as an NBSlike disorder.
21
S2/4
ROLE OF THE BREAST CANCER SUSCEPTIBILITY GENE Brca2 AND THE
Rad51C GENE IN PROVIDING GENOME STABILITY AND PROTECTION
AGAINST DNA DAMAGE
M.Z. Zdzienicka
Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center,
Wassenaarseweg 72, 2333 AA Leiden, NL
Homologous recombination plays an essential role in the cellular responses to DNA damage and in
the maintenance of genome integrity. The Rad51 gene product is a key protein in this process. Rad51
interacts with many proteins including the proteins encoded by the breast cancer susceptibility genes,
BRCA1 and BRCA2. In addition, the following gene products: XRCC2, XRCC3, Rad51B, Rad51C, and
Rad51D are involved in the formation of the Rad51protein complex, in response to DNA damage.
Recently we identified two mitomycin C (MMC) -sensitive hamster cell mutants, V-C8 and CL-VC4,
that are defective in the Brca2 and Rad51C genes, respectively. The characterization of these mutants
sheds new light on the function of these genes in the cellular response to DNA damage. V-C8 mutant
represents the XRCC11 group of X-ray-sensitive mammalian mutants, while CL-V4B is the only
mammalian mutant known to be defective in the Rad51C gene. In addition to hypersensitivity to a wide
variety of DNA damaging agents, V-C8 cells show radioresistant DNA synthesis following irradiation,
suggesting that Brca2 is involved in several pathways, including cell cycle checkpoint regulation.
Interestingly, we found that V-C8 cells have a 2.5-fold higher mutation rate than wild-type V79 cells,
revealing why Brca2-deficiency can contribute to cancer progression. Both mutants, V-C8 and CLV4B, are very sensitive to cross-links and also display a tremendous spontaneous chromosomal
instability, indicating important roles of Brca2 and Rad51C in the stabilization of the genome and
protecting cells against DNA cross-links.
22
Workshop 1
Mitosis versus meiosis
W1/1 – W1/4
23
W1/1
MITOSIS : A
TRANSITION
UNIFYING
MODEL
FOR
THE
METAPHASE/ANAPHASE
M. Kirsch-Volders and I. Decordier
Vrije Universiteit Brussel, Laboratorium voor Cellulaire Genetica, Pleinlaan 2,
B-1050 Brussel, Belgium.
The term mitosis actually covers a complex sequence of events at the level of the cell membrane, the
cytoplasm, the nuclear membrane and the chromosomes; recently attention has been focused more
and more on the checkpoints that control their orderly progression and in particular the
metaphase/anaphase transition. Accurate segregation of sister chromatids between the daughter cells
is dependent on coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibres,
topoisomerases, proteolytic processes and motor proteins. A review paper by us (Kirsch-Volders et al.,
1998) indicated that sister chromatid separate independently of the tubulin fibres, as a result of
proteolytic processes controlled by the anaphase promoting complex. During metaphase the activity of
separin, which contributes to the sister-chromatid separation, is blocked by securin. Activation of the
anaphase promoting complex causes the degradation of securin and the release of separin resulting
in the loss of cohesion between the sister chromatids (Orr-Weaver, 1999).
The spindle fibres are necessary to move the separated chromatids to the spindle poles but probably
not to initiate separation. Deficiencies in or impairment of any of these structures or in their control
systems may lead to a more or less important genomic imbalance. Moreover, recently apoptosis was
shown to be induced by tubulin poisons in a p53-dependent and independent manner (Casenghi et al.,
1999). A number of remaining questions will also be highlighted.
References:
Casenghi et al. (1999) Exp. Cell Res., 250, 339-350
Kirsch-Volders et al. (1998) Mutagenesis, 13, 321-335.
Orr-Weaver (1999) Science, 285, 344-345
24
W1/2
CELL CYCLE INSIGHTS OF MOUSE PRIMARY SPERMATOCYTES
P. de Boer and O. van den Broek*
Department of Obstetrics and Gynaecology, University Medical Center St Radboud, Nijmegen, The
Netherlands; *Wageningen Institute of Animal Sciences, Wageningen, The Netherlands
Once in their lifetime, germ cells that survive multiplication by mitosis have to make the decision to go
meiotic. In this survey, an attempt will be made to delineate the first signals that forecast this change in
behaviour, with mouse primary spermatocytes as the model system. After these first signals, the
spermatogonial and primary spermatocyte cell cycles differ vastly, due to the requirements for
homologous chromosome pairing, chromosome synapsis and homologous recombination. These
factors lead to a more than 30 times prolonged duration of the interval end S-phase to metaphase in
the primary spermatocyte, taking the G2 phase of the B spermatogonium as the reference.
Questions to be asked: a) is from the perspective of cell cycle regulation all of meiotic prophase
equivalent to somatic prophase, and b) what step in recombination determines the meiotic fate of the
cell. Sex differences in first meiotic prophase that allow for the preparation of spermiogenesis, will be
taken into account. Restrictions that meiotic recombination may impose upon DNA repair during first
meiotic prophase will likewise be addressed.
25
W1/3
26
W1/4
DIFFERENCES IN THE PROCESS OF MITOTIC AND MEIOTIC DIVISIONS AND
THEIR CONSEQUENCES FOR CHEMICAL EFFECTS.
Ilse-Dore Adler
GSF-Institute of Experimental Genetics, D-85758 Neuherberg, Germany.
Mitotic divisions distribute exact numbers of chromosomes to daughter cells while during meiosis the
number of chromosomes is reduced to one half. These processes are ensured by various
mechanisms such as cell cycle checkpoints, chromosome or chromatid pairing, spindle formation from
microtubules, their controlled breakdown and the action of motor proteins. The process of mitotic
chromosome distribution is characterized by centromere separation and distribution of chromatids to
opposite spindle poles. The process of meiotic chromosome distribution is characterized by
homologous chromosome pairing and distribution of entire chromosomes to opposite spindle poles.
Meiotic prophase in male germ cells takes weeks rather than hours and in female germ cells it takes
between months (mouse) and years (humans). Mitotic spindles are bipolar with two microtubule
organizing centres (MTOC). The female meiotic spindle is barrel shaped with multiple MTOC. The
formation of spindle polarity differs between somatic and female meiotic spindles and the
metaphase/anaphase checkpoint in female germ cells is permissive. These differences explain why
somatic cells show different sensitivity to aneuploidy induction by chemicals. However, so far there
seem to be no germ cell specific aneugens. Instead, the biologically relevant thresholds may differ
between somatic and germinal cells. Sensitivity differences also exist between sexes. Often, female
germ cells are more sensitive than male germ cells (e.g. to griseofulvin or vinblastine). An examples
for male germ cells being more sensitive than female germ cells is diazepam. The difference reflects
the mode of action of the aneugen, e.g. for diazepam the inhibition of centriole separation. Since
different biological activity of an aneugen and multiple targets characterize aneugenic events,
systematic comparative studies are required.
Supported by EU-contract QLK4-CT-2000-00058
27
Symposium 3
Ecogenotoxicology
S3/1 – S3/5
28
S3/1
TOXICITY AND GENOTOXICITY OF HYDROPHOBIC CHEMICALS SAMPLED
WITH SEMIPERMEABLE MEMBRANE DEVICES (SPMDs) IN THE AQUATIC
ENVIRONMENT
1D.
Sabaliunas; J. Lazutka2; I. Sabaliuniene2
1Procter&Gamble
2Faculaty
Technical Centres Ltd., Staines, Middlesex, UK
of Nature Sciences, Vilnius University, Lithuania
Semipermeable membrane devices (SPMDs) are passive samplers capable of pre-concentrating
hydrophobic chemicals from water, sediments, soil and air. They consist of layflat polymeric
membrane such as polyethylene containing a thin film of synthetic lipid such as triolein. The transport
of hydrophobic chemicals through the membrane into the lipid is governed by the process of passive
diffusion, whereas the molecular size exclusion limit of the polyethylene membrane is similar to that of
biological membranes. Therefore, SPMDs sample bioavailable chemicals in a way similar to
organisms helping to reveal their true exposure to hydrophobic substances.
We have used SPMDs in the monitoring of concentrations and effects of organic pollutants in the
aquatic environment. In laboratory flow-through studies, we compared the uptake of model
compounds (various pesticides) by SPMDs and bivalves. Mixtures of chemicals accumulated by
SPMDs and mussels were tested in standard toxicity and genotoxicity assays (Microtox, Mutatox,
invertebrate toxicity tests, the Ames test, sister chromatid exchange test). To further validate the
method, SPMDs were deployed in surface, ground water sources and sediments known to be
contaminated by hydrophobic pollutants. Bioassay-directed fractionation and chemical analysis
methods were used to identify the substances sampled (PAHs, PCBs, organochlorines) and their
effects were evaluated in bioassays. SPMDs proved to be useful tools in monitoring of organic
pollutants under the field conditions. Potentially, they can be used in the Toxicity Identification
Evaluation (TIE) procedures, site-specific environmental risk assessment and, eventually, risk
management.
Sabaliunas et al., 1997, Environmental Pollution, 96, 195-205.
Sabaliunas et al., 1998, Environmental Toxicology and Chemistry, 17, 1815-1824.
Sabaliunas et al., 1999, Ecotoxicology and Environmental Safety, 44, 160-167.
Sabaliunas et al., 2000, Environmental Pollution, 109, 251-265.
29
S3/2
CYTOGENETIC STUDIES ON THE EFFECT OF IONISING RADIATION BY SOLIDSTAIN AND FISH TECHNIQUES.
J.F. Barquinero1; S. Cigarrán1; A. Duran2; M.R. Caballín1, M. Ribas3; L. Barrios2.
1Unitat
d’Antropologia, Dpt. Biologia Animal, Biologia Vegetal i Ecologia. 2Unitat de Biologia Cel.lular,
Dpt. Biologia Cel.lular, Fisiologia i Immunologia. 3Servei de Radiofísica i Radioprotecció de l’Hospital
de la Santa Creu i Sant Pau. Universitat Autònoma de Barcelona, 08193 Bellaterra. Spain
The effect of overexposures to very low doses of ionising radiation (IR) have been analysed to
evaluate the basal frequency of aberrations using conventional solid-stain and FISH painting
techniques. In these studies a significantly higher background frequency respect to a control group
was only observed for acentric fragments. For translocations FISH detected, in spite of an slight
increase in the exposed population, no significant differences were observed. This result is related to
the wide range in the basal frequency of translocations, that contrasts with the reported observations
for dicentrics. The implications in biodosimetry studies using tanslocations is discussed. Other studies
carried out to evaluate the effect of low dose exposures have been focussed to evaluate the existence
of an adaptive response induced by occupational exposures. For this reason lymphocyte treatments in
G0 with IR and in G2 with bleomycin have been studied. In these studies significant lower frequencies,
in the occupationally exposed group respect to controls, were observed for dicentrics and chromatid
breaks respectively. Implications on the inter-individual susceptibility to clastogenic agents, and
implications on the biodosimetry studies are discussed.
Before the elaboration of dose-effect curves using FISH painting techniques, it was analysed the
relative chromosome involvement in the radio-induced chromosome aberrations. For this purpose
samples from a female and a male were irradiated at 3 and 5 Gy respectively, in both experiments
chromosomes were analysed one by one independently. The results indicated that smaller
chromosomes are more involved and larger less than expected due to their relative DNA content.
Taking into account these results and the different chromosome morphology, to obtain calibration data
using FISH techniques, chromosomes 1, 4 and 11 were selected. The coefficients obtained are similar
with those obtained by solid stained dicentric analyses, indicating that for short term biodosimetry, in
spite of that the technique to choice is the conventional solid-stain, the use of FISH painting will give
similar dose estimations. This could be of particular interest in follow-up studies. Is in this sense an
study, in which 20 partial irradiations were simulated, on the suitability of FISH painting techniques to
assess partial irradiations, for both just after the exposure and after a long post-irradiation time is also
discussed.
30
S3/3
ASSESSMENT OF AIR QUALITY IN SILESIA, POLAND BASED ON CHEMICAL
ANALYSES AND SALMONELLA ASSAY – NOW AND 5 YEARS AGO
D. Mielżyńska; E. Siwińska; A. Bubak; L. Kapka
Institute of Occupational Medicine and Environmental Health, Sosnowiec, Poland
For many years ambient air pollution has been a very important contributor to the
environmental pollution in Silesia province - a region of highest urbanization and industrialization in
Poland, characterized by high cancer and infant mortality. During the last 10 years the concentration of
some chemical air pollutants has decreased due to the fact that many noxious industrial plants have
been closed down. In 1994/95 we performed a project supported by the Polish Committee of Scientific
Research (grant no. 4P05D 014113) including the determination of concentrations of airborne
particulate matter (PM10), 4 polycyclic aromatic hydrocarbons (PAHs): pyrene, benzo(a)anthracene
(BAA), benzo(a)pyrene (BAP) dibenzo(a,h)anthracene (DAHA) and mutagenic effect of airborne
particles. PAHs were determined by HPLC method and mutagenic effect by the plate incorporation
Salmonella/mammalian-microsome assay with standard strains TA98S9, TA100S9 and the strain
YG1041S9 with elevated level of nitroreductase and O-acetylotransferase enzymes. The purpose of
this study was to compare these results with new investigations supported by the National Fund for
Environmental Protection and Water Management which were carried out in 1999/2000 in order to find
whether the exposure to PAHs and other mutagenic chemical compounds has also changed to better.
The assessment of exposure involved the same methods than in the previous study. 24-hour samples
of airborne particulate matter were collected 2 times per week during three winter months at the same
17 measurement points. Mutagenic effect was expressed as mutagenicity index (MI/m 3) - relative
measure including the number of revertant colonies in the appropriate control. Selected results
(arithmetic means with standard deviation) are presented in the following table.
Strain
MI 1994/95
MI 1999/00
PAHs
1994/95
1999/2000
TA98
1,90,8
3,00,6
Pyrene
22,78,7
37,612,8
TA98+S9
3,91,1
4,91,8
BAA
30,212,3
15,48,4
YG1041
5,71,9
2,00,6
BAP
24,89,8
33,311,0
YG1041+S9
9,22,5
1,90,9
DAHA
8,03,0
13,84,5
Generally it was found that the exposure to PAHs and other mutagenic compounds exceeded the level
assessed 5 years ago. Lower values of MI detected with YG1041 may indicate lower contribution of
nitro aromatic compounds, aromatic amines and hydroxyloamines to the total mutagenic effect of
airborne particles due to different weather conditions in examined periods or other factors that should
be examined in future. The investigations involving samples collected in summer are being continued.
31
S3/4
ASSESSMENT
OF
TOXICITY
AND
GENOTOXICITY
OF
INDUSTRIAL
EFFLUENTS IN SOUTHERN ITALY
A. Dell’Aquila; A. Diodati; M. Isidori; M. Lavorgna; A. Mancini
Dipartimento di Scienze della Vita – Seconda Università di Napoli – Via Vivaldi, 43 – 81100 Caserta –
Italy
The increase of industrial sites triggered the need to assess the release of countless man-made
chemicals to the biosphere and the potentially harmful impact they can determine alone or in
combination toward biological integrity.
Effluents from the factories of the Industrial Development Area of Caserta (Southern Italy) were tested
to evaluate their toxic and genotoxic effects on biota of receiving environments.
Toxicity tests were performed on reducers (the bacterium Vibrio fischeri), producers (the alga
Selenastrum capricornutum) and consumers including a rotifer (Brachionus calyciflorus), a cladoceran
(Daphnia magna), an anostracan (Thamnocephalus platyurus) to evaluate effects on freshwater
organisms from different trophic levels.
Furthermore SOS Chromotest, a bacterial colorimetric assay with E.coli PQ37, was carried out to
assess genotoxic activity of these industrial effluents, detecting DNA-damaging agents.
Results showed that some industrial effluents are only toxic for organisms of the aquatic chain
whereas others are highly genotoxic for E.coli PQ37.
The present study suggests that different kinds of bioassays such as toxic and genotoxic tests offer
complementary tools and approaches that can be applied to measure hazard and risk of xenobiotics
on the environment.
1) Blaise C. (2000). Canadian application of microbiotests to assess the toxic potential of comlex liquid
and solid media. New Microbiotests for Routine Toxicity Screening and Biomonitoring, 3-12.
2) Persoone G. (1998). Development and validation of Toxicity microbiotests with invertebrates, in
particular crustaceaus. Microscale Aquatic Toxicology, 311.320.
3) Quillardet P. and Hofnung M. (1985). The SOS Chromotest, a colorimetric bacterial assay for
genotoxins: procedures. Mutation Research, 147, 65-78.
4) Sbrilli G., Bucci M., Brilli L., Gambassi F. (1995). Utilizzazione di test di tossicità nel controllo degli
scarichi industriali. Acqua Aria, 539-548.
32
S3/5
EMOTIONAL STRESS MODIFY GENOME SENSITIVITY TO ENVIRONMENTAL
MUTAGENS.
PARALLELS
IN
RESULTS
ON
MICE
AND
HUMAN
INVESTIGATIONS
F. Ingel
A.N.Sysin Research Institute of Human Ecology and Environmental Health RAMS
Moscow, Russia e-mail: [email protected]
Human emotional stress, giving a significant deposit into morbidity, usually doesn’t taking into account
in genetical studies. Here are presented results of 3 single and 2 repeated complex ecologicalgenetical-psychological studies carried out in 4 Russian industrial towns (totally 184 persons). Groups
for the investigations were formed in: Diadkovo Town – workers of Crystal plant and citizens (men and
women); Chapaevsk Town – workers of agricultural poisons plant and 2 groups of citizens, (women);
Jaroslavl Town – workers of oil refinery and machinery plants (men); Moscow – workers of oil refinery
and citizens (men); Moscow (scientists, toxicologists (women). Program of the investigations included:
1. air samples of working places and living zones total toxicity and total mutagenisity evaluation or
evaluation of dioxin in blood and /or
heavy metals contents in hairs; 2. level of chromosome
aberration (CA) and coefficient of UV-induced DNA reparation (Cuv UDS) in human blood
lymphocytes; 3. evaluation of
human emotional stress level (ESL) by standard psychological
questionnaires detected different kinds of stress – psychological depression, anxiety and overfatigue.
Results of the study demonstrated that people, living and working in pollutant zones, more often were
in stress then the ones living in cleaner arias and working in plants with lowest level of air pollution.
Human ESL correlated with level of CA (P≤0,05) , with Cuv UDS (P≤0,01) and with blood dioxin
contents (P≤0,001). Moreover, people in stress, as a rule, had highest level of CA and abnormal DNA
reparation in blood cells than ones in state of psychological comfort. Repeated studies showed that
people in stress had highest sensitivity of genome to environmental mutagens as well as their blood
lymphocytes - to in vitro mutagenic loads, what is in good agreement with results of our prolonged
experiments on mice.
So, our data demonstrate that stress significantly modifies human genome sensitivity to environmental
mutagens and, consequently, is an important risk factor, which have to be included into system of
genetical monitoring: 1. people with high level of chronicle stress have to be included into groups of
high genetical risk; 2. genetical parameters detection only for people in state of psychological comfort
allow to evaluate levels of genetic damage and DNA reparative activity being free of social-economical
factors influence. This procedure can be useful for correct comparison of genetical effects between
groups, departments, plants, and ets.
33
Symposium 4
Molecular epidemiology & biomonitoring
S4/1 – S4/4
34
S4/1
DNA ADDUCTS IN PAH EXPOSED PERSONS MODULATED BY GENETIC
POLYMORPHISMS IN CARCINOGEN METABOLIZING ENZYMES
F.J. Van Schooten
Dept Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands
During the past decade considerable improvements in methodologies have been achieved in the
sensitive and specific quantitation of carcinogen-DNA adducts. Thereupon significant levels of DNA
adducts have been detected in humans as a result of exposure to several exogenous as well as
endogenous sources. At present the most widely used approaches are the postlabelling assay,
immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA
adducts or modified DNA, and adduct identification using physico-chemical instrumentation.
Application of these techniques for biomonitoring purposes in human populations has been hampered
by the inaccessibility of target tissues such as lung. Consequently more readily available surrogate
tissues have been studied such as white blood cells or sputum cells. Especially much work has been
done on human populations exposed to polycyclic aromatic hydrocarbons (PAH) via cigarette
smoking, medicinal treatment, occupation and/or diet. The results of most studies show that PAH-DNA
adducts have been associated with exposure in a variety of human tissues, including target organs of
PAH- and tobacco-associated cancers. Furthermore, studies indicate that DNA adduct levels are
modulated by host polymorphisms in genes that code for metabolizing enzymes. DNA adduct
measurements may be a suitable biomarker to be used in molecular epidemiological studies. Future
biomonitoring studies in human populations exposed to complex mixtures should combine DNA
adduct formation with genetic markers of (cancer) susceptibility in a number of cancer predisposing
genes.
35
S4/2
TOBACCO
SMOKE-INDUCED
GENOTOXICITY
IN
LARYNGEAL
CANCER
SUBJECTS
K.Szyfter1,2, P.Jałoszyński1, J.Banaszewski2, M.Pabiszczak2, L.Möller3, W.Szyfter2
1. Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland
2. Department of Otolaryngology, Umiversity of Medical SCiences, Poznań, Poland
3. Department of Biosciences, Centyer for Nutrition and Toxicology, Huddinge, Sweden
Tobacco smoking is the unquestionable primary causative factor of laryngeal cancer. DNA lesions
induced in laryngeal mucosa by tobacco smoke carcinogens are at the beginning of multistep
carcinogenesis terminated by squamous cell carcinoma of the larynx.
DNA adducts are recognised as a marker of exposure to exogenous environmental mutagens.
Aromatic DNA adducts and N7-alkyl-dGMPs were already analysed in the target tissue (laryngeal
tumour and non-tumour) by the relevant variants of
32P-postalebelling
assay. The levels of DNA
adducts were found dependent on exposure, patient's sex and moderately on age. A significance of
genetic factor was established only for multiple defects in genes coding detoxifying enzymes.
Recently an analysis was extended for oxidative DNA damage. Six oxidative DNA base
modifications (5-OH-Ura, 5-OH-Cyt, 8-oxoGua, 8-oxo-Ade fapyguanine and fapyadenine) were
estimated in 68 subjects using gas chromatography/isotope dilution mass spectroscopy. A weak, but
still distinct effect of tumour grading and metastatic status was observed in both kinds of tissue for
oxidised pyrimidines and purines but not for ring-opened purines. Since the levels of oxidative DNA
damage tended to increase with tumour aggressiveness, we postulate that oxidative DNA lesions
increase genetic instability and thus contribute to tumour progression in laryngeal cancer.
No association between aromatic DNA adduct level and oxidative DNA lesion was found,
suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in
larynx cancer. Hence, it seems that oxidative DNA damage is mostly connected with endogenous but
not environmental exposure.
A pattern of DNA lesions induced by PAH (aromatic DNA adducts), N-nitrosamines (N7-alkyldGMPs) and reactive oxygen species (oxidative DNA base lesions) are fairly independent. The
findings in relation to staging and grading of laryngeal tumour seem to indicate that the occurrence of
the first two types of lesions in connected with laryngeal cancer initiation and (?) promotion. The
current oxidative DNA damage appears to be involved rather in progression of laryngeal cancer.
36
S4/3
MICRONUTRIENTS, GENE-DIET INTERACTION AND GENOMIC STABILITY
Michael Fenech
CSIRO Health Sciences and Nutrition, Adelaide, Australia.
Several micronutrients (vitamins and minerals) are required as co-factors in DNA synthesis, DNA
repair, DNA methylation and apoptosis. Some notable examples include (a) folic acid and vitamin B12
required for maintenance methylation of DNA and the synthesis of dTTP from dUTP , thus prevent the
misincorporation of uracil into DNA, a highly mutagenic and chromosome-breaking event, (b) niacin,
is essential in the form of the coenzymes NAD and NADP which act as a substrate for polyADPribose
polymerase (PARP), an enzyme thought to facilitate efficient DNA repair and telomere length
regulation and (c) zinc, apart from its antioxidant role as a co-factor in Cu/Zn SOD, it is required in its
stabilizing role of
the DNA-binding domain of p53 (residues 102-292)
and thus is essential for
apoptotic response to DNA damage. Optimal levels in a test-tube or in tissue culture have been
defined for some of these micronutrients with regard to prevention of oxidative damage to DNA,
optimal DNA repair or apoptotic activity. However, the real challenge is to define the level of intake of
these micronutrients to prevent DNA damage in vivo. Recent studies in humans, including those from
our laboratory on (a) folate/vitamin B12
and genomic stability and (b) polymorphisms in folate
metabolising enzymes and genomic stability, will be reviewed. Some of these studies suggest that our
ability to damage genes by inappropriate diet may be as important as mutation induced by exogenous
chemicals and radiation. The current recommended dietary allowances (RDAs) of minerals and
vitamins are designed for the prevention of diseases of deficiency. It is time for a concerted research
effort to define RDAs on the basis of optimal genomic stability because the link between DNA damage
and degenerative disease is becoming more evident. Reference: Fenech M. (2001) Recommended
dietary allowances for genomic stability. Mutation Res. ( in press).
37
S4/4
EFFECT OF METABOLIC GENOTYPES ON THE FORMATION OF BUTADIENEHEMOGLOBIN ADDUCTS
M. Warholm; P. Begemann; A. Colombi; S. Fustinoni; H.G. Neumann; A. Rannug; L. Soleo; J.
Swenberg; L. Vimercati
Institute of Environmental Medicine, Karolinska Institutet, and National Institute for Working Life,
Stockholm Sweden
1,3-Butadiene (BD) has been classified as a carcinogen or probable carcinogen by regulatory
agencies. BD is genotoxic and carcinogenic in rodents and forms DNA and protein adducts following
cytochrome P450 catalysed metabolic oxidation to epoxides. The expoxides (monoepoxybutene,
epoxybutanediol, diepoxybutane) are detoxified either through further hydrolysis catalysed by epoxide
hydrolase (mEH) or through glutathione transferase (GST)-mediated conjugation with glutathione. In
the present study biomarkers of BD exposure were studied in 30 workers (exposed to BD in monomer
and polymer production) and 10 controls (clerks). Occupational BD exposure, monitored by personal
sampling, ranged between 4 and 200 µg/m 3. Hemoglobin N-(2,3,4-trihydroxybutyl)-valine (THBVal)
adducts, formed from the diolepoxide metabolite, as well as urinary excretion of 1,2-dihydroxy-4-(Nacetylcysteinyl)-S-butane, derived from GST-mediated metabolism of BD, were found in all samples
investigated. Although the levels of these biomarkers were slightly higher in the workers than in the
controls, the differences were not statistically significant. It was therefore considered appropriate to
analyse the total group with regard to the influence of biotransformation genotypes on the levels of
THBVal adducts. Polymorphisms in cytochrome P450 (CYP) 2E1 and 2A6, mEH, GSTM1, GSTP1 and
GSTT1 were analysed. We found that the G–35T (5’-flanking region) polymorphism of CYP2E1
influenced the THBVal adduct levels. Individuals with the GG genotype had higher levels (39.4±8.8
pmol/g globin) than individuals with at least one T-allele (31.7±9.8 pmol/g globin) (p= 0.04). The
THBVal adduct levels were also found to be higher in individuals lacking GSTT1 or GSTM1 compared
to those possessing these isoenzymes (p=0.001 and 0.08, respectively), which is in accordance with
the detoxifying function of GSTT1 and GSTM1. The effects of the genotypes were similar in BDexposed workers and controls. Furthermore, smoking positively influenced the THBVal adduct levels
(p = 0.017). In summary, genotypes of CYP2E1, GSTM1 and GSTT1, as well as smoking, influence
the hemoglobin THBVal adduct levels. It is unclear if this effect is primarily associated with BD
exposure, or whether endogenous materials may also cause this adduct.
38
Symposium 5
Low doses & thresholds
S5/1 – S5/4
39
S5/1
THE RISK ASSESSMENT OF GENOTOXIC CHEMICALS
James M. Parry
Centre for Molecular Genetics and Toxicology
University of Wales Swansea
Swansea, SA2 8PP, U.K.
The currently recommended testing packages for genotoxic chemicals involve the evaluation
of DNA reactivity and the induction of point and chromosomal mutations both in vitro and in vivo.
Positive results in the tests allow the identification of mutagenic hazard and within the European Union
(EU) can result a hazard classification.
When genotoxic potential is identified (particularly when
associated with rodent carcinogenicity) then risk assessment is performed which generally involves
the application of linear stochastic models.
However, our increasing understanding of the mechanisms by which chemicals produce
genomic change suggests that the simplistic application of linear dose response models may produce
assessments which over-estimate risks at low exposure doses for some compounds. Factors which
modify linear risk models at low doses include chemical detoxification, DNA repair activity and multiple
targets for modification leading to mutation. In the later case, the concept of thresholded non-linear
dose response relationships for spindle damaging aneugens has been proved and accepted by
regulators within the EU.
The human genome is continually exposed to endogenous genotoxins with the consequence
of a background of “spontaneous” mutations. The level of spontaneous mutations and their specificity
of both type and position can provide critical information when estimating the risks of individual
genotoxins. The increasing volume of information on mutation specificity available from both tumours
and experimental systems allows the determination of both spontaneous and unique chemical
mutation profiles which can be applied to the risk assessment process.
40
S5/2
LOW DOSES AND THRESHOLDS IN HEPATOCARCINOGENESIS
G. Williams, M.D.
Department of Pathology, New York Medical College, Valhalla, New York, U.S.A.
A series of studies has been conducted on quantitative hepatocarcinogenesis with the DNA-reactive
carcinogens, 2-acetylaminofluorene and diethylnitrosamine (Williams et al., 2000).
Using precise
dosing in sensitive F344 male rats, critical effects underlying liver carcinogenesis have been examined
and liver tumors were quantified using phenobarbital as a promoting agent. The following findings will
be reported:
(1)
formation of hepatic DNA adducts can be nonlinear, with a plateau at toxic
exposures; (2) liver cell cytotoxicity appears to be proportional to exposure; (3)
compensatory
hepatocyte proliferation can show no-effect levels and can be supralinear at cytotoxic exposures; (4)
formation of preneoplastic hepatocellular altered foci can be
supralinear at exposures that elicit
compensatory cell proliferation; (5) no-effect levels can exist for liver tumor development and the
exposure-response can be supralinear at high exposures, corresponding to induction of preneoplastic
lesions. Thresholds for carcinogenesis are difficult to establish using only tumors as the endpoint, but
the demonstration of no-effect levels for effects underlying tumor development support the conclusion
of thresholds for carcinogenesis, even for genotoxic agents.
Williams, G.M., Iatropoulos, M.J. and Jeffrey, A.M. (2000) Mechanistic basis for non-linearity and
thresholds in rat liver carcinogenesis by the DNA-reactive carcinogens 2-acetylaminofluorene and
diethylnitrosamine. Toxicologic Pathology, 28:388-395.
41
S5/3
SPONTANEOUS MUTATION RATE • THE APPARENT THRESHOLD
R.C. von Borstel
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Tazima suggested the name "apparent threshold" for the spontaneous mutation frequencies that are
normally subtracted from induced mutation data before the data are plotted. For micro-organisms or
cultured cells, if at least seven different cultures are grown, and the median spontaneous mutation
frequency taken, this then can serve as the apparent threshold without there being an actual
determination of the spontaneous mutation rate. If possible, the median culture also should be used
for the induced mutation frequencies for the experiment under consideration. For higher organisms,
such as Drosophila or the mouse, the spontaneous mutation rate is calculated as mutations per
organism per sexual generation; the results of the zero exposure control, even historical results, can
serve as the apparent threshold.
When the data for exposures of different mutational measurement systems are plotted with the
apparent threshold included, the point of extrapolation through the apparent threshold is indeed the
true threshold for observation of a mutation or a cancer induced by mutagens or carcinogens.
Moreover, it is a truism that the more sensitive the system for recognition of a low exposure event, the
higher the apparent threshold. Thus, the apparent threshold is inversely related to the biological
importance of the system being tested.
42
S5/4
FISH-DETECTED ASYNCHRONOUS REPLICATION IN HUMAN CELLS
EXPOSED TO GENOTOXIC AGENTS
C.Z. Cotrim1; A. Brás1; I.Vasconcelos1 ;M. Sá da Costa2;J.Rueff1
1-Department of Genetics, Faculty of Medical Sciences, New University of Lisbon
Lisbon, Portugal.
2- Service of Radiology, Santa Maria Hospital, Lisbon, Portugal.
Recent studies using FISH analysis of the replication timing of several human genes have shown
differences between normal and mutant cells. In this study we applied this technology for human
lymphocytes exposed to X-rays (1 and 2 Gy), mitomycin C (0.3 and 1.5M) and H2O2 (15 and 20mM)
using the TP53 and Rb-1 probes. We have previously demonstrated a significant induction of
chromosomal aberrations with the same concentrations1.
After harvesting the cultures, slide spreads were prepared according to standard cytogenetics
procedures, denaturated for 1.5 min. in 70% formamide/2xSSC (pH 7.5) at 73ºC, and dehydrated in a
graded ethanol series. Probe hybridization was performed for 20 hours at 37ºC in a moist chamber.
Slides were then washed, counterstained with DAPI and analysed for Cy3 and DAPI (Vysis). Nonreplicated sequences were detected as two single hybridization signals (SS), sequences that have
completed replication were detected as two double-hybridization signals (DD). Sequences undergoing
non-synchronous DNA replication appeared as one singlet and one doublet (SD). Two hundred nuclei
were scored for each donor, for each genotoxic agent and for each probe.
All the genotoxic agents produced a significant increase (p<0.05; Student´s t test) in SD nuclei
percentage for both probes: (i) using TP53: X-rays (2 Gy - 27.284.53); mitomycin.C (0.3 M –
26.505.81; 1.5M - 28.032.37); H2O2 (15mM – 25.331.31; 20mM - 28.050.19) (ii) using Rb-1: Xrays (2Gy - 33.675.05); mitomycin C (0.3 M – 26.5 3.95; 32.67 2.2), H2O2 (15mM – 28.005.14;
20mM - 29.007.58). The results obtained further indicate that DNA damage hampers replication and
represent a fast and accurate method of assessing that phenomenon (Our current research is
supported by European Commission, Luso-American Foundation for Development and “Liga
Portuguesa contra o Cancro”).
1 - Rueff J., Brás A., Cristóvão L., Mexia J., Sá da costa M. and Pires V.: DNA strand breaks and
chromosomal aberrations induced by H2O2 and 60Co -radiation. Mutation Res. 1993; 289: 197-204.
43
Workshop 2
Electromagnetic fields
(parallel session)
W2/1 – W2/4
44
W2/1
ARE EXTREME LOW AND RADIOFREQUENCY FIELDS HAZARDOUS TO
HUMANS? AN INTRODUCTION.
L. Verschaeve
Vlaamse Instelling voor Technologisch Onderzoek, Milieutoxicologie,
Boeretang 200, B-2400 Mol, BELGIUM
In 1979 Wertheimer and Leeper provided evidence suggesting an association between exposure to
extreme low frequency (ELF) electromagnetic fields (high tension power lines) and childhood
leukemia. Several other investigations did corroborate their results, but others did not. Up to now there
still is a lot of controversy about the possible ELF-cancer association, also in adults.
In the early nineties a man claimed that his wife’s brain tumor was linked to her constant cellularphone use. This was the starting point for many other claims on “mobile phone frequency”-induced
adverse health effects. As the mobile phone technology and human exposure to this kind of
radiofrequency fields (handset and base station antennas) are quite recent, epidemiological evidence
in favor or against a cancer-link is very limited so far and hence the debate is still very lively.
Present day knowledge suggests that if electromagnetic fields (EMF) do cause an increased cancer
risk, this risk is only modest and it is unlikely that epidemiology alone will be able to clearly establish
such an effect. Therefore laboratory investigations are certainly needed. With cancer in mind,
investigations on genetic effects are certainly of great importance.
The present workshop is an attempt to provide up to date information on the genetic toxicology of
extreme low frequency and radiofrequency electromagnetic fields. Two lectures will focus on results
from recent research involving the above-cited EMF’s, also in combination with exposure to chemical
mutagens. A third lecture will report on the IARC-evaluation of “static and extreme-low frequency
electromagnetic fields” that is presently being performed in conjunction with WHO (EMF project).
45
W2/2
ELECTROMAGNETIC FIELDS AND CARCINOGENESIS: INTERACTION OF
GENOTOXIC AND NON-GENOTOXIC FACTORS
J. Juutilainen
Department of Environmental Sciences, University of Kuopio, Finland
Research on cancer-related biological effects of electromagnetic fields (EMF), including both
extremely low frequency (ELF) magnetic fields and radiofrequency (RF) fields, is discussed in the light
of current understanding of carcinogenesis as a multistep process of accumulating mutations. There is
little experimental or theoretical evidence that cancer could be intiated by direct effects of EMF on
DNA. Therefore, the presentation focuses on possible “non-genotoxic” and cocarcinogenic effects.
Different animal models and study designs have been used to address possible cocarcinogenic
effects. The results of such studies are discussed, as well as other relevant in vivo and in vitro results,
and possible mechanisms of EMF effects on carcinogenesis, and the adequacy of the classical twostep intiation/promotion animal experiments for simulating human exposure to the complex mixture of
environmental carcinogens. Analogies to known “non-genotoxic” and cocarcinogenic agents are
discussed. It is concluded that very little is currently understood of
cocarcinogenesis and “non-
genotoxic” carcinogenesis, and that experiments designed according to the two-step paradigm may
not be sufficient for studying the possible role of MF in carcinogenesis (1).
