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80 Cytogenetic biomarkers and genetic polymorphisms
Symposium S9. Genetic susceptibility towards genotoxic agents
S9 Genetic susceptibility towards genotoxic agents 80
CYTOGENETIC BIOMARKERS AND GENETIC POLYMORPHISMS
H. Norppa. Finnish Institute of Occupational Health, Helsinki, Finland Cytogenetic biomarkers have long been applied in surveillance of human genotoxic exposure and early effects of genotoxic carcinogens. Due to their wide use, it has been possible to evaluate, in international collaborative studies, if a high level of these biomarkers in peripheral lymphocytes is predictive of cancer risk. Thus far, such an association has been observed for chromosomal aberrations (CAs), but not for sister chromatid exchanges (SCEs) or micronuclei (MN). The cancer risk predictivity of CAs did not appear to be explained by tobacco smoking or occupational exposure to carcinogens, but was seen in unexposed non-smokers as well. This suggests a role for individual susceptibility factors. Genetic polymorphisms of various xenobiotic-metabolising enzymes, influencing the metabolic activation and detoxification of carcinogens, have been associated with cancer risk and some of them also appear to affect cytogenetic biomarkers. The lack of glutathione S-transferase M1 (GSTM1 null genotype) appears to be associated with increased sensitivity to genotoxicity of tobacco smoke, and GSTM1 null smokers show an increased frequency of CAs. N-acetyltransferase (NAT2) slow acetylation genotype and glutathione S-transferase T1 (GSTT1) null genotype seem to elevate the baseline level of CAs and SCEs, respectively - possibly because of reduced capacity to detoxify some wide-spread genotoxins. For some chemicals, in vitro cytogenetic studies with lymphocyte donors representing different genotypes have been able to predict a differential in vivo response. The in vitro genotoxicity of styrene and epoxide metabolites of 1,3-butadiene is modified by GSTM1 and GSTT1 genotypes - which also influence the excretion of specific mercapturic acids in humans exposed to butadiene and styrene. Polymorphisms of DNA repair and folate metabolism are expected to be of special importance in modulating genotoxic effects, but there is presently inadequate information about their possible effect on cytogenetic biomarkers. (Supported by EU QLK4–2000–00628) 81
CHROMOSOMAL ABERRATIONS AND INDIVIDUAL SENSITIVITY TOWARDS IONISING RADIATION.
A. Vral, A. Baeyens, H. Thierens, L. De Ridder. Department of Anatomy, Embryology, Histology and Medical Physics, University of Ghent, Ghent, Belgium The development of biomarkers of risk or susceptibility to identify individuals who are at increased risk for the development of cancer is an important issue. Possible candidate susceptibility markers are biomarkers that measure the sensitivity of chromosomes to ionising radiation. Several studies have indeed confirmed that chromosomal radiosensititvity is a hallmark not only for a certain number of genetic disorders but also for a significant proportion of breast cancer patients. These findings which favour the application of chromosomal aberration assays, such as G2 assay and micronucleus (MN) assay, as biomarkers of susceptibility induced us to start a thorough study of the reliability and sensitivity of the G2 and micronucleus assay. Before these assays can be used for individual risk assessment it is indeed essential to know how specific, sensitive and reliable they are. For this, the G2 assay and the MN assay were performed on repeated occasions on blood samples of healthy individuals and intra- and interindividual variabilities were analysed. For the MN assay Go lymphocytes were exposed to 3.5 Gy Co γ-rays either at high dose-rate (HDR) or at low dose-rate (LDR). For the G2 assay lymphocytes were irradiated with a dose of 0.4 Gy Co γ-rays in G2 phase of the cell cycle. The repeat experiments on blood samples of the same donor revealed that the intraindividual coefficients of variation were not significantly different from the interindividual coefficients of variation in both G2 and MN assay. As the intraindividual variability determines the assay reproducibility this would indicate that the assays are not able to
s25
detect real, reproducible differences in radiation sensitivity between normal individuals in the population. The repeat experiments further revealed that for some healthy donors in one or two occasions high values were obtained while at all the other time points the values were within the normal range (non-sensitive). Although a normal mean value was obtained for these healthy individuals, they could have been regarded as sensitive based on one occasional high value. We conclude that results obtained with the G2 and MN assay that are based on single measurements are insufficient to determine the susceptibility of an individual. To yield reliable conclusions, the results should be based on multiple measurements from multiple blood samples. 82
GENETIC POLYMORPHISM AND CYTOGENETIC DAMAGE IN HUMANS EXPOSED TO URBAN AIR POLLUTION
