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“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 5 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 6 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 7 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 9 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 TA98S9, TA100S9 and the strain YG1041S9 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 1,90,8 3,00,6 Pyrene 22,78,7 37,612,8 TA98+S9 3,91,1 4,91,8 BAA 30,212,3 15,48,4 YG1041 5,71,9 2,00,6 BAP 24,89,8 33,311,0 YG1041+S9 9,22,5 1,90,9 DAHA 8,03,0 13,84,5 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 1.5M) 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 - 27.284.53); mitomycin.C (0.3 M – 26.505.81; 1.5M - 28.032.37); H2O2 (15mM – 25.331.31; 20mM - 28.050.19) (ii) using Rb-1: Xrays (2Gy - 33.675.05); mitomycin C (0.3 M – 26.5 3.95; 32.67 2.2), H2O2 (15mM – 28.005.14; 20mM - 29.007.58). 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 P1/8 82 P1/9 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 P1/10 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: 445AG in exon 3 causing Lys117Arg and 1299GT in exon 8 causing Gly402Cys. Both variants are in residues highly conserved through evolution. One common sequence variant in hHR23B was found: 1059CT 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 P1/11 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 P1/13 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 P1/15 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 P1/17 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 P1/18 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 P1/19 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 P1/20 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 P1/21 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 P1/24 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 37C 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 P1/25 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: 6.784.92 and 5.542.99, respectively). Interestingly, smoking status significantly raised chromosomal damage among the exposed workers (smokers: 8.835.94 MN/1000 BN, no-smokers: 4.842.61 MN/1000 BN; p= 0.011 Wilcoxon test), but not among controls (smokers: 6.001.94 MN/1000 BN no-smokers: 5.153.67 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