References
(1) Juutilainen J, Lang S, Rytömaa T (2000) Bioelectromagnetics, 21, 122-128.
46
W2/3
GENOTOXIC EFFECTS OF BOTH ELF AND RADIOFREQUENCY ALONE AND IN
COMBINATION WITH EXPOSURE TO CHEMICALS
MR. Scarfì
CNR-Institute for Electromagnetic Sensing of Environment; Naples, Italy.
Due to widespread use of electromagnetic sources in industry, medicine and every day life, a great
concern exists in biological effects of such non ionising radiation. A great variety of studies both in vivo
and in vitro on different biological systems have been published by evaluating several biological
functions but the results are not yet conclusive. Among the possible biological effects induced by
electromagnetic fields (EMFs), cancer occurrence is one of the most investigated but epidemiological
evidence is not univocal. Regarding the genotoxic potential of EMFs, most of the data available in
literature indicates absence of effects even if some positive findings have been reported. More
recently, following the increasing use of EMFs and the large diffusion of chemical pollution it may be
questionable whether combined exposures to such agents could induce cooperative effects on living
organisms which, in the real life situation, are exposed every day to more than one chemical and/or
physical agents. Moreover it is likely that combined action of different agents is involved in
cancerogenesis and the EMFs are suggested to act as co-carcinogens if given in combination with
genotoxic and/or non genotoxic carcinogens.
The present work deals with more recent results
reported in literature on the in vitro investigation of genotoxic effects following exposure to both
extremely low frequency and radiofrequency, alone and in combination with chemical treatments.
47
W2/4
STATIC AND EXTREMELY LOW FREQUENCY ELECTROMAGNETIC FIELDS:
EVALUATION OF CANCER HAZARDS
R. A. Baan
Unit of Carcinogen Identification and Evaluation
WHO - International Agency for Research on Cancer, Lyon, FRANCE
Exposure to electric and magnetic fields arises from a wide variety of sources that use electrical
energy at various frequencies. The generation, transmission and use of electric power is associated
with the production of weak electric and magnetic fields which oscillate 50 times (Europe) or 60 times
(USA) per second. These frequencies are in the extremely low frequency (ELF) region of the
electromagnetic spectrum (30-300 Hz). At these frequencies man-made fields are orders of magnitude
stronger than the natural fields arising from the Sun and the Earth. Apart from exposure in the vicinity
of overhead power lines, electromagnetic fields in the home arise from the electric wiring system and
from the use of electric appliances such as hair dryers, electric blankets and televisions.
Expert committees in the USA and the UK have recently reviewed the evidence for a possible
association of exposure to extremely low frequency electromagnetic fields with an increased risk for
cancer (National Institute of Environmental Health Sciences, NIEHS, 1998; National Radiological
Protection Board, NRPB, 2001). The conclusions of these reviews will be discussed, and compared
with the outcome of the evaluations of an international Working Group of experts that recently
convened at IARC to discuss this topic (IARC, 2001).
NIEHS (1998) Assessment of health effects from exposure to power-line frequency electric and
magnetic fields. National Institute of Environmental Health Sciences, Research Triangle Park, NC,
USA. NIH Publication No. 98-3981.
NRPB (2001) ELF Electromagnetic Fields and the Risk of Cancer. Documents of the NRPB, Vol. 12,
No. 1. National Radiological Protection Board, Chilton, UK.
IARC (2001) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 80, NonIonizing Radiation, Part 1: Static and extremely low frequency electromagnetic fields, Lyon,
IARCPress, in preparation.
48
Workshop 3
Microarray systems
(parallel session)
W3/1 – W3/4
49
W3/1
TOXICOGENETICS AND TOXICOGENOMICS: PROMISES AND REALIZATIONS.
J.H.M. van Delft
Nutrition and Toxicology Research Institute Maastricht, University of Maastricht, Maastricht, The
Netherlands
As of the mid nineties, DNA-hybridisation based methods have been developed for simultaneous
analysis of 100n to 10,000n sequences on DNA-microarrays or DNA-chips. This enables down-scaling
of the classical hybridisation assays to small sample sizes (few DNA or RNA targets required) and upscaling to large numbers of sequences. Application of microarrays can roughly be divided into two
areas: quantification of targets and mutation detection. When incorporated in toxicology these termed
toxicogenomics and toxicogenetics.
In toxicogenomics most attention goes to multiplex gene expression analysis. Microarrays allow the
detection of mRNA levels for of 100 n to 10,000n genes simultaneously. Questions that are addressed
are: Induce specific classes of toxicants specific gene expression patterns, in vitro and/or in vivo? Can
modulation of gene expression patterns be used for toxicity screening and mode-of-action
identification?
Can
toxicogenomics
improve
interspecies
extrapolation
and
dose-response
relationships and predict long-term effects in short-term assays? Are gene expression patters suitable
biomarkers for biological monitoring studies? Etc. Besides this focus on mRNA, analysis of
chromosomal DNA for amplification or loss of specific genes or chromosome regions is also
introduced. For instance in tumours.
In toxicogenetics, oligonucleotides are used as capture probes for the array-based detection of
mutations. Generally, these oligos are 15- to 25-mers with in their middle a variable base, resulting in
either the wild-type sequence or one of the three possible mutants. When the oligos are designed for
the polymorphic sequences of normal variant alleles, this enables the screening of very many genetic
polymorhisms simultaneously in population-based studies. The on-chip synthesis technology from
Affymetrix providing >100,000 probes per chip, allows to re-sequence an entire gene. Currently this is
available for p53.
During the presentation an overview will be presented on application of microarrays/chips in the area
of toxicology and chemical carcinogenesis.
50
W3/2
Identification of gene expression profiles in different mouse tissues using
cDNA MicroArray.
P. Van Hummelen1; J. Mathys2; K. Marchal2; P. Glenisson2; Y. Moreau2.
1
MicroArray Facility, Flanders Institute of Biotechnology (VIB), Leuven, Belgium;
2
Dept Electrical Engineering (ESAT), KU Leuven, Leuven, Belgium
We have used a high-density cDNA microarray to analyze 8 different mouse tissues for gene
expression profiling. The arrays contained 9,216 cDNA fragments representing approximately 4,000
randomly chosen mouse genes. cDNA was prepared from kidney, heart, liver, lung, skeletal muscle,
spleen, brain and testis and hybridized each time against spleen. Spleen, heart, brain and testis were
repeated and in total 12 slides were analyzed. All experiments showed good fluorescent signals
ranging from 2.5 to 3 orders of magnitude between the lowest and highest spot intensity after
thresholding. The threshold was set at the local background of the spot added up with 2 standard
deviations of the mean spot intensity. On average for any given tissue, 35-40 percent of the clones
present on the array had significant fluorescent intensities.
To assess tissue-specific gene expression a standard concept in data mining, discretization, was
used. Discretization means that, based on predefined thresholds, decisions are made about when a
gene is ON, OFF or differentially expressed.
Using this approach 4 main groups of genes could be defined. Group 1 contained 602 genes that were
ON in every tissue and showed a 2 fold differentially expression in at least one tissue. This group of
genes could be further subdivided using hierarchical cluster analysis into tissue-dependent
differentially expressed genes. Group 2 contained 84 genes that were also ON in every tissue but did
not show any differentially expression. These genes are potential ‘house-keeping’ genes that are
tissue or organ independent. Interestingly, the most commonly used housekeeping genes,
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin or alpha-tubulin are not members of this
group but members from group 1. The third group contained the true tissue specific genes as they
were only found ON in one specific tissue and OFF in all others. Except for heart, for each tissue such
a group ranging from 5 to 63 members could be found. The last group contained genes for which the
spots never had fluorescent signals above threshold (N=629). These genes were not expressed in any
of the tissues or were in such low copy numbers that they were below the detection limit of our
technology.
In a final analysis the tissues were clustered according to the expression profiles of group 1 genes.
The repeat experiments clustered nicely together as expected, but also, heart and skeletal muscle,
and liver and kidney, showed highly comparable profiles, respectively. In summary, using a simple
discretization process, the initially large data set can be subdivided into smaller groups for further
analysis.
51
W3/3
NORMALIZATION AND ERROR ANALYSIS OF DNA MICROARRAY DATA:
AFFECT ON INTERPRETATION OF BIOLOGICAL RESULTS
Roger E. Bumgarner
Department of Microbiology, University of Washington, Seattle Washington, USA
While discussions of error analysis for microarray data are abundant in the literature, very few
publications exist in which error bars are placed on the gene expression measurements. In addition,
there is a plethora of literature on various normalization techniques that can be applied to microarray
data but very little consistency in the approaches used in published experiments. This talk will discuss
the methods we use for both statistical analysis and normalization of microarray data and will compare
these methods to those commonly used in the literature. We will focus on a number of data sets
produced at the University of Washington and discuss the impact of different methods of analysis on
the interpretation of the biological results. In particular, we will discuss the impact of different methods
on the selection of false positives and the subsequent effort that a lab may undergo in following up on
spurious data. We will conclude with recommendations on both methods of data analysis and the
number of replicate experiments one may need to perform in different situations.
52
W3/4
POTENTIAL APPLICATION OF DNA-CHIP TECHNOLOGY (MICROARRAY) IN
TOXICOLOGY
Silvio Albertini and Laura Suter-Dick
Pharmaceutical Division, Non-Clinical Development - Drug Safety, F. Hoffmann-La Roche AG, CH4070 Basel (Switzerland)
Toxicogenomics is a new scientific discipline that combines the emerging technologies of genomics and
bioinformatics to identify and characterise the mechanism of action of known and suspected toxicants based on
gene expression patterns. Currently widely used toxicogenomics tools are DNA microarrays able to monitor the
expression level of thousands of known genes simultaneously and differential gene expression technologies, able
to detect differentially expressed genes independently of their sequences being known or novel. Several research
institutes and pharmaceutical companies are now exploring these technologies in order to better understand and
in the long term possibly predict toxicity of compounds in discovery and/or development (1).
We are currently creating a comprehensive database of transcript and protein expression patterns associated with
known toxins that will greatly enhance our ability to predict safety and to recognise specific toxicological liabilities
of compounds under consideration. Availability of such a database can then be considered in an accelerated
process of compound selection for entry into humans with a higher likelihood of detection of problem areas, thus
increasing the overall success of development.
In a first step we focus on hepatotoxicity and the analysis of the influence of chosen well characterised model
compounds on gene expression (Affymetrix gene chip technology and kinetic PCR) and protein patterns.
Compounds were investigated in vivo (rat) and in vitro (primary rat hepatocytes) evaluating several doses (toxic to
non toxic) as well as different time points (short to long). The obtained results were compared with conventional
endpoints of toxicity (clinical pathology and histopathology). Model compounds known to induce cholestasis,
steatosis or inducing liver damage by a direct mode of action in man were evaluated.
The analysis of gene expression patterns seems suitable to detect possible liver toxins at doses that are not
sufficient to trigger obvious hepatotoxicity detectable by conventional endpoints (clinical pathology and
histopathology). Many of the regulated genes are more or less related to the pharmacological effects of the
compound. Gene expression profiles (fingerprints) characteristic for known hepatotoxicity mechanisms (e.g.
cholestatsis) were identified and are currently used as working hypothesis to test compounds potentially acting via
the same mechanism.
Very promising results using toxicogenomics have been obtained. Nevertheless, these represent just the
beginning and substantially more data need to be collected in order to validate this technology as a tool for
prediction of possible toxicants, especially in man.
Last, but not least, the major issue currently facing the field of toxicogenomics relates to the interpretation of the
obtained data in order to transform the huge amount of information into knowledge.
No doubt, toxicogenomics represents an exciting, new approach and has a great potential to influence positively
the predictability of preclinical safety assessments. Nevertheless, this new approach represents an immense task
and will require considerable amounts of time, manpower, money and effort.
References
Corton JC, Anderson SP, Stauber AJ, Janszen DB, Kimbell JS, Conolly RB: Entering the era of toxicogenomics
with DNA microarrays. CIIT 19 (2): 1-9 (1999).
53
Symposium 6
Regulations update & genotoxicity screening
S6/1 – S6/4
54
S6/1
GENOTOXICITY ASSESSMENT IN DRUG DISCOVERY AND EARLY
DEVELOPMENT
L. Müller
NOVARTIS PHARMA AG, Toxicology/Pathology, Basel, Switzerland
55
S6/2
UK COM GUIDANCE ON A STRATEGY FOR TESTING CHEMICALS FOR
MUTAGENICITY
David J Tweats
GlaxoSmithKline, Ware,Herts, UK.
New technical knowledge and scientific understanding in the field of genetic toxicity has advanced
rapidly in the last decade. In the light of this, the UK Committee on Mutagenicity, which is an advisory
body for UK Government Departments, revised and re-issued its guidance document in December
2000, the previous issue was in 1989. The main changes in the document include the need to screen
for aneugenicity in what it terms Stage 1 testing and the development of tests in addition to the rat liver
UDS assay for in vivo genotoxicity testing (Stage 2) when there is a need to investigate tissues other
than the bone marrow. The COM decided that there is sufficient evidence to recommend the use of
the in vitro micronucleus test, despite the fact that there is no internationally accepted protocol, in view
of its versatility as a screen for both clastogenicity and aneugenicity. The guidance document gives
some information on what would be regarded as an acceptable protocol. The provision of more
options for in vivo screening takes in to account the development of in vivo assays for DNA damage
(e.g. the COMET assay) and mutations (e.g. rodent transgenic assays) since 1989. Although the first
choice in vivo assays continues to be the rodent bone marrow assays for chromosome damage, the
new assays are better placed for detecting for example, site of contact effects.
As the role of COM is advisory, the guidance document has no formal regulatory status. In addition its
remit is to advise on all chemicals , not just a subset of compounds e.g. pharmaceuticals. The
Committee recognises that it will be some time before the strategy outlined will be reflected in
mandatory guidelines of the various agencies, but it is hoped that the recommendations made will
prompt an update of internationally harmonised guidelines in this field.
56
S6/3
PROGRESS
TOWARDS
HARMONISATION
IN
GENOTOXICITY
TESTING
THROUGH THE INTERNATIONAL WORKSHOPS (IWGT)
D. Kirkland
Covance Laboratories Ltd, Otley Road, Harrogate, UK
Two international workshops aiming to harmonise procedures for a range of commonly used and new
genotoxicity tests have been held (Melbourne 1993, Washington 1999).
The output from these
workshops has influenced ICH and OECD recommendations, and helped to speed up agreement in
these initiatives.
The International Workshops on Genotoxicity Testing (IWGT) are now being
incorporated into a Foundation of the IAEMS that will focus on a variety of applied activities. As a
result, regular IWGT workshops are anticipated in the future.
A third workshop will take place as a satellite of the Shizuoka ICEM in October 2001. Test procedure
issues still to be resolved (left over from Washington) are:
Mouse lymphoma assay – measures and levels of cytotoxicity, use of conditioned medium, cleansing
of cells, statistics, use of microwell vs agar techniques.
In vitro micronucleus assay – use of cytochalasin B, primary cells vs established cell lines,
measures and levels of cytotoxicity, discrimination of aneugens and clastogens, equivalence of
different cell types.
Transgenic genotoxicity models – treatment schedule, sampling times, size of experiment, data
evaluation.
New working groups will address topics not so readily associated with procedures for genotoxicity
tests:
P53 and Hras2 transgenic tumour models – genetic/molecular characterisation required in animals,
tissues and tumours before and after treatment.
Strategy and classification – development of an IARC-like classification system for genotoxins, a
decision-tree approach to testing and classification, definition of risk categories.
It is hoped that the success of the previous workshops will be repeated here, and that constructive,
useful recommendations will be agreed by the >50 invited experts.
57
Symposium 7
Polymorphism in risk assessment and therapy
S7/1 – S7/4
58
S7/1
POLYMORPHISM OF DRUG METABOLISING ENZYMES AND XENOBIOTIC
TOXICITY
Magnus Ingelman-Sundberg
Division of Molecular Toxicology, IMM, Karolinska Institutet, SE 171 77 Stockholm, Sweden.
Xenobiotic metabolism is carried out to a great extent by polymorphic phase I and phase II enzymes.
The majority of human xenobiotic metabolism is catalysed by polymorphic and inducible enzymes,
which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. Stable
duplication, multiduplication or amplification of active genes has been described. In mice models it is
apparent that inactivation of specific enzymes active in xenobiotic metabolism can affect the risk for
cancer development in relation to specific xenobiotic exposure, whereas the situation in humans is by
far much more complex. The polymorphism of CYP enzymes is expected to influence the individual
sensitivity and toxicity for different environmental agents, although there is yet no real consensus in
the literature about specific firm relationships in this regard. The incidence of serious and fatal adverse
drug reactions (ADRs) has been found to be very high among hospitalised patients, and ADRs cost
the society a lot and is responsible for 5-10 % of all hospital admissions. It is likely that predictive
genotyping could avoid 10-20 % of the ADRs. In the present lecture an overview is presented
regarding our present knowledge about the polymorphism of drug metabolising enzymes with
emphasis on xenobiotic metabolising CYPs and the importance for metabolic activation of xenobiotics.
59
S7/2
GENETIC
POLYMORPHISM
IN
GLUTATHIONE
S-TRANSFERASE
AND
RESPONSE TO XENOBIOTICA
H.Autrup
Department of Environmental Medicine, University of Aarhus, Denmark
Glutathione-S-transferases (GST) are a group of enzymes involved in the detoxification of many
environmental toxicants and in the defense against oxidative stress. The effect of GST activity can
either be direct i.e. detoxification of the active species, or indirect i.e., modifying the level of
compounds in the diet with anti-carcinogenic properties.
The homocygote deletion of the GSTM1 has been linked to a slight increased risk of e.g. lung and
bladder cancer. It has been estimated that the lack of GSTM1 activity independent of smoking status
is accounting for approx. 20% of lung cancer cases.
It is assumed, that the lack of the enzyme activity is especially relevant at low exposure situations,
such as exposure to passive smoke and air pollution. The results on the interaction of smoking and
GSTM1 genotype are conflicting. In general, a slightly lower risk for lung cancer in people with
GSTM1*0/0 was observed at lower dose (pack-year of exposure), whereas the null genotype was
associated with an increased risk for larynx cancer (g tobacco/day) and gastric adenocarcinoma
(pack-year of exposure), but not for the higher doses. Increased level of biomarkers, i.e. carcinogenDNA adducts and rate of p53 mutations has been reported in people deficient of GSTM1 activity.
An interaction between the GSTM1 genotype and the dose of antracycline on survival of patients with
acute myeloid leukemia has also been reported, the rate of survival in the low dose group being higher
in patients with GSTM1*0/0.
In addition to gene-environment interaction, it is also important to study gene-gene interaction when
assessing risk. An additive risk of GSTM1*0/0 and GSTT1*0/0 has been reported for the same types
of cancers, making it even more complex to study the effect of metabolic genotype at low dose
exposure situations.
60
S7/3
INTERINDIVIDUAL VARIABILITY IN RESPONSE TO RADIATION EXPOSURE:
ANALYSIS OF DNA REPAIR IN CANCER PATIENTS.
C. Alapetite
Département de Radiothérapie Oncologique - Section Médicale and UMR 218 - Section Recherche;
Institut Curie, Paris, France
Exposure to therapeutic doses of ionising radiation reveals an inter individual heterogeneity of normal
tissue sensitivity in cancer patients. Profiling the individual risk before planning treatment might avoid
the emergence of complications in the subgroup of hypersensitive patients. Association of cancer
predisposition and hypersensitivity to radiation is well established in some rare genetic syndromes
such as ataxia telangiectasia and Nijmegen breakage syndrome whose gene products (Atm, Nbs1)
are involved in maintenance of the genomic integrity .Other actors of the DNA repair networks have
recently been related to clinical hypersensitivity (DNA ligase IV, hMRE11). This raises the question of
the possible role of alterations and polymorphisms of such genes in the observed sporadic cases of
hyper-radiosensitivity.
In order to examine the hypothesis of DNA repair as a biological marker of the individual risk
associated to radiation we documented, using the comet assay, the response to in vitro irradiation of
cancer patients cells with different hypersensitivity. This assay, in alkaline conditions, is applicable to
lymphocytes and offers the practical advantages of a predictive test. It measures discontinuities in
relation to single, double-strand breaks and alkali-labile sites induced by radiation either directly or
indirectly through enzymatic processing of the DNA lesions. This assay detects the defects in
nucleotide excision repair and base excision repair, but defects in fidelity of DNA double strand break
repair are not recognised. Both among patients having developed secondary thyroid tumours after
radiotherapy at a young age and breast cancer patients with severe early or late effects, a subgroup of
patients demonstrated elevated residual DNA strand breaks. In the last subset of patients such
uncomplete repair was correlated with the most severe degree of adverse reactions to radiotherapy.
These results favour the hypothesis of a contribution of DNA repair impairment in aggravating the
prognosis of adverse response of normal tissue to radiation exposure and encourages to further
explore the individual DNA repair competency before applying treatments based on genotoxic agents.
61
S7/4
IMPACT OF POLYMORPHISMS IN PHASE I OR PHASE II ENZYMES ON
LYMPHOCYTE DNA ADDUCTS IN POPULATIONS SUFFERING MEDIUM TO
LOW EXPOSURE TO PAH.
P. Georgiadis1 ; J. Topinka2, M. Stoikidou3; S. Kaila1; M. Gioka3, K. Katsouyianni3, R. Sram2; H.
Autrup4 ; A. Kyrtopoulos1
1National
Hellenic Research Foundation, Athens, Greece
2Laboratory
of Genetic Ecotoxicology, Institute of Experimental Medicine Acad. Sci. C.R. and Regional
Institute of Hygiene of Central Bohemia, Prague, Czech Republic
3Laboratory
of Hygiene and Epidemiology, University of Athens Medical School
4Department
of Environmental Medicine, Univ. of Aarhus, Denmark
The levels of bulky DNA adducts were measured by
32P-postlabelling
(nuclease P1 enrichment) in
lymphocytes of 194 non-smoking technical institute students living in the city of Athens, and the rural
region of Halkida, Greece, in whom personal exposure to PAH was also measured. Genotype analysis
of various polymorphic genes which encode biotransforming enzymes involved in the activation (phase
I) or detoxification (phase II) of xenobiotics was performed and the effects on adduct levels was
assessed. Genetic polymorphisms were examined in cytochromes P450 1A1, 1B1 and 2E1, in the
GSTM1, GSTP1 and GSTT1 as well as in the NAT2, the NQO1, and microsomal epoxy hydrolase
(mEH) genes.
Higher DNA adduct levels were observed in individuals carrying the CYP1A1 Ile/Val genotype
compared to CYP1A1 Ile/Ile carriers (p=0.033). mEH wild type homozygotes (Arg/Arg in exon 4)
were shown to have elevated adduct levels compared to the mutant homozygotes (His/His), while
heterozygotes had intermediate levels (p=0.006 for linear trend). In the subgroup of ETS exposed
individuals the effect of mEH polymorphism was more potent (p=0.001 for linear trend). Higher adduct
levels were also found in carriers of the combined CYP1A1 (Ile/Val)/GSTM1 (null) or CYP1A1
(Ile/Val)/mEH (Arg/Arg) genotype compared to CYP1A1 (Ile/Ile)/GSTM1 (wild type) or CYP1A1
(Ile/Ile)/mEH (His/His) genotype carriers, respectively. Carriers of all the other genotype combinations
had intermediate DNA adduct levels (p=0.026 and p=0.001 for linear trends respectively). The effect
of the combined CYP1A1/mEH genotype was found to be significant during both seasons (winter –
summer), both years (1997-1998) and in both locations of the study.
These results suggest that CYP1A1 and mEH variants might have an impact on the DNA adduct
levels in environmentally exposed populations.
Acknowledgements: This work was supported by an EU grant (contract no. ENV4V-96-0203)
62
Symposium 8
Genotoxicology of metals
S8/1 – S8/4
63
S8/1
METAL- AND REDOX- REGULATION OF P53 PROTEIN FUNCTIONS.
P.Hainaut
Group of Molecular Carcinogenesis, International Agency for Research on Cancer, WHO, Lyon,
France
The p53 protein is a tumor suppressor often inactivated in cancer, which is induced in response to
many forms of cellular stress, genotoxic or not, and controls cell proliferation and survival through
several coordinated pathways. The p53 protein is a zinc-binding protein containing several reactive
cysteines, and its key biochemical property, sequence-specific DNA binding, is dependent upon metaland redox-regulation in vitro. Down-regualtion of zinc levels using metal chelators in intact cells
inactivated p53 by altering protein conformation. After chelation, folding back into active conformation
requires addition of zinc into the culture medium. Control of p53 folding involves at least two important
metal/redox regulators, metallothioneins and thioredoxin. Moreover, p53 acts as a transactivator or
transrepressor of several genes involved in the production and control of reactive oxygen
intermediates (ROI). Overall, these data indicate that p53 lies at the center of a network of complex
redox interactions. In this network, p53 can control the timely production of ROI (e.g. to initiate
apoptosis) but this activity is itself under the control of changes in metal levels and in cellular redox
status. This redox sensitivity may be one of the biochemical mechanisms by which p53 acts as a
“sensor” of multiple forms of stress.
Perturbation of the metal/redox equilibrium that controls p53 may lead to its inactivation. There is
evidence that agents such as Nitric Oxide or Cadmium directly affect the conformation of the protein,
turning wild-type p53 into a form functionally identical to mutant p53. This mechanism may contribute
to inactivation of p53 in cancer. In contrast, we have shown that the redox-active drug amifostine can
induce changes in p53 protein conformation and activate p53. These observations provide support to
the notion that it may be possible to design drugs that specifically regulate p53 function by acting on
the redox-sensitivity of p53 protein conformation.
64
S8/2
DETERMINANTS OF THE GENOTOXICITY OF METALLIC COMPOUNDS.
D. Lison
Unit of Industrial Toxicology and Occupational Medicine, Catholic University of Louvain, Brussels,
Belgium.
Excessive occupational or environmental exposure to several metallic and metalloid compounds is
associated with an increased risk of cancer. For most of these compounds, direct or indirect genotoxic
properties have been reported to contribute to the carcinogenic potential but the exact mechanisms
remain, however, incompletely understood.
The objective of this presentation will be to highlight some essential physico-chemical and biological
determinants of the genotoxicity of the metallic compounds.
Questions that will be addressed include :
Are the genotoxic effects of metallic compounds
necessarily mediated by the ionic form ? How do we express the dose of a(n in)soluble metallic
compound ? How do we take into account the importance of speciation and oxidation state ? Is there
evidence of interaction of metallic compounds with other elements or agents that elicit new genotoxic
properties ? Is it still acceptable in 2001 to generically classify metals for their genotoxic potential?
How do we need to integrate our knowledge of species differences in bioavailability or activity ?
The examples given during the presentation will illustrate the necessity of a better consideration of
these determinants that will undoubtedly contribute to improve our understanding of the mode of
action of the metallic compounds and refine our assessment of their toxicity.
65
S8/3
CARCINOGENIC METAL COMPOUNDS: INTERFERENCE WITH DNA REPAIR
PROCESSES AND CELL CYCLE CONTROL
A. Hartwig1, M. Asmuss1, A. Buerkle2, I. Ehleben1, D. Kostelac1, A. Pelzer1, T. Schwerdtle1
1)Institute
2)Dept.
of Food Chemistry and Toxicology, University of Karlsruhe, Karlsruhe, Germany;
of Gerontology, University of Newcastle, Newcastle upon Tyne, UK
Nickel, cadmium, cobalt and arsenic compounds are well known carcinogens to humans and
experimental animals. Even though their DNA damaging potentials are rather weak, they interfere with
different DNA repair systems at low, non-cytotoxic concentrations. Thus, Ni(II) and Cd(II) impair the
removal of oxidative DNA lesions by base excision repair. Furthermore, Ni(II), Cd(II), Co(II) and As(III)
inhibit nucleotide excision repair involved in the elimination of bulky DNA adducts induced by
environmental mutagens. For example, both water soluble Ni(II) and particulate black NiO greatly
reduce the repair of DNA adducts induced by benzo[a]pyrene, an important environmental pollutant.
As potential mechanism we observed an inactivation of different zinc finger proteins involved in DNA
repair, namely the bacterial formamidopyrimidine-DNA glycosylase (Fpg protein), the mammalian XPA
protein and the poly(ADP-ribose)polymerase (PARP). While all proteins were inhibited by Cd(II) and
Cu(II), XPA and PARP but not Fpg were inhibited by Co(II) and Ni(II); As(III) inactivated only PARP,
but did so at very low concentrations. Besides DNA repair processes, one other important protective
event after DNA damage is cell cycle control, and the effects of Ni(II), As(III) and Co(II) on cell cycle
progression, also in response to UVC radiation, were investigated. Both Ni(II) and Co(II) alone induced
an arrest in the G1 phase of the cell cycle, while As(III) provoked an G 2/M arrest. After UV-irradiation,
we observed an S phase arrest, which was diminished in the presence of all three metals. Thus, metal
ions interfere with DNA repair processes as well as with cell cycle control mechanisms. In case of
DNA repair enzymes, zinc finger structures may represent sensitive targets for some toxic metals; the
molecular mechanisms leading to altered cell cycle progression following DNA damage are currently
investigated. Since DNA is permanently damaged by endogenous and environmental factors,
functioning processing of DNA lesions is an important prerequisite for maintaining genomic integrity;
its inactivation by metal compounds may therefore constitute an important
mechanism of metal-
related carcinogenicity.
66
S8/4
ELUCIDATION OF THE BIOCHEMICAL PATHWAYS INVOLVED IN THE
MUTAGENICITY, ASSOCIATED WITH ISOLATED PARTICULATE DEBRIS FROM
THE PERI-PROSTHETIC TISSUE OF FAILED PROSTHESIS.
S.Clerkin, C.P.Case.
Bristol Implant Research Centre, Dept. of orthopaedic surgery, Southmead Hosp., BS10 5NB,
England.
Total hip arthroplasty is the second most performed operation within the United Kingdom. However,
some 20% of prostheses will fail within 20 years. Failed prostheses generate wear debris, which is
systemically disseminated within the human body (Case,C.P. et al). Little is known about the
physiological effect of exposure to these orthopaedic debris. This is becoming increasingly important
to understand, with regard to changes in orthopaedic practices, in that more young people are
receiving joint replacements, increasing the time of exposure to this particulate debris.
Previous studies have shown an increase in genotoxicity in peripheral lymphocytes from revision
patients. It was also shown that there was a much higher percentage of aneuploidy in the peripheral
lymphocytes of those patients with a failed titanium vanadium prosthesis, while those with a cobalt
chrome prosthesis were more susceptible to chromosomal aberrations (A. Doherty et al. in press).
In vitro, cultured amniocytes where exposed to particulate metal debris extracted from the periprosthetic tissue of both failed titanium vanadium alloy hips
and cobalt chrome alloy hips. The
mutagenicity was then either potentiated or attenuated using antioxidants, metal chelators and antiapoptotic agents, and measured using the micronucleus assay. Preliminary data point to both the
antioxidants and the metal chelators giving a reduction in the frequency of micronuclei, while the antiapoptotic agent demonstrated an induction.
Further experiments using a DNA microarray were used to determine the differential gene expression
pattern of the amniocytes when exposed to either the titanium vanadium particulate debris, or the
cobalt chrome particulate debris. Further work is currently been planned using other techniques,
including northern blotting and fluorimetry, to further elucidate and confirm the biochemical pathways
involved in the differential mutagenicity of these particles.
A.T. Doherty, B. Lewis, R.T.Howell, G. Langkamer, C.P.Case. (2001) JBJS (in press)
C.P.Case, D. Path, V.C.Langkamer, at al. (1996) CORR, no329S, S269-S279.
67
Symposium 9
Chromosomal sensitivity towards genotoxic agents
S9/1 – S9/5
68
S9/1
69
S9/2
CHROMOSOMAL ABNORMALITIES IN MITOMYCIN C-SENSITIVE CHINESE
HAMSTER CELLS DEFECTIVE IN THE Brca2 AND Rad51C GENES
P.P.W. van Buul; A. van Duijn-Goedhart; M. Kraakman-van der Zwet; B.C. Godthelp; M.Z. Zdzienicka
Dept. Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden, The
Netherlands
Recently, it has been shown that genes involved in the formation of the Rad51 protein complex play
essential roles in repair of DNA double-strand breaks (DSB) via homologous recombination (HR). We
identified two Chinese hamster cell mutants, V-C8 and CL-V4B, both hypersensitive for cell killing by
mitomycin C (MMC), which are deficient in Rad51 foci formation in response to DNA damage. Lately,
we found that V-C8 and CL-V4B are defective in Brca2 and Rad51C, respectively (data not published).
To investigate effects of these genes on the chromosomal level, we studied structural chromosomal
aberrations (CA) and sister chromatid exchanges (SCE’s) in these mutants. In both cell lines the
frequencies of spontaneous CA were greatly enhanced, in V-C8 about 20 times and in CL-V4B 6
times, above control level. Aberrations were predominantly of the chromatid-type indicating their
formation during DNA replication. The frequency of MMC-induced aberrations were also extremely
high, they were about 600 times and 300 times higher in V-C8 and CL-V4B, respectively, when
compared to that observed in wild type cells. MMC-induced CA were as well, nearly all chromatid-type.
Since it is well established that DNA DSB form the primary lesion leading to structural chromosomal
aberrations, our results suggest that repair of MMC-induced DNA damage results in DNA doublestrand breaks, that are normally processed via Brca2 and Rad51C mediated HR. Analysis of SCE’s in
the V-C8 and CL-V4B mutants showed slightly decreased spontaneous levels in both, but more
importantly no induction after MMC treatment was found. Thus, it seems that repair of MMC induced
DNA damage in normal cells results in Brca2 and Rad51C dependent SCE formation. These results
indicate that both genes are of great importance for the repair of spontaneous and MMC-induced
chromosome breaks. They demonstrate the significance of HR for maintaining the integrity of the
genome by stabilising chromosome structure, in this way asserting an essential role of these genes in
preventing tumour formation.
70
S9/3
HOW
RELIABLE
ARE
CHROMOSOMAL
ABERRATION
ASSAYS
AS
BIOMARKERS OF INDIVIDUAL SENSITIVITY TOWARDS IONISING RADIATION?
A. Vral*, H. Thierens°, A. Baeyens* and L. De Ridder*
Department of Anatomy, Embryology, Histology* and Medical Physics°, University of Gent,
L. Pasteurlaan 2* and Proeftuinstraat 86°, 9000 Gent, Belgium.
E-mail: [email protected]
Abstract: Biomarkers of susceptibility or sensitivity towards ionising radiation can be important for the
identification of individuals that may be at increased risk for the development of cancer after
occupational, environmental or medical exposures. It is essential that these biomarkers have certain
traits in order to be effective indicators of sensitivity. They should be specific, sensitive and reliable.
Possible candidates for biomarkers of radiosensitivity are chromosomal aberrations. It has been
shown that the induction of chromatid aberrations after irradiation of lymphocytes in G2 phase of the
cell cycle and the induction of MN after irradiation in Go both allow discrimination between normal
individuals and patients with cancer prone genetic diseases.
In this study we investigated the inter- and intra- individual variation of the MN assay and the G2 assay
in irradiated lymphocytes to assess their suitability as biomarkers of susceptibility. For this, the G2
assay and the MN assay were performed on blood samples of 10 healthy individuals. For the MN
assay Go lymphocytes were exposed to 3.5 Gy Co -rays either at high dose-rate (HDR) and
stimulated immediately or with 6h delay (DS) or at low dose-rate (LDR). For the G2 assay lymphocytes
were irradiated with a dose of 0.4 Gy Co -rays in G2 phase of the cell cycle. Two individuals were
assayed 9 times each in nine different experiments over a time period of 1 year. All samples were
analysed by 2 scorers and no significant differences between them were observed using a paired ttest. The repeat experiments on blood samples of the same donor revealed that the inter-experimental
/ intra-individual coefficients of variation were not significantly different from the inter-individual
coefficients of variation in both G2 and MN assay. As the intra-individual variability determines the
assay reproducibility this would indicate that the assays are not able to detect real, reproducible
differences in radiation sensitivity between normal individuals in the population. The repeat
experiments further revealed that for some healthy donors a high value, defined as sensitive taking the
90th percentile as cut-off point to define sensitivity, is obtained only at one time point while the values
obtained at the other time points were within the normal range (non-sensitive). Based on this one time
point the individual would have been regarded as sensitive. To conclude, our results show that a
chromosomal aberration assay based on one blood sample may lead to erroneous conclusions with
respect to the individual radiosensitivity of workers.
71
S9/4
DISAPPEARENCE OF CHROMOSOMAL ABERRATIONS FROM THE BLOOD
CIRCULATION DEPENDS ON THE LOCATION OF IRRADIATED LYMPH NODES
S. Gundy, G. Székely, Zs. Kelecsényi, O. Ésik
National Institute of Oncology, Budapest, Hungary
Peripheral blood lymphocytes (PBLs) are extensively used objects of human genotoxicology studies
in the measurement of genetic damage in such forms as chromosomal aberrations (CAs), SCEs,
micronuclei etc. Following irradiation CAs disappear from the blood circulation gradually, however,
the time-course of the elimination is still not clear. Dicentric- and ring (dic+ring) aberrations have
been used so far to estimate the life-span of PBLs, and approximately 700-1500 days were
considered as the time of disappearence of 50% of dic+ ring aberrations from the peripheral blood.