P. Hrelia. Department of Pharmacology, University of Bologna, Bologna,Italy Urban air pollution is considered a major hazard to human health. Benzene, a recognized hematotoxin and leukemogen, might be responsible for adverse health effects in population exposed to high level of air pollution. The aim of this study was to investigate the relationship between benzene exposure and biomarkers of exposure (personal passive samplers, µg/m3 ), effect (micronuclei (MN)) and susceptibility (MPO, GSTP1 and GSTT1 genotypes). Samples of peripheral blood were collected from 49 policemen (32 no-smokers and 17 smokers mean age:39.53±7.14), carrying on different outdoor activities in high traffic area of Bologna, and 36 indoor workers enrolled as controls (23 no-smokers and 13 smokers, mean age: 40.13±7.22). The average benzene exposure during the work-shift was significantly higher in the traffic policemen compared to the control population (24.32±14.38 and 4.39±0.99 µg/m3 , P= 0.001). MN frequency was significantly increased in policemen as compared with controls (MN/1000 binucleated (BN) cells: 7.06±2.87 and 4.61±2.04, P=0.001). Multiple regression analysis showed MN frequency was significantly modulated by age (P= 0.001) whereas smoke habits and gender did not have any influence. All the fortynine policemen were genotyped. Preliminary results showed higher frequency of MN in subjects with GSTT1 null genotype compared with those with positive genotype (8.30±4.64 and 6.79±2.15 MN/BN); on the other hand no association was observed with any of the other selected genes. Results support the idea that long-term exposure to urban air pollution increases DNA damage and that polymorphisms analysis should be taken into account in understanding benzene-induced adverse health effects. The integration of biomarkers of exposure, effect and susceptibility in population studies may contribute significantly to the establishment of risk profiles of individuals in given exposure situations. 83
IMPACT OF PHASE I OR PHASE II ENZYME POLYMORPHISMS ON LYMPHOCYTE DNA ADDUCTS OF SUBJECTS EXPOSED TO AMBIENT AIR POLLUTION AND ENVIRONMENTAL TOBACCO SMOKE
P. Georgiadis 1 , J. Topinka 2 , M. Stoikidou 3 , M. Bekyrou, K. Katsouyianni 3 , R. Sram 2 , H. Autrup 4 , A. Kyrtopoulos 1 . 1 National Hellenic Research Foundation, Athens, Greece; 2 Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine Acad. Sci. C.R. and Regional Institute of Hygiene of Central Bohemia, Prague, Czech Republic; 3 Laboratory of Hygiene and Epidemiology, University of Athens Medical School; 4 Department of Environmental Medicine, Univ. of Aarhus, Denmark The levels of bulky DNA adducts were measured by 32 P-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. Individuals were sampled twice, during two consecutive winter and summer periods. 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
S9 Genetic susceptibility towards genotoxic agents 80
CYTOGENETIC BIOMARKERS AND GENETIC POLYMORPHISMS
H. Norppa. Finnish Institute of Occupational Health, Helsinki, Finland Cytogenetic biomarkers have long been applied in surveillance of human genotoxic exposure and early effects of genotoxic carcinogens. Due to their wide use, it has been possible to evaluate, in international collaborative studies, if a high level of these biomarkers in peripheral lymphocytes is predictive of cancer risk. Thus far, such an association has been observed for chromosomal aberrations (CAs), but not for sister chromatid exchanges (SCEs) or micronuclei (MN). The cancer risk predictivity of CAs did not appear to be explained by tobacco smoking or occupational exposure to carcinogens, but was seen in unexposed non-smokers as well. This suggests a role for individual susceptibility factors. Genetic polymorphisms of various xenobiotic-metabolising enzymes, influencing the metabolic activation and detoxification of carcinogens, have been associated with cancer risk and some of them also appear to affect cytogenetic biomarkers. The lack of glutathione S-transferase M1 (GSTM1 null genotype) appears to be associated with increased sensitivity to genotoxicity of tobacco smoke, and GSTM1 null smokers show an increased frequency of CAs. N-acetyltransferase (NAT2) slow acetylation genotype and glutathione S-transferase T1 (GSTT1) null genotype seem to elevate the baseline level of CAs and SCEs, respectively - possibly because of reduced capacity to detoxify some wide-spread genotoxins. For some chemicals, in vitro cytogenetic studies with lymphocyte donors representing different genotypes have been able to predict a differential in vivo response. The in vitro genotoxicity of styrene and epoxide metabolites of 1,3-butadiene is modified by GSTM1 and GSTT1 genotypes - which also influence the excretion of specific mercapturic acids in humans exposed to butadiene and styrene. Polymorphisms of DNA repair and folate metabolism are expected to be of special importance in modulating genotoxic effects, but there is presently inadequate information about their possible effect on cytogenetic biomarkers. (Supported by EU QLK4–2000–00628) 81
CHROMOSOMAL ABERRATIONS AND INDIVIDUAL SENSITIVITY TOWARDS IONISING RADIATION.