The main objective of our 7-year-follow-up study was to investigate the life-span of human
lymphocytes in patients who had received radiation therapy in 5 different volumes and locations of
the body. CAs were studied during and immediately after the termination of treatments in thyroid
cancer patients irradiated either with external doses of 50 Gy (25x2Gy) in two different volumes of
the neck area (parajugular lymph nodes - PLN- and PLN+upper mediastinum -UM- in a ratio of
volumes of 1:3 ), or with radioisotope
131-I
following total thyroidectomy (whole body radiation load).
Furthermore, testicular cancer patients were examined who were irradiated with either 26 Gy
(13x2Gy) or with 41 Gy (27x1.5 Gy) in two fields of pelvic (PAO-PIL) region in volumes exceeding
the PLN volumes 10 and 15 times. The yield of dicentrics/cell showed 3-fold individual variability in
each group regardless on doses, volumes and locations of lymph nodes. During the courses of
radiotherapy at fixed doses and volumes the variability of parameters of linear dose-effect curves
also differed among donors. The pelvic area volumes (1:10 and 1:15) and locations did not, but the
neck area volumes (1:1 and 1:3) did influence the yields and elimination rate of dic+rings regardless
the size of these volumes which were much smaller than those in pelvic region. The highest rate of
CAs was found when UM was included as an irradiated field, and the lowest rate of CAs occurred
when radioiodine isotope was internally used.
The cytogenetic follow-up data up to 7 years after the termination of treatment show that the average
half-time of lymphocytes depends on the location of lymph nodes, but in general it is much shorter
(1 year) than the usually accepted value of 2-4 years reported in the literature. The rate of the
disappearence of dic+rings from the peripheral blood is 88-95% by the end of 7th year after the
termination of the treatment.
This work was supported by grant US-Hungarian Joint Fund No. 391
72
S9/5
ARA A ENHANCEMENT OF CHROMATID BREAKS IN IRRADIATED CHO
CELLS: LACK OF CORRELATION WITH DSB REJOINING
P. E. Bryant and C. Finnegan*,
School of Biology, University of St Andrews, St Andrews KY16 9TS, Scotland .
*Present address: School of Biomedical Sciences, University of Ulster, at Jordanstown, Newtonabbey,
Co Antrim, BT37 0QB, UK.
The purpose of our experiments was to examine the relationship between rejoining of DNA double-strand breaks
(dsb) and kinetics of chromatid breaks (cb) in the same cell systems, following treatment with araA (9- -Darabinofuranosyladenine). Cb in rodent and human G2 cells disappear with time following irradiation, with
apparently exponential (first-order) kinetics (1-4) and evidence that this process represents a type of rejoining
mechanism, and not simply
a change in chromosomal radiosensitivity during the G2 phase, comes from
experiments with nucleoside analogues such as araA or araC (1- -D-arabinofuranosyl-cytosine). These
analogues are powerful inhibitors of semi-conservative DNA replication (5) and in murine Ehrlich ascites tumour
cells they act as inhibitors of dsb rejoining (6,7). AraA and araC block the disappearance of chromatid breaks and
in the case of araC an increase in frequency of cb with time after irradiation is observed (8,9). We therefore
decided to re-examine the effects of one of these inhibitors (araA) on the dsb rejoining and cb kinetics in the same
three cell systems (Chinese hamster ovary CHOK1 cells, a normal murine CB17 cell line and murine SCID cells).
For cb assays, exponentially growing cultures of CHOK1, CB17 and SCID cells were treated after irradiation with
araA at 100 M (and without, as controls). Chromosome spreads prepared and stained by standard procedures.
Dsb were measured using constant-field gel electrophoresis.
nes the
frequency of cb remained constant in irradiated and araA treated cells, while in controls the cb disappeared with
be found on dsb rejoining in any of the cell lines tested.
We conclude that the lack of correspondence between dsb rejoining and cb kinetics during araA treatment argues
strongly against a “breakage-first” interpretation for chromatid breaks, and is important evidence for the
dissociation of initiating dsb from the process of formation of a chromatid break. The presence of colour-switches
at chromatid breakage sites in harlequin-stained cells suggests that the process of cb formation involves rearrangements at crossover points of chromatin loop domains either within or between chromatids.
1. Mozdarani, H., and Bryant, P.E. (1987) Mutagenesis 2, 371-374.
2. Mozdarani, H., and Bryant, P.E., (1989) Int. J. Radiat. Biol., 55, 71-84.
3. Macleod, R.A.F., and Bryant, P.E. (1992) Mutagenesis, 7, 285-290.
4. Bryant, P.E. and Slijpecevic, P. (1993) Env. and Mol. Mutagenesis, 22, 250-256.
5. Furth, J.J. and Cohen, S.S. (1968) Cancer Research, 28, 2061-2067.
6. Bryant, P.E., and Blöcher, D. (1982) Int. J. Radiat. Biol., 42, 385-394.
7. Iliakis, G., and Bryant, P.E. (1983) Anticancer Res., 3, 143-150.
8. Preston, R.J. (1980) Mutat. Res., 69, 71-79.
9. Mozdarani, H. and Bryant, P.E. (1989) Mutat. Res. Letters, 226, 223-228.
73
Poster session 1
Major topics:
Genetic susceptibility,
DNA repair,
Ecogenotoxicology
P1/1 – P1/34
74
P1/1
DIFFERENCES IN MECHANISMS OF APOPTOSIS IN THE HL60 CELL LINE AND
SYRIAN HAMSTER EMBRYO (SHE) CELLS.
Alexandre S., Rast C. and Vasseur P.
EBSE, University of Metz, France.
In a parallel study on the HL60 cell line and primary Syrian hamster embryo (SHE) cells, we have
demonstrated that apoptosis may proceed through mechanisms varying according to cell type or
apoptosis inducer. In addition, markers which are generally considered to be hallmarks of apoptosis
may fail to appear in some cell types. Indeed, topoisomerase inhibitors, which were shown to be
potent apoptosis inducers in the HL60 cell line, induced only a weak apoptotic response in SHE cells.
On the other hand, a serum-free medium, that rapidly induced apoptosis in SHE cells, did not affect
the HL60 cell line.
In both cell types, apoptosis was expressed by condensed chromatin, fragmented nuclei and DNA
laddering on electrophoretic gels. In apoptotic HL60 cells, the cleavage of 113-kDa poly(ADPribose)polymerase (PARP) resulted in the "so-called" apoptotic 89-kDa fragment and was associated
with increased caspase-3 activity. In apoptotic SHE cells, PARP degraded early but the degradation
profile was not characterized by the appearance of a stable 89-kDa fragment. Moreover, no activation
of caspase-3 was noted. Apoptosis induced by serum deprivation was linked with c-myc negative
regulation in SHE cells without p53 protein accumulation, while topoisomerase inhibitors led to p53
stabilization without any change in c-myc expression. Serum-free medium and topoisomerase
inhibitors did not modify c-myc expression in HL60.
75
P1/2
DEVELOPMENTAL ABNORMALITIES INDUCED BY X-IRRADIATION IN P53
DEFICIENT OR HETEROZYGOUS MICE.
S. Baatout; P. Jacquet; A. Michaux; J. Buset; W. Schoonjans; J. Yan; A. Benotmane; L. de SaintGeorges; C. Desaintes; M. Mergeay
Laboratory of Radiobiology, Belgian Nuclear Research Center, SCK/CEN, Mol, Belgium
In order to assess the influence of a p53 mutation on radiation-induced developmental effects, males
heterozygous for the p53 mutation (mimicking the human Li-Fraumeni syndrome) were crossed with
C57BL females. Their heterozygous p53+/- progeny was mated with each other, in order to obtain
p53+/- (50%), p53-/- (25 %) and p53+/+ (25 %) embryos. Pregnant females were X-irradiated with 0.5 Gy
on days 1 (pre-implantation period), 8 or 11 (organogenesis period) of gestation. Dissection of the
pregnant females occurred on day 19 of gestation. P53 genotype was determined by PCR from small
pieces of soft foetal tissues. In non-irradiated animals, slightly less p53-/- foetuses were found upon
dissection than expected, probably reflecting a predominant elimination of these embryos during
gestation. Exencephaly was the only external malformation found in foetuses from non-irradiated
females, affecting as much as 5 of the 91 living foetuses of this series. Four of those were p53 -/- and
one was p53+/-. In animals irradiated on day 1 of pregnancy, prenatal mortality was increased,
predominantly affecting the p53-/- embryos. Among the 100 living foetuses obtained in this series, 2
showed exencephaly, both of them being p53-/-. This lower frequency of malformed foetuses
compared to non-irradiated animals could be due to an increased elimination of p53-/- foetuses or
embryos after irradiation. The proportion of living p53-/- foetuses that were obtained after irradiation on
day 8 was also lower than expected, and elimination preferentially affected female foetuses.
Malformations were twice as frequent as in the non-irradiated group, and predominantly affected
female foetuses (73%). Abnormal foetuses were either p53 -/- (6/94) or p53+/- (5/94). Interestingly, in
addition to exencephaly, other various external malformations were found in this group, including
cephalic oedema, gastroschisis, polydactyly and cleft palate. In foetuses irradiated on day 11 of their
development, preferential elimination of p53-/- female foetuses was observed. To date, the
malformations found were exencephaly (1/51), gastroschisis (1/51), polydactyly (2/51) and tail
anomaly (1/51). These malformations affected only p53-/- foetuses. Overall, these results point to the
importance of the p53 tumour-suppressor protein for normal development. They strongly suggest that
homozygous p53-/- (or heterozygous p53+/- at a lesser extent) foetuses may be more at risk for
radiation-induction of external malformations during the organogenesis period, but might be
preferentially eliminated by irradiation at the preimplantation stage.
76
P1/3
77
P1/4
THE P53 CODON 72 SNP AND LUNG CANCER
E. Biros1; I. Biros2; A. Kohut3; I. Kalina3; E. Bogyiova; J. Stubna
1Institute
of Experimental Medicine, Acad. Sci. CR, Prague, Czech Republic;
2Department
of Physiology and Pharmacology, AEGRC, The University of Queensland, Brisbane,
QLD 4072, Australia;
3Department
of Medical Biology and Department of Pharmacology, School of Medicine, P.J. Šafárik
University, Košice, Slovak Republic
The tumor suppressor gene p53 encodes nuclear protein that acts as a transcription factor.
Normally, p53 controls passage of the cells from G1 into S phase of the cell cycle. It is estimated that
a wide range of cancers are associated with somatic mutations of p53, including approximately 50% of
non-small cell lung cancer (NSCLC) and 80% of small cell lung cancer (SCLC) cases.
Wild-type p53 tumor suppressor gene exhibits several common single nucleotide
polymorphisms (SNP) both in coding and non-coding regions. We tested the codon 72 single
nucleotide polymorphism (SNP) of the p53 gene for an association with lung cancer. This polymorphic
site within the p53 gene represents an amino acid replacement of Pro (CCC, A1 allele) by Arg (CGC,
A2 allele), though the functional difference between A1 and A2 allele is still unclear.
In our hospital-based case-control study, 168 lung cancer patients (134 males and 34
females) and 148 controls without malignant diseases were recruited. The genotype characteristics
were determined by PCR-based RFLP method using DNA extracted from peripheral blood. We found
only in lung cancer patients but not in the controls both a significant decrease of A1 allele of the p53
codon 72 (P=0.024, OR 0.56, 95% CI 0.43-0.72) and A1/A1 homozygous genotype (P=0.006, OR
0.27, 95% CI 0.15-0.51). The results of this study suggest a protective effect of A1 allele against lung
cancer.
Supported by the grant of the Slovak Ministry of Health (contract KLV-44/97) and by EC / RD-G /
ICA1-CT-200070028.
78
P1/5
ASSESSMENT OF CHEMOTHERAPY-INDUCED DNA DAMAGE IN PERIPHERAL
BLOOD LEUKOCYTES OF CANCER PATIENTS USING THE ALKALINE COMET
ASSAY
N. Kopjar1; V. Garaj-Vrhovac1; I. Milas2
1 Laboratory
2
of Mutagenesis, Institute for Medical Research and Occupational Health, Zagreb, Croatia
The University Hospital for Tumors, Zagreb, Croatia
The alkaline comet assay was employed to assess the pre- and post-treatment levels of in vivo DNA
damage in peripheral blood leukocytes of twelve cancer patients. During study all patients were given
antineoplastic drugs, mainly as polychemotherapy. To quantify the DNA damage the comet tail length
and the tail moment were evaluated. Our results indicate marked interindividual variations between
baseline DNA damage in peripheral blood leukocytes recorded among cancer patients prior to the
chemotherapy. After intravenous administration of various antineoplastic drugs a significantly
increased level of DNA damage in all cancer patients compared to their pre-treatment values was
recorded. The highest level of DNA damage was pronounced in a patient received antineoplastic
drugs according to COPP/ABV protocol. Despite of their limitations, our results confirm the usefulness
of the alkaline comet assay as a sensitive biomarker of exposure that enables rapid and simple
detection of primary DNA damage in peripheral blood leukocytes of cancer patients. Together with
standard cytogenetic endpoints comet assay provides a powerful technique for the routine detection of
critical DNA lesions produced after administration of antineoplastic drugs in the clinical settings.
References
1. Olive PL: The comet assay in clinical practice. Acta Oncol 38(7):839-844, 1999.
2. Rigaud O, Guedeney G, Duranton I, Leroy A, Doloy MT, Magdelenat H: Genotoxic effects of
radiotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. II
Alteration of DNA repair and chromosome radiosensitivity. Mutat Res 242:25-35, 1990.
79
P1/6
GENETIC SUSCEPTIBILITY TO BLADDER CANCER IN SLOVAK-CAUCASIANS:
ROLE OF NAT2 POLYMORPHISM
V. Habalová; L. Klimčáková; J. Šalagovič; I. Kalina; M. Hrivňák*; H. Schneider*
Department of Medical Biology, School of Medicine, P.J.Šafárik University, Košice, Slovakia
* Department of Urology, University Hospital, Košice, Slovakia
The acetylation polymorphism, discovered 40 years ago, holds a special place as one of the
first described examples of a pharmacogenetic defect affecting xenobiotic biotransformation capacity
in human populations. The genetically determined N-acetyltransferase activity is involved in
activation/inactivation reactions of numerous xenobiotics. Polymorphic substrates can be used to
phenotype individuals as “slow” or “rapid” acetylators. The slow phenotype is inherited as an
autosomal recessive trait. The association between acetylation phenotype and cancer or toxicity has
received considerable attention over the years.
A genotyping approach has been used to investigate NAT2 type with putative relevance in
bladder cancer in population of 74 Slovak-Caucasians patients attending a clinic at a hospital. Results
have been compared to 176 non-malignant individuals from the same region. Genomic DNA was
prepared from a blood sample. The PCR-RFLP-based genotyping method was used to detect four
most common alleles (NAT2*4, NAT2*5, NAT2*6, NAT2*7).
The proportion of putative risk phenotype (slow acetylator) in case group (71,62%) was
significantly higher compared to in control group (53,98%) (Fishers exact test, p=0,01). Individuals with
slow acetylator phenotype are at an approximately 2,1-fold higher risk (OR = 2,15 95 %CI 1,13-4,20)
of developing bladder cancer in comparison with individuals with rapid acetylator phenotype.
Our results suggest that the polymorphism of the N-acetyltransferase 2 is one genetic factor in
the origin of bladder carcinoma in Slovak region such as in most populations studied to date.
80
P1/7
POLYMORPHISM OF THE GSTM1 GENE ASSOCIATED WITH SUSCEPTIBILITY
TO LUNG CANCER IN RELATION TO THE DURATION OF SMOKING IN SLOVAK
POPULATION
J. Šalagovič ; J. Štubňa* ; I. Kalina; L. Klimčáková; V. Habalová
Department of Medical Biology, Medical Faculty, University P. J. Šafárik, Košice, SK
*Department of Tuberculosis and Respiratory Diseases , University Hospital, Košice, SK
Glutathione S-transferases (GSTs) are known to take part in detoxification of many potentially
carcinogenic compounds. Therefore, polymorphisms of GST genes have been considered as
potentially important modifiers of individual risk of environmentally induced cancers. Tobacco use is an
established main cause of lung cancer, resulting in a increased risk among individuals who have ever
smoked. The duration and intensity of smoking (expressed usually in pack-years) are the most
important determinant for the development of lung cancer.
In this study, we determined the genotype distribution of GSTM1 and GSTT1 genes among 338
Slovak lung cancer patients and 313 population healthy controls. GSTM1 and GSTT1 genotypes were
determined using multiplex PCR. Interactions between pack-years smoked (the amount of cigarette
consumption during lifetime), duration of smoking and intensity of smoking (cigarettes/day), GSTM1
and GSTT1 genotypes, and case status were evaluated.
In general, none of the GSTM1 and GSTT1 genotypes had a statistically significant effect on lung
cancer risk. For the group of individuals with lung cancer as a whole, or in subsets of histological
subtypes, our data for the Slovak population did not show a positive correlation between the null
genotypes and the neoplasm. However, we observed that lung cancer risk greatly associates with the
smoking history but neither does with combined indicator - pack-years, nor with the intensity of
smoking, but merely yes with the duration of smoking. Significantly increased lung cancer risk
(OR=6.3, 95% CI=1.9-26.7) associated with GSTM1 0/0 genotype was found only among smokers
smoking less than 20 years, independently on intensity of smoking. We did not found any significant
association related to GSTM1 null genotype between lung cancer risk and cigarette dose (pack-years)
or intensity of smoking. GSTT1 polymorhism had no significant impact on lung cancer risk associated
with smoking.
In conclusion, our results indicate that GSTM1-mediated susceptibility to lung cancer is highly related
to duration and not intensity of smoking. Potential risk GSTM1 null genotype significantly increase risk
for earlier development of lung cancer among smokers.
81
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82
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A
YEAST
GENOTOXICITY
AND
CYTOTOXICITY
ASSAY
FOR
HIGH-
THROUGHPUT SCREENING
N. Billinton ; P. Cahill ; A. W. Knight ; R. M. Walmsley
Gentronix Ltd, Fairbairn Building, 72 Sackville Street, Manchester, M60 1QD, UK.
Short-term
genotoxicity
assays
have
changed
little
in
the
last
30
years.
The
Ames/Salmonella/microsome test is still the regulatory benchmark in terms of microbial assays.
Current microbial genotoxicity assays monitor either mutagenicity or induction of DNA repair in
prokaryotes such as Salmonella, Escherichia and Vibrio. These bacterial cells are not particularly
robust, and they differ from (eukaryotic) mammalian cells in such factors as uptake, metabolism,
chromosome structure and DNA repair processes.
Such tests can be a misleading source of
information on the mutagenic and carcinogenic potency of a substance in mammals and can take days
to perform. We are developing a rapid genotoxicity and cytotoxicity test using yeast cells, which are
both robust and eukaryotic. Up-regulation of DNA repair activity at the transcriptional level is linked to
synthesis of the Green Fluorescent Protein (GFP), which is stable and can be estimated noninvasively. A preliminary validation study of 80 compounds has revealed a strong correlation between
positive results and mammalian carcinogenicity.
Our assay correctly gave positive results for a
number of compounds, in the absence of the exogenous metabolic activation required by other tests.
We have also demonstrated that the inherent metabolic competency of yeast can be enhanced by the
expression of human cytochrome P450 genes. Such modifications allow the activation of known promutagens to DNA-damaging species, hence permitting the detection of their genotoxicity. The test
can be automated for use in medium- and high-throughput screening using standard laboratory
robotics.
83
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POLYMORPHISMS IN DNA REPAIR GENES, XPB, XPC AND hHR23B, IN
POLISH POPULATION – A PRELIMINARY STUDY.
D. Butkiewicz; M. Rusin; M. Pawlas; M. Chorazy
Department of Tumor Biology, Center of Oncology – M.Sklodowska-Curie Memorial Institute,
Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland
A deficiency in DNA repair is associated with an increased cancer risk, e.g. an inherited defect in
nucleotide excision repair (NER) genes leads to cancer-prone syndrome - xeroderma pigmentosum
(XP). Polymorphisms in DNA repair genes may be linked to person-to-person variations in DNA repair
efficiency. Recently several polymorphisms in various repair genes were identified. Although the
biological function of these polymorphisms has not yet been elucidated, some of these variants may
be associated with a reduced repair capacity and increased cancer susceptibility. We searched for
new polymorphisms in coding regions of two NER genes – XPB (ERCC3), encoding 3’>5’ helicase
subunit of TFIIH, and hHR23B, coding for a protein complexed with XPC and participating in early
recognition of various lesions (e.g. photoproducts, DNA adducts). The known polymorphism Val499Ala
in exon 8 of XPC was also studied. We investigated the frequency of those polymorphisms in 96 nonsmall cell lung cancer patients and 96 healthy controls - all inhabitants of a highly industrialized and
polluted region of Upper Silesia, Poland. New polymorphic variants were searched in 35 randomly
selected individuals by RT-PCR and cDNA sequencing and then PCR-RFLP analysis was used to
genotype cases and controls. Two single nucleotide substitutions causing amino acid exchanges in
XPB and one in hHR23B were detected. For the XPB only single heterozygote was found for each
variant: 445AG in exon 3 causing Lys117Arg and 1299GT in exon 8 causing Gly402Cys. Both
variants are in residues highly conserved through evolution. One common sequence variant in
hHR23B was found: 1059CT substitution causing Ala249Val. The 249Val allele frequency was 0.26
in cases and 0.29 in controls. The XPC-499 Ala allele was found to be more frequent in our group
(80%). The frequency of Ala/Ala genotype was slightly higher in younger patients (below 56 y), in
never smokers and patients having increased aromatic adduct levels in lung tissue. Our preliminary
results suggest that polymorphisms in NER genes are worth to be further investigated.
84
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INFLUENCE OF VHL EXPRESSION ON DNA REPAIR
C. Flohr 1; E. Weidt 2; J. Decker 2; B. Epe 1
1
Institute of Pharmacy, University of Mainz, Staudinger Weg 5, 55099 Mainz, Germany
2
Hematology, Third Department of Medicine, University of Mainz, 55131 Mainz, Germany
The VHL tumor suppressor gene is supposed to have a critical role in various processes that are
central to carcinogenesis, including cell-cycle control, differentiation and angiogenesis. The
degradation of the HIF protein (hypoxia-inducible-factor) seems to depend on VHL, indicating a role of
VHL in the signaling of oxygen tension and possibly in the response to oxidative stress.
The wild-type VHL gene was transfected into a renal carcinoma cell line (786-O) lacking functional
pVHL to examine the effects of the VHL gene product on (i) the steady-state (background) levels of
oxidative DNA modifications, (ii) the susceptibility of the cell lines to DNA damage by exogenous
oxidants and (iii) the repair kinetics of oxidative DNA modifications. Steady-state levels of oxidative
DNA damage, quantified by means of the alkaline elution assay in combination with various repair
endonucleases (Fpg protein, T4endonucleaseV, exonucleaseIII) were found to be similar in VHLexpressing and control cells. Moreover, the susceptibility to the induction of oxidative DNA damage by
the photosensitizer RO 19-8022 plus visible light was the same in VHL-expressing and deficient cells.
Surprisingly, the repair kinetics of Fpg-sensitive oxidative base modifications in the two cell lines
differed considerably. No repair was observed in the cells lacking functional pVHL after 48 h. In VHLexpressing cells, repair of Fpg-sensitive modifications was significant, although still slower than in wildtype kidney cells used as a control. In contrast, the repair of pyrimidine dimers was similar in all cell
lines (t 1/2 8-9 h).
The results might indicate a role for VHL in the sensing and the repair of oxidative DNA damage.
85
P1/12
DNA REPAIR CAPACITY AND EXPRESSION PROFILES OF DNA REPAIR
GENES IN RESTING AND PHA-STIMULATED HUMAN PERIPHERAL BLOOD
LYMPHOCYTES
C. Mayer1, O. Zelezny1, M.C. von Brevern2, A. Bach2, H. Bartsch1 and P. Schmezer1
1German
Cancer Research Center, 2BASF-LYNX Bioscience AG, Heidelberg, Germany.
DNA repair plays an important role in maintaining genomic integrity, and deficiencies in this system
likely lead to the development of cancer. Several studies have explored whether the individual
capacity to repair DNA damage can be used in molecular epidemiological studies as cancer risk
marker. Hereby, in general, peripheral blood lymphocytes (PBLs) are challenged by a genotoxic
treatment and the proportion of DNA damage removed over time is measured. As the cell’s ability to
remove DNA damage may be correlated with proliferative activity, it is important whether quiescent or
dividing cells are used for such studies. PBLs are resting in G 0 of the cell cycle but can be efficiently
stimulated by mitogens to divide in vitro. Our study was aimed to compare DNA repair capacity and
expression profiles of 70 DNA repair genes, both in resting and phytohemagglutinine (PHA) stimulated
PBLs.
PBLs from 9 individual donors were irradiated with 5Gy, and DNA repair was measured by alkaline
comet assay up to 60 min following treatment. There was no difference, neither in radiation sensitivity
nor DNA repair capacity between PHA stimulated and non-stimulated PBLs. Stimulated cells,
however, showed always elevated (1.4 to 1.7-fold) tail moment values.
Transcriptional profiles of repair genes were analysed using a custom made cDNA array.
Hybridisation experiments were performed with mRNA isolated both from unstimulated and PHA
stimulated (24-72h) PBLs. Detectable signals were found for more than 60% of the genes. Excluding
genes with a constant expression level over time, we identified 15 repair genes varying 3- to more
than 18-fold. In these cases maximal induction was observed 72h after stimulation. Most of the repair
enzymes which were upregulated during PHA-stimulation, also play a role in replication and/or
transcription. Enzymes involved in DNA mismatch repair and homologous recombination as well as
DNA glycosylases were not affected by the mitogenic stimulus.
In conclusion we found no evidence for an increased DNA repair capacity in stimulated vs. nonstimulated cells using the comet assay. We found up-regulation only for a few specific repair enzymes,
which are also involved in transcription/replication. Our results do not support a general increase in
DNA repair of PBLs by PHA stimulation.
86
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87
P1/14
PARP INHIBITOR 3-AMINOBENZAMIDE DOES NOT INCREASE THE YIELDS OF
CHROMOSOMAL
ABERRANT
CELLS
INDUCED
BY
BORON
NEUTRON
CAPTURE REACTION IN V79 CHINESE HAMSTER CELLS
N.G. Oliveira1,2; M. Castro2,3; A.S. Rodrigues1,4; I.C. Gonçalves5; R. Cassapo1; A.P. Fernandes5; T.
Chaveca1,2; J.M. Toscano-Rico3 and J. Rueff1
1Department
of Genetics, FCM UNL, Lisbon, Portugal; 2FFUL, Lisbon, Portugal; 3CFEC, FML, Lisbon,
Portugal; 4University Lusófona, Lisbon, Portugal; 5Nuclear and Technological Institute, Portuguese
Research Reactor, Sacavém, Portugal
Mechanistic knowledge on DNA and cell damage induced by alpha-particles remains limited. It is well
known that high-LET radiation induces both DNA single (ssb) and double strand breaks (dsb), being
the latter frequently associated with cell death. The repair of these DNA lesions and specially dsb are
thus fundamental for the understanding of high-LET radiation effects. Poly (ADP-ribose) polymerase is
a nuclear enzyme, which detects and signals DNA strand breaks (ssb and dsb). The important role of
this enzyme in the maintenance of DNA integrity has been extensively studied for genotoxic chemicals
and low-LET ionizing radiation. Nevertheless, sparse information concerning the role of PARP in highLET radiation effects is available. The purpose of this work is to examine whether the PARP inhibitor
3-aminobenzamide (3-AB) enhances the yields of chromosomal aberrations induced by the boron
neutron capture (BNC) reaction in V79 Chinese hamster cells. Wild-type V79 cells were pre-incubated
for 48 hours with different concentrations (0.48–2.4 mM) of the boron delivery agent 4-borono-Lphenylalanine (BPA) and then irradiated for different periods of time with thermal neutrons. In the 3-AB
treated cultures, four hours before the irradiation the cells were incubated with different concentrations
of this inhibitor (1.5-10 mM) which remained in culture until colchicine was added. The chromosomal
aberrations assay was performed according to standard protocol. A clear dose-response in the
frequencies of chromosomal aberrant cells excluding gaps (%CAEG) induced by the BNC reaction
was observed for both BPA concentration and thermal neutron fluence. There was no evidence of an
increase in the % CAEG induced after incubation with 3-AB. Some cytoxicity was observed (mitotic
index) after 3-AB incubation in BPA irradiated cells. In conclusion, the clastogenic potential of the
alpha-particles generated through the BNC reaction was not affected by using a classic PARP
inhibition approach.
88
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INFLUENCE OF NITRIC OXIDE ON DNA REPAIR
N. Phoa , B. Epe
Institute of Pharmacy, University of Mainz, Staudinger Weg 5, 55099 Mainz, Germany
We have investigated the effects of endogenously and exogenously generated nitric oxide (NO) in
cultured mammalian cells on (i) the levels of oxidative base modifications (8-oxoG), (ii) the repair
kinetics of various additonal lesions and (iii) the nature of modifications induced by exogenously
derived NO. Steady state levels of oxidative DNA base modifications, measured by means of an
alkaline elution assay in combination with the repair endonuclease Fpg protein, were similar in NOoverproducing B6 mouse fibroblastes (stably transfected with an inducible NO synthetase) and in
control cells. Increased oxidative damage was observed only after exposure to high (toxic)
concentrations of exogenous NO. The modifications induced were mainly oxidative purines recognised
by Fpg protein and only a few AP sites and single-strand breaks. The repair rate of additional oxidative
DNA base modifications induced by photosensitization was not affected by the endogenous NO
generation, but was completely blocked by exogenous NO at concentratons which did not cause any
oxidative DNA damage by themselves. In contrast, the repair of pyrimidine dimers induced by UVB
and the repair of single-strand breaks was not influenced by exogenously derived NO at
concentrations which completely blocked the repair of the oxidative lesions.
These results indicate that NO generates DNA damage only inefficiently, but is able to inhibit the
repair of oxidative DNA base modifications. This might contribute to the generation and progression of
cancer in vivo.
89
P1/16
DNA DAMAGE AND REPAIR EFFICIENCY IN SCHIZOPHRENIC PATIENTS
D. Psimadas 1, 3, N. Messini-Nikolaki 3, A. Fortos 2, S. Tsilimigaki 1
and S.Μ. Piperakis 1
1DNA
Repair Laboratory, Institute of Biology, National Center of Scientific Research
“Demokritos”, Athens, Greece. E-mail : [email protected]
2Department
3Division
of Geriatrics, Dromokaitio Psychiatric Hospital, Athens, Greece.
of Cell Biology and Biophysics, Department of Biology, University of Athens,
Athens, Greece.
Schizophrenia is a relative common debilitating, chronic, psychotic disorder. Its lifetime prevalence is
approximately 0.85% in the general population. There is very little evidence up to today whether
individuals suffering from schizophrenia have a defective DNA repair system (Magin et al 1991).
In the present study we examined the sensitivity to DNA damage and the repair efficiency of a
schizophrenic patients population. The patients that participated in this study were selected with the
help of a detailed questionnaire containing questions on their health, age, diet, smoking habits, genetic
background, medication etc. The DNA damage, the effects of external factors (H2O2 and γ-radiation)
and the repair efficiency of the population was estimated with the comet assay technique which
detects DNA breaks (Piperakis et al 1998, Piperakis et al 1999). The induced DNA breaks were
evaluated with a suitable program (kinetic image analysis) as well as with visual scoring. The statistical
analysis was performed with a non-parametric test (Kruskal-Wallis).
Our preliminary results suggest a difference in the effectiveness of the DNA repair systems of the
schizophrenic patients if compared to normal population.
Magin G.K, Robison S.H, Breslin N, Wyatt R.J. and Alexander R.C. DNA repair and
mutant frequency in schizophrenia. Mutation Res. 1991, 255, 241-246.
Piperakis S.M, Visvardis E.E, Sagnou M, and Tassiou A.M. Effects of smoking and aging on
oxidative DNA damage of human lymphocytes. Carcinogenesis, 1998, 19,
695-698.
Piperakis S.M, Visvardis E-E, Sagnou M, Tassiou A.M. Comet assay for nuclear DNA
damage. Methods in Enzymology. 1999, 300, 184-194.
90
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SEARCHING FOR REPAIR COMPETENCE OF CELLS OF LARYNX CANCER
PATIENTS – GENETIC INSTABILITY AND ALLELIC LOSSES IN GENES
CONTROLLING CELL CYCLE AND DNA REPAIR.
Stembalska – Kozłowska A. 1, R. Smigiel 2, T. Kręcicki 3, M. Blin 4, F. Mirghomizadeh 4, K. Bartusiak1,
M. Sasiadek 1.
1
Department of Genetics, 2 Department and Clinic of Otolaryngology, Medical University of Wrocław,
Poland, 3 Institute of Anthropology and Human Genetics, Tuebingen, Germany
Introduction: The aetiology of larynx cancer is complex with both genetic factors and mutagenic
exposure involved. It has been hypothesised that HNSCC development is related to the widespread
genomic instability such as chromosomal and microsatellite instability, allelic imbalance / allelic loss,
as well as to the exposure to biological (e.g., papilloma-viruses) and chemical (e.g., tobacco and
alcohol) carcinogens.
The aim of the present study was to investigate (i) “hidden chromosome instability” (ii) microsatellite
instability (MSI) and (iii) loss of heterozygosity (LOH) in chosen genes controlling cell cycle and DNA
repair, as possible characteristics of the tumours of larynx.
Material and methods: The study group comprised 20 patients, diagnosed with the primary
squamous cell carcinoma of larynx. None of the patients had a family history of cancer. To test for
genes important in carcinogenesis [MLH1, HPC1, APC, an unknown tumor suppressor in 8p22,
MSH2, MET, P53, nm23 (M1, M2, M3)] 10 microsatellite markers were applied. MSI was monitored by
using markers: BAT26, BAT25, and BAT40. For searching for „hidden chromosome instability” the
bleomycin test was carried out.
Results: LOH was most frequent in the MLH1, the unknown tumor suppressor in 8q22 and nm23. The
analyses of BAT25, BAT26, and BAT40 showed no evidence of MSI. Statistically significant (p<0.05)
increase in all parameters of hypersensitivity to bleomycine was noted in the group of cancer patients,
in comparison to the controls. No correlation was discernible between the frequency of LOH or
markers of “hidden chromosome instability” and the stage of disease.
References: 1. Vokes EE, et al. N Eng J Med 328, 1993; 2. Papadimitrakopoulou VA: Curr Opinion
Oncol 12: 240-245, 2000; 3. Fan CY: Curr Oncol Rep 3: 66-71, 2001; 4. Gleich LL et al.: Arch
Otolaryngol Head Neck Surg 125: 949-952, 1999; 5. Hsu TC et al.: Cancer Genet Cytogenet 17: 307313, 1985; 6. Cawkwell L et al.Br J Cancer 67: 1262-1267, 1993.
91
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COMPARISON OF DIETHYL SULFATE MUTAGENICITY ON FEMALE AND MALE
GERM CELLS OF DROSOPHILA MELANOGASTER UNDER DIFFERENT REPAIR
CONDITIONS.
J. Hernando; M. A. Comendador; L. M. Sierra.
Dpto. Biología Funcional e Instituto Universitario de Oncología. Área de Genética. University of
Oviedo, 33006. Spain.
The mutagenicity of diethyl sulfate (DES) was analysed on female germ cells of D. melanogaster,
using the sex linked recessive lethal test (RL) and the mutagenicity index, under different repair
conditions: efficient and deficient for the nucleotide excision repair (NER), and for a bypass-mediated
tolerance mechanism (BTM).
The results of this work show that, in all repair conditions, DES is mutagenic in premeiotic female germ
cells, as it is in postmeiotic male germ cells, although the RL frequency is much higher in males than
in females.
Analysis of NER influence confirms that this mechanism repairs at least part of the DES-induced
damages, because the mutability index (4.3) is statistically higher than 1. In addition, the effect of NER
is larger in females than in males (4.3 versus 2.2), probably reflecting the short time period for the
maternal repair, in the case of postmeiotic male germ cells.
With respect to the BTM mechanism, although previous results showed a clear influence on males, no
effect was found in females. This system has been involved on the bypass of persistent and difficult
repaired damages, to increase the time available for their repair. According to our results then, part of
the DES-induced damage is substrate of this tolerance system, and the lack of effect on females, is
probably because those cells have got much more time, than postmeiotic male cells, to repair this
damage by other repair mechanisms, such as NER.
In summary, apart from being the first evidence of DES mutagenicity on premeiotic germ cells of D.
melanogaster, this work reveals the importance of analysing different cell types on mechanistic studies
involving repair analysis.
92
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EXCISION
OF
PYRIMIDINE
RING-RUPTURED
1,N6-ETHENOADENINE
BY
THYMINE GLYCOL-DNA GLYCOSYLASE
M. Bajek, J.M. Cieśla, B. Tudek
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106
Warsaw, Poland
A highly mutagenic DNA lesion, 1,N6-ethenoadenine (A) is chemically unstable and either
depurinates or converts to pyrimidine ring-opened product of water molecule addition to C(2)-N(3)
bond in dA (compound B). Compound B subsequently undergoes deformylation to yield compound
C, which depurinates in the final step of A rearrangement pathway. We have previously shown that
A rearrangement products are not repaired by human N-methylpurine-DNA-glycosylase, which
excises parental A. Compound B was found to be eliminated from B:T pair by E.coli
formamidopyrimidine-DNA glycosylase (Fpg), which removes B also from B:C pair, as well as from
pairs B:A and B:G but much less efficiently than when it is paired with pyrimidines (1). Here we show
that compound B is also recognized by E.coli endonuclease III (Nth protein). The efficiency of excision
depends on the opposite base pair. Most efficient repair is observed when this derivative is paired with
dT (Km= 30 nM, kcat= 14), but it is less favorable when paired with dC (Km= 40 nM, kcat= 3.5) and dG
(Km= 18 nM, kcat= 2.2). Compound B is also removed from single-stranded DNA and with a similar
efficiency from B:dA pair. A similar opposite base specificity was found for excision of compound B by
yeast nuclear homolog of thymine glycol-DNA glycosylase, Ntg2 protein.