A. Vral, A. Baeyens, H. Thierens, L. De Ridder. Department of Anatomy, Embryology, Histology and Medical Physics, University of Ghent, Ghent, Belgium The development of biomarkers of risk or susceptibility to identify individuals who are at increased risk for the development of cancer is an important issue. Possible candidate susceptibility markers are biomarkers that measure the sensitivity of chromosomes to ionising radiation. Several studies have indeed confirmed that chromosomal radiosensititvity is a hallmark not only for a certain number of genetic disorders but also for a significant proportion of breast cancer patients. These findings which favour the application of chromosomal aberration assays, such as G2 assay and micronucleus (MN) assay, as biomarkers of susceptibility induced us to start a thorough study of the reliability and sensitivity of the G2 and micronucleus assay. Before these assays can be used for individual risk assessment it is indeed essential to know how specific, sensitive and reliable they are. For this, the G2 assay and the MN assay were performed on repeated occasions on blood samples of healthy individuals and intra- and interindividual variabilities were analysed. For the MN assay Go lymphocytes were exposed to 3.5 Gy Co γ-rays either at high dose-rate (HDR) or at low dose-rate (LDR). For the G2 assay lymphocytes were irradiated with a dose of 0.4 Gy Co γ-rays in G2 phase of the cell cycle. The repeat experiments on blood samples of the same donor revealed that the intraindividual coefficients of variation were not significantly different from the interindividual coefficients of variation in both G2 and MN assay. As the intraindividual variability determines the assay reproducibility this would indicate that the assays are not able to
s25
detect real, reproducible differences in radiation sensitivity between normal individuals in the population. The repeat experiments further revealed that for some healthy donors in one or two occasions high values were obtained while at all the other time points the values were within the normal range (non-sensitive). Although a normal mean value was obtained for these healthy individuals, they could have been regarded as sensitive based on one occasional high value. We conclude that results obtained with the G2 and MN assay that are based on single measurements are insufficient to determine the susceptibility of an individual. To yield reliable conclusions, the results should be based on multiple measurements from multiple blood samples. 82
GENETIC POLYMORPHISM AND CYTOGENETIC DAMAGE IN HUMANS EXPOSED TO URBAN AIR POLLUTION
P. Hrelia. Department of Pharmacology, University of Bologna, Bologna,Italy Urban air pollution is considered a major hazard to human health. Benzene, a recognized hematotoxin and leukemogen, might be responsible for adverse health effects in population exposed to high level of air pollution. The aim of this study was to investigate the relationship between benzene exposure and biomarkers of exposure (personal passive samplers, µg/m3 ), effect (micronuclei (MN)) and susceptibility (MPO, GSTP1 and GSTT1 genotypes). Samples of peripheral blood were collected from 49 policemen (32 no-smokers and 17 smokers mean age:39.53±7.14), carrying on different outdoor activities in high traffic area of Bologna, and 36 indoor workers enrolled as controls (23 no-smokers and 13 smokers, mean age: 40.13±7.22). The average benzene exposure during the work-shift was significantly higher in the traffic policemen compared to the control population (24.32±14.38 and 4.39±0.99 µg/m3 , P= 0.001). MN frequency was significantly increased in policemen as compared with controls (MN/1000 binucleated (BN) cells: 7.06±2.87 and 4.61±2.04, P=0.001). Multiple regression analysis showed MN frequency was significantly modulated by age (P= 0.001) whereas smoke habits and gender did not have any influence. All the fortynine policemen were genotyped. Preliminary results showed higher frequency of MN in subjects with GSTT1 null genotype compared with those with positive genotype (8.30±4.64 and 6.79±2.15 MN/BN); on the other hand no association was observed with any of the other selected genes. Results support the idea that long-term exposure to urban air pollution increases DNA damage and that polymorphisms analysis should be taken into account in understanding benzene-induced adverse health effects. The integration of biomarkers of exposure, effect and susceptibility in population studies may contribute significantly to the establishment of risk profiles of individuals in given exposure situations. 83
IMPACT OF PHASE I OR PHASE II ENZYME POLYMORPHISMS ON LYMPHOCYTE DNA ADDUCTS OF SUBJECTS EXPOSED TO AMBIENT AIR POLLUTION AND ENVIRONMENTAL TOBACCO SMOKE
P. Georgiadis 1 , J. Topinka 2 , M. Stoikidou 3 , M. Bekyrou, K. Katsouyianni 3 , R. Sram 2 , H. Autrup 4 , A. Kyrtopoulos 1 . 1 National Hellenic Research Foundation, Athens, Greece; 2 Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine Acad. Sci. C.R. and Regional Institute of Hygiene of Central Bohemia, Prague, Czech Republic; 3 Laboratory of Hygiene and Epidemiology, University of Athens Medical School; 4 Department of Environmental Medicine, Univ. of Aarhus, Denmark The levels of bulky DNA adducts were measured by 32 P-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. Individuals were sampled twice, during two consecutive winter and summer periods. 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