1. Speina, E., Cieśla, J.M., Wójcik, J., Bajek, M., Kuśmierek, J.T., Tudek, B. (2001)
J.Biol.Chem., 276, (in press).
93
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UNSCHEDULED DNA SYNTHESIS: MEASUREMENT OF DNA REPAIR IN A
HUMAN HEPATOMA CELL LINE (HEPG2 CELLS)
I. VALENTIN*; Y. LOSSOUARN+; V. THYBAUD+; J-C. LHUGUENOT* and M-C. CHAGNON*
* Laboratoire de Sécurité Alimentaire – UMR 0938 – 1, esplanade Erasme, 21000 Dijon, France.
+ Aventis Pharma, Paris Research Center, Drug Safety Evaluation– 13, quai Jules Guesde, 94100
Vitry sur Seine, France.
Numerous in vitro test systems measuring different genotoxic endpoints have been developed during
the last 20 years for assessment of genotoxic hazard and risk. However, there is still a need for the
development of test systems more relevant to human. In this study, an established cell line derived
from a human hepatoma, HepG2 cells which shows many morphological and biochemical
characteristics of normal hepatocytes and drug metabolising capacity (Knowles et al., 1980; Rueff et
al., 1996) was chosen. Only limited data were reported in the literature on DNA repair capacity in this
cell line. Therefore, the evaluation of unscheduled DNA synthesis (UDS) was carried out to measure
the DNA repair activity in response to the induction of DNA damage by many classes of chemicals
(Mitchell et al.,1983). To avoid false positive response, the cytotoxicity of the compounds was first
evaluated using a sensitive assay, the measurement of RNA synthesis (Valentin et al., 2001). The
UDS activity was measured by monitoring the uptake of [ 3H]-thymidine incorporated in the DNA of non
S phase cells either by autoradiography (AutoR) (Stich and San, 1970) or by liquid scintillation
counting (LSC) (Trosko and Yager, 1974). The autoR UDS assay seems to be the best method to
measure UDS on a cell. It is more sensitive (6 to 15 fold) than LSC approach and the use of DNA
synthesis inhibitors like hydroxyurea is not necessary. AutoR UDS on HepG2 cells was carried out
with some well known mutagenic compounds. 4-nitroquinoline-N-oxide greatly induced UDS activity in
HepG2 cells (8.8-fold increase at 0.1 µM, respectively). All the three pro-mutagen compounds tested
clearly induced UDS activity. 15.8- and 7-fold increases in UDS activity were measured after treatment
with 250 mM dimethylnitrosamine and 0.5 µM 2-acetylaminofluorene, respectively. Benzo[a]pyrene
was the most potent UDS inducer (12-fold increase at 0.1 µM). In conclusion, the autoR UDS test on
human HepG2 provides a useful tool for genotoxicity assessment and in particular the detection of
pro-mutagens.
References :
Knowles et al. (1980). Science. 209, 497-499. Mitchell et al. (1983). Mutat. Res. 123, 363-410 Rueff et
al. (1996). Mutat. Res. 353, 151-176. Stich, H.F., and San, R.H.C. (1970). Mutat. Res. 10, 389-404.
Trosko, J. E., and Yager, J. D. (1974). Exp. Cell Res. 88, 47-55. Valentin et al. (2001). Toxicology.
158, 127-139. Williams et al. (1982). Mutat. Res. 97, 359-370.
94
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95
P1/22
96
P1/23
GENOTOXICITY DETECTED WITH COMET ASSAY AND MICRONUCLEUS TEST
IN CYPRINUS CARPIO SPECIMENS EXPOSED IN SITU TO TRASIMENO LAKE
WATERS TREATED WITH DISINFECTANTS FOR POTABILIZATION.
Buschini A.*, Martino A.*, Gustavino B.**, Monfrinotti M.***, Poli P.* , Rossi C.*, Santoro M.** & Rizzoni
M.**..
* Dipartimento di Biologia – Università di Parma, Italy.
** Dipartimento di Biologia – Università degli Studi di Roma “Tor Vergata", Roma, Italy.
** Laboratorio di Ecologia Sperimentale e Acquacoltura - Dipartimento di Biologia – Università degli
Studi di Roma “Tor Vergata”, Roma, Italy.
The aim of the present work was to detect the possible genotoxic effect of water treated with
disinfectants for potabilization using Comet assay and micronucleus test in circulating erythrocytes of
Cyprinus carpio. Young specimens (20-30 gr) were exposed in vivo & in situ in experimental basins
within the potabilizatin plant of Castiglione del Lago (Perugia, Italy), in which the water of the
Trasimeno Lake are treated and disinfected before it is put in the distribution net of drinkable water.
Basins were filled with water in a continuous flow at a constant rate, each basin with water treated
continuously at a constant concentration with one of the three tested disinfectants (sodium
hypochlorite, peracetic acid and chloride dioxide), being one control basin supplied with untreated
water. Three sampling campaigns are here described: July 2000 (preliminar), October 2000 and
Febuary 2001. Repeated blood intracardiac samplings allowed to follow the same fish populations
after different exposure tumes: sample were taken before disinfectant input (for both tests), 3 hours
after (for Comet assay), 10 days after (for micronucleus test only) and 20 days after (for both tests).
Results showed a DNA damage in fish exposed to water disinfected with sodium hypochlorite and, at
a lesser extent, chloride dioxide with a different time course for the two tests: while Comet assay gives
an immediate response for a damage occurred directly in circulating erythrocytes, micronuclei reach
their higher frequencies at the latest sampling times, when a DNA damage in stem cells of the
cephalic kidney are expressed in circulating erythrocytes.
97
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EFFECTS
OF
BRUSSELS
SPROUTS
EXTRACTS
AND
THE
ACTIVE
CONSTITUENTS ON OXIDATIVE DNA DAMAGE AND THE ACTIVITY OF
NAD(P)H: QUINONE REDUCTASE IN HEPA 1c1c7 CELLS
C.Y. Zhu; S. Loft
Institute of Public Health, Faculty of Health Science, University of Copenhagen, Copenhagen,
Denmark
We have studied the effect of aqueous extracts of cooked and autolysed Brussels sprouts, sinigrin
(found high concentration in Brussels sprouts) and the myrosinase-catalysed products as well
isothiocyanates, on the activities of NAD(P)H: quinone reductase (QR) and the strand breaks of
hydrogen peroxide-induced DNA damage in Hepa 1c1c7 murine hepatoma cells.
Hepa 1c1c7 cells were plated at a density of 10 000 cells/well for 24 h and then incubated with
tested samples in -minimal essential medium at concentrations 1-50 µg/ml in a 37C incubator for 24
h. For quinone reductase assay, the cells were lysed by 0.8% digitonin and then exposed to a
complete reaction mixture. The activity of quinone reductase were determined by a Mutiskan Ascent
system. For the Comet assay, Hepa 1c1c7 cells were detached by trypsin/EDTA and then exposed to
100 µM H2O2 for 5 min on ice. The induced DNA strand breaks in Hape 1c1c7 cells were evaluated by
mean of the Comet assay.
Results showed that the maximum increase in quinone reductase activity of sinigrin and its
myrosinase-catalysed products was 1.2 and 1.4 times at concentration 2.5 µg/ml, and 1.1 of the
aqueous extract of cooked Brussels sprouts and the myrosinase-catalysed products at concentration
10 and 1 µg/ml, respectively. Isothiocyanates (ally ITC, propyl ITC and benzyl ITC) did not influence
the QR activities of Hepa 1c1c7 cells.
The extracts of cooked and autolysed Brussels sprouts and sinigrin inhibited the hydrogen peroxide
induced DNA strand breaks in Hepa 1c1c7 cells, the maximum inhibition was 38% and 34% at
concentration 10 µg/ml of extract powders, and 21% of sinigrin at concentration 2.5 µg/ml.
The effects of protection against the oxidative DNA damage and enhancement of the activity of
quinone reductase could explain the suggested cancer preventive effect of cruciferous vegetables.
98
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THE USE OF IN VITRO ASSAYS TO TEST SOUTH AFRICAN MEDICINAL PLANT
EXTRACTS FOR MUTAGENIC ACTIVITY
E.E. Elgorashi(1,2); J.L.S. Taylor(1,2); L. Regniers(1); L. Verschaeve(1); A. Maes(1); N. De Kimpe(2); J. van
Staden(3); A. Fossey(4)
1. Environmental Toxicology, Flemish Institute for Technological Research, Boeretang 200, B-2400
Mol, BELGIUM
2. Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences,
University of Gent, B-9000 Gent, BELGIUM
3. Research Centre for Plant Growth and Development, University of Natal Pietermaritzburg, Private
Bag X01, Scottsville 3209, SOUTH AFRICA
4. School of Molecular and Cellular Biosciences, University of Natal Pietermaritzburg, Private Bag
X01, Scottsville 3209, SOUTH AFRICA
The majority of the world’s population, which is situated predominantly in developing countries, relies
on plants and plant-derived products as their primary health care resource. In South Africa, over 60%
of the population consults one of an estimated 200 000 traditional healers, in preference to, or addition
to western medical doctors, especially in rural areas of the country. There have been many validations
of traditional remedies through scientific research. The potential risk from long term usage of such
remedies has not, however, been fully investigated, especially in terms of potential carcinogenic
activity. Various South African plant species were thus selected on the basis of their use in traditional
medicine. Crude extracts, prepared from the dried plant material using dichloromethane and
methanol/water, were tested for activity in the Ames test, the Vitotox  test, and the Micronucleus test.
These tests aimed to identify potential safety risks for the continued use of the plants in traditional
medicine. Preliminary screening results indicate the formation of micronuclei in human lymphocytes by
some plant extracts. No positive genotoxicity results were obtained for the Vitotox  test, but these
results indicated that many of the plant extracts were toxic at high concentrations.
99
P1/26
GENOTOXICITY EVALUTATION OF TRITERPENES FROM SAMBUCUS NIGRA
T. Cangianoa, M. Della Grecab, A. Fiorentinoa, A. Gentilia, M. Isidoria
a Dipartimento
di Scienze della Vita, Seconda Università di Napoli, Caserta, Italy
b Dipartimento
di Chimica Organica e Biochimica, Università Federico II, Napoli, Italy
High plants produce a large variety of secondary metabolites, which could be involved in different
interactions. When these substances are released from plants in the environment, they can interfere
with the growth of other plants or act as mycotoxins and antimicrobials 1. In research of bioactive
secondary metabolites we have investigated Sambucus nigra L. (Caprifoliaceae), a shrub widely
spread in all the Mediterranean region.
Ten triterpenes were isolated and identified from this plant, showing a strong in vitro cytotoxic activity
on the brine shrimp Artemia salina2.
R2
R2
HO
HO
R1
1
2
3
4
R1 = H R2= H
R1 = H R2= CHO
R1 = CH2OH R2= COOH
R1
HO
5
6
7
8
9
10
R1 = H R2= H
R1 = CH2OH R2= H
R1 = CHO R2= H
R1 = COOH R2= H
R1 = COOH R2= OH
R1 = COOMe R2= OH
In the light of these results we have also investigated about a potential genotoxic activity of these
compounds. The genotoxicity was tested using SOS Chromotest on Escherichia coli PQ37 with and
without S93. Only compoud 4 showed a strong genotoxic activity. This result is according to its high
citotoxic activity.
1Rice
E.F.(1984) Allelopathy, Academic Press, NewYork.
2Meyer
B.N., Ferrigni N.R., Putnam J.E., Nicholas D.E., Mclaughlin J.L. (1982) Brine
shrimp: a
convenient geneal bioassay for active plant constituents. Planta Medica. 45: 31-34.
3Quillardet
P. and Hofnung M. (1985) The SOS Chromotest, a colorimetric bacterial assay for
genotoxins: procedures. Mutation Research.147: 65-78.
100
P1/27
101
P1/28
INDUCTION OF ANEUPLOIDY IN HUMAN CELL LINES BY HORMONES
Mahmood A. Kayani, James, M. Parry
Center for Molecular Genetics and Toxicology,
School of Biological Sciences,
University of Wales, Swansea.
Hormones play certain important roles in the development of hormone dependent types of cancers
such as breast cancer, ovarian cancer, cervical cancer, etc. The mechanism of carcinogenesis due to
steroid hormones is still not clear. The available data on the genotoxicity and mechanisms of action
are either insufficient or often misinterpreted. Experiments were carried out using seven diverse
hormones
namely,
17-B
Estradiol,
Progesterone,
Testosterone,
Megesterol
Acetate,
Diethylstilboestrol, Trenbolone Acetate and Zearanol. To evaluate their genotoxic potential the
hormones were screened using cytokinesis blocked micronucleus assay (CBMN). Once found positive
for micronucleus induction, kinetochore labeling was employed to see whether the micronuclei were
formed due to aneugenic or clastogenic mechanism using antikinetochore antibodies. The hormones
found positive in kinetochore labeling assay were screened for non-disjunction potential for
chromosomes 10, 17 and 18, using flouresence in situ hybridisation technique. In this study 17-
Estradiol, Diethylstilboestrol, Testosterone, Progesterone and Trenbolone acetate were found to be
significantly positive (P<0.001) for micronucleus induction. Kinetochore labeling revealed that 17-
Estradiol and Diethylstilboestrol produce aneugenic effects, Trenbolone acetate was clastogenic,
whereas, Testosterone and Progesterone were both aneugenic and clastogenic. The chromosome
non-disjunction results show that 17- Estradiol, Diethylstilboestrol and Testosterone produce
significantly high increase (P<0.001) in the frequency of non-disjunction for chromosomes 10, 17 and
18, which might provide understanding of the mechanism of carcinogenic action of these hormones.
102
P1/29
INDUCTION OF MICRONUCLEI AND CHROMOSOME NON-DISJUNCTION
AFTER SHORT-TERM EXPOSURE TO CARBENDAZIM IN CULTURED HUMAN
LYMPHOCYTES
Mahmood. R1, 2 and Parry. J. M1
1
Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales,
Singleton Park, Swansea SA2 8PP. U.K.
2
Department of Biotechnology, Kuvempu University, Gnanasahyadri, Shankarghatta-577 451.
Shimoga Dist. Karnataka, India.
Chromosome non-disjunction is the major cause of aneuploidy, which may lead to a number of genetic
diseases. Several environmental chemicals such as drugs, pesticides, etc., have been shown to
induce aneuploidy. There is considerable interest in the assessment of clastogenic and aneugenic
effects, which may be due to pesticide exposure. It is therefore important to evaluate the aneugenic
potential of widely used chemical compounds by using sensitive techniques. In the present study we
assessed the aneugenic ability of the carbamate fungicide-carbendazim, by employing five different
doses, ranging from 0.5–8.0 g/ml at two short-term exposures of 60 min and 120 min in cultured
human peripheral blood lymphocytes. The test chemical was added following 24h of the initiation and
washed after the respective exposure times. Cells were harvested at 72h after initiation. The
cytokinesis blocked micronucleus assay was used for the detection of micronuclei. To detect
chromosome non-disjunction, the fluorescent in situ hybridisation (FISH) technique was performed by
using three centromere specific probes for chromosomes 10, 17 and 18 in accordance with the
recommendations of Parry et. al., (1994). It was observed that all the doses of the chemical tested
could effectively induce micronucleus and non-disjunction at both the exposure times employed. The
incidence of micronuclei and non-disjunction at all doses was significantly higher (p<0.05) than the
controls. The results suggested that the longer exposure of 120 min produces higher frequencies of
micronuclei and non-disjunction, when compared to 60 min exposure. Furthermore, the incidence of
non-disjunction for chromosomes 17 and 18 is found to be higher when compared to chromosome 10.
This indicates that carbendazim could more effectively induce aneuploidy of chromosome 17 and 18
even at a short exposure time of 60 min in human lymphocytes. It is obvious from the present study
that the test chemical carbendazim is a potent aneugen even at low exposures. Therefore, it is
imperative to pay much attention towards the safe handling of the chemical in view of its high activity
and potent aneugnic ability.
103
P1/30
IN SITU MONITORING
WITH TRADESCANTIA- MICRONUCLEUS ASSAY ON
THE GENOTOXICITY OF URBAN AIR IN SOUTHERN ITALY
F. Cundari b; M. Isidori a; A. Nardelli a; A. Parrella a; O. Pepe a
a
Dipartimento Scienze della Vita – Seconda Università di Napoli – Via Vivaldi, 43
81100 Caserta, Italy
b
ACSA CE3 – Azienda Consortile Servizi Ambientali
This study concerns the assessment of genotoxicity in the urban air of Caserta, a town with heavy car
traffic in Southern Italy. Tradescantia-micronucleus (Trad-MCN) assay was applied to establish the
urban gradient of pollutants in the air matrix. The in situ monitoring was carried out by exposing young
inflorescences of the plant cuttings for 24h at seventeen sampling points in two different seasons of
the year. Pollutants induced chromosomic aberrations become micronuclei in the synchronized tetrads
and they were easily identified and scored. The increase in frequency of micronuclei was expressed in
terms of MCN/100 tetrads.
Collected data were processed using Dunnett’s test to determine the level of significance against the
negative control values in each experimental series.
The results were closely associated with the weather, the velocity of wind, the temperature and the
relative humidity. Significant increases in the frequency of micronuclei were observed in different
points of the town grid indicating the good applicability of Trad-MCN for monitoring the presence of
hazardous air contaminants.
1) T.H. Ma, C. Xu, S. Liao, H. McConnell, B.S.Jeong, C.D. Won (1996). In situ monitoring with the
Tradescantia bioassays on the genotoxicity of gaseous emissions from a closed landfill site and an
incinerator. Mutation Research 359, 39-52.
2) E.T. Guimaraes, M. Domingos, E.S. Alves, N. Caldini Jr, D.J.A. Lobo, A.J.F.C. Lichtenfels, P.H.N.
Saldiva (2000). Detection of the genotoxicity of air pollutants in and around the city of Sao Paulo
(Brazil) with the Tradescantia-micronucleus (Trad-MCN) assay. Environmental and Experimental
Botany 44, 1-8.
3) Te-Hsiu Ma (1981). Tradescantia Micronucleus Bioassay and pollen Tube Chromatid Aberration
Test for in situ monitoring and mutagen screening. Environmental Health Perspectives 37, 85.90.
104
P1/31
THE MICRONUCLEUS ASSAY IN HAEMOCYTES OF Dreissena polymorpha
FOR THE DETECTION OF GENOTOXICITY IN FRESHWATER ENVIRONMENTS
M. Pavlica; G.I.V. Klobučar; R. Erben; D. Papeš
Department of Biology, Faculty of Science, University of Zagreb
Rooseveltov trg 6, Zagreb, Croatia
The micronucleus (MN) assay was performed on zebra mussels, Dreissena polymorpha Pallas, to
evaluate the genotoxic effect of freshwater environments. Caged zebra mussels were transplanted to
five monitoring sites: one in the artificial lake (control), one upstream and three downstream of
municipal and industrial wastewater outlets. Mussels collected from their natural habitat in the river
Drava (uncontaminated site) were also used as control. Micronuclei were detected after bisbenzimide
fluorescent staining. Positive response was obtained at three investigated sites downstream from the
municipal and industrial wastewater outlets, while other monitoring sites gave negative response. The
mean MN frequency ranged from 0.50‰ (spontaneous level at reference sites Drava and Jarun) to
3.16‰ for the most contaminated site.
These results confirmed the sensitivity and usefulness of zebra mussel and MN assay in rapid
screening of genotoxic compounds in polluted freshwater environments.
105
P1/32
EVALUATION OF GENOTOXIC ACTIVITY OF OXIDIZING TREATMENTS TO
REMOVE SIMAZINE FROM WATER
Sueiro R.A.; Suárez S.; Rubio A., Araujo M., Garrido M. J.
Instituto de Investigación e Análises Alimentarias, Laboratorio de Microbioloxía, Universidade de
Santiago, Santiago de Compostela, Spain
The presence of simazine residues, a triazine herbicide, in water has important social and economic
repercussions in Extremadura (South-West of Spain).
There are some treatments that could remove this herbicide from water. However, the very same
treatments used to eliminate the herbicide, could very well be toxic of themselves because of the
potential generation of reactive compounds with such activity. Because of this, in the study we
evaluated the genotoxic activity of three oxidizing treatments. The selected methods were: ozonation
(ozone alone), combined ozonation with hydrogen peroxide and Fenton oxidation (ferrous salts with
hydrogen peroxide).
Prokaryotic and eukaryotic assays were used to evaluate the genotoxic potential of treated water.
Specifically, the Salmonella typhimurium and Escherichia coli mutagenicity tests and, the sister
chromatid exchange (SCE) and micronucleus test (MN) in human peripheral lymphocytes were
performed.
Negative results were obtained with the three types of water resulting the different treatments.
Under the conditions tested, our results indicate that the oxidation methods tested are not a risk for
public health.
This investigation received financial support from FEDER and CICYT Founds (Project Nº 1FD972222-C03-03)
106
P1/33
UTILIZATION OF THE VITOTOX GENOTOXICITY TEST AND ACUTE AND
CHRONIC MICROBIOTESTS TO ASSESS THE ENVIRONMENTAL RISKS OF
SOLID INDUSTRIAL WASTES.
A. Van Cauwenberge, P. Bouviez and E. Noël
Institut Provincial d’Hygiène et de Bactériologie du Hainaut, Département d’Ecotoxicologie et
Département d’Environnement, Boulevard Sainctelette 55, B-7000 MONS, BELGIUM.
Chemical properties of four different industrial solid wastes (respectively (1) resins, (2) demolition
wastes, (3) industrial sludges, and (4) slags) were analysed in order to find the most appropriate way
to manage each of them. As the Walloon government legislation requires to take into consideration the
H14 criteria concerning the impact on the ecological systems, biological assays were performed to
evaluate the intrinsic ecotoxicological risks of the wastes analyzed.
Leachates from the four wastes were performed following the DIN 38414-S4 guidelines in order to
apply biological tests requiring an aqueous phase. The cytotoxicity and genotoxicity of each sample
were examined using the Vitotox test based on bioluminescence emitted by the bacteria Salmonella
thiphimurium. These results were compared with those obtained directly on the solid wastes, using a
direct contact microbiotest based on the observation of mortality or growth inhibition of the ostracod
crustaceans Heterocypris incongruens (Ostracodtoxkit test) when placed in contact with toxicants.
The advantage of this new chronic test is to be directly performed on the solid phase of the wastes
tested, avoiding any loss of potentially toxic elements during the sample preparation or extraction. The
results obtained with the Vitotox test for waste (1) clearly show a genotoxic effect after four hours of
contact, despite the fact that the chemical analysis did not predict any major toxicity problem regarding
its very low concentrations of formalin and phenol (<0,1%). The results of this preliminary study also
show a good correlation between the different biological tests used. This study clearly demonstrates
the usefulness of associating ecotoxicological tests with chemical analysis in waste management.
107
P1/34
COMPARATIVE ASSESSMENT OF WEAK GENOTOXIC PESTICIDE EFFECTS IN
PLANTS AND HUMAN CELL CULTURES
M. Wilder°; B. Volkmer*; E. A. Sanders°; R. Greinert*; E.W. Breitbart*; D. Pollet°
°University of Applied Sciences, Department of Applied Natural Sciences, Hamburg, Germany;
*Dermatologisches
Zentrum, Buxtehude, Germany
Compounds showing only weak effects in short-term genotoxicity tests may nevertheless have
significant ecotoxicological implications when intentionally or accidentally released into the
environment. It has been suggested that long-term exposure to low-level genotoxic pesticides might
adversely affect the survival of natural populations, particularly in aquatic ecosystems where foodchains are closely linked to human nutrition and health [1]. In this context, sufficiently sensitive
genotoxicity test methods are of crucial importance for an appropriate risk assessment. Due to their
versatile applicability, the micronucleus test and more recently the comet assay have gained
increasing use in environmental biomonitoring. For comparative experiments involving different tester
organisms, carboxin (Cx) was chosen as a representative test compound. Cx is an anilide-type
fungicide which recently has been released as a General Use Pesticide onto the market. This
compound is regarded as a very weak mutagen by USEPA, based on data from several mutagenicity
assays with bacteria and mammalian cells [2]. Our own results with micronucleus tests in human cell
cultures initially confirmed these findings. The highest non-cytotoxic dose (100 ppm for 6 hrs) resulted
in a reproducible but weak increase in micronucleus frequency of 1.57fold over solvent control.
Moreover, no increased DNA migration was detectable in the comet assay. However, if similar
treatment conditions were applied in the Tradescantia-micronucleus assay, micronucleus frequency
was significantly increased 3.25fold. In this test system micronuclei formed in pollen mother cells of
developing inflorescences from exposed Tradescantia plants serve as indicators of mutagenicity [3].
Staining of kinetochores in human cells with CREST antiserum revealed that Cx acts clastogenic as
no increase in CREST+ micronuclei was found after treatment. Taken together, the results obtained so
far suggest differences in the metabolism of this compound between plant and mammalian cells.
Therefore, Cx may exert adverse long-term effects in ecosystems due to its genotoxic action in plants,
despite its very weak mutagenicity in mammalian cells.
[1] Awadhesh N.J. et al. (2000), Mutat. Res. 464: 213-228
[2] EXTOXNET Pesticide Information Profiles, http://ace.orst.edu/cgi-bin/mfs/01/pips
[3] Ma T.-H. et al. (1994), Mutat. Res. 310: 221-230
108
Poster session 2
Major topics:
Molecular epidemiology and biomonitoring,
Low doses and thresholds,
Genotoxicology of metals,
Polymorphism in risk assessment and therapy,
Misc.
P2/1 – P2/36
109
P2/1
POTENTIAL GENOTOXIC RISK FOR HUMANS BY THE ENVIRONMENTAL
CONTAMINANT 3-NITROBENZANTHRONE
V.M. Arlt1,2 ; C.A. Bieler1 ; M. Wiessler1 ; D.H. Phillips2 ; H.H. Schmeiser1
1Division
of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany, 2Institute
of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK.
Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in
humans. Of the compounds found in diesel exhaust and in airborne particulate matter 3nitrobenzanthrone (3-NBA) is a particularly powerful mutagen and was shown to be genotoxic in vitro
and in vivo in rats by forming DNA adducts. In this study a panel of V79 Chinese hamster fibroblast
cell lines, expressing various human cytochrome P450 (CYP) enzymes (CYPh1A1, CYPh1A2,
CYPh3A4) and/or human NADPH:CYP oxidoreductase (CYPhOR) was used to identify enzymes
involved in the metabolic activation of 3-NBA in humans. We analysed the formation of specific 3NBA-adducts by
32P-postlabelling
after exposing cells to 1 µM 3-NBA. In all cell lines tested, an
identical pattern with a total of four distinct 3-NBA-adducts was found similar to those found in vitro
using xanthine oxidase or rat liver S9 as the activating system and in rats in vivo. On TLC plates all
adducts migrated primarily along a diagonal zone, typical for DNA adducts derived from extracts of
airborne particulate matter. Total adduct levels ranged from 75 to 220 adducts per 108 nucleotides
using either the nuclease P1 or butanol enrichment, respectively. Comparison of activation of the
parental cell line V79MZ with activation in cells expressing CYPhOR alone or expressing both
CYPhOR and CYPh3A4 demonstrated that both enzymes were involved in the metabolic activation of
3-NBA. Furthermore, in V79NH cells expressing high activities of nitroreductase and N,Oacetyltransferase (NAT2), high adduct levels of up to 1 adduct per 10 4 nucleotides were detected.
When patterns produced in V79MZ cells were compared to those in V79NH cells no additional adducts
were obtained. Our results demonstrate that 3-NBA binds covalently to DNA after metabolic activation,
forming multiple DNA adducts that are all products derived from reductive metabolites. These results
further suggest that nitroreduction is the major pathway in the bioactivation of 3-NBA. Moreover,
acetylation of the initially formed N-hydroxy arylamine intermediates may contribute to the high
genotoxic potential of 3-NBA.
110
P2/2
CHINESE HERBS NEPHROPATHY AND UROTHELIAL CARCINOMA: AN OUTBREAK IN BELGIUM
V.M. Arlt1,3 ; J.L. Nortier2 ; J.-L. Vanherweghem2 ; H.H. Schmeiser1
1Division
of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany,
2Department
of Nephrology, Hôpital Erasme, Brussels, Belgium, 3Present address: Institute of Cancer
Research, Haddow Laboratories, Sutton, Surrey, UK.
Chinese herbs nephropathy (CHN), a unique type of nephropathy, associated with the prolonged
intake of Chinese herbs during a slimming regimen, was reported for the first time in Belgian women in
1993. From about 1500-2000 patients treated with this regimen already more than 100 CHN cases
have been identified, half of whom needing renal transplantation. The toxic effects have been traced to
Aristolochia fangchi containing nephrotoxic aristolochic acid (AA) inadvertently included in the weightreducing pills. In a group of 39 CHN patients with end-stage renal disease we have now found 18
cases with urothelial carcinoma (UC) (prevalence 46%) and 19 cases showed mild to moderate
dysplasia. Logistic regression analysis predicted that the cumulative dose of Aristolochia fangchi was
associated with a significantly higher probability of developing UC (P=0.045). Thus, CHN patients with
a mean intake of 200 g of Chinese herbs had a 50% higher risk of urothelial cancer. In contrast we
found no evidence for confounding effects of concomitantly administered medication on the
development of UC (specifically, appetite suppressant drugs, analgesic use and cigarette smoking).
Using the
32P-postlabelling
method we found specific AA-DNA adducts, described biomarkers of AA
exposure and associated with AAs carcinogenic and mutagenic activity, in all urothelial tissues
analyzed. The major adenosine adduct of aristolochic acid I (dA-AAI), the major component of the
plant extract AA, was detectable in renal (RAL from 0.12 to 16.5 per 10 8 nucleotides, n=61) and
ureteral (RAL from 0.25 to 3.0 per 108 nucleotides, n=17) tissues even 7 years after the patients
stopped taking the herbal drugs. However, no quantitative relationship was found between mean dAAAI-levels in renal tissue from CHN patients who developed UC and those from tumor-free CHN
patients (RAL 2.9  0.9 vs 3.1  0.8 per 108 nucleotides, P=0.91). In conclusion our results
demonstrate that AA is the causal factor in CHN. Our data clearly indicate that exposure to AA and in
particular the formation of AA-DNA adducts is involved in the development of urothelial cancer in CHN
patients. Furthermore, our results highlight the need for vigilance to ensure that AA is not present in
herbal medicinal remedies.
111
P2/3
THE CHANGES OF SPONTANEOUS FREQUENCY OF CHROMOSOMAL
ABERRATIONS IN THE 20 YEARS PERIOD IN THE CZECH REPUBLIC
POPULATION GROUPS
1H.
Bavorova; 1D. Ocadlikova; 1P. Rössner; 2R. J. Sram
1National
Institute of Public Health, Prague, Czech Republic;
2Laboratory
of Genetic Ecotoxicology AS CR, Prague, Czech Republic
Spontaneous level of chromosome aberrations in different age groups in the Czech Republic was
determined to evaluate the significance of occupational and non-occupational exposure. The
knowledge of spontaneous level of chromosomal aberrations of non-exposed population groups had
been used to evaluate possible health effects of industry contamination of the Czech Republic by
genotoxic factors. Cytogenetic analysis from whole blood was carried out in short-term cultures. At the
time of blood drawing a questionnaire was administered. The questions covered a brief medical and
family history including age, sex, medication, infectious diseases, smoking habits, X-ray examinations,
alcohol consumption etc. The cultivation time was 52 hours with all cells being in the first mitosis. A
total of 100 well-spread metaphases containing 46  1 centromeres were examined per donor on
coded slides. The four categories of chromosome aberrations were evaluated: chromatid and
chromosome breaks, chromatid and chromosome exchanges. Cells bearing breaks or exchanges
were classified as aberrant cells. Gaps were recorded but not scored as aberrations. The obtained
data represent a basis for quantification of exposure and for preventive measure application.
Laboratories of Genetic Toxicology obtained presented cytogenetic analysis data in the course of last
20 years.
Results of the cytogenetic analysis from control individuals
(N = 6 810) indicated elevation of
spontaneous frequency of aberrant cells (AB.C.) with age. The mean levels in the period 1977 - 1999
were 1.11 % AB.C. (N = 763) in newborns; 0.86 % AB.C.(N = 134) in the group 5 – 6 yr.; 1.47 % (N
= 2 078) in the group 7 - 15 yr.; 1.62 % AB.C. (N = 390) in the group 16 - 19 yr. and 1.60 % (N =3
445) in the group 20 - 59 yr. Interesting results seem to be the fact that level of AB.C. decreased in all
age groups (except newborns) since 1994.
This work was also supported by Contract EC – QLK4-2000-00628 – Cytogenetic Biomarkers and
Human Cancer Risk.
112
P2/4
MICRONUCLEI IN UNCULTURED T-LYMPHOCYTES OF RAILROAD WORKERS
EXPOSED TO TRANSIT CHEMICALS
G. Falck1 ; H. Järventaus1 ; T. Kallas1 ; J. Catalán2, ; L. Pitkämäk3i ; and H. Norppa1
1Finnish
3VR
Institute of Occupational Health, Helsinki, Finland; 2University of Zaragoza, Zaragoza, Spain;
Ltd, Kouvola, Finland
Railway transport of complex chemical mixtures from Russia to Finland for further industrial use in
Finland or in central Europe may involve occupational exposure to genotoxic agents such as PAHs,
benzene, and styrene. Previous studies showed that railway workers in contact with transit chemical
wagons have an increased frequency of chromosomal aberrations in their peripheral lymphocytes.
Four years after starting a campaign aiming at reducing the chemical exposure, a group of tank wagon
inspectors, still considered potentially exposed, were examined for cytogenetic damage. We present
here the results of micronucleus (MN) analysis of immunomagnetically isolated T-lymphocytes from 17
inspectors and 14 referents, all non-smoking men. Uncultured T lymphocytes were examined to
assess genotoxic damage that had occurred in vivo. Preliminary results suggested similar total
frequencies of micronucleated cells among the exposed workers and the referents. When the MN
found were characterised by fluorescence in situ hybridisation (FISH), there were no clear differences
between the exposed and referents in the frequency of centromere-positive or -negative MN. The
centromeric label was observed in 2/3 of all MN, indicating that most MN in T-lymphocytes of men in
vivo contain whole chromosomes (or chromatids). The occurrence of both the X and Y-chromosomes
in MN was higher than would be expected assuming equal contribution by all chromosomes. The
negative findings concerning MN induction by the occupational exposure agree with a parallel analysis
of chromosomal aberrations. Sex chromosomes appear to be over-represented in lymphocyte MN of
men in vivo, confirming previous results obtained in vitro.
113
P2/5
DIFFERENCES IN SMOKING-RELATED DNA ADDUCT LEVELS IN TUMOROUS
AND NON-TUMOROUS TISSUES FROM LUNG CANCER PATIENTS
E. Győrffy 1; Z. Győri2; I. Soltész3; S. Kostič3; A. Csekeő3; J. Minárovits2,; B. Schoket1
1József
Fodor National Center for Public Health; 2Béla Johan National Center for Epidemiology;
3Korányi
National Institute of Pulmonology; Budapest, Hungary
The correlation between levels of biomarkers of exposure in the target and surrogate tissues is an
important issue in human genotoxic exposure and risk assessment. The aim of the present study was
to obtain knowledge of the formation of aromatic DNA adduct levels in various tissues from smokers
and non-smokers. Tumorous and non-tumorous peripheral lung tissue samples, pieces of normal
bronchial tissue and peripheral blood lymphocytes were obtained from patients undergoing lung
surgery. Aromatic DNA adduct levels were measured by the
32P-postlabelling
method with nuclease
P1 adduct enrichment. Smoking caused significantly higher adduct levels for each type of tissues,
except peripheral blood lymphocytes, as compared to non-smokers (P≤0.02). In smokers, DNA adduct
levels were 1.6 to 1.8-fold higher in normal lung and bronchial tissues than in tumorous lung tissues
and peripheral blood lymphocytes (P≤0.02). There was a strong correlation between adduct levels in
the normal bronchial and peripheral lung tissues. Differences of the mean levels in tissue samples
from non-smokers were of borderline or not statistically significant. The tissue-specific variations in
DNA adduct levels may originate from different internal doses of cigarette smoke-derived components
reaching the tissues, differences in metabolic activation and detoxification and the capacity of DNA
repair processes.
114
P2/6
BIOLOGICAL SAMPLE COLLECTION AND PROCESSING IN AN ON SITE
LABORATORY IN ESTONIAN SHALE OIL MINE - BIOMODEM STUDY
L.E. Knudsen; A. Jensen; J. Kusova; J. Kubackova; V. Muzyka; R. Anzion; P. Scheepers
Institute of Public Health, University of Copenhagen, Denmark
Regional Institute of Hygiene, Ostrava, Czech Republic
Institute of Experimental and Clinical Medicine, Tallinn, Estonia
University Medical Centre St Radboud, Nijmegen, The Netherlands
The BIOMED-concerted action “BIOMarkers for Occupational Diesel exhaust Exposure Monitoring
(BIOMODEM)” project implied biological sampling from 100 workers of an Estonian shale oil mine.
Experiences from pilot studies in Ostrava in Czech Republic and Kotla-Järve in Estonia stressed the
need of fast processing and storage of biological samples Therefore, in the main study a field
laboratory was set up in a building at the mine. Urine samples were collected pre-shift and post-shift
after one workday and after 3-4 workdays. Urine samples were seperated into 6 aliquots and stored at
–20°C.
Blood samples were collected in CPT-tubes and processsed with isolation of lymphocytes,
erythrocytes and plasma. The detailed steps in the processing will be presented together with yields of
cell populations compared with amount of blood processed. The samples were stored in liquid nitrogen
on site and transported on dry ice to laboratories in the United Kingdom, Denmark and Tallinn. The
transport of processed samples was taken care of by the project participants to avoid delay with
consequent thawing.
115
P2/7
URINARY MUCONIC ACID AND PHENYL MERCAPTURIC ACID EXCRETION IN
ESTONIAN SHALE OIL MINE WORKERS DEPEND ON GST - GENOTYPES
L.E. Knudsen; A. Jensen; S.Loft; H.Autrup; J.Poole
Institute of Public Health, University of Copenhagen, Denmark
Institute of Environmental Medicine, University of Århus, Denmark
MRC Environmental Epidemiology Unit, University of Southhampton, UK
The BIOMED-concerted action BIOMarkers for Occupational Diesel exhaust Exposure Monitoring
(BIOMODEM) project implied biological sampling from 100 workers (50 underground and 50 surface)
in an Estonian shale oil mine (shale with oil with up to 3% of benzene). Urine samples were collected
pre-shift and post-shift after one workday and after 3-4 workdays. Urine samples were analysed for
metabolites of benzene bioactivated by CYP2E1, of trans-trans muconic acid
(MA) and
phenylmercapturic acid (PMA). Genotypes of Glutathione-S-transferase M1, T1 and P1 were
determined. The comet assay was used for assessment of DNA damage.
Urinary levels of MA and PMA were increased in smokers. Both levels increased from baseline to
post-shift samples in underground but not in surface workers. Higher levels of PMA were found in the
urine of GSTT1 positive genotype workers compared with GSTT1 null, significantly higher (p<0.05) for
post-shift samples after adjusting for smoking. GSTM1 positive genotype was also associated with
increased level of PMA. The interactions between genotype, exposure and DNA damage will be
further discussed.
116
P2/8
LEUKOCYTE
DNA
DAMAGE
IN
FIBERGLASS-REINFORCED
PLASTIC
WORKERS MEASURED BY THE COMET ASSAY
B. Laffon1,2; E. Pásaro2; J. Méndez1
1Dept.
Cell and Molecular Biology and 2Health Sciences Institute, University of A Coruña, Campus A
Zapateira s/n, 15071-A Coruña, Spain.
Styrene is an important industrial chemical, widely used in the production of plastics, resins and
synthetic rubber. The highest human exposure to styrene take place by inhalation during the
manufacture of fiberglass-reinforced plastics, specially large items such as boats that involve
lamination by hand procedures (Miller et al., 1994). Styrene and its main reactive metabolite styrene7,8-oxide (SO) have been demonstrated to be genotoxic in vitro (Scott and Preston, 1994; Laffon et
al., 2001).
In this work, we have evaluated DNA damage in peripheral leukocytes of a group of fiberglassreinforced plastic workers by means of the comet assay, in comparison with a control population, in
order to enlarge the knowledge about the genotoxic risk associated to in vivo styrene exposure. A
significant increase in comet tail length was found in the exposed population, according to the data
described by Vodicka et al. (1995) and Somorovská et al. (1999) for styrene exposure levels between
7 and 65 ppm.
DNA damage and length of exposure have been shown to be correlated, indicative of an increment in
individual sensitivity with time as a consequence of the chronic exposure to styrene. Moreover, the
influence of smoking habit on DNA damage has been detected in the exposed population, probably
associated to an increase in sensitivity to tobacco smoke components in these subjects due to the
continuous styrene exposure. Nevertheless, a solid statistical evaluation on the connection between
GSTM1 and GSTT1 genotypes and the comet assay parameter could not be performed.
References
Laffon, B.; Pásaro, E. and Méndez, J. (2001) Mutat. Res. 491: 163-172.
Miller, R.R.; Newhook, R. and Poole, A. (1994) Crit. Rev. Toxicol. 24: S1-S10.
Scott, D. and Preston, R.J. (1994) Mutat. Res. 318: 175-203.
Somorovská, M.; Jahnová, E.; Tulinská, J.; Zámecníková, M.; Sarmanová, J.; Terenová, A.;
Vodicková, L.; Lísková, A.; Vallová, B.; Soucek, P.; Hemminki, K.; Norppa, H.; Hirvonen, A.;
Tates, A.D.; Fuortes, L.; Dusinská, M. and Vodicka, P. (1999) Mutat. Res. 428: 255-269.
Vodicka, P.; Bastlová, T.; Vodicková, L.; Peterková, K.; Lambert, B. and Hemminki, K. (1995)
Carcinogenesis 16: 1473-1481.
117
P2/9
ANALYSING MUTATION SPECTRA
P. D. Lewis and J. M. Parry
Mammalian Gene Mutation Database, University of Wales Swansea, UK
Over the last decade there has been a dramatic increase in the number of publications of mutation
data for a wide range of agents. This data has been obtained using a number of different gene
mutation detection systems in both bacterial and eukaryotic cells as well as transgenic rodent models.
Generally, the mutation data for the DNA sequence under investigation is presented as a mutation
spectrum. A mutation spectrum is a simple bar chart of the DNA sequence showing where the
mutations were observed, at what frequency and, importantly, reveal mutation prone sites (hotspots).
Mutation spectra have been widely used to determine the specificity of mutagen interaction with a
target nucleotide sequence (Lewis et al., 2000) and in molecular epidemiological studies to reveal
disease linked mutation hotspots such as at codon 273 of the p53 gene in lung cancer. In the
literature, statistical analysis of mutation spectra for mutagens has generally involved a comparison of
the mutagen and control (spontaneous) spectra using the hypergeometric test. The rapid accumulation
of mutation data for chemical and physical mutagens has led to the construction of publicly accessible
internet resources such as The Mammalian Gene Mutation Database (Lewis et al., 2000), itself
containing data for over 30 000 mutants derived from over a hundred different chemicals. This has
ultimately led to the requirement for multiple comparisons of mutation spectra and the development of
new methods and software to perform the task of grouping mutagens by their 'mutation fingerprints'.
We tested existing methods such as Multivariate Statistics and Similarity Pattern Analysis for
comparing multiple mutation spectra and designed new approaches and a software package that will
assist in the visualisation and pattern identification of large groups of mutation spectra.
P.D. Lewis, J.S. Harvey, E.M. Waters and J.M. Parry (2000). Mutagenesis, 15, 411-414
118
P2/10
MICRONUCLEI ANALYSIS IN PERIPHERAL LYMPHOCYTES OF HOSPITAL
WORKERS OCCUPATIONALLY EXPOSED TO IONIZING RADIATIONS.
F. Maffei1, S. Angelini1, G. Cantelli Forti1, V. Lodi2, F.S. Violante2, S. Mattioli3, P. Hrelia1.
1Department
of Pharmacology, University of Bologna, Bologna, Italy 2Occupationally Medicine Unit,
Sant’Orsola Malpighi Hospital Bologna, Italy, and 3Health Documentation Center of Regional Agency
for Health Care of Emilia Romagna Region.
In medical surveillance programs devised for workers regularly exposed to low levels of ionizing
radiations, risk evaluation is usually done by physical dosimetry. However, this approach does not
provide any information on the late effects of ionizing radiation, such us carcinogenesis. Genetic
markers could be the biological end-point of choice for assessing the effects of ionizing radiations in
exposed populations.
In the present study the micronuleus (MN) assay was used as a biomarker to investigate chromosomal
damage in peripheral lymphocytes from hospital workers exposed to low-level of ionizing radiations.
Moreover, the influence of confounding factors like smoking status, age and gender on MN frequency
was investigated by multiple regression analysis. Sample of peripheral blood were collected from 37
subjects (19 no-smokers, and 18 smokers; mean age: 43.7±8.9), working in radiodiagnostic, and 37
matched controls (20 no-smokers, and 17 smokers; mean age: 41.6±8.3), working in the same
hospital. The results obtained indicated that, overall, the MN frequencies of exposed workers did not
differ from those of controls (MN/1000 binucleated (BN) cells: 6.784.92 and 5.542.99, respectively).
Interestingly, smoking status significantly raised chromosomal damage among the exposed workers
(smokers: 8.835.94 MN/1000 BN, no-smokers: 4.842.61 MN/1000 BN; p= 0.011 Wilcoxon test), but
not among controls (smokers: 6.001.94 MN/1000 BN no-smokers: 5.153.67 MN/1000 BN). This
suggests that smoking and ionizing radiation might exert a synergistic influence on chromosomal
damage. Among both exposed workers and controls, MN frequency was found to increase with age.
On the other hand, no relationship between gender and MN frequency emerged in either group. The
information obtained in the study indicates that the effect of cigarette smoking should be carefully
factored in to genetic monitoring studies assessing the risks associated with low-level radiation
exposure.
This work was jointly supported by a MURST (grant ex-60%) and a special grant to Francesca Maffei
from University of Bologna for the project “Giovani Ricercatori”
119
P2/11
INFLUENCE OF PHASE II ENZYMES ON BIOMARKERS OF EXPOSURE AND
EFFECT IN SILESIAN CHILDREN EXPOSED TO POLYCYCLIC AROMATIC
HYDROCARBONS
D. Mielżyńska, K. Szyfter*, E. Siwińska, L. Kapka, R. Jaskuła –Sztul*
Laboratory of Environmental Mutagenesis, Institute of Occupational Medicine and Environmental
Health, Sosnowiec, Poland
*Laboratory of Mutagenesis, Institute of Human Genetics, Polish Academy of Sciences, Poznań,
Poland
Individual variation in genetic suseptibility to environmental exposure to PAHsis recogized as a factor
in chemical carcinogenesis. The objective of our study is to assess of influence of polymorphic
detoxification genes like GSTM1, GSTM3, GMST1, GSTP1, EPHX and NAT2 on biomarkers of
exposure (urinary mutagenicity and 1-hydroxypyrene, aromatic
DNA adducts)
and effect
(chromosomal aberrations, micronuclei and sister chromatid exchange) in children living in two towns
in Silesia Region. GST, EPHX and NAT metabolic genotypes were determined on white-cell DNA from
peripheral blood using the PCR technique. Urinary mutagenicity was tested by the plate incorporation
Ames test using conventional strain TA 98 and other Salmonella typhimurium strains with elevated
levels of nitroreductase and/or O-acetylotransferase enzymes YG: 1021, 1024, 1041 with metabolic
activation. Urinary 1-hydroxypyrene concentration was determined by the HPLC-method. DNA adduct
analysis was performed by the
32P-postlabelling
assay. Chromosomal aberrations, the level of
micronuclei and the number of sister chromatid exchanges were evaluated in children lymphocytes
according to the routine procedures. We evaluated the influence of single polymorphism of all
determed genes and combined genotypes: GSTM1/GSTT1 and GSTM1/NAT2. We found that
polymorphism of GSTM3 did not influence any biomarkers. Urinary mutagenicity was influenced by
polymorphism of other genes but the results depended on applied strains. Only slow acetylators had
statistically lower levels of 1-hydroxypyrene. Statistically higher levels of aromatic DNA adducts were
observed in children with genotypes GSTM1 (0) and GSTT1 (0). Single polymorphism of analysed
enzymes did not influence any biomarker of effect: CA, MN, SCE and HFC. Children with deficient
genotypes of genes GSTM1/GSTT1 excreted less mutagens in urine tested by TA98+S9 and reveled
3 time higher levels of aromatic DNA adducts in PBL and higher SCE. Deficiency of combined genes
GSTM1/NAT2 resulted only in increase of DNA adducts.In conclusion we think the increase of SCE in
PBL may depend on exposure to PAHs and combined deficiency of genes GSTM1/GSTT1.
120
P2/12
BIOMONITORING STUDY OF A GROUP OF WORKERS OCCUPATIONALLY
EXPOSED TO PAINTS AND SOLVENTS.
Migliore L., R. Bibbiani, Z. Ricevuto, M. Vicentini*, N. Serretti*, G. Loprieno**.
Dipartimento di Scienze dell’Uomo e dell’Ambiente, Università di Pisa,
*Azienda U.S.L.5, Pisa,.
**Dipartimento della Prevenzione, A.S.L.2, Lucca, Italy
Car painters should be exposed to an extensive variety of hazardous substances such as ketone,
aliphatic, aromatic and ester solvents, organic and inorganic pigments and several types of resins,
which may cause damage to human health, even if in the last years there has been a gradually
technological improvement of the productive processes and a better attention for the production, for
the choice of the employing materials, and in particular the use of suitable hygienic measures.
We employed biomarkers of exposure and genotoxicity to perform a biomonitoring study of a group
of car painters. The analysis of chemicals was performed using active (environmental) and passive
(personal) sampler, and HPLC chromatography frequencies; lung function parameters were also
evaluated. The human lymphocyte micronucleus assay coupled with fluorescence in situ hybridization
(FISH) analysis using a pancentromeric probe, was carried out on 45 male car painters aged 25 to 55
years who had been working for a period of at least 5 years, employed into automobile body and
painting shops located in Pisa. The control group consisted of 36 healthy (unexposed) males of
similar age and smoking habit. The chemical
analyses confirmed the presence of 62 different
compounds which are present in more than 103 final products; only 25 chemicals were used in all the
workplace considered. The data were grouped by individual workplace and relevant working phase
(car preparation, dying, and drying). According to the working-phase analysis, a relevant exposure to
toluene was found during transparent dye drying phase (40,98 mg/m³) but levels are under the
correspondent TLV, as well as those of styrene found during part preparation (6,47 mg/m³). Our
results show that the micronucleus frequency in peripheral blood lymphocytes does not increase
linearly with exposure (either using individual data or workplace average data); age, on the contrary, is
confirmed to be an important confounding factor. The same negative finding was found using
spyrometer evaluation and MN/chemical data. A series of multivariate correlation and statistical
analysis were performed using the questionnaire, but no significant correlations were found.
121
P2/13
CYTOGENETIC STUDIES IN SOMATIC AND GERM CELLS
INDIVIDUALS EXPOSED TO STYRENE IN THE WORKPLACE.
OF
MALE
Naccarati A.; A. Zanello; R. Scarpato; L. Lastrucci*; L. Migliore
Dipartimento di Scienze dell’Uomo e dell’Ambiente, Pisa University and *Dipartimento di
Prevenzione U.O. Igiene e Medicina del Lavoro, Azienda USL 12, Versilia, Italy
A study employing different biomarkers of exposure and genotoxicity in somatic and in germ cells was
carried out in a group of subjects occupationally exposed to styrene. The biomonitoring included 48
exposed male individuals workers and 31 male controls of comparable mean age, recruited from three
different areas of the Tuscany region.
Urinary mandelic acid (MA) was measured as indicator of external exposure. Blood samples were
assayed for the presence of micronuclei (MN) in lymphocytes (cytokinesis block method), FISH
technique for the detection of structural or numerical chromosome damage by means of a
pancentromeric DNA probe was also performed.
There was a significant increase in the frequency of MN in the exposed workers compared to controls
(13.7‰ ± 5.0 vs. 6.0‰ ± 4.8 , p<0.001).
To evaluate primary DNA damage in germ cells we employed the single cell gel electrophoresis
(SCGE), or comet assay and performed three-colour fluorescence in situ hybridisation (FISH) with
centromere-specific DNA probes to study aneuploidy and diploidy of both sex chromosomes and
chromosome 2 simultaneously in decondensed sperm nuclei. We found a significant difference in
sperm DNA integrity between the two groups (mean comet tail DNA percentage being 10.9 ± 3.0 and
7.4 ± 2.3 in exposed and controls, respectively). The comet assay resulted thus sensitive in the
detection of a significant effect of occupational exposure in DNA integrity of germ cells. The incidence
of aneuploidy for all tested chromosomes did not result statistically different between the two groups of
exposed and controls, with exception that the frequency of nullisomy for sex chromosomes observed
in sperm nuclei was higher in exposed individuals than in controls (0.24  0.15 versus 0.15  0.10,
p<0.05). In general a statistically significant difference (p<0.001) was observed in the rate of total
aneuploidy for the sex chromosomes (0.43  0.14) in comparison with that of the autosome (0.21 
0.10).
The concentrations of MA in the urine were not significantly correlated with genotoxic damage,
however multiple regression analysis revealed a positive correlation between the biomarkers of
genotoxicity in somatic and in germ cells evaluated by MN assay and comet assay, respectively.
122
P2/14
A BIOMARKER APPROACH TO DETECT EARLY DNA DAMAGE AND
GENOTOXIC
RISK
OF
COMMON
ENVIRONMENTAL
POLLUTANTS
IN
ADOLESCENTS
T Nawrot1; E Den Hond1; HA Roels2; L Verschaeve3; G Koppen3, JA Staessen1
1Studiecoördinatiecentrum,
Departement Moleculair en Cardiovasculair Onderzoek, Katholieke
Universiteit Leuven, Faculteit Geneeskunde, Leuven, Belgium.
2Unité
de Toxicologie Industrielle et de Médecine du Travail, Université catholique de Louvain,
Bruxelles, Belgium.
3Vlaamse
Instelling voor Technologisch Onderzoek, Mol, Belgium.
Biomarkers of DNA damage were studied in relation to common environmental pollutants in 200
adolescents of 16-18 years old. The study participants [80 boys and 120 girls: mean age of 17.4 (SD
0.8)] were recruited in Belgium from a rural control area (Peer; n = 100) and from two polluted suburbs
(Wilrijk; n = 42 and Hoboken; n = 58). We measured (1) as biomarkers of oxidative DNA damage the
urinary concentration of 8-hydroxy-2-deoxyguanosine (8-OH-dG) and the percent DNA in the tail area
of the comet assay; (2) as biomarkers of genotoxic risk we used chromatid breaks, chromosome
aberrations and micronuclei (in 100 randomly selected subjects); (3) the biomarkers of exposure to
genotoxic pollutants were t,t-muconic acid (for benzene), 1-hydroxypyrene (for PAHs), and o-cresol
(for toluene) in urine; and (4) the plasma/serum concentrations of antioxidants (vitamins A and E) and
selenium (component of glutathione peroxidase) were used as biomarkers of individual susceptibility.
The atmospheric ozone concentration during the week proceeding the day of blood sampling was also
taken into account. Stepwise multiple regression analysis showed that the comet assay results were
significantly and positively correlated with the urinary concentrations of 1-hydroxy-pyrene (partial
r=0.15; p=0.017), o-cresol (partial r=0.25; p<0.001), and mean atmospheric ozone concentrations
(partial r=0.46; p<0.001).
Urinary levels of 8-OH-dG were positively related to o-cresol (r=0.30;
p<0.001). Relative risk of chromatid breaks was 1.74 (p=0.01) for a two-fold increase in the urinary
concentration of t,t-muconic acid or 1-hydroxypyrene (1.58; p=0.01).
Probability of chromosome
aberrations was 1.56 (p=0.02) for a doubling of the urinary 1-hydroxypyrene concentration.
No
relation was found between micronuclei and the biomarkers of exposure. None of the measured
antioxidants was significantly correlated with any of the biomarkers of DNA damage. In conclusion,
oxidative DNA damage and markers of genotoxity were found to be associated with environmental
pollution.
123
P2/15
INFLUENCE
OF
CYP1A2
AND
NAT2
PHENOTYPES
ON
URINARY
MUTAGENICITY IN CIGARETTE SMOKERS.
S.Pavanello1, P. Simioli2, S. Lupi2, P. Gregorio2, and E. Clonfero1
1
Section of Occupational Health, Department of Environmental Medicine and Public Health University
of Padova, Via Giustiniani 2 Padova Italy.
2Section
of Hygiene and Occupational Medicine, Department of Clinic and Experimental Medicine,
University of Ferrara, Italy.
The influence of metabolic phenotype NAT2 and CYP1A2 on urinary mutagenicity of 73 smokers (at
least 10 cigarettes /day) was studied. In the late-afternoon urine samples, mutagenicity, by Ames test
(preincubation plate incorporation assay on YG1024 Salmonella typhimurium strain in presence of S9
mix), and nicotine plus metabolites were determined. The NAT2 and CYP1A2 phenotypes were
measured by the molar ratio of urinary caffeine metabolites, determined by HPLC analysis, as follow:
CYP1A2 = (AFMU+1X+1U)/17U and NAT2= AFMU/(AFMU+1X+1U). An AFMU/ (AFMU+1X+1U) ratio <
0.3 defined NAT2 slow acetylators, while (AFMU+1X+1U)/17U ratio < 5.0 defined CYP1A2 poor
metabolizers (PM). On the other hand ratios > 0.3 and >0.5 defined rapid acetylators and extensive
metabolizers (EM), respectively.
Frequencies of slow/rapid NAT2 and PM/EM CYP1A2 phenotypes were 63%, 37% and 37%, 63%,
respectively.In smokers with high urinary mutagenicity corrected by nicotine plus metabolites (>2000
net revertants /mg of nicotine plus metabolites), the EM frequency was significantly higher than those
EM with low urinary mutagenicity (70% vs 59%; chi square =9.29, p<0.0023). In the present study,
while NAT2 phenotype alone did not have any influence on urinary mutagenicity of smokers, the
frequency of combined phenotype NAT2 rapid/ CYP1A2 EM was found significantly higher in smokers
with high mutagenic activity, than in those with low mutagenic activity (25%vs16%; chi square 11.76,
p=0.0006).
In conclusion, the CYP1A2 dependent increased levels of urinary mutagens suggest that phenotypic
differences in metabolic activation/detoxification of tobacco smoke mutagens are able to modulate the
presence of promutagens in urine of cigarette smokers. The influence of NAT2 phenotype that, alone or in
combination even with GSTM1 null genotype, has already been found by other Authors to modulate
smokers urine mutagenicity, require further elucidation.
124
P2/16
A STUDY ON THE EFFECTS OF SEASONAL SOLAR RADIATION ON EXPOSED
POPULATIONS
S. Tsilimigaki 1,3, N. Messini-Nikolaki 3, M. Kanariou 2, and S.Μ. Piperakis.1
1DNA
Repair Laboratory, Institute of Biology, National Center of Scientific Research
‘Demokritos’, Athens, Greece.
E-mail : [email protected]
2Department
of Immunology & Histocombatibility, Agia Sofia Hospital, Αthens, Greece.
3Department
of Cell Biology, School of Biology, University of Athens, Athens, Greece.
Sunlight is a significant human carcinogen (IARC 1992, Longstreth et. al. 1998) and in terms of
absolute numbers of cancers is one of the most significant carcinogens. There has been an enormous
increase in all forms of skin cancer in Europe over the last 40 years due to sun exposure. (Longstreth
et. al. 1998).
In the present study the effects of seasonal solar radiation in exposed populations of two different age
groups (20-25 and 40-55 years old) were evaluated. The populations examined were selected with the
use of a detailed questionnaire containing questions on health, lifestyle, diet, age, smoking habits,
hours of exposure under the sun etc. The damage in DNA was estimated with the comet assay
technique which is a sensitive and rapid method for the detection of DNA breaks (Piperakis et. al.
1999). These breaks migrate faster towards the anode giving thus the impression of comets, which
can be observed under a fluorescence microscope after staining with a fluorescent DNA binding stain.
Evaluation of the induced DNA breaks was done with an image analysis system connected to a
computer with a suitable program (kinetic image analysis). Statistical analysis was performed with a
non parametric test (Kruskal-Wallis). In addition the effects of external factors e.g. H 2O2 , γ-radiation
and the DNA repair efficiency in these populations were also examined.
Our results show significant effects of the solar radiation on the exposed populations during the
summer months if compared with winter. This was also influenced by the age group.
IARC Solar and ultraviolet radiation, IARC Monograph, Lyon, 1992.
Longstreth J, De Gruijl F.R, Kripke M.L, Abseck S, Arnold F, Slaper H.I, Velders G, Takizawa Y, and van der Leun J.C. Health Risks. J. Photochem. Photobiol. B. Biol. 1998, 46,
20-39.
Piperakis S.M, Visvardis E-E, Sagnou M, and Tassiou A.M. Comet assay for nuclear DNA
damage. Methods in Enzymology. 1999, 300, 184-194.
125
P2/17
DNA DAMAGE-REPAIR IN A POPULATION WITH CHRONIC PSYCHOGENIC
STRESS
E. Dimitroglou
1,3,
M. Zafiropoulou 2, N. Messini-Nikolaki 3, S. Ntountounakis 4, S. Tsilimigaki
1
and
S.Μ. Piperakis.1
1DNA
Repair Laboratory, Institute of Biology, National Center of Scientific Research
‘Demokritos’, Athens, Greece.
2Developmental
E-mail : [email protected].
Psychology and Psychopathology Laboratory, School of Human
Studies, University of Thessaly, Volos, Greece.
3Dιvision
of Cell Biology and Biophysics , Department of Cell Biology,
University of Athens, Athens, Greece.
4Department
of Cystic Fibrosis, Agia Sophia Hospital, Athens, Greece.
Stress is defined as a negative emotional experience accompanied by predictable biochemical,
physiological, cognitive, and behavioural changes that are directed either towards altering the stressful
event or accommodating to its effects.
From studies with people
and animals it was found that it causes harmful effects, including
immunological defects (Fischman et al 1996). In the present study we examined the effects of chronic
stress in an exposed group of people compared to a non-stressed sample. The stress levels of our
sample was determined using
the State Trait Anxiety Scale. With the use of the comet assay
technique (Piperakis et al 2000) we measured the background level of DNA damage as well as the
sensitivity of the lymphocytes of this population to external factors (H 2O2, γ-radiation) and its DNA
repair efficiency. The evaluation of the DNA breaks was done by visual scoring and with the image
analysis system. Statistical analysis was performed with a non parametric test (Kruskal-Wallis).
Preliminary results show that stress appears to have some effect on DNA damage-repair processes.
Fischman H.K, Pero R.W, and Kelly D.D. Psychogenic stress induces chromosomal
and DNA damage. Int. J. Neurosci. 1996, 219-227.
Piperakis S.M, Petrakou E, and Tsilimigaki S. Effects of air pollution and smoking on
DNA damage of human lymphocytes. Environm. Molec. Mutagenesis. 2000, 36,
243-249.
126
P2/18
BIOLOGICAL DOSIMETRY IN A GROUP OF NUCLEAR POWER PLANT
WORKERS IN BELGIUM BY MEANS OF CHROMOSOME PAINTING.
Roncancio C.L.1; Laurent C.1.; Thierens H.2 & Lambert V.3.
1. Oncology, Radiobiology and Experimental Mutagenicity Laboratory, University of Liège, Liège.
Belgium. 2. Department of Biomedical Physics and Radiation Protection, University of Ghent, Ghent.
Belgium. 3 Laboratoire de biologie des tumeurs et du développement, University of Liège, Liège.
Belgium
Fluorescence in situ hybridization (FISH) with chromosome specific DNA probes is useful for
quantifying genetic damage, induced by radiation. We analyzed the incidence of chromosomal
aberrations in peripheral blood lymphocytes from 30 occupationally exposed people in a nuclear
power plant in Belgium, involved in the recycling of nuclear fission products, and 20 controls, from the
administrative area. FISH was performed with whole chromosome specific probes for chromosomes
2, 4 and 8 and the genomic translocation frequencies were calculated based on Lucas formula; and
absorbed radiation dose was calculated by using in vitro dose response curve established for
60Co

rays. The presence of dicentric, acentric fragments, insertions, rings and the one way and two ways
translocations have been scored. The frequency of one way translocations were 63% and 70% in the
exposed and control groups, respectively. Individual dose estimates for the exposed populations
ranged between 0,07 and 0,61Gy. Lifestyle factors have been taking into account in the analysis of the
data. Our results confirmed the usefulness of FISH for an accurate estimation of different types of
cytogenetic damage.
127
P2/19
THE INFLUENCE OF OCCUPATIONAL EXPOSURE TO PAHs ON THE
EXPRESSION OF p53 AND p21/WAF1 PROTEINS
P. Rössner Jr.; B. Binková; R. J. Šrám
Regional Institute of Hygiene of Central Bohemia and Institute of Experimental Medicine AS CR,
Prague, Czech Republic
Polycyclic aromatic hydrocarbons (PAHs) seem to be the main source of carcinogenic risk among
coke oven workers. p53 is a tumour supressor protein that is induced after DNA damage and
regulates the transcription of a number of genes responsible for cell cycle arrest and apoptosis.
p21/WAF1 protein is a downstream effector of p53 responsible for G1 or S-phase arrest. In in vitro
studies was shown that PAHs are able to induce the expression of both the p53 and p21/WAF1
proteins.
The effect of occupational exposure to carcinogenic PAHs (cPAHs) on the plasma level of p53 and
p21/WAF1 proteins in coke oven workers was studied. The samples from coke oven workers (N=66)
and matched controls (N=50) were collected during the years 1995-1996. The expression of both the
proteins was determined by ELISA immunoassays.
No difference in the expression of either p53 or p21/WAF1 protein between coke oven workers and
controls was found. No difference between smokers and nonsmokers within the groups was observed.
However, after stratification of all subjects into two groups according to the exposure to cPAHs
(<
1 g/m3 or > 1 g/m3), higher expression of p53 protein was found in the group exposed to cPAHs < 1
g/m3 (P=0.044). Similarly, the expression of p53 protein was higher in coke oven workers from
Košice (Slovakia) when compared with coke oven workers from Ostrava (the Czech Republic;
P=0.004). The personal exposure to cPAHs was lower in Košice as compared with Ostrava. The
expression of p21/WAF1 protein was higher in exposed group from Ostrava, but the difference was
not significant (P=0.056). In the overall study negative correlation between the expression of p53
protein and the levels of cPAHs was found. The expression of p21/WAF1 protein negatively correlated
with vitamin E levels, and in the exposed group (PAHs > 10 g/m3) a positive correlation with the level
of cPAHs was found.
Our results suggest the inhibitory effect of cPAHs on the expression of p53 protein. We can
hypothesize that the lower expression of p53 protein could reduce the repair ability of cells, leading to
the formation of mutations. On the other hand, the higher exposure to cPAHs induce the expression of
p21/WAF1 protein. No correlation between p53 and p21/WAF1 expression was found, probably other
mechanism regulating p21/WAF1 expression may be involved.
The study was supported by the grant of Ministry of Environment of the Czech Republic VaV/340/2/00
and the grant of Grant Agency of the Czech Republic 310/01/P030.
128
P2/20
SPERM CHROMATIN STRUCTURE IN WORKERS EXPOSED TO LOW LEVELS
OF INORGANIC LEAD
M. Spano1, F. Caruso1, G. Leter1, E. Cordelli1, M. Joffe2, P. Apostoli3, S. Porru3, P. Kiss4, M.
Vanhoorne4, F. Comhaire5, A. Giwercman6, L. Bisanti7, W. Zschiesche8, J.P. Bonde9
1
Section of Toxicology and Biomedical Sciences, ENEA Casaccia, Rome, Italy; 2Department of Epidemiology and Public Health,
Imperial College School of Medicine, London, United Kingdom; 3Department of Occupational Medicine and Industrial Hygiene,
University of Brescia, Italy; 4Section of Occupational and Environmental Health, Ghent University, Belgium; 5Laboratory of
Andrology, Gent University, Belgium; 6Department of Urology, Malmö University, Sweden; 7Local Health Authority, Department
of Epidemiology, Milano, Italy;
8
Department of Social and Occupational Medicine, University of Erlangen, Germany;
9
Department of Occupational Medicine, University Hospital of Aarhus, Denmark
Several occupational surveys have linked exposure to inorganic lead with male reproductive toxicity
but adverse effects in the low dose range in humans need to be fully characterized.The flow cytometric
Sperm Chromatin Structure Assay (SCSA), which evaluates chromatin abnormalities in terms of an
increased susceptibility to acid induced in situ DNA denaturation, can detect derailments from the
correct sperm chromatin packaging due to the occurrence of DNA breaks, insufficient protamination
and abnormal -SH expression level. The SCSA is particularly fit for large scale epidemiological
surveys and its results are independent from the parameters of the conventional WHO semen quality
assessment (3). Interestingly, the SCSA was the only assay able to detect reproductive damage after
lead low dose chronic exposure in primates (4). Within the framework of the Asclepios project, an
European Concerted Action on Occupational Hazards to Male Reproductive Capability, the SCSA has
successfully been applied to detect sperm chromatin abnormalities induced by occupational exposure
to pesticides (1) and styrene (2). In this study, the SCSA was selected as biomarker of lead effects in
a cross-sectional semen survey of 503 men employed at 10 companies in England, Italy and Belgium.
The average blood lead concentration was 31.0 g/dl in 362 lead exposed workers and 4.4 g/dl in
141 reference workers. Each man provided one fresh semen sample at enrolment. Lead levels were
measured in the blood and, in a subgroup of 165 individuals, also in seminal plasma and in
spermatozoa. Damage to sperm chromatin structure was not related to blood lead level. However, the
values for the SCSA parameters resulted elevated in men with the highest spermatic concentrations of
lead. Taking also into account the results of the conventional semen quality assessment, we conclude
that lead, at blood levels >50 g/dl, can adversely affect directly the mature sperm integrity as a result
of the competition for Zn-binding sites on protamine 2 (5), offering a sound hypothesis for the known
male fertility impairment after exposure to this gonotoxin.
1) Larsen et al. (1998) Reprod Toxicol 12: 581-589
2) Kolstad et al. (1999) Int Arch Occup Environ Health 72: 135-141
3) Spanò et al. (1998) Hum Reprod 13: 2495-2505
4) Foster et al. (1996) Toxicol Ind Health 12: 723-735
5) Quintanilla-Vega et al. (2000) Am J Ind Med 38: 324-329
129
P2/21
BIOMONITORING OF HUMAN POPULATION EXPOSED TO SIMAZINE IN TAP
WATER
Suárez S., Rubio A., Sueiro R.A., Garrido M. J.
Instituto de Investigación e Análises Alimentarias, Laboratorio de Microbioloxía, Universidade de
Santiago, Santiago de Compostela, Spain
In recent years, the use of pesticides in agriculture has been increasing steadily. This practise could
conduce to the introduction of any pesticides in the trophic chain. An example of this event is the
presence of the herbicide simazine in some rivers that supply water for human consumption in
Extremadura region (South-West of Spain).
The aim of this study was to determine the potential hazard to human populations exposed to
simazine in the water supply as well as than exposed through the consumption of dietary products. For
this purpose, we analyse some cytogenetic markers from two human populations, one exposed and
another one as a control. The exposed group was formed by males living in a town with high levels of
simazine in tap water and the control group included healthy males who, to the best of our knowledge,
had never been exposed to pesticides.
The assays used were the sister chromatid exchange (SCE) and micronucleus test (MN) in human
peripheral blood lymphocytes.
The results obtained showed no differences in the markers studied between the two populations
analysed. In this way, our finding suggests the absence of genotoxic hazard due to simazine
exposure.
This investigation received financial support from FEDER and CICYT Founds (Project Nº 1FD972222-C03-03)
130
P2/22
SISTER CHROMATID EXCHANGE AND PROLIFERATIV RATE INDEX IN A
CROATIAN POPULATION OCCUPATIONALY EXPOSED TO PESTICIDES
D. Želježić and V. Garaj-Vrhovac
Laboratory of Mutagenesis, Institute for Medical Research and Occupational Health, Zagreb, Croatia
At present, there are more than 1000 chemicals classified as pesticides and many reports have shown
that some of them have genotoxic properties. In the present longitudinal study possible genetic
damage on a population of workers occupationally exposed to a mixture of pesticides by using sister
chromatid exchange analysis have been evaluated. As an additional cytogenetic parameter, the
proportion of lymphocytes that undergo one, two or three cell divisions as well as proliferative rate
index have been determined. This study was performed on the exposed group of workers employed in
pesticide production, simultaneously exposed to a complex mixture pesticides (atrazine, alachlor,
cyanazine, 2,4-dichlorophenoxyacetic acid, malathion). The blood samples of the exposed subjects
were collected in three different periods: before the beginning of the new pesticide production period,
after 8 months of everyday work in the pesticide production, and 8 months after the removal of
subjects out of the production. In all three samplings, the mean value of SCE and number of HFCs in
the exposed group was significantly higher in the comparison with the control group. There were no
differences in the PRI between the control and exposed group, no regards to sampling period. In both
groups examined, the majority of lymphocytes were found in the second cell division, following
cultivation. These results suggest that the increase in the number of SCE found in the exposed
subjects is not the results of neither cytotoxic nor epigenetic action of pesticide mixture, but chronic
occupational exposure to mixture of pesticides.
References
1. Latt, S.A., 1981. Sister chromatid exchange formation. Annu. Rev. Genet. 15, 17-62.
2. Ponzanelli, I., Landi, S., Bernacchi, F., Barale, R., 1997. The nature of high frequency sister
chromatid exchange cells (HFCs). Mutagenesis 12(5), 329-333.
131
P2/23
COMPARISON OF Saccharomyces cerevisiae TEST AND COMET ASSAY ON
HUMAN LEUKOCYTES IN THE LOWEST EFFECTIVE DOSE OF CHLORINE
DISINFECTANTS
Annamaria Buschini, Paola Poli, Luca Pasini, Chiara Alessandrini, Carlo Rossi
Istituto di Genetica, Università di Parma, Parma, IT
Many studies have detected the presence of mutagens in drinking water by means of short-term
mutagenicity tests in vitro. Mutagenicity of drinking water is due not only to industrial, agriculture and
urban pollution but also to disinfection treatment using sodium hypochlorite (NaClO) or chlorine
dioxide (ClO2) and especially to their disinfection by-products. All disinfection methods provide a
disinfectant residual dose on the distribution system. The aim of the present research is to evaluate
the different sensitivity against these world-wide used disinfectants in a standardised “short-term” test
on Saccharomyces cerevisiae and the Comet assay on human leukocytes.
Materials and Methods:
Human leukocytes: SCGE was performed basically according to Singh et al., 1988. The blood of
healthy non-smoker donors was treated with different concentrations of disinfectants (ph=13;
unwinding 20’, electrophoresis 20’ at 0.78Vcm -1 and 300mA).
S.cerevisiae D7 (Zimmermann et al.,1975) was used to determine the frequencies of mitotic gene
conversion (GC), point mutation (PM) and mitochondrial DNA mutability.
Results and Conclusion:
The results on S.cerevisiae D7 show a genotoxic response on GC and PM endpoints with
effect from 10-30ppm ClO2 or NaClO, while the disinfection standards are 1-2ppm. It’s worth
noting a mitochondrial genotoxic effect with NaClO without endogenous metabolic activation
(stationary phase cells) and with ClO2 with endogenous metabolic activation (growing cells).
The DNA damage was measured by Comet assay on human leukocytes after 1hour’s treatment
in PBS (37°). The statistic analysis of the mean tail moment on 100 cells, a parameter that
incorporates both the amount of damaged DNA in the tail and the distance of migration, shows
0.5ppm as the lowest effective dose for NaClO and 0.2ppm for ClO2 (Student’s t, p<0.001). The
values exceeding the 95th percentile of the reference distribution, a relevant index of DNA
damage, confirmed the genotoxic effect at concentrations lower than standard ones.
References:
Zimmermann, F.K., Kern, R., Rosenberg, H., 1975. Mutat.Res. 28, 381-388.
Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988.. Exper.Cell Res. 175, 184-191.
132
P2/24
DEVELOPMENT OF AN IN SITU METHOD FOR MICRONUCLEUS ASSAY WITH
HEP G2 AND CHO CELLS.
Fessard V.1; Valentin I.2; Mourot A.1 ; Chagnon M.C.2; Poul J.M.1; Lhuguenot J.C.2
1. AFSSA, Laboratoire d'Etudes et de Recherches sur les Médicaments Vétérinaires et les
Désinfectants, La Haute Marche, BP 90 203, 35 302 Fougères Cedex, FRANCE
2. ENSBANA, Laboratoire de Toxicologie, 1 place Erasme, 21 000 Dijon, FRANCE
In vitro micronucleus test with HepG2 cells was developed by Darroudi et al. (1996). At the end of the
cytochalasin block, cells are trypsined, spread on a slide and stained with Giemsa. However,
development of an in situ method appears attractive in order to get ride of the trypsination and
spreading steps. Considering the difficulty to distinguish between cells within Hep G2 monolayers, in
situ Giemsa staining, as performed with CHO cells, did not give good results. Therefore, we have
developed a more appropriate method using a double labelling : one for nuclei with DAPI staining and
the other one with phalloidin–TRITC. This latter compound binds to F actin and stains the cortical actin
cytoskeleton which delimits the border between two cells.
Using trypsination, spreading and Giemsa straining, good results were obtained with more of 60%
binucleated cells in control HepG2 cultures. Induction of micronuclei was observed with mitomycin C
as a positive control. In vitro micronucleus assay using fluorescence staining gave more reliable
results as previously shown by other authors (Surrallés et al., 1995). A comparison of both methods
will be presented including the results obtained with CHO cells
Adding a further step for centromere staining by FISH or CREST antibody would improve this method
and permit the detection of aneugens or clastogens on the same slide. However, an important
technical problem to solve will be the autofluorescence of phalloidin-TRITC into the green spectrum.
Darroudi F., Meijers C.M., Hadjidekova V., Natarajan A.T. 1996 Mutagenesis, 11(5) : 425- 433
Surrallés J., Catalan J., Creus A., Norppa H., Xamena N., Marcos R. 1995 Mutagenesis, 10 (5) : 417423
133
P2/25
GRISEOFULVIN : DOSE-RESPONSE STUDIES IN HEPATOCARCINOGENESIS
MEDIUM-TERM ASSAY AND IN IN VITRO ANEUPLOIDY INDUCTION IN SPLEEN
LYMPHOCYTES IN THE RAT
K. Labay 1; M. Ould Elhkim 2; M. Poul 1; G. Jarry 1; S. Marteau 1; J.M. Poul 1; P. Sanders 1
1 AFSSA
2
Fougères, Unité de Toxicologie Alimentaire, BP 90203, 35302 Fougères Cedex, France
AFSSA-DERNS, Unité d’évaluation des risques physico-chimiques, 23 avenue du Général de
Gaulle, BP 19, 94701 Maisons Alfort Cedex, France
We recently showed that griseofulvin (GF), an aneugenic compound, had a tumor promoting activity in
rat liver. In order to estimate the threshold of GF-induced hepato-promoting effect, male F344 rats
were given a single dose of diethylnitrosamine (DEN, 200 mg/kg body weight, ip), then were subjected
to a two-thirds partial hepatectomy, followed by an oral administration of GF (50; 100; 250; 500; 1000
and 2000 mg/kg body weight), daily for 12 weeks. Livers were examined immunohistochemically for
expression of glutathione S-transferase placental form (GST-P). A statistically significant increase in
the relative area of GST-P-positive foci (mm² / cm² of liver section) was observed from the dose of 500
mg/kg body weight when compared to rats treated with DEN alone.
To correlate the induction of the GST-P-positive foci to blood concentrations of GF in rats, a
pharmacokinetic study was performed on animals treated orally with the same dose range. GF was
assayed in the plasma using a HPLC method. Mean plasma concentrations of GF ranged from 1 to 3
µg/ml for oral doses ranging from 50 to 2000 mg/kg body weight. The lowest effective plasma
concentration was estimated to be about 2.7 µg/ml. A dose-response study for in vitro aneuploidy
induction by GF is currently in progress on lymphocytes isolated from rat spleen, using the cytokinesis
blocked micronucleus test and FISH staining with a centromeric DNA probe. In vitro and in vivo doseresponse curves will be analysed and both thresholds of GF effects will be compared.
134
P2/26
LOW DOSES OF GAMMA RAYS: MOLECULAR ALTERATION INDUCED IN
HUMAN TUMOR CELLS
M. Osmak; A. Brozović
Department of Molecular Genetics, Ruđer Bošković Institute, Zagreb, Croatia
We have shown previously, that human cervical carcinoma cells irradiated with low doses of gamma
rays (0.5 Gy x 30 fractions; HeLa1500 cells), changed their sensitivity to the subsequent treatment
with various structurally and functionally unrelated anticancer drugs (1). Several possible mechanisms
were examined that could explain the drug-resistance of preirradiated cells (2,3). However, the
determined alterations were not sufficient to explain the level of drug-resistance. Therefore, the aim of
the present study was to investigate further molecular mechanisms that are involved in this
phenomenon. The interest was focused on the genes involved in the repair of DNA damage and
apoptosis. The expression of corresponding genes was analysed by Western blot method. The kinetic
of apoptosis was followed under the fluorescent microscope. The activity of caspases was determined
by a commercial kit. Our results show that the constitutive levels of the proteins involved in mismatch
repair, as well as ERCC1, were not altered in HeLa1500 cells. The induction of apoptosis (following
the treatment with cisplatin) was inhibited in HeLa1500 cells. Preliminary results show that in
HeLa1500 cells (as compared to control cells), the expression of BCL-2 was increased, the expression
of caspase 8 was decreased, and caspase 9 was increased. In conclusion, low doses of gamma rays
may change the sensitivity of irradiated cells to the subsequent treatment with drugs due to the
inhibition of apoptosis.
References
1. Osmak M., Perović S., Int. J. Radiat. Oncol. Biol. Phys., 16 (1989) 1537-1541.
2 . Osmak M., Neoplasma, 40 (1993) 97-101.
3. Osmak M., Matulić M., Sorić J., Neoplasma, 40(1993) 359-362.
135
P2/27
136
P2/28
AN INVASTIGATION OF THE MUTAGENIC DAMAGE THAT IS CAUSED IN
HUMAN
CELLS
BY
DEBRIS
FROM
WORN
ORTHOPAEDIC
JOINT
REPLACEMENTS
W. Niedzwiedz, C.P. Case
Bristol Implant Research Centre. University of Bristol, Southmead Hospital, BS10 5NB.
Orthopaedic joint replacement is the second most commonly performed surgical operation in
the UK. However, the joint replacement becomes loose in approximately 20% of patients, resulting in
the generation of particular and soluble wear debris. This wear debris contains metals, including Ni,
Cr, Co, Vi and Ti, which are known carcinogens or mutagens as well as plastic and cement. The wear
debris is systematically disseminated to the bone marrow and lymph nodes. Previous studies have
shown an increase in chromosomal aberrations in the bone marrow of patients exposed to wear debris
(Case et. al, 1996). Furthermore, Doherty et. al. (2001) observed a 5-fold increase in aneuploidy in the
peripheral lymphocytes of patients with titanium prostheses and a 3.5-fold increase in chromosomal
translocations in the peripheral blood of patients with cobalt chrome prostheses. Increasing numbers
of young patients are undergoing joint replacement surgery; one-third of patients are now under 60
years of age, and 10 % are under 40. Therefore, there is concern for the health of these patients and
for their offspring after long term exposure.
The aim of this study was to examine the mechanism of the genetic damage observed in vivo
using an in vitro system analysed by the comet assay and flow cytometry. Pooled human amnion cells
in tissue culture were exposed to wear debris extracted from patients with failed prosthesis, using a
dose of wear debris that give a statistically significant induction of micronuclei after 24 h of exposure.
The results show that Co/Cr wear debris causes a progressive increase in DNA damage with time,
and initial decrease in cell viability up to 6 h . In contrast, no DNA damage was detected in cells
exposed to titanium wear debris even after 9 h of treatment, and cell viability was unaffected. Both,
Co/Cr and Ti wear debris caused the arrest of cells in S-phase after 24 h of treatment. The normal
kinetics of the cell cycle was restored following a further 24 h exposure to Ti, but not Co/Cr. The result
show that the different mutagenic effects of titanium (aneuploidy) and Co/Cr (chromosomal
translocations) wear debris are accompanied by differences in the cell cycle kinetics, cell viability and
DNA damage. Further experiments are in progress to reveal the precise nature of induced DNA
damage, the mechanisms responsible for alteration in the cell cycle and the genes involved in the
chromosomal alterations observed.
A.T. Doherty, B. Lewis, R.T.Howell, G. Langkamer, C.P.Case. (2001) JBJS (in press)
C.P.Case, D. Path, V.C.Langkamer, at al. (1996) CORR, no329S, S269-S279.
137
P2/29
138
P2/30
APPLICATION OF THE MICRONUCLEUS ASSAY IN PATIENTS TREATED WITH
RADIONUCLIDE THERAPIES: RESULTS AND LIMITATIONS.
M. Monsieurs, A. Vral, B. Brans, L. De Ridder, RA Dierckx and H. Thierens.
Faculty of Medicine, University Ghent
The aim of the present study was to determine the equivalent total body dose (ETBD) using the
cytokinesis blocked micronucleus assay (CBMN) in 76 patients treated with
131I-labeled
radiopharmaceuticals for radionuclide therapy in nuclear medicine.
39 patients were treated with
131I
for thyrotoxicosis (TT, n = 31, mean 0.7 GBq, SD: 0.2) or thyroid
carcinoma (TCA, n = 8, mean 2.5 GBq, SD: 0.5), 22 patients were treated with
131I-MIBG
for
neuroblastoma (NB, n = 18, mean 5.1 GBq, SD: 1.6) or carcinoid tumors (CA, n = 4, mean 7.7 GBq,
SD: 0.5) and finally 16 patients were treated with
131I-lipiodol
for hepatocellular carcinoma (HCC) with
a mean of 1.9 GBq (SD: 0.2).
For each patient, blood samples were taken immediately before and 1 week after therapy. The first
blood sample was irradiated in vitro with
60Co
-rays to determine the dose response curve.
Micronuclei were scored in 1000 binucleated cells. From the increase in micronucleus yield after
therapy (second blood sample) the ETBD was derived using the dose response curve.
Based on 3 consecutive biplanar scans, taken after therapy, the total body dose following the MIRD
formalism was calculated for patients treated with 131I-MIBG and 131I-lipidol.
For
131I
therapy, the calculated ETBD values for TT and TCA (respectively 0.34 Gy and 0.32 Gy) were
not significantly different despite the large difference in administered activity. These low dose values
explain the lack of reports of late detrimental effects after
The CBMN was evaluable in only 14 out of 22
131I
131I-MIBG
therapy.
therapies 11 out of 16
131I-lipiodol
therapies
due to cell division inhibition caused by previous chemotherapy treatments, lymphocyte dilution due to
blood transfusions given closely after therapy and lymphocyte break down caused by hypersplenism.
A mean ETBD of 0.95 Gy (SD: 0.55) for NB and a mean of 0.46 Gy (SD: 0.09) for CA was calculated.
A reasonable correlation (R = 0.87) between the ETBD and the MIRD dose was obtained. The slope
value of 0.75 can be explained by the low dose rate effect. The same effect was observed for
131I-
lipiodol therapy where the mean ETBD was 0.99 Gy (SD : 0.47) and the mean MIRD dose was 1.33
Gy (SD: 0.28).
The authors therefore conclude that the CBMN can be used to determine the ETBD after radionuclide
therapies. However, in case of previous chemotherapy treatment, frequent blood transfusions or
hypersplenism, the results have to be evaluated carefully.
139
P2/31
IN
VITRO
APOPTOSIS
TESTING
COMPARED
TO
THE
CLINICAL
CHARACTERISTICS
J. Philippé, A. Janssens, F. Offner, H. Thierens
Dept. Of Clinical Biology, Hematology and Medical Physics, Ghent University, Belgium
Introduction:
Deregulation of apoptosis in B-CLL plays a major role in the pathogenesis of this disease. We have
examined the correlation of in vitro apoptosis with clinical staging (RAI classification) and lymphocyte
doubling time. We also explored whether different apoptosis responses could be identified in
differently treated patient groups. Finally, we also looked for a concordance between in vitro apoptosis
and the in vivo response to chemo- and/or radiotherapy.
Materials and methods:
Peripheral blood from 91 typical B-CLL patients was examined. In vitro apoptosis from the
mononuclear cell fraction was measured after a 24 hr culture period. We applied the flow cytometric
method using annexin V binding combined with 7-amino actinomycin D for staining of late apoptotic
cells. Cells were cultured in RPMI supplemented with glutamin, antibiotics and 10% FCS. Fractions of
cells were irradiated or incubated with chlorambucil (2.5 µM), fludarabin (5 µM) or methylprednisolon
(10 µM).
Results and Discussion:
We observed a variable apoptotic response. Spontaneous apoptosis scored 30.5 % on
average.Irradiation, and chemotherapy increased the apoptosis to 45% and up to 51%, respectively.
Clinical staging and lymphocyte doubling time did not appear to be related with in vitro apoptosis.
There was not any change in apoptosis when prior treatment with chlorambucil had been applied. A
subgroup of ten patients had received several treatments in the past and we saw a trend to a lower
spontaneous apoptosis and a lower induced apoptotic response by chemo- or radiotherapy in these
patients.
However, a good correlation and predictive value was observed between in vitro apoptosis and a
subsequent in vivo therapeutic response. A sensitivity of 89%, specificity of 78%, a PPV of 94% and a
NPV of 64% were noted. These results show that the applied assay offers a tool to predict therapy and
is useful as a drug sensitivity test.
140
P2/32
MODULATION OF MUTAGENIC ACTIVITY IN MEAT SAMPLES DEEP-FRIED
UNDER DIFFERENT CONDITIONS
C. Perez; A. Lopez de Cerain ; J. Bello
Dpt. of Food Sciences & Toxicology, University of Navarra, Pamplona, Spain.
Mutagenic heterocyclic amines (HAs) appear during the cooking of protein-rich foods at normal
cooking temperatures. Previous studies have been carried out on the influence of frying fats on the
formation of food mutagens, but most of them have been performed on model systems or under
cooking conditions that are more frequent in northern countries.
Purpose:
The objective of the present work was to study the overall mutagenic activity generated in hamburgers
and commercial hot dogs, deep-fried in a large volume of several oil classes used in the
Mediterranean diet, in order to verify if there was any modulation of the mutagenic activity with respect
to other cooking conditions previously studied.
Material and Methods:
Hamburgers were prepared from beef meat purchased in a butcher’s shop. Commercial hotdogs as
well as the oils (olive, marc (“orujo”) olive, sunflower) were purchased in a local supermarket. The
general composition of the samples was determined before cooking by standard analytical techniques.
The samples were fried in a teflon-coated frying pan with 200 mL of oil or without any fat at 170-180ºC
during 10-20-30 min. The mutagens were extracted by a method based on that of Bjeldanes and
Grosse (1982). The mutagenic activity has been evaluated using the Salmonella mammalian
microsome assay with the strain TA 98 and 10% Aroclor induced S9 mix (Maron and Ames, 1983).
Two independent assays were carried out for each experimental condition. The statistical analysis was
performed by one-way and two-way ANOVA and the Tukey test a posteriori.
Results and Conclusions:
All the hamburgers fried in oil showed a mutagenic activity that was more than 4 times higher than that
of the controls; hamburgers fried in olive oil for 10 min showed a significant increase in the number of
revertants with respect to the other oils, probably due to the fact that the temperature reached was
approximately 10ºC higher. Longer frying times increased significantly the number of revertants in
samples fried in oils, except in olive oil, probably due to it lower content in polyunsaturated fatty acids.
Hot dogs showed a lower mutagenic activity than hamburgers fried in the same conditions because
they have a lower content of protein and a higher content of fat.
References:
Bjeldanes et al., Mutat. Res. 105:43-49,1982; Maron & Ames, Mutat. Res. 113:173-.215, 1983.
141
P2/33
INDUCTION
OF
MICRONUCLEI
BY
COMBINED
TREATMENTS
OF
HETEROCYCLIC AMINES (HAs) IN V79 CELLS
C. Perez; A. Lopez de Cerain ; L. Alvarez; J. Bello
Dpt. of Food Sciences & Toxicology, University of Navarra, Pamplona, Spain
Several epidemiological studies have shown a relationship between the consumption of meat and an
increased risk of some cancers. The formation of mutagenic/carcinogenic heterocyclic amines (HAs)
during the cooking of protein-rich products may be responsible for part of the observed increased risk.
Purpose:
The objective of the study was to investigate the induction of micronuclei (MN) by several HAs, added
to V79 cells in combined treatments, in order to see if there was an additive toxic effect, an inhibition of
the mutagenicity or other kind of interaction.
Material and Methods:
The food-related mutagens IQ, IQx and PhIP were obtained from
Toronto Research Chemicals
(Ontario, Canada). V79 cells (ECACC) were grown in EMEM medium supplemented with 10% fetal
calf serum, 1% L-glutamine and 1% streptomycin/penicilline, at 5% CO 2 and 37ºC. The products were
added 24 h after setting up the cultures, both in the presence and in the absence of 10% S9, in
independent or in combined treatments, and retired after 2h. Then, cytocalasin B (Sigma) at a final
concentration of 2 µg/mL, was added and maintained during 21h. Cells were harvested and stained
with Giemsa 10%, 4 min. The MN were scored following the criteria of Fenech (1993), in at least 1000
binucleated cells per condition. Statistical analysis was performed using one-way ANOVA and Tukey
test to determine the differences between the doses. A Chi-squared test was applied to verify the
additive effect.
Results and Conclusion:
The three HAs have been tested at five doses between 50 and 2100 µM first in independent assays
and afterwards, in combination: PhIP-IQ, PhIP-IQx and IQ-IQx. Positive results in the presence of S9
were obtained for the three HAs in independent assays, with the genotoxic potency being similar (PhIP
= 37 MN/100µM; IQ = 27 MN/100 µM; IQx = 26 MN/100µM). PhIP also increased the number of MN
with respect to the control without metabolic activation, but the difference was not statistically
significant. In combined treatments, the number of MN obtained was related to an additive effect in all
of the cases: PhIP-IQ 2 = 1.678, p=0.432 ; PhIp-IQx 2 = 5.813, p=0.121 ; IQ-IQx 2 = 0.219,
p=0.896.
References:
Fenech, M. Mutat. Res. 285: 33-44 , 1993.
142
P2/34
DEVELOPMENT OF AN IN VITRO ASSAY TO DETECT MUTATION INDUCTION
IN THE LACZ TRANSGENE OF MUTATMMOUSE CULTURED SPLENOCYTES
M. Ballantyne1; R. Marshall1; A. Wolfreys2; G. Ellis2
1Department
2SEAC
of Genetic and Molecular Toxicology, Covance Laboratories Limited, Harrogate, UK and
Toxicology Unit, Unilever Research Colworth, Sharnbrook, Bedford, UK
Due to a failure to detect mutation induction in the oral mucosa following treatments of Muta TMMice
with cumene hydroperoxide, investigations were made to determine whether the lacZ transgene
system per se is sensitive to the type of mutations induced by oxidative mutagens. This was
addressed by development of an in vitro method to detect mutation at the lacZ transgene. Splenocytes
were extracted from MutaTMMice and cultured for approximately 48 hours prior to treatment, followed
by a 24 hour treatment and expression period. DNA was extracted from the treated cells and mutation
frequencies (MF) at the lacZ transgene calculated. This assay methodology was used to assess
mutation induction by a known in vivo mutagen; 4-nitroquinoline 1-oxide (NQO), and by two oxidative
mutagens; cumene hydroperoxide (CHOP) and hydrogen peroxide (H 2O2). The mean MF value for
NQO at 0.02 µg/ml was 919.7 x 10-6, compared with a mean mutation frequency in the solvent controls
of 44.5 x 10-6. CHOP treatments resulted in mean MF values for each dose tested (1-30 µg/ml) that
were in the range 14.4-76.4 x 10-6, and H2O2 treatments at 0.5-10 µg/ml provided MF values in the
range 18.4-44.7 x 10-6. The maximum concentration assessed for each chemical was governed by
toxicity. The in vitro method was considered to have indicated its ability to detect mutation induction by
NQO, but failed to provide a clear indication of the oxidative mutagenic activity of CHOP or H 2O2. This
preliminary evaluation suggests that oxidative mutagens may fail to induce mutations that are
detectable using the lacZ transgene and indicates that this in vitro method may be valuable for
mechanistic investigations of transgenic systems.
143
P2/35
IN
VITRO
DNA
ADDUCT
FORMATION
BY
HETEROCYCLIC
AMINES,
ACTIVATED VIA DIFFERENT METABOLIC PATHWAYS
H.J.J. Moonen ; T.M.C.M. de Kok ; J.C.S. Kleinjans
Department of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, The
Netherlands
Background:
Heterocyclic amines (HCA) are formed during the preparation of food at high temperatures. The
bioactivation of HCA to colon carcinogens is hypothesized to occur via N-oxidation to N-hydroxy
metabolites followed by O-acetylation to form N-acetoxy arylamines which can form DNA adducts.
These steps are catalyzed by hepatic cytochrome P4501A2 (CYP1A2) and colonic acetyltransferase-2
(NAT-2). Prostaglandine H synthase (PHS) may be of interest for the extrahepatic formation of
reactive intermediates of heterocyclic amines since it occurs in most mammalian tissues, with
relatively high activities in the gastrointestinal tract.
Objective:
We investigated the activation of the two heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) by two different enzyme systems,
by establishing differences in DNA adduct levels and profiles.
Design and methods:
Both single-stranded and double-stranded salmon testes DNA was incubated with either PhIP or IQ.
Rat liver S9-mix or PHS was used as the activating system, representing the hepatic or extrahepatic
metabolic pathway, respectively. The DNA was isolated and used for DNA adduct measurement by
means of TLC-32P-postlabelling.
Results: For IQ, both pathways lead to adduct formation, where PHS-activation appears more effective
compared to S9-activation. Furthermore, metabolic activation of IQ by S9 and PHS results in different
DNA adduct spots. For PhIP, in contrast, only PHS activation leads to adduct formation, whereas S9
activation does not result in adduct spots. Under identical incubation circumstances, adduct levels are
found to be higher in single stranded DNA as compared to double-stranded DNA. Conclusions:
Different enzyme systems representing different metabolic pathways result in different levels and
profiles of DNA adducts induced by heterocyclic amines.
144
P2/36
HISTORY OF THE CYTOGENETIC ANALYSIS IN THE CZECH REPUBLIC
Z. Smerhovsky(1); P. Rossner(1); R.J. Sram (2), and K. Landa(1).
1 National
2
Institute of Public Health, Czech Republic
Lab. of Genetic Ecotoxicology, Institute of Experimental Medicine AS, Czech Republic
Cytogenetic analysis (CA) in PBL has an outstanding history in the Czech Republic. It can be
thought to be a tremendous success of the Czech Hygienic Service. There is no other country in the
world, which has been using the CA to monitor occupational and environmental exposures so
systematically as the Czech Republic. Since very beginning, the CA has been conceptualized and
implemented into a common practice as a method capable of the detection of genetic damage to
somatic cells caused by mutagens and carcinogens. This approach has facilitated the implementation
of the CA as a part of periodic checkups in selected working places. As a result, there are
occupational groups, which have been systematically monitored and thousands of tests were
performed. Furthermore, the CA has been used not only for a passive monitoring of selected
occupational groups but also for an active intervention. There are two groups of examples: First one is
related to setting of maximal allowable concentration, second one is related to chemoprophylaxis of
cancer. The history of the CA has been started in 1974, when the Czech Hygienic Service was short of
adequate methods to supervise working conditions in newly emerged chemical industries. An effort to
implement an effective instrument to monitor health risks related to exposure to known carcinogens
resulted in establishing of the first cytogenetic laboratory, which was used, in first phase, to measure
occupational exposures to BCME and ECHH. The success of the first laboratory initiated the
establishment of the other laboratories; finally, there were 22 cytogenetic laboratories in the country in
the middle of 80s. Soon it became apparent, that their activities must be coordinated and unified. So,
in 1977 the National Reference Laboratory was established at the NIPH (former IHE). Since founding
this laboratory has played the pivotal role in the organization of the cytogenetic monitoring. Its
responsibilities include training of staff, development of unified methods, distribution of standard
laboratory chemicals, quality control etc. The accumulation of approximately 20 000 cytogenetic
examinations performed by the same methods made it possible to carry out the epidemiologic studies
on the long term effects of elevated frequencies of chromosomal aberrations in PBL on human health.
At present, the Czech cytogenetic data represents the largest cytogenetic database available in the
world.
Supported by the EU contract No QLK4-2000-00628.
145
Poster session 3
Major topics:
Chromosomal sensitivity towards genotoxic agents,
Mitosis versus meiosis,
Electromagnetic fields,
Microarray systems,
Misc.
P3/1 – P3/35
146
P3/1
A COMPARISON OF THE CYTOGENETIC RESPONSE TO IRRADIATION OF
RESTING PERIPHERAL BLOOD LYMPHOCYTES AND EPSTEIN-BARR VIRUS
TRANSFORMED LYMPHOBLASTOID CELLS.
A.Baeyens*, A. Vral*, H. Thierens° and L. De Ridder*
Department of Anatomy, Embryology, Histology* and Medical Physics°, University of Gent, L.
Pasteurlaan 2* and Proeftuinstraat 86°, 9000 Gent, Belgium.
E-mail: [email protected]
Ionising radiation induces chromosomal damage. An enhanced chromosomal radiosensitivity has not
only been demonstrated in a large number of patients with cancer prone genetic diseases (e.g. AtaxiaTelangiectasia(A-T)) but has also been observed in a significant proportion of sporadic breast cancer
patients and other cancers with no obvious family history (head and neck, colorectal,...). To investigate
the chromosomal radiosensitivity of lymphocytes in cancer patients the G2-assay and MN-assay are
often used. Several radiosensitivity studies with AT-patients are done in Epstein-Barr virus (EBV)
transformed lymphoblastoid cell lines. The advantage of EBV cell lines for this kind of tests in cancer
patients is that the tests can be easily repeated without any further blood sampling.
The question is whether the radiation response of Epstein-Barr virus transformed lymphoblastoid cells
is the same as in resting peripheral blood lymphocytes.
In our study we have used peripheral blood lymphocytes and lymphoblastoid cell lines derived from 5
healthy individuals, 3 AT-patients and 5 breast cancer patients with an increased radiosensitivity. For
the G2-assay blood lymphocytes were irradiated with a dose of 0.4 Gy 60Co -rays after 71h incubation
in a CO2 incubator at 37°C. The lymphoblastoid cell lines were irradiated 24h after subdivision from
multiwell plates to tubes. At 30 min post-irradiation colcemid was added and 60 min later the cultures
were harvested and chromatid breaks were analysed in 50 well spread metaphases. For the MNassay blood lymphocytes and lymphoblastoid cells were exposed to 2 Gy
60Co
-rays. The blood
cultures were stimulated with PHA immediately after irradiation and 24h later cytochalasine B was
added. 72h post-irradiation the blood cultures were arrested. For the lymphoblastoid cellines
cytochalasine B was added immediately after irradiation and the cells were arrested 48h postirradiation. Micronuclei were scored in 1000 binucleate cells.
The results of this study are still in progress and will be presented during the congress.
147
P3/2
148
P3/3
GENOTOXIC EFFCTS OF TWO PESTICIDES AND THEIR MIXTURES:
IN-VIVO CHROMOSOMAL ABERRATIONS AND MICRONUCLEUS ASSAY
E.N. El-Khatib*; and H.A. Rokaya**
* Department of Mammalian Toxicology, Central Agricultural Pesticides Laboratory, Dokki, Giza,
Egypt. E-mail: [email protected]
** Lecturer of Genetics, Department of Zoology, Girls College for Art, Science and Education, Ain
Shams University, Cairo, Egypt. E-mail: [email protected]
Epidemiologic data showed an increase in the number of cancer cases in persons involved in
agricultural production using pesticides. Synthetic pyrethroids have been extensively used because of
their short half-lives and broad spectrum of activity. Alpha-cypermethrin is one of the widely used
synthetic pyrethroid pesticides. On the other hand, diazinon is an organophosphate pesticide, one of
the most commonly found pesticides in rivers and streams in urban areas. The present study was
carried out in order to assess the genotoxicity of the insecticides diazinon and alpha-cypermethrin
using the rat bone-marrow cells chromosomal analysis and the micronucleus test. The pesticides were
tested both separately and in combination. Male Swiss albino rats were treated orally for 48 hours.
Three dose levels were used at (1/2 high ‘H’, 1/4 median ‘M’ and 1/10 low ‘L’ LD 50). Commercial
formulations of the tested pesticides were used and dissolved in corn oil. Groups of five animals /
treatment were used in the present study. Animals received single oral administration of each
pesticide singly and in combination “HH, MM and LL”. The obtained data indicated that both
compounds induced dose dependent increases in the structural and numerical chromosomal
aberrations. While diazinon significantly increased the number of structural aberrations, alphacypermethrin significantly increased the number of numerical aberrations. But the combined exposure
showed no additive effects. The obtained results revealed that both tested pesticides induced
significant increase in the micronucleus frequency and reduced mitotic index in all of the treated
groups compared with control.
149
P3/4
THE PROTECTIVE EFFECT OF VITAMIN C AGAINST CYFLUTHRIN INDUCED
CLASTOGENICITY IN RAT BONE MARROW CELLS
H. N. EL-KHATIB
Department Of Mammalian Toxicology, Central Agricultural Pesticides Laboratory, Agricultural
Research Center, Ministry of Agriculture, Dokki, Giza, Egypt.
(Tel/ Fax: 202 3845 609 / 202 7428 514 - E-mail : [email protected].)
According to IARC, more than 25% of pesticides are classified as oncogens. It was reported that
vitamin C, when administered concurrently with a pesticide may decrease the frequency of pesticideinduced clastogenic and mitosis-disruptive changes in the bone marrow cells of albino rat. So, in the
present study the clastogenic effect of the synthetic pyrethroid “Cyfluthrin” was studied after five days
of daily exposure using mitotic index, micronuclei induction, and chromosome abnormalities assays in
Swiss albino rat bone marrow cells.
And the protective role of vitamin C was tested after daily
administrating of the vitamin concurrently with the pesticide. Three dose levels (five successive doses)
were used for the evaluation of the subchronic genotoxic potentiality. Cyfluthrin was found to reduce
the mitotic index, and induce significant increase in the frequency of micronuclei and chromosome
abnormalities. A dose-response relationship was observed. It was also observed that administrating of
vitamin C significantly minimized the pesticide- induced genotoxicity.
150
P3/5
CYTOGENETIC BIOMONITORING OF EGYPTIAN WORKERS EXPOSED TO
PESTICIDES:
MICRONUCLEI
ANALYSIS
IN
PERIPHERAL
BLOOD
LYMPHOCYTES
H. N. EL-KHATIB and F. M. Hammam
Department Of Mammalian Toxicology, Central Agricultural Pesticides Laboratory, Agricultural
Research Center, Ministry of Agriculture, Dokki, Giza, Egypt.
(Tel/ Fax: 202 3845 609 / 202 7428 514 - E-mail : [email protected].)
Cytogenetic biomarkers in peripheral blood lymphocytes have for over 30 years been used to
assess carcinogenic or mutagenic exposures and early effects in occupational and environmental
settings. In the present study, we evaluate whether or not occupational exposure to a complex mixture
of pesticides results in a significant increase of micronuclei (MN) in peripheral blood lymphocytes. The
lymphocytes were cultured for analysis of micronuclei (MN) in cytochalasin B-induced binucleated
cells. Twenty smokers and twenty non-smokers pesticide applicators from a private farm south
Alexandria (Egypt), together with ten men from the same area, without indication of exposure to
pesticides, that served as controls were used in this investigation. The obtained results indicated that
there are statistically significant differences in the MN frequencies between the three groups. These
results suggest that the human lymphocyte micronucleus test can be used to assess genotoxic injuries
due to environmental effects in human lymphocytes.
151
P3/6
PHENOLIC COMPOUNDS FROM RED WINE ARE PROTECTIVE AGAINST THE
DNA DAMAGING EFFECT OF IONISING RADIATION EX VIVO
W. Greenrod1,2; C. Stockley3; M. Abbey1; M. Fenech1
1CSIRO
Health Sciences and Nutrition, Adelaide, Australia;
2Clinical
& Experimental Pharmacology, University of Adelaide, Adelaide, Australia; 3Australian Wine
Research Institute, Adelaide, Australia.
Using the cytokinesis-block micronucleus assay we (a) investigated which compounds in red wine can
prevent oxidative damage to DNA in vitro and (b) performed in vivo interventions with red wine (RW),
dealcoholised red wine (DEALC) and alcohol (ALC) to distinguish the effects of alcohol from the other
fractions of RW in prevention of oxidative damage to DNA ex vivo. Cells were either challenged with
ionising gamma radiation or hydrogen peroxide, two different forms of oxidative stress. The relative
contribution of ethanol, glycerol, tartaric acid, catechin + caffeic acid, a mixture of all of these and
phenolic stripped RW at in vivo relevant concentrations on spontaneous and oxidative stress induced
DNA damage was evaluated in vitro. The results from these studies have shown that (a) only ethanol
significantly increases spontaneous DNA damage, but this effect is eliminated when ethanol is
included in a mixture of all the other wine components (P<0.05), (b) the strongest and only significant
protective effect against hydrogen peroxide induced DNA damage was observed for the catechin +
caffeic acid mixture (P<0.05) and (c) all compounds tested were significantly protective against
ionising radiation-induced DNA damage in a dose dependent manner with the strongest protection
being observed for the catechin + caffeic acid mixture and a mixture of all components (P < 0.0001). In
the in vivo intervention studies with ex vivo challenge of whole blood showed that 1-2h after
consumption of 300ml DEALC produced a significant protection against the DNA damaging effects of
ionising radiation (P = 0.0002) but ALC significantly enhanced the damaging effects of ionising
radiation (P = 0.0002) while RW produced an intermediate effect. The data from these studies are
particularly interesting because they clearly demonstrate the potential beneficial effects of wine
phenolics in counteracting the harmful effects of ionising radiation. Furthermore they demonstrate that
the potential of alcohol to exacerbate the DNA damaging effects of oxidative stress is neutralised in
the presence of the other RW components.
152
P3/7
INDIVIDUAL VARIABILITY IN THE YIELD OF CHROMOSOMAL ABERRATIONS
AFTER LOW DOSE GAMMA-RAY IRRADIATION
A. Kiuru ; C. Lindholm ; A. Koivistoinen ; R. Mustonen
Radiobiology Laboratory, STUK-Radiation and Nuclear Safety Authority, Helsinki, Finland
Factors such as DNA repair, chromatin structure, cell cycle control and apoptosis can modify the
response of mammalian cells to ionising radiation. Consequently, genetic differences underlying these
phenomena may affect individual susceptibility to ionising radiation1. In the present study
interindividual differences in dose response of chromosomal aberrations at low doses of gamma-rays
were examined.
Peripheral lymphocytes from ten healthy males were isolated from a sample of whole blood. Doses
of 0, 0.1, 0.25, 0.5, 0.75 and 1.0 Gy at a dose rate of 0.8 Gy/min were given using a
60Co
source. The
cells were incubated at +37 C for 48 hours, the last 4 hours in the presence of 0.2 g/ml Colcemid.
The cells were treated with hypotonic solution (0.075 M KCl) and fixed in methanol - acetic acid (3:1).
A cocktail of biotin-labelled whole-chromosome probes for chromosomes 1, 2 and 4 and a
digoxigenin-labelled pan-centromeric probe were used.
Detection and amplification of the
chromosome cocktail and the centromere probe were performed simultaneously by three layers of
antibodies: 1) avidin-FITC and anti-digoxigenin, 2) biotin-labelled anti-avidin and AMCA anti-mouse, 3)
avidin-FITC and AMCA anti-rat. Translocations, dicentrics, acentics, insertions and painted ring
chromosomes were scored. The observed frequencies of painted translocations and dicentrics were
converted into genomic frequencies by the formula established by Lucas et al. 2, and using the lengths
of chromosomes 1, 2 and 4 given by Morton3. The results will be described in detail and the individual
variability will be discussed.
1
Scott D. et al. - Int. J. Rad. Biol. 75: 1-10, 1999
2
Lucas J.N. et al. - Int. J. Rad. Biol. 62: 53-63, 1992
3
Morton N.E. - Proc. Natl. Acad. Sci. USA 88: 7474-7476, 1991
153
P3/8
INFLUENCE
OF
AGE
ON
VINBLASTINE-INDUCED
CHROMOSOME
MALSEGREGATION IN PERIPHERAL LYMPHOCYTES OF FEMALE DONORS.
P. Leopardi, R. Crebelli, F. Marcon, A. Zijno, G. Dobrowolny
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di sanità, Rome (Italy)
Spontaneous chromosome malsegregation in human lymphocytes is influenced by donor age and by
specific chromosome features. The age-related factors decreasing the fidelity of chromosome X
segregation are partially defined. On the other hand, the role of age and chromosomal features on
chemically induced malsegregation have been not yet elucidated, even though recent data suggest
that mitotic machinery
ageing
leads
to the
downregulation
of genes
involved
in mitotic
checkpoints (1).
In an attempt to gain more information on the matter, a study was carried out on twenty healthy female
donors belonging to two different age classes. The young group included ten individuals aged under
30 years, the elderly class ten individuals of over 50 years. Whole blood cultures from all subjects
were set up for a total of 60 h. For the last 18 h a low vinblastine (VBL) concentration (7.5 ng/ml) and
cytochalasin B (6 g/ml) were present. Binucleate cells were harvested and micronuclei and cell-cycle
indices, as MI and NDI, were analyzed on Giemsa stained slides. FISH technique, using centromeric
probes, was applied to analyze both spontaneous and induced malsegregation (loss and nondisjunction) of chromosomes X and 8 in binucleate cells and polyploidy in mononucleate lymphocytes
(cells containing four signals for each chromosome).
In untreated cells no statistically significant differences for MI, NDI, chromosome 8 malsegregation and
polyploidy were observed between the two age classes. On the other hand, significantly higher
frequencies of micronuclei and X chromosome malsegregation were observed in the lymphocytes of
elderly donors, confirming previous information.
VBL treatment significantly affected all parameters, except X and 8 chromosome losses. Results
obtained in the two age classes show a slightly higher damage in lymphocytes of aged subjects
relative to the younger. However, due to the high interindividual variability in the response to the VBL
treatment, such difference did not attain statistical significance. This suggests that factors leading to
spontaneous chromosome X malsegregation in aged individuals do not synergistically interact with
chemical factors.
In spite a similar relative increase in non-disjunction was induced by VBL in both chromosomes,
chromosome X resulted 4 fold more susceptible than the autosome 8. This difference was observed in
both age classes.
1) Ly et al. (2000) Science 287, 2486-2492.
154
P3/9
MUTAGENICITY OF AMOSITE FIBRES IN THE LUNG OF lacI TRANSGENIC
RATS (A REPORT FROM THE FIBRETOX PROJECT)
P. Loli1; J. Topinka2; M.Hurbankova3; P. Georgiadis1; T. Wolff2; S. A. Kyrtopoulos1
1Chemical
Carcinogenesis Laboratory, Institute of Biological Research and Biotechnology, National
Hellenic Research Foundation, Athens, Greece
2GSF,
3
National Center for Health and Environment, Institute of Toxicology, Neuherberg, Germany
Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic
Amosite asbestos is classified as carcinogenic to humans and animals. In addition, amosite along with
most of the types of asbestos fibres is suggested to be genotoxic, causing chromosomal aberrations
and DNA damage. However, the evidence on amosite’s ability to bring about gene mutations is limited.
The presented work is part of an E.U. funded project (FIBRETOX project), whose purpose is to
investigate the mechanisms of cytotoxicity and genotoxicity of man-made mineral fibres in comparison
to amosite, and to provide reliable biomarkers of fibre toxicity.
In order to investigate the mutagenicity of amosite fibres, we employed the Big Blue Transgenic Rat
Mutation Assay. Male homozygous lacI transgenic Fisher 344 rats were intratracheally instilled with a
single dose of 1 & 2mg/animal of amosite fibres (75% of fibres were 20-30µm of length, average
diameter 0.71µm) or with multiple doses of 2 mg administered weekly on 4 consecutive weeks. The
animals were sacrificed 4 or 16 weeks after the final instillation. Subchronic exposure to amosite for 16
weeks significantly increased MF in the lung in a dose-dependent manner (p=0.035). Four weeks after
instillation, neither the single nor the multiple doses of amosite significantly increased the mutation
frequency (MF) of the transgene in the lung as compared to controls, although there was a tendency
of increase after the multiple dose.
In an attempt to examine the interaction of benzo[a]pyrene (B[a]P) with amosite, 2 consecutive daily
i.p. injections of B[a]P (40 mg/kg b.w.) were given to the animals immediately after the last fibre
administration and sacrificed 4 weeks later. Preliminary data analysis indicated an increase in the MF
of B[a]P treated rats (p=0.04). However, no significant additive or synergistic effect of amosite was
observed. A possible explanation of the weak mutagenic response observed is that the fibres cause
mutations in certain proliferating cell types that are mixed with a majority of non-target cells during the
lung tissue homogenisation.
The work was supported by an E.U. grant, contract No. QLK4-CT-1999-01629.
155
P3/10
LYMPHOCYTES FROM IODINE-131 TREATED THYROID CANCER PATIENTS
UNDERGO
A
TRANSIENT
ADAPTATION
TOWARDS
MITOMYCIN
C
GENOTOXICITY
O. Monteiro Gil1,2; N.G. Oliveira1,3; A.S. Rodrigues1,4; A. Laires5; T.C. Ferreira6; E. Limbert6; J. Rueff1
1Dep.
Genetics, FCM - UNL, Lisbon, Portugal; 2Nuclear and Technological Institute, Dep. Radiological
Protection
and
Nuclear
Safety,
Sacavém,
Portugal;
3Faculty
of
Pharmacy,
UL,
Lisbon,
Portugal;4University Lusófona, Lisbon, Portugal; 5Faculty of Sciences and Technology, UNL;
6
IPO
Lisbon, Portugal.
Radioactive iodine
131I
has been extensively used to treat thyroid cancer patients. The doses used for
the treatment are usually in the range of 30-100 mCi. Biological dosimetry studies on thyroid cancer
patients submitted to
131I
therapy have been performed and pointed out that radiation doses in these
patients are rather small in the order of some hundreds mGy. Doses of this order could possibly
induce an adaptive response in cells exposed in vivo.
The aim of the present study was to assess the induction of an adaptive response in circulating
lymphocytes of 11 thyroid cancer patients, having undergone therapy with a dose of
131I
(70 mCi),
towards a challenging dose of mytomicin C (MMC, 0.75 M) in vitro. The endpoint chosen to assess
DNA damage was the induction of micronuclei in citokinesis-blocked peripheral blood lymphocytes
(MNCB). The sampling times were immediately before treatment, one, six and twenty-four months after
therapy.
The results obtained after challenging lymphocytes of these patients in vitro with MMC showed a
reduction in the micronuclei frequency (‰ MNCB) one-month after treatment (34.4  25.7, mean  SD)
when compared to the micronuclei presented before treatment (52.1  16.7, mean  SD). In 7 of the 11
patients studied this reduction was significant (p<0.001; 2 test) and around 50%. Six months after
treatment, however, there was a complete disappearance of the adaptive response presented and
indeed a clear increase in the genotoxicity induced by MMC was observed (133.1  45.3, mean  SD).
Two years afterwards, the frequencies of MMC induced micronuclei were lower when compared to six
months results (84.0  16.0, mean  SD) but were still higher than the initial results (before treatment).
An overall analysis of these results suggests the possible existence of an adaptation induced by iodine131 treatment, with some degree of inter-individual heterogeneity, and show clearly its transient nature.
156
P3/11
CYTOGENETIC BIOMONITORING OF EXTERNAL WORKERS IN THE NUCLEAR
INDUSTRY: STUDY OF EXPOSURE EFFECTS AND SUSCEPTIBILITY
H. Thierens 1; A. Vral1 ; M. Barbé2; A. Baeyens1; L. De Ridder1
Dept.Anat. Embr. Hist. & Med.Phys – Univ. Ghent1, Occup. Med. Serv. NPP Doel2, Belgium
External workers receive the highest dose at a relatively short period in the Belgian nuclear industry (
up to 15 mSv in less than one month). Their activities are mainly cleaning, maintenance and repairing
jobs. A cytogenetic biomonitoring of 40 external workers, involved in the revision of the four reactors of
the Electrabel Nuclear Power Plant Doel, with respect to exposure effects and susceptibility was
performed during the year 2000. A comparison of the results of the in vitro micronucleus assay on
blood samples before and after the revision allowed an evaluation of the cytogenetic effect induced by
the short time exposure. An assessment of individual chromosomal radiosensitivity of the workers was
performed before and after the revision using the HDR-LDR micronucleus assay and the G2 assay.
For this cytogenetic susceptibility monitoring, blood samples were irradiated in vitro with 3.5 Gy Co-60
-rays at low and high dose rate in G0 and with 0.4 Gy Co-60 -rays in G2.
A first analysis of the data shows an increase in the micronucleus yield by the exposure for the group
of external workers, receiving a dose between 3 and 15 mSv within one month according to the
electronic personnel dose monitoring, whereas no increase is present for workers receiving no
significant dose ( less than 1 mSv).
Using the 90th percentile as cut-off point determined in control populations, none of the workers
showed an abnormal high radiosensitivity status, systematically present as well before as after the
revision of the reactors. This conclusion was obtained for all susceptibility endpoints used: the G2
assay, the HDR-micronucleus assay, the LDR-micronucleus assay and the dose rate sparing from the
HDR-LDR combination. A comparison of the HDR-micronucleus data before and after the in vivo
exposure for the group of exposed workers shows any effect of the exposure on the radiosensitivity
status. On the other hand lower in vitro induced micronucleus yields and G2 indexes were obtained
after the in vivo exposure, pointing to a possible adaptive response effect.
157
P3/12
INTERFERING WITH HISTONE DEACETYLATION WILL CAUSE ANEUPLOIDY IN
MAMMALIAN CELLS
D. Cimini, D. Fioravanti, F. Degrassi
Centre for Evolutionary Genetics, Department Genetics and Molecular Biology,
"La Sapienza" University, Rome, Italy
Chromosome segregation at anaphase is the crucial event for maintenance of the correct
chromosome number at each mitotic division. While mitotic spindle poisons are well known inducers of
chromosome malsegregation, only recently it has been pointed out that chemicals interfering with
chromosome or kinetochore structure, such as topoisomerase inhibitors, may be important cause of
aneuploidy.
In this work we have investigated whether interfering with the physiological pattern of histone
deacetylation that occurs prior to mitosis may cause chromosome malsegregation in human cells. To
this end we have shortly treated primary human fibroblasts with the histone deacetylase inhibitor
Trichostatin A (TSA) and examined chromosome segregation at anaphase in treated cells. Both
lagging chromosomes and chromatin bridges were efficiently induced by TSA , indicating that an
altered chromosome segregation intervenes when histones are not properly underacetylated prior to
mitosis. Mitotic chromosome condensation is accompanied by phosphorylation of histones H1 and H3,
being S-10 H3 the critical residue for mitotic and meiotic associated phosphorylation. To investigate
whether the presence of acetylated histones interferes with S-10 H3 phosphorylation and chromosome
condensation we made use of anti-acetylated and anti-phospho H3 antibodies. Immunostaining of
TSA-treated cells with these antibodies showed that treated cells enter mitosis with elevated levels of
acetylated H3 histones, lower reactivity to the phospho H3 antibody and a normal response to MPM-2
antibody. Furthermore, we observed an incomplete chromosome condensation, when in vivo
progression of mitosis was followed in PTK-1 cells expressing an Histone H2B-GFP construct to label
chromosomes, suggesting that persistence of acetylated histones in G2-prophase prevents proper
chromosome condensation. Taken together, our results indicate that underacetylation of histones is
required for proper chromosome condensation and dynamics during mitosis.
158
P3/13
APPLICATION OF THE ALKALINE SINGLE CELL GEL ELECTROPHORESIS
(SCGE) ASSAY IN ASSESSMENT OF DNA DAMAGE IN PERIPHERAL BLOOD
LEUKOCYTES OF RADAR-FACILITY WORKERS
V. Garaj-Vrhovac, N. Kopjar, D. Želježić
Laboratory of Mutagenesis, Institute for Medical Research and Occupational Health, Zagreb, Croatia
The alkaline single cell gel electrophoresis (SCGE) assay was employed to assess the levels of DNA
damage in peripheral blood leukocytes of radar-facility workers daily exposed to microwave radiation
and unexposed control subjects. To quantify the DNA damage the comet tail length and the tail
moment were evaluated. In peripheral blood leukocytes of the exposed subjects the alkaline SCGE
assay showed larger amount of DNA migrated, expressed by tail length and tail moment. Between the
levels of DNA damage recorded in exposed subjects interindividual variations were also observed. Our
results suggest that long time occupational exposure to microwave radiation could be able of causing
genome damages in somatic cells and therefore it may represent a potential hazard to human health.
The results obtained confirm the usefulness of the alkaline SCGE assay as a sensitive biomarker of
exposure. Together with different cytogenetic techniques it provides a powerful technique for rapid
detection of primary DNA lesions in peripheral blood leukocytes of population occupationally exposed
to microwave radiation.
References
1. Lai H, Singh NP: Single- and double-strand DNA breaks in rat brain cells afrer acute exposure to
radiofrequency electromagnetic radiation. Int J Radiat Biol 69(4):513-521, 1996.
2. Garaj-Vrhovac, V: Micronucleus assay and lymphocyte mitotic activity in risk assessment of
occupational exposure to microwave radiation. Chemosphere 39(13):2301-2312, 1999.
159
P3/14
CYTOGENETIC EFFECTS OF HIGH FREQUENCY ELECTROMAGNETIC FIELDS
ON HUMAN LYMPHOCYTES IN VITRO
I.-L. Hansteen ; E. H Kure;
Telemark Central Hospital, Skien, Norway
The aim of the study is to test whether frequencies used in the next generation of radio communication
systems will give chromosome and chromatid type aberrations in human lymphocytes in vitro.
Lymphocytes from 4 non-smoking blood donors, 2 females and 2 males, were cultured for 53 hours.
Half of the cultures for each person were cultured under continuous EMF exposure, to ensure
exposure during the whole cell cycle. As a positive control Mitomycin C was added to half of the
cultures with and without
EMF exposure after 30 hours to test if a combination of
a known
clastogenic agent together with EMF exposure would be more detrimental to the cells.
Results from exposure to 18 GHz radiation will be presented.
160
P3/15
EFFECTS OF LOW-FREQUENCY ELECTROMAGNETIC FIELDS IN HUMAN
LYMPHOCYTES
J. Delimaris 1,3, S. Tsilimigaki 1, N. Messini-Nikolaki 2, G. Ziros 3 and S.M. Piperakis 1
1DNA
Repair Laboratory, Institute of Biology, National Center of Scientific Research
'Demokritos' , Athens, Greece. E-mail : [email protected]
2Division
of Cell Biology and Biophysics , Department of Biology,
University
of Athens, Athens, Greece.
3 Laboratory
of Microbiology , 1st I.K.A. Health Institution , Athens, Greece.
Some epidemiological studies have suggested that exposure to ambient low-level 50/60 Hz electric
and magnetic fields (EMFs) increases risk of disease (Valberg et al 1997, Stepansky et al 2000).
In the present study lymphocytes from normal healthy individuals were exposed to several different
doses of EMFs. DNA damage and repair efficiency as well as the effects of external factors (γradiation, H2O2) were investigated. The damage in DNA was estimated using the comet assay
technique, a rapid and sensitive method for the detection of DNA breaks (Piperakis et al 1999,
Piperakis et al 2000). The evaluation of the DNA breaks was done with an image analysis system as
well as with visual scoring.
For the statistical analysis a non-parametric test (Kruskal-Wallis) was
used.
Our results indicate that EMFs have an effect on the DNA of the exposed lymphocytes.
Piperakis S.M, Visvardis E-E, Sagnou M, and Tassiou A.M. Comet assay for nuclear
DNA damage. Methods in Enzymology. 1999, 300, 184-194.
Piperakis S.M, Petrakou E, and Tsilimigaki S. Effects of air pollution and smoking on
DNA damage of human lymphocytes. Environm. Molec. Mutagenesis. 2000, 36,
243-249.
Stepansky R, Jahn O, Windischbauer G, and Zeithofer J. Electromagnetic fieldseffects on health. Acta Med. Austriaca 2000, 27, 69-77.
Valberg P.A, Kavet R, and Rafferty C.N. Can low-level 50/60 Hz electric and
magnetic fields cause biological effects? Radiat. Res. 1997, 148, 2-21.
161
P3/16
ABSENCE
OF
COOPERATIVE
EFFECTS
IN L929
CELLS
FOLLOWING
COMBINED EXPOSURE TO MX AND A 50 Hz SINUSOIDAL MAGNETIC FIELD
O. Zeni, A. Perrotta, P. Pisani, M.R. Scarfì
CNR-Institute for Electromagnetic Sensing of Environment; Naples, Italy.
The interest in the evaluation of biological effects induced by electromagnetic field exposures has
widely raised in last decades. Recently the attention focuses on the possibility that such non ionising
radiation could enhance the effect of chemical pollutants whose action is well known.
Aim of this study was to investigate the genotoxic effects (micronucleus formation) induced in L929
cells by
3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) treatment, a drinking water
mutagen [1], in presence and in absence of a 50 Hz sinusoidal magnetic field. 3x10 5 cells were
cultured in 3 ml Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS and 5 µg/ml
Penicillin-Streptomycin, at 37°C in humidified 5% CO2 atmosphere. For each experiment 6 cultures
were set up, 3 of them treated with MX (200µM final concentration) and 3 untreated, and 3 conditions
were tested: unexposed, magnetic field exposed and sham exposed. During the first 2 hours of growth
cells were exposed and/or MX treated. In chemically treated samples MX was removed by washing
cells twice with Versene. Helmholtz
coils were used to obtain both magnetic field and sham
exposures. In the first case samples were exposed to 10 Gauss field intensity [2], while in the second
one cultures were positioned inside an identical coil which, opportunely powered, realised the same
thermal increase, if any, but in absence of magnetic field. In order to block cytokinesis, cytochalasin-B
(6 µg/ml final concentration) was added after 22 hours of growth. At the end of culture period (42
hours) cells were collected and slides prepared and for each condition 1000 binucleated cells were
scored to evaluate MN frequency. Cell proliferation was also calculated by classifying 500 cells
according to the number of nuclei. [3]. The results obtained on 3 independent experiments (two tailed
paired Student’s t test) indicate that MX treatment induces an increase in MN frequency and a slight
decrease in cell proliferation, as expected, but such an increase was not enhanced by 50 Hz
sinusoidal magnetic field exposure, suggesting that, in the experimental condition adopted, no
cooperative effects are induced.
References
[1] Brunborg G. et al., 1991, Mutation Research, 260: 55-64.
[2] Scarfì M.R. et al, 1999, Health Physics, 76 (3): 244-250.
[3] Surralles J. Et al, 1995, Mutation Research, 341: 169-184.
162
P3/17
GENOTOXIC EFFECTS OF 50 Hz MAGNETIC FIELDS ON HUMAN BLOOD CELLS
A. Testa, L. Stronati, D.Conti, P. Villani, , A. M. Fresegna, F. Russo, G. Lovisolo, C. Marino and E. Cordelli
Section of Toxicology and Biomedical Sciences. ENEA CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy
The possible health hazard of exposure to extremely low frequency magnetic fields (ELFMFs)
has became an issue of considerable public concern. Although many epidemiological studies
have been done, a definite correlation between exposure to environmental ELFMFs and cancer
has not been found.. According to several investigations, ELF fields cannot directly damage
DNA or cause mutations (NRPB,1992; Verschaeve 1995). On the other hand few studies have
addressed the possibility that ELF magnetic fiels could be able to enhance the genotoxic effect
of known chemical or physical mutagens (Hintenlang, 1993; Maes et al., 2000). The aim of the
present study is to investigate the potential genotoxic effect of ELFMFs alone or in
combination with X rays on human blood cells. Four different cytogenetic tests (chromosome
aberration, cytokinesis-block micronucleus, sister chromatid exchange and comet assay) have
been applied.
Human blood samples were exposed to 50 Hz, 1 mT uniform magnetic field generated by a Helmholtz coil system,
in incubator. Whole blood samples from three healthy donors (30-40 years) were exposed to ELF MFs for 2 h and
further blood samples from other three donors were diluted in culture medium in absence of phytoemoagglutinin
and then exposed for 48 h to ELF fields. A sham control consisting on a same current system, with the coils
activated in anti-parallel direction was used. A positive control (1 Gy of X-rays) were also included. A potential
synergistic effect between ELF and X-rays (1 Gy) exposures was also investigated.
Results did not show any significant differences between 2 h ELFMFs-exposed and unexposed samples for each
cytogenetic endpoints analyzed. Similarly, the combined treatments failed to indicate the presence of any
synergistic effect between the ELF magnetic fields and the physical mutagen. Results from the 48 h exposure time
are currently being processed.
References
NRPB (1992) Electromagnetic fields and the risk of cancer .NRPB document 3(1):1-138
L. Verschaeve (1995) Can non-ionizing radiation induce cancer? Cancer J. 5:237-249
D.E.Hintenlang (1993) Sinergistic effects of ionizing radiation and 60 Hz Magnetic fields.
Bioelectromagnetics 14, 545-551
A.Maes, M.. Collier, S.Vandoninck, P.Scarpa and L.Verschaeve (2000) Cytogenetic potential effects of 50
Hz magnetic fields of different magnetic flux densities. Bioelectromagnetics 21, 589-596
This project is partially supported by Ministry of Environment (4.4)
163
P3/18
NO INTERACTION OF ELF MAGNETIC FIELDS WITH A CHEMICAL ANEUGEN
ON MICRONUCLEUS INDUCTION IN HUMAN LYMPHOCYTES.
G.R. Verheyen; G. Pauwels; L. Verschaeve; G. Schoeters
Center of Expertise in Environmental Toxicology, Flemish Institute of Technological Research (VITO),
Mol, Belgium
Epidemiological studies have suggested a possible association between
50 Hz Extreme Low
Frequency (ELF) fields and an increased risk of cancer (e.g., Verschaeve et al., 1995). However,
contradictory findings are reported in the literature, suggesting that if there is a hazardous effect of
ELF on human health, that it is a complex multifactorial process.
Here, we tested if 50 Hz magnetic fields can interfere with the action of a known aneugen (Vinblastine
- VBL) on micronucleus formation in lymphocyte cultures.
Isolated lymphocyte cultures were prepared in duplicate from 18 individuals. Three groups of 6
individuals were exposed to 50 Hz magnetic fields of respectively 0, 80 and 800 µT during the 72
hours incubation period. Twenty-four hours after culture initiation, each individual within each ELF
group was exposed to a concentration gradient (0, 5, 10, 15 ng/ml) of VBL (Marshall et al., 1996).
Cytochalasine was added after 44 hours, and the cells were harvested at 72 hours. Isolated
lymphocyte cultures were scored for the presence of micronuclei, the nuclear division index and
apoptosis. Data were analysed using ANOVA and repeated measures ANOVA.
As expected, increased VBL concentration resulted in an increased micronucleus and apoptosis
frequency and in a decreased NDI. We observed no effect of ELF on micronucleus induction or
apoptosis frequency. In the absence of VBL, the NDI was significantly higher in the 800 µT group
compared to the other groups, suggesting an effect of ELF on cell proliferation. No interaction between
ELF and VBL was observed.
Marshall R. R., Murphy M., Kirkland D. J., Bentley K. S. (1996) Mutation Research 372: 233-245.
Verschaeve L. (1995) Cancer J. 5: 237-249.
164
P3/19
NEW MOLECULAR TOOLS FOR PREDICTIVE TOXICOLOGY: THE ROLE OF
MOLECULAR DATABASES AND COMPREHENSIVE MICROARRAYS
P. Alen, K. Schmeiser, W. Whitford, S. Hicken and G. Farris
PHASE-1 BioResearch N.V., Technologiepark 4, B-9052 Zwijnaarde, Belgium
Although several microarrays are available to analyze rat gene expression, we saw the need for a chip
containing specific genes selected for toxicologic response. Two approaches were used to identify
genes whose expression is markedly affected by toxic insults.
First, animals were treated with a set or known toxicants. The response of 17,500 genes to each of
these treatments was analyzed. The second approach was the evaluation of the responsive genes in
the liver and kidney of rats exposed to cisplatin of aflatoxin using transcriptome profiling. Thus, a total
of 700 genes were chosen as toxicology responsive and used to build a microarray for comprehensive
toxicity testing (rat CT array).
The CT array was used to build a database of gene expression information. Rat TOXbank presently
contains gene expression data for several tissues from rats exposed to > 100 compounds, with a total
of approximately 190 gene expression hybridizations per compound. Apart from gene expression data,
TOXbank also contains histopathology images and results from urine and serum chemistry and
hematological analysis. The database will grow to include over 100 compounds during the next year.
In combination with Phase-1’s MATRIXexpress software package, the CT array and TOXbank offers
toxicologists tools to discover mechanisms of toxicity and to predict toxicological profiles for new
drugs.
165
P3/20
DNA ADDUCTS, MUTANT FREQUENCY AND GENE EXPRESSION PROFILES IN
BPDE-EXPOSED TK6 CELLS
1S.M.
Morris,
1O.E.
Domon,1L.J. McGarrity,
1S.J.
Culp,
1L.
Blankenship,
1J.T.
MacGregor,
2B.
Rosenzweig and 2F.D. Sistare
1NCTR/FDA,
Jefferson, AR, USA and 2CDER/FDA, Laurel, MD, USA
Our laboratories are interested in determining if carcinogen-induced alterations in gene expression
profiles relate to measures of genetic toxicity such as DNA adduct formation, mutation induction or cell
death. Thus, TK6 cells were exposed to 0.0, 0.1, 0.2, or 0.3 M BPDE for 4 hours, the carcinogen
removed and the cells either subcultured for an additional 20 hours or seeded for mutation analysis.
At 4 and 24 hours, aliquots of the cell suspension were used to (1) measure the formation of specific
DNA adducts by
32P-postlabeling,
(2) define the cell death pathways by flow cytometry and (3)
establish gene expression profiles by cDNA microarray analysis utilizing a 350 gene human array.
The dG-N2-BPDE adduct was identified at both 4 and 24 hours after exposure and may account for
the significant increase in the mutant frequency at the Thymidine Kinase locus and at the
Hypoxanthine Phosphoribosyl Transferase locus. Molecular analysis of expanded clones is currently
being conducted to confirm the nature of the mutagenic lesion. Cell death occurred primarily by
apoptosis at all concentrations of BPDE. In order to be classified as a positive in the gene expression
analysis, the following criteria were applied.
The fluorescence intensity was at least 4 standard
deviations above background, the change in expression was at least 1.5X above or below the control,
and the change in expression must have been in the same direction when the dye labels were
reversed (T/C = Cy3/Cy5  T/C = Cy5/Cy3). Positive changes in expression, as defined by the above
criteria, were detected in 8 of 350 genes at 4 hours and 16 genes at 24 hours. A separate set of
genes (4 genes at 4 hours and 7 genes at 24 hours) were initially classified as positive but were
eliminated when the dye labels were reversed and the direction of expression changed. Further, no
increase or decrease in expression greater than 3X over control was detected. Experimental variables
are currently being systematically examined in order to enhance the induction of gene expression
associated with BPDE treatment.
166
P3/21
DIFFERENTIAL GENE EXPRESSION IN RATS AFTER INJECTION WITH KIDNEY
TOXICANTS
K. Schmeiser, P.Alen, G. Farris and L. Kier
Phase-1 BioResearch, Technologiepark 4, B-9052 Zwijnaarde, Belgium
Gene expression profiling using the Phase-12 rat CT microarray was performed on kidney samples
obtained from male rats treated with 21 compounds. Kidney samples were also examined for
histopathological changes and serum chemistry. Overall perturbation of the tissues, as evidenced by
the number of up or down-regulated genes, correlates with histopathology. Determination of
correlation between individual gene expression and histopathology indicated a number of genes
whose induction or repression in the kidney specifically correlated with kidney histopathology.
Correlating genes at earlier times tended to be more reflective of damage and compound-specific
while genes correlating at the later time point tended to be responsive to all compounds. These
patterns are consistent with sequential transition from active damage processes to repair processes.
The use of subsets of correlating genes in a correlation matrix analysis enhanced discrimination
between compounds producing kidney histopathology. This also identified gentamicin as producing
expression changes consistent with kidney toxicity. These data clearly indicate that gene expression
is correlative with toxicity in the kidney and can provide robust, quantitative information on the
biological processes underlying the toxicity.
The identification of sets of specific genes whose
expression correlates with toxic endpoints appears to have significant predictive value.
167
P3/22
IDENTIFICATION
OF
MECHANISMS
AT
THE
GENOME
LEVEL
FOR
PROTECTION AGAINST COLON CANCER BY VEGETABLES
S.G.J. van Breda1; J.H.M. van Delft1; L.G.J.B. Engels2; J.C.S. Kleinjans1
1Department
2Department
of Health Risk Analysis and Toxicology, University of Maastricht, Maastricht, NL;
of Gastroenterology, Maasland Hospital, Sittard, NL
Globally, cancer of the colon and rectum is the fourth most common incident cancer and cause of
death from cancer. Particularly in modern societies and urbanising areas in the developing world,
incidence and deaths from this type of cancer are increasing. In the Netherlands, colorectal cancer
accounts for approximately 10% of total cancer cases. It is commonly accepted that food preparation
and dietary habits are the most relevant exogenous factors affecting colorectal cancer risk.
Epidemiological studies have demonstrated that particularly the consumption of raw, green and
cruciferous vegetables is associated with reduced cancer risk. Several types of plant food components
have been identified, e.g. flavonoids, carotenoids, glucosinulates, vitamins and fibers, that may explain
the observed protective effects via various different mechanisms. However, the involved molecular
processes at the genome level are mostly unknown. Furthermore, most studies focus on surrogate
tissue rather than on the ultimate target organ. Therefore, a human dietary intervention study is started
to identify the genes whose expression is modified in vivo in human colon epithelium by vegetables. A
study population of female patients with colorectal adenomas, and healthy controls is subjected to
either a low (75 g per day) or high (300 g per day) vegetable diet consisting of carrots, cauliflower,
peas and unions for a period of two weeks. Before and after the dietary intervention, biopsies are
obtained from the sigmoid colon. Using micro-array technologies, colonic messenger RNA levels of
approximately 4000 genes will be analysed. The selected genes will include oncogenes, tumorsuppressor genes and biotransformation genes. Comparison of pre- and post-intervention values as
well as comparison of the experimental groups will identify those genes whose expression is
modulated by vegetables. Comparison of gene expression in patients and controls will reveal genetic
susceptibility factors for colorectal cancer.
168
P3/23
CHROMOSOME
ABERRATIONS,
GENOTOXICITY
AND
CYTOTOXICITY
INDUCED BY SELECTED CHEMICALS
P. Arni and M. Kiffe
Syngenta AG, Health Assessment and Environmental Safety, CH-4332 Stein, Switzerland
Several non-genotoxic chemicals are known which induce chromosomal aberrations in vitro at
cytotoxic concentrations only. The general relevance of clastogenic effects, which occur only together
with cytotoxicity, is therefore questionable. In the present work, the chromosome aberration (CA) test
and the single cell gel electrophoresis (comet) assay were used on the same cell line to investigate,
whether the latter could assist in the interpretation of questionable results obtained with the CA test.
The following compounds were tested: Menthol a non-mutagen and non carcinogen, sodium iodoacetate, a metabolic poison, valinomycin, which is known to induce apoptosis, chloral hydrate and
hydroquinone, both potential aneugenic chemicals.
Both, the CA test and the comet assay were performed on CHO cells K5 cloned in our laboratory. The
CA test was performed in situ; the cells were treated 3h (+ 18 h recovery), 21h or 24h. In the comet
assay treatment was for 3 or 24 h. With sodium iodoacetate an additional test with lysed cell was
performed. Cytotoxicity was assessed by measurement of the inhibition of the mitotic index and with
trypan blue or propidium iodide staining (flow cytometry).
Menthol was negative in both test systems. Sodium iodoacetate was also negative in a series of
cytogenetic experiments, but revealed positive effects in the comet assay. When tested in lysed cells,
it was negative. Valinomycin induced cytological detectable apoptosis, but was negative in both test
systems. Chloral hydrate clearly induced CAs at cytotoxic concentrations. In the comet assay it was
negative, however, at highly toxic concentrations an effect was seen after treatment for 24h. The
results obtained with hydroquinone did not meet the criteria for a positive response in the CA test. It
was considered negative in the comet assay. Effects were seen at highly toxic concentrations only.
The comet assay is a useful tool in the interpretation of effects obtained in the CA test, although it
does not reveal the same results. The test variant with lysed cells offers a possibility to assess indirect
genotoxic effects. With the comet assay it was not possible to detect apoptosis with valinomycin,
which is known to induce this effect. Some published cytogenetic results with CHO cells were not
reproducible.
169
P3/24
HIGH THROUGHPUT SINGLE CELL QUANTIFICATION OF DNA DAMAGE
BASED ON CONFOCAL IMAGING OF VERTICAL COMETS
Ph. Baert; P. Van Oostveldt
Dept. Molecular Biotechnology, FLTBW, Ghent University, Belgium
Single cell gel electrophoresis or 'Comet assay' is a rapid and sensitive fluorescent microscopic
method to examine DNA damage and repair at individual cell level. Since the introduction of the
Comet assay, a number of advancements have greatly increased the flexibility and utility of this
technique in diverse research fields ranging from fundamental DNA repair to ecotoxicological studies
(Fairbairn et al., 1995).
However, major drawbacks of conventional non automated CCD based microscopic systems are (1)
the quality of the comet images, (2) the correct sampling of the DNA content and (3) the cumbersome
and time consuming procedures encountered when recording the comets. We therefore developed an
innovative approach of the comet assay that greatly reduces the time of analysis while enhancing the
sampling of the DNA content. The approach is based on 3D confocal imaging of vertical oriented
comets at high density. By this way a large number of comets within a microscopic field can be
obtained without DNA overlap of multiple nuclei. An experimental setup enabling observation of
vertical comets while using conventional alkaline comet procedures (Olive et al., 1990) was developed
together with dedicated software algorithms to retrieve quantitative data in the form of classic comet
parameters at single cell level.
Results show that when using a 60 fold objective, the whole procedure of comet analysis can be
reduced by a factor 10 since one stack of confocal images can contain as much as 10 different comets
while maintaining sufficient resolution. Furthermore confocal microscopy provides images with high
contrast in which the object can be clearly separated from background while comet pixel intensities
remain in a broad dynamic range of the detector. The sampling of DNA at different focal distances
enables also a much better discrimination between comet head and tail since the origin of scattered
DNA can be elucidated at every distance.
In conclusion, this novel approach of comet analysis has important advantages over classical
methods, not only in the field of speed, but also in the field of sensitivity.
Fairbairn, D. W., Olive, P. L. and O’Neill, K. L. (1995). Mutation Research, 339, 37-59.
Olive, P. L. Banath, J. P. and Durand, R. E. (1990). Radiation Research, 122, 86-94.
170
P3/25
GENOTOXICITY OF
ORGANIC EXTRACTS
DERIVED FROM
AIRBORNE
PARTICULATE MATTER IN FLANDERS, BELGIUM
E. Brits1, G. Schoeters1, L. Verschaeve1, E. Roekens2, E. Muylle2
1 Flemish
Institute for Technological Research (Vito), Environmental Toxicology, Mol, Belgium
2 Flemish
Environmental Agency (VMM and MIRA), Erembodegem, Belgium
Hazard characterisation of airborne particulate matter in Flanders is based on chemical compound
analysis. Considering the complexity of these environmental mixtures, the chemical approach alone is
insufficient. In order to perform an effect-orientated evaluation of the air quality, the genotoxic potency
of airborne particulate matter originating from various sites in Flanders was tested. The particles
contain many chemicals, including polycyclic aromatic hydrocarbons (PAHs), which have been
suggested to increase lung cancer risk in humans. In this study, bioassay genotoxicity was conducted
with organic extracts of PM10, collected on PTFE filters in urban, industrial and rural sites in Flanders,
Belgium. The genotoxic activity of the organic extracts was assessed using a battery of 4 in vitro
genotoxicity tests. The most widely used genotoxicity test, performed using bacteria as target cells,
was employed to analyse the extracts: the Ames reverse mutation test with Salmonella typhimurium
TA98 tester strain. Recently, several new bacterial tests have been developed that are less labour
intensive compared to the Ames test. For this study we selected the Vitotox test which measures the
bioluminescence activity from an integrated lux-operon reporter system, based on the activation of the
SOS recN gene in the Salmonella typhimurium TA104. In addition, two tests using human blood cells
as target cells were utilised. The Comet assay employs electrophoresis of leukocytes embedded in
agarose on a microscopic slide resulting in DNA-comets where the tail-DNA-content reflects the
amount of DNA-damage. Finally, in the Micronucleus test, the incidence of micronuclei in binucleated
lymphocytes serves as an index of genetic damage. PM10 organic extracts proved to be potentially
genotoxic, depending on the origin of the particles and on the bioassay used for the screening. PM10
organic extracts from industrial sites and 1 urban site did not induce micronuclei, but were positive in
the other three tests. For the rural sites, the effects of PM10 organic extracts were small or nonexistent in all bioassays. The PM10 organic extract of 1 urban site showed a significant dose-response
relationship for the micronucleus test and the Ames test, and positive results in the Vitotox test and
Comet assay. Adding S9 to the bacterial tests increased the effect of all extracts. The Ames test was
more appropriate for the genotoxicity assessment of organic extracts in comparison with the Vitotox
test, due to the toxicity of organic solvents to the Vitotox bacteria. The comet assay showed that DNA
damage is caused by organic compounds adsorbed onto PM10, but it was not possible to establish a
dose-response curve for all samples.
171
P3/26
INDUCTION OF ATHEROSCLEROSIS IN APOE-KNOCKOUT MICE EXPOSED TO
BENZO[A]PYRENE
D.M.J. Curfs1 ; E. Lutgens2; M.J.A.P. Daemen2; F.J. van Schooten1
1Department
of Health Risk Analysis and Toxicology,
2Department
of Pathology, University of
Maastricht, Maastricht, The Netherlands
Background:
In the early 70s it was recognized that chemicals like benzo[a]pyrene (B[a]P), which can induce
tumour development by damaging the DNA, are also able to initiate/promote atherosclerotic plaques.
However, the mechanisms responsible for developing chemically induced atherosclerosis are still not
well understood.
Objective:
We performed a pilot study to investigate the effects of B[a]P treatment on the induction and
promotion of plaque formation in atherosclerosis susceptible mice (apoE-knockout mice) as well as the
influence on plaque composition and stability.
Design & Methods:
Twenty male apoE-knockout mice were orally treated once per week during 12 weeks with 5
mg/kg.bw. B[a]P or with vehiculum only. After completion of the experiment animals were sacrificed .
Blood was drawn from the caval vein for the assessment of lipid profile. Subsequently, the complete
arterial tree was taken out and used for histological examination and
32P-postlabeling.
Additionally,
organs like lung, liver and heart were collected.
Results:
In the exposed animals mean adduct levels in the aorta were higher than those in lung (respectively
38.9±6.8 and 28.5±11.0 adducts per 108 nucl., n=9). Histological examination showed more advanced
lesions in the aortic arch of the exposed group (4 out of 5 animals) compared to the control group (2
out of 4). Also the mean advanced lesion area per aortic arch was larger in exposed animals
compared to the controls (128351±38413 and 44981±39153 m2). Moreover, phenotypical differences
were observed; the fibrous caps of the advanced lesions in the exposed animals contained more
infiltrated cells and were covered with macrophage-foam-cells.
Conclusions:
B[a]P treated animals showed a considerable amount of B[a]P-DNA adducts in the arterial tree.
Correspondingly, the aorta of exposed animals showed increased plaque formation in a more
advanced stage compared to the control group. At present we are performing a larger study to confirm
the above mentioned results.
172
P3/27
APOPTOSIS INDUCTION IN HUMAN LYMPHOCYTES AFTER IN VITRO
EXPOSURE TO COBALT/HARD METAL COMPOUNDS
1
M. De Boeck , I. Decordier1, N. Lombaert1, E. Cundari1, D. Lison2 and M. Kirsch-Volders1
1Vrije
Universiteit Brussel, Laboratorium voor Cellulaire Genetica, Brussel, Belgium.
2Université
catholique de Louvain, Unité de Toxicologie industrielle et Médecine du Travail,
Bruxelles, Belgium.
An increased risk of lung cancer is associated with occupational exposure to mixtures of cobalt metal
(Co) and tungsten carbide (WC) particles, but apparently not when exposure is to cobalt alone. The
mechanism for this increased cancer risk is not fully understood. The evaluation of the in vitro
genotoxic effects in lymphocytes exposed to varying cobalt species demonstrated that the WC-Co
hard metal mixture is more genotoxic (DNA damage, chromosome/genome mutations) than metallic
Co alone. WC alone was not genotoxic. Thus, WC-Co represents a specific (geno)toxic entity.
In order to assess the survival of human lymphocytes after in vitro exposure to metallic Co, CoCl2, WC
and the WC-Co mixture, two apoptosis/necrosis detection methods were applied (annexin V staining
and flow cytometry). Annexin-V staining of early apoptotic cells demonstrated a dose- and time
dependent induction of apoptosis by metallic Co, CoCl 2, WC and the WC-Co mixture. The time course
of the process varied according to the metal species tested. Metallic Co and CoCl 2 caused a gradually
increasing frequency of apoptotic cells with time (up to 24 h). WC-induced apoptosis displayed a
typical 6 hour peak, which was not the case for the WC-Co mixture or for Co. Apoptosis induction by
the WC-Co mixture was intermediate between that induced by Co and WC separately. Analysis of
propidium iodide stained cells by flow cytometry was performed as a later marker for apoptosis
induction. Preliminary data indicate similar tendencies of apoptosis induction as those detected by
annexin-V. Identification of the apoptotic pathway triggered by the metal compounds was studied by
inhibition of the ceramide-apoptosis pathway by fumonisin causing reduction of apoptosis induction for
all compounds, but strongest after 6 hour exposure to WC. The use of specific caspase inhibitors will
allow to further elucidate the different pathways involved. The current data demonstrating in vitro the
apoptosis induction by metal compounds, in addition to their in vitro genotoxic activity may help to
explain their in vivo carcinogenicity in humans.
173
P3/28
RELATION BETWEEN THE INDUCTION OF APOPTOSIS AND THE INDUCTION
OF
MICRONUCLEI
AFTER IN VITRO
EXPOSURE
TO THE
ANEUGEN
NOCODAZOLE.
I. Decordier1, M. Kirsch-Volders1 and E. Cundari2.
1Vrije
Universiteit Brussel, Laboratorium voor Cellulaire Genetica, Pleinlaan 2,
B-1050 Brussel, Belgium.
2
Centro di Genetica Evoluzionistica CNR, Via degli Apuli, 4, 00185 Roma, Italy.
Our interest for the consequences of dysfunction of the microtubules is based on previous studies of
the laboratory on the mechanisms of induction of aneuploidy
and apoptosis after exposure to
microtubule inhibitors such as nocodazole, a chemotherapeutic agent that inhibits microtubule
polymerisation (Elhajouji et al., 1997; Verdoodt et al., 1999). The demonstrations by our previous
studies showing not only the induction of aneuploidy and apoptosis by nocodazole, but also that
threshold values exist for the induction of chromosome non-disjunction and chromosome loss may
have important implications for hazard and risk assessment. It is therefore crucial to determine
whether around these threshold values, apoptosis might be induced by the aneugen, eliminating cells
containing premutagenic/mutagenic lesions, if this would be the case the defined thresholds would not
be applicable to apoptosis deficient cells. The main objectives of this study were the confirmation of a
nocodazole triggered apoptotic signal and the investigation whether micronuclei induced by
nocodazole could be eliminated by apoptosis. Therefore we aimed at the identification of the caspase
pathway(s) activated in microtubule-induced apoptosis and at the investigation of the consequences of
caspase inhibition on the frequencies of micronucleated lymphocytes. Three specific caspase
inhibitors Ac-DEVD-CHO, Boc-AEVD-CHO and Ac-LEHD-CMK, a caspase-3, caspase-8 and
caspase-9 specific inhibitor respectively, were used to answer these questions. Our results showed
the involvement of the initiator caspases 8 and 9 and the effector caspase-3 in the apoptotic process
triggered by microtubule inhibiton after exposure to the aneugen nocodazole. The obtained data
indicated that in vitro in human lymphocytes apoptosis is also induced in interphase. Furthermore
these results suggest that micronuclei can be eliminated by apoptosis.
References:
Elhajouji et al. (1997) Mutagenesis, 12, 133-140.
Thornberry and Lazebnik (1998) Nature, 281, 1312-1316.
Verdoodt et al. (1999) Mutagenesis, 14, 513-520.
Acknowledgements:
This study was supported by the EU research program ENV4-CT97-0471.
174
P3/29
THE USE OF IN-SILICO STRUCTURE ACTIVITY-BASED PREDICTION SYSTEMS FOR
GENOTOXICITY AT NOVARTIS
S. Glowienke ; HJ. Martus ; G. Bold, L. Mueller
Genetic and Experimental Toxicology, NOVARTIS Pharma AG, CH-Basel, Switzerland
In silico predictions for toxicity based on the evaluation of structure-activity-relationships (SAR) are
increasingly gaining importance owing to the needs imposed by combinatorial chemistry and high
throughput pharmacological target screening. Various in silico systems are available to predict toxicity,
in particular genotoxicity (e.g. DEREK, Multicase, Topkat, Toxsys). Naturally, the predictions from all
of these systems have to rely on the openly available literature and confidential data shared with the
system owners. Recently, we have tested 20 different simple anilines in the Ames test, using strains
TA98 and TA100. With the same molecules, a SAR prediciton of mutagenicity was done using the
above SAR systems. Our comparison showed that no single system has major advantages over the
others with regard to prediction accuracy. From our limited data, we conclude that anilines with two
halogen-containing substituents exhibit no mutagenicity. Hence, we can use the data to improve the
prediction accuracy of the SAR systems.
We are currently in the process of validating DEREK, Version 4.0.1, for our in-house use as an
advisory system for the prediction of genotoxicity. In our initial selection of 515 Novartis compounds
(93 Ames test positives, 410 Ames test negatives), DEREK predicted Ames positivity with a sensitivity
of only 29% and a specificity of 87%. Subsequently, the sensitivity of the system could be improved
up to more than 82% by the inclusion of 3 additional rules that would give alerts for Novartis-specific
genotoxic structures and that have been thus far not included in DEREK.
175
P3/30
THE STUDIES OF FLUAZIFOP FROM FENOXY ACID DERIVATIVES AS
PEROXISOME PROLIFERATOR IN RAT LIVER
G. Kostka; J.K. Ludwicki; D. Palut; K. Lembowicz; B. Wiadrowska
Department of Environmental Toxicology, National Institute of Hygiene, Warsaw, Poland
In our previous study (1) we have fully demonstrated that diclofop [2-[4-(2,4-dichlorophenoxy) phenoxy
]propionic acid], introduced to the environment as herbicide, exhibits the properties of peroxisome
proliferators (PPs). It was therefore considered to be of particular interest to establish to what degree
other phenoxy propionic acid herbicides share these properties.
The
effect
of
fluazifop
[2-[4-(5-trifluoromethyl-2-piridyloxy)phenoxy]propionic
acid]
on
hepatomegaly, peroxisome proliferation and DNA synthesis in hepatocytes of male Wistar rats.
Fluazifop was administered by oral gavage in an olive oil suspension at daily dose of 445
mg/kg b.w. x day-1 for 2, 4, 7 and 14 days. DNA synthesis measured by [3H] thymidine incorporation
into nuclear DNA was determined by scintillation counting and was expressed in dpm per mg DNA.
Peroxisome proliferation was determined by electron microscopy JEOL 100 C in preparations stained
with uranyl acetate and lead citrate.
The results of our study demonstrate that fluazifop significantly increased relative liver weigh
(RLW) to 130, 133, 142 and 162% above the control value, after administration 2, 4, 7 and 14 oral
dose of 445 mg/kg b.w. x day-1. Ultrastructural examination of liver section from rats treated with
fluazifop, demonstrated the increase in number of peroxisomes. After 2, 4, 7 and 14 days treatment,
fluazifop induced 1,5-fold (p< 0,05), 2,0-fold (p< 0,01), 2,9-fold (p< 0,001) and 2,6-fold (p< 0,001)
increase in number of peroxisomes, respectively. The parallel biochemical measurements showed that
was an increase in peroxisomal palmitoyl-CoA oxidation and catalase activity (markers of peroxisome
proliferation) in rats treated with fluazifop. Treatment of rats with fluazifop resulted in increase in DNA
synthesis, although to a relative minor degree; there was 198 (p< 0,05), 200 (p < 0,01) and 216% (p<
0,05) of control after 2, 4 and 7 days of dosing. After prolonged administration (for 14 days) of the
above dose DNA synthesis declined to control level.
In conclusion, the results of our study demonstrate that fluazifop exhibits the properties PP s.
1. Palut D., Ludwicki J.K., Kostka G., Kopeć-Szlęzak J., Wiadrowska B., Lembowicz K.,2001.
Studies of early hepatocellular proliferation and peroxisomal proliferation in Wistar rats treated
with herbicide diclofop. Toxicology, 158, 119-126.
176
P3/31
ANEUPLOIDY AND TUMOUR PROGRESSION IN BARRETT’S OESOPHAGUS
Elizabeth M Parry, Jeanette Croft and Shareen Doak
Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales
Swansea, Singleton Park, Swansea SA2 8PP, UK.
Genetic instability is a characteristic feature of cancer cells. Additionally, most cancers are clonal and
heterogeneous with regard to karyotype. Therefore, it is often difficult to establish the relative timing of
events such as point mutation and chromosome instability that may be driving cancer progression. To
determine the frequency of aneuploidy in tumour progression we have examined biopsy specimens
from Barrett’s oesophagus. In this condition the stratified squamous epithelium of the distal
oesophagus is replaced by a columnar epithelium and the progression to malignancy occurs according
to a multistep process: metaplasia, low and high grade dysplasia, and adenocarcinoma. The
segregation of chromosomes 1, 4, 9, X and Y have been measured in the interphase nuclei of imprint
preparations made from biopsy samples by using chromosome specific probes and in situ
hybridisation techniques. Copy number of gene specific probes for p53, Rb and p16 have also been
studied in the same cells. A clear relationship between aneuploidy and tumour progression was seen
for all chromosomes although differences in frequency were also observed.
177
P3/32
TESTING MELANOIDIN FRACTIONS FOR MUTAGENICITY - THE USE OF IN
VITRO TESTS
J.L.S. Taylor(1,2); L. Regniers(1); L. Verschaeve(1); A. Maes(1); C. Arribas Olave(2); K. AbbaspourTehrani(2); E. Elgorashi(1,2); N. De Kimpe(2); J. van Staden(3); A. Fossey(4)
1. Vlaamse Instelling voor Technologisch Onderzoek, Environmental Toxicology, Boeretang 200, B2400 Mol, BELGIUM
2. Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences,
University of Gent, B-9000 Gent, BELGIUM
3. Research Centre for Plant Growth and Development, University of Natal Pietermaritzburg, Private
Bag X01, Scottsville 3209, SOUTH AFRICA
4. School of Molecular and Cellular Biosciences, University of Natal Pietermaritzburg, Private Bag
X01, Scottsville 3209, SOUTH AFRICA
Melanoidins comprise a complex, and largely undefined mixture of compounds formed by the
interaction of free amino groups and monosaccharides, a reaction that occurs during the processing,
cooking and storage of food. The series of interactions resulting finally in melanoidins is also referred
to as the Maillard reaction. The composition of melanoidins varies greatly with the reaction conditions,
including temperature and reaction time. These products have been reported in various articles to
possess mutagenic and anti-mutagenic activity. In effect, many compounds are formed in the Maillard
reaction - some mutagenic and some anti-mutagenic. These cannot be differentiated, and it is thus the
net influence of the combined mutagenic and anti-mutagenic compounds that is tested in the assays.
The current study involved testing melanoidins produced using the standardised COST protocol for the
Maillard reaction, in three mutagenicity assays. The starting products used were glucose and glycine.
The melanoidins were separated using dialysis into high and low molecular weight fractions, and
these, as well as the volatile fraction collected during the reaction, were tested for the presence of
potential mutagens using the Ames test, the Vitotox  test, and the Micronucleus test (conducted using
human lymphocytes).
In addition to detecting mutagenic activity, the Vitotox  test also gives an
indication of the potential toxicity of the test substances.
178
P3/33
179
P3/34
SIMULTANEOUS ASSESSMENT OF GENOTOXICITY AND CYTOTOXICITY IN A
DOWNSCALED IN VITRO MICRONUCLEUS TEST
F. Van Goethem, V. Van Hoof, E. Hansen, K. Cools and P. Vanparys
Dept. of Genetic & In Vitro Toxicology, Janssen Pharmaceutica N.V., B-2340 Beerse, BELGIUM
The in vitro micronucleus test (MNT) is a well established assay to evaluate the genotoxic (clastogenic
and aneugenic) properties of new chemical entities during the safety assessment phase of drug
development. However, the increasing numbers of new molecules in the pharmaceutical, chemical
and cosmetic industry requires an optimized and standardized approach providing rapid test results
and the use of small amounts of test compound. Although several initiatives to increase the throughput
already have shown very promising results, it is a well known fact that the cytotoxic potential of a test
compound can cause chromosomal damage. In this case, a confounding interpretation of the
genotoxic potential might occur since the observed micronuclei can be the result of cell death-induced
DNA cleavage.
For these reasons, we developed a down-scaled version of the MNT in 96-well microplates, hereby
simultaneously assessing cytotoxicity and genotoxicity. To prevalidate this approach, V-79 Chinese
hamster cells were treated with typical clastogens (Mitomycin C and Cyclophosphamide) with and
without a metabolic activation system (liver S9-homogenate). Further, non-genotoxic compounds
(Ethanol and DMSO) were used up to cytotoxic concentrations. Cytotoxicity of each test compound
was assessed by a fluorometric procedure with Alamar Blue, and this in the same target cell
population in which MN analysis will occur. Once the cytotoxicity profile was determined, cells were
isolated, fixed and stained with a differential fluorescent dye solution (DAPI/SR101).
Results show that the experimental set-up required only 5 mg of test compound, and that a
simultaneous cytotoxicity evaluation was able to identify false positive results interfering with the final
outcome of genotoxicity testing.
When this screening methodology can be implemented during the early phases of preclinical drug
development, the selection process will be improved and lead compounds can be optimized.
180
P3/35
DEVELOPMENT
OF
A
DISSOCIATION
METHOD
TO
OBTAIN
SINGLE
COLUMNAR EPITHELIAL COLON CELLS TO BE USED IN THE COMET ASSAY
A. Vanhauwaert
Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Faculteit Wetenschappen
Pleinlaan 2 - B 1050 BRUSSEL - Belgium
Previous work concentrated on the in vivo gut micronucleus test and how it is able to detect
clastogens and aneugens administered orally and was described in Vanhauwaert et al., 2001. To have
a biologically more relevant assessment of the carcinogenic risk at the level of the gut (large intestine),
it is essential not only to quantify the mutations that arose during the first cell division after exposure,
but also to take the survival rate (apoptosis/necrosis) of these genetic aberrations into account, and to
have an idea about exposure at the level of the DNA. The laboratory aims at the development of a
sensitive test model for per os mutagens/carcinogens. In this model several tests would be used: a
viability test to assess cytotoxicity, the Comet assay to detect DNA-damage and alkali labile sites, and
the previously mentioned gut micronucleus test to assess both chromosome and genome mutations
(with or without application of the FISH-technique).
It is interesting to use the comet assay because it is a technique which allows to assess exposure
(because of the detection of, not only double and single strand breaks, but also open repair sites). The
test can be applied on all cell types when they are available as a single cell suspension. So, to be able
to apply the comet assay on gut cells, a method needed to be found to isolate the colon cells. Several
methodologies were tested.
Cold and warm trypsinization lead to suspensions of nuclei which had a TD (tail DNA) of respectively
34.7% and 17.8% (1h lysis, 40 min denaturation, 20 min electrophoresis). A homogenizing technique
using a manual Potter-type homogenizer also gave a suspension of nuclei which showed a lot of DNA
damage in the Comet assay (1h lysis, 40 min denaturation, 20 min electrophoresis). The problem with
suspensions of nuclei is that one cannot be sure that the obtained nuclei are the nuclei from columnar
epithelial cells (our cells of interest) and not the nuclei from for instance cells from the surrounding
muscle layer. Finally, several methods using EDTA solutions were used (30 mM, 100 mM, 150 mM)
combined with different comet assay protocols (1 h lysis, 40 min denaturation, 20 min electrophoresis /
1 h lysis, 20 min denaturation, 15 min electrophoresis / 3 h lysis, 20 min denaturation, 10 min
electrophoresis), which seem to be good methods firstly because columnar epithelial cells are present
in the suspension, and secondly because the comet assay results are more acceptable (mean TD of
12.2%).
Annelies Vanhauwaert, Philippe Vanparys, Micheline Kirsch-Volders (2001) The in vivo gut
micronucleus test detects clastogens and aneugens given by gavage. Mutagenesis, 16(1), 39-50.
181
Author Index
182
Abbaspour-Tehrani K.
P3/32
Bibbiani R.
P2/12
Abbey M.
P3/6
Bieler C.A.
P2/1
Abbondandolo A.
S1/1
Billinton N.
P1/9
Adler I.-D.
W1/4
Binková B.
P2/19
Alapetite C.
S7/3
Biros E.
P1/4
Albertini S.
W3/4
Biros I.
P1/4
Alen P.
P3/19; P3/21
Bisanti L.
P2/20
Alessandrini C.
P2/23
Blankenship L.
P3/20
Alexandre S.
P1/1
Blin M.
P1/17
Alvarez L.
P2/33
Bogyiova E.
P1/4
Angelini S.
P2/10
Bold G.
P3/29
Anzion R.
P2/6
Bonde J.P.
P2/20
Apostoli P.
P2/20
Bouviez P.
P1/33
Araujo M.
P1/32
Brans B.
P2/30
Arlt V.M.
P2/1; P2/2
Brás A.
S5/4
Arni P.
P3/23
Breitbart E.W.
P1/34
Arribas Olave C.
P3/32
Brits E.
P3/25
Asmuss M.
S8/3
Brozović A.
P2/26
Autrup H.
S7/2; S7/4;
Bryant P.E.
S9/5
P2/7
Bubak A.
S3/3
Baan R. A.
W2/4
Buerkle A.
S8/3
Baatout S.
P1/2
Bumgarner R.E.
W3/3
Bach A.
P1/12
Buschini A.
P1/23; P2/23
Baert Ph.
P3/24
Buset J.
P1/2
Baeyens A.
S9/3; P3/1;
Butkiewicz D.
P1/10
P3/11
Caballín M.R.
S3/2
Bajek M.
P1/19
Cabral-Neto J.
S2/2
Ballantyne M.
P2/34
Cahill P.
P1/9
Banaszewski J.
S4/2
Cangiano T.
P1/26
Barbé M.
P3/11
Cantelli Forti G.
P2/10
Barquinero J.F.
S3/2
Caruso F.
P2/20
Barrios L.
S3/2
Case C.P.
S8/4; P2/28
Bartsch H.
P1/12
Cassapo R.
P1/14
Bartusiak K.
P1/17
Castro M.
P1/14
Bavorova H.
P2/3
Catalán J.
P2/4
Begemann P.
S4/4
Cerosaletti K.
S2/3
Bell, D.A.
S1/3
Chagnon M.C.
P1/20; P2/24
Bello J.
P2/32; P2/33
Chaveca T.
P1/14
Benotmane A.
P1/2
Chorazy M.
P1/10
183
Cieśla J.M.
P1/19
Dimitroglou E.
P2/17
Cigarrán S.
S3/2
Diodati A.
S3/4
Cimini D.
P3/12
Doak S.
P3/31
Citterio E.
S2/1
Dobrowolny G.
P3/8
Clerkin S.
S8/4
Domon O.E.
P3/20
Clonfero E.
P2/15
Duran A.
S3/2
Colombi A.
S4/4
Ehleben I.
S8/3
Comendador M. A.
P1/18
Elgorashi E.E.
P1/25; P3/32
Comhaire F.
P2/20
El-Khatib E.N.
P3/3
Concannon P.
S2/3
El-Khatib H. N.
P3/4; P3/5
Conti D.
P3/17
Ellis G.
P2/34
Cools K.
P3/34
Engels L.G.J.B.
P3/22
Cordelli E.
P2/20; P3/17
Epe B.
P1/11; P1/15
Cotrim C.Z.
S5/4
Erben R.
P1/31
Crebelli R.
P3/8
Ésik O.
S9/4
Croft J.
P3/31
Falck G.
P2/4
Csekeő A.
P2/5
Farris G.
P3/19; P3/21
Culp S.J.
P3/20
Fenech M.
S4/3; P3/6
Cundari E.
P3/27; P3/28
Fernandes A.P.
P1/14
Cundari F.
P1/30
Ferreira T.C.
P3/10
Curfs D.M.J.
P3/26
Fessard V.
P2/24
Daemen M.J.A.P.
P3/26
Finnegan C.
S9/5
De Boeck M.
P3/27
Fioravanti D.
P3/12
de Boer J.
S2/1
Fiorentino A.
P1/26
de Boer P.
W1/2
Flohr C.
P1/11
De Kimpe N.
P1/25; P3/32
Fortos A.
P1/16
de Kok T.M.C.M.
P2/35
Fossey A.
P1/25; P3/32
De Ridder L.
S9/3; P2/30;
Fresegna A. M.
P3/17
P3/1; P3/11
Fustinoni S.
S4/4
de Saint-Georges L.
P1/2
Garaj-Vrhovac V.
P1/5; P2/22;
Decker J.
P1/11
Decordier I.
W1/1; P3/27;
Garrido M. J.
P1/32; P2/21
P3/28
Gennery A.
S2/3
Degrassi F.
P3/12
Gentili A.
P1/26
Delimaris J.
P3/15
Georgiadis P.
S7/4; P3/9
Dell’Aquila A.
S3/4
Gioka M.
S7/4
Della Greca M.
P1/26
Girard P.
S2/3
Den Hond E
P2/14
Giwercman A.
P2/20
Desaintes C.
P1/2
Glenisson P.
W3/2
Dierckx RA.
P2/30
Glowienke S.
P3/29
P3/13
184
Godthelp B.C.
S9/2
Kalina I.
P1/4; P1/6;
Gonçalves I.C.
P1/14
Greenrod W.
P3/6
Kallas T.
P2/4
Gregorio P.
P2/15
Kanariou M.
P2/16
Greinert R.
P1/34
Kapka L.
S3/3; P2/11
Gundy S.
S1/2; S9/4
Katsouyianni K.
S7/4
Gustavino B.
P1/23
Kayani M. A.
P1/28
Győrffy E.
P2/5
Kelecsényi Zs.
S9/4
Győri Z.
P2/5
Kier L.
P3/21
Habalová V.
P1/6; P1/7
Kiffe M.
P3/23
Hainaut P.
S8/1
Kirkland D.
S6/3
Hammam F. M.
P3/5
Kirsch-Volders M.
W1/1; P3/27;
Hansen E.
P3/34
Hansteen I.-L.
P3/14
Kiss P.
P2/20
Hartwig A.
S8/3
Kiuru A.
P3/7
Hernando J.
P1/18
Kleinjans J.C.S.
P2/35; P3/22
Hicken S.
P3/19
Klimčáková L.
P1/6; P1/7
Hirvonen A.
S1/2
Klobučar G.I.V.
P1/31
Hoeijmakers J.H.J.
S2/1
Knight A. W.
P1/9
Hoogstraten D.
S2/1
Knudsen L.E.
P2/6; P2/7
Houtsmuller A. B.
S2/1
Kohut A.
P1/4
Hrelia P.
P2/10
Koivistoinen A.
P3/7
Hrivňák M.
P1/6
Kopjar N.
P1/5; P3/13
Hurbankova M.
P3/9
Koppen G.
P2/14
Ingel F.
S3/5
Kostelac D.
S8/3
Ingelman-Sundberg M.
S7/1
Kostič S.
P2/5
Isidori M.
S3/4; P1/26;
Kostka G.
P3/30
P1/30
Kraakman-van der Zwet M.
S9/2
Jacquet P.
P1/2
Kręcicki T.
P1/17
Jałoszyński P.
S4/2
Kubackova J.
P2/6
Janssens A.
P2/31
Kure E. H
P3/14
Jarry G.
P2/25
Kusova J.
P2/6
Järventaus H.
P2/4
Kyrtopoulos A.
S7/4
Jaskuła –Sztul R.
P2/11
Kyrtopoulos S. A.
P3/9
Jeggo P.
S2/3
Labay K.
P2/25
Jensen A.
P2/6; P2/7
Laffon B.
P2/8
Joffe M.
P2/20
Laires A.
P3/10
Juutilainen J.
W2/2
Lambert V.
P2/18
Kaila S.
S7/4
Landa K.
P2/36
Lastrucci L.
P2/13
P1/7
P3/28
185
Laurent C.
P2/18
Mergeay M.
P1/2
Lavorgna M.
S3/4
Messini-Nikolaki N.
P1/16; P2/16;
Lazutka J.
S3/1
Le Page F.
S2/2
Michaux A.
P1/2
Lembowicz K.
P3/30
Mielżyńska D.
S3/3; P2/11
Leopardi P.
P3/8
Migliore L.
P2/12; P2/13
Leter G.
P2/20
Milas I.
P1/5
Lewis P. D.
P2/9
Minárovits J.
P2/5
Lhuguenot J-C.
P1/20; P2/24
Mirghomizadeh F.
P1/17
Limbert E.
P3/10
Möller L.
S4/2
Lindholm C.
P3/7
Monfrinotti M.
P1/23
Lison D.
S8/2; P3/27
Monsieurs M.
P2/30
Lodi V.
P2/10
Monteiro Gil O.
P3/10
Loft S.
P1/24; P2/7
Moonen H.J.J.
P2/35
Loli P.
P3/9
Moreau Y.
W3/2
Lombaert N.
P3/27
Morris S.M.
P3/20
Lopez de Cerain A.
P2/32; P2/33
Mourot A.
P2/24
Loprieno G.
P2/12
Müller L.
S6/1; P3/29
Lossouarn Y.
P1/20
Mustonen R.
P3/7
Lovisolo G.
P3/17
Muylle E.
P3/25
Ludwicki J.K.
P3/30
Muzyka V.
P2/6
Lupi S.
P2/15
Lutgens E.
P3/26
Naccarati A.
Nardelli A.
P2/13
P1/30
MacGregor J.T.
P3/20
Nawrot T.
P2/14
Maes A.
P1/25; P3/32
Neumann H.G.
S4/4
Maffei F.
P2/10
Niedzwiedz W.
P2/28
Mahmood. R.
P1/29
Noël E.
P1/33
Mancini A.
S3/4
Norppa H.
S1/2; P2/4
Marchal K.
W3/2
Nortier J.L.
P2/2
Marcon F.
P3/8
Ntountounakis S.
P2/17
Marino C.
P3/17
O’Driscoll M.
S2/3
Marshall R.
P2/34
Ocadlikova D.
P2/3
Marteau S.
P2/25
Offner F.
P2/31
Martino A.
P1/23
Oliveira N.G.
P1/14; P3/10
Martus HJ.
P3/29
Osmak M.
P2/26
Mathys J.
W3/2
Ould Elhkim M.
P2/25
Mattioli S.
P2/10
Pabiszczak M.
S4/2
Mayer C.
P1/12
Palut D.
P3/30
McGarrity L.J.
P3/20
Papeš D.
P1/31
Méndez J.
P2/8
Parrella A.
P1/30
Parry E. M.
P3/31
P2/17; P3/15
186
Parry J. M.
S5/1; P1/28;
Rossi C.
P1/23; P2/23
P1/29; P2/9
Rössner Jr. P.
P2/19
Pásaro E.
P2/8
Rössner P.
P2/3; P2/36
Pasini L.
P2/23
Rubio A.
P1/32; P2/21
Pastoriza M.
S2/2
Rueff J.
S5/4; P1/14;
Pauwels G.
P3/18
Pavanello S.
P2/15
Rusin M.
P1/10
Pavlica M.
P1/31
Russo F.
P3/17
Pawlas M.
P1/10
Sá da Costa M.
S5/4
Pelzer A.
S8/3
Sabaliunas D.
S3/1
Pepe O.
P1/30
Sabaliuniene I.
S3/1
Perez C.
P2/32; P2/33
Šalagovič J.
P1/6; P1/7
Perrotta A.
P3/16
Sanders E. A.
P1/34
Philippé J.
P2/31
Sanders P.
P2/25
Phillips D.H.
P2/1
Santoro M.
P1/23
Phoa N.
P1/15
Sarasin A.
S2/2
Piperakis S.Μ.
P1/16; P2/16;
Sasiadek M.
P1/17
P2/17; P3/15
Scarfì M.R.
W2/3; P3/16
Pisani P.
P3/16
Scarpato R.
P2/13
Pitkämäki L.
P2/4
Scheepers P.
P2/6
Poli P.
P1/23; P2/23
Schmeiser H.H.
P2/1; P2/2
Pollet D.
P1/34
Schmeiser K.
P3/19 ; P3/21
Poole J.
P2/7
Schmezer P.
P1/12
Porru S.
P2/20
Schneider H.
P1/6
Poul J.M.
P2/24; P2/25
Schoeters G.
P3/18; P3/25
Poul M.
P2/25
Schoket B.
P2/5
Priestly A.
S2/3
Schoonjans W.
P1/2
Psimadas D.
P1/16
Schwerdtle T.
S8/3
Rannug A.
S4/4
Serretti N.
P2/12
Rast C.
P1/1
Sierra L. M.
P1/18
Regniers L.
P1/25; P3/32
Simioli P.
P2/15
Ribas M.
S3/2
Sistare F.D.
P3/20
Ricevuto Z.
P2/12
Siwińska E.
S3/3; P2/11
Rizzoni M.
P1/23
Smerhovsky Z.
P2/36
Rodrigues A.S.
P1/14; P3/10
Smigiel R.
P1/17
Roekens E.
P3/25
Soleo L.
S4/4
Roels HA
P2/14
Soltész I.
P2/5
Rokaya H.A.
P3/3
Spano M.
P2/20
Roncancio C.L.
P2/18
Sperling K.
S2/3
Rosenzweig B.
P3/20
P3/10
187
Sram R. J.
S7/4; P2/3;
Van Oostveldt P.
P3/24
P2/19; P2/36
van Schooten F.J.
S4/1; P3/26
Staessen J.A.
P2/14
van Staden J.
P1/25
Stembalska – Kozłowska A.
P1/17
van Staden J.
P3/32
Stockley C.
P3/6
Vanhauwaert A.
P3/35
Stoikidou M.
S7/4
Vanherweghem J.-L.
P2/2
Stronati L.
P3/17
Vanhoorne M.
P2/20
Štubňa J.
P1/4; P1/7
Vanparys P.
P3/34
Suárez S.
P1/32; P2/21
Vasconcelos I.
S5/4
Sueiro R.A.
P1/32; P2/21
Vasseur P.
P1/1
Suter-Dick L.
W3/4
Verheyen G.R.
P3/18
Swenberg J.
S4/4
Vermeulen W.
S2/1
Székely G.
S1/2; S9/4
Verschaeve L.
W2/1; P1/25;
Szyfter K.
S4/2; P2/11
P2/14; P3/18;
Szyfter W.
S4/2
P3/25; P3/32
Taylor J.L.S.
P1/25; P3/32
Vicentini M.
P2/12
Testa A..
P3/17
Villani P.
P3/17
Thierens H.
S9/3; P2/18;
Vimercati L.
S4/4
P2/30; P2/31;
Violante F.S.
P2/10
P3/1; P3/11
Volkmer B.
P1/34
Thybaud V.
P1/20
von Borstel R.C.
S5/3
Topinka J.
S7/4; P3/9
von Brevern M.C.
P1/12
Toscano-Rico J.M.
P1/14
Vral A.
S9/3; P2/30;
Tsilimigaki S.
P1/16; P2/16;
P3/1; P3/11
P2/17; P3/15
Walmsley R. M.
P1/9
Tudek B.
P1/19
Warholm M.
S4/4
Tuimala J.
S1/2
Weidt E.
P1/11
Tweats D.J.
S6/2
Whitford W.
P3/19
Valentin I.
P1/20; P2/24
Wiadrowska B.
P3/30
van Breda S.G.J.
P3/22
Wiessler M.
P2/1
van Buul P.P.W.
S9/2
Wilder M.
P1/34
Van Cauwenberge A.
P1/33
Williams G.
S5/2
van Delft J.H.M.
W3/1; P3/22
Wolff T.
P3/9
van den Boom V.
S2/1
Wolfreys A.
P2/34
van den Broek O.
W1/2
Yan J.
P1/2
van der Horst G.T.J.
S2/1
Zafiropoulou M.
P2/17
van Duijn-Goedhart A.
S9/2
Zanello A.
P2/13
Van Goethem F.
P3/34
Zdzienicka M.Z.
S2/4 ; S9/2
Van Hoof V.
P3/34
Zelezny O.
P1/12
Van Hummelen P.
W3/2
Želježić D.
P2/22 ; P3/13
188
Zeni O.
P3/16
Zhu C.Y.
P1/24
Zijno A.
P3/8
Ziros G.
P3/15
Zschiesche W.
P2/20
189