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P XIII.93 Analysis of BCL-2 translocation in human TK6 lymphoblastoid cells following exposure to 1,2,3,4-diepoxybutane
S-XIII: Identification and evaluation of environmental mutagens, ecogenotoxicology
Ip XIII.91!
Blomonltorlng studies on an buman population exposed to pesticides
Helena Borba, Margarida Monteiro, Maria Jose Proenca, Jose Rueff. Department of Genetics, Faculty of Medical Sciences UNL, Rua da Junqueira, 96, 1300 Llsboa, Portugal We are studying a population of farmers occupationally exposed to pesticides. Our aim is to help validate and optimise the use of a combination of methods applied to human populations exposed to possible genotoxic compounds. The biomarkers studied until now were: · the urinary genotoxicity using the Ames test (TA98 and YGI024); different urinary fractions were prepared using the XAD-2 extraction procedure previously or after hydrolysis of the glucuronide conjugates excreted. · an indicator of lipid peroxidation - using the erytrocitary and urinary MDA-TBA reactive products formed measured by the conventional spectrophotometric method and by an HPLC analytical technique. · an indicator ofthe induction levels of P4S0 - evaluated using the inhibition of f3-glucuronidase by the urinary excreted d-glucaric acid hydrolised in the corresponding lactones. From the 31 farmers who applied pesticides and 16 controls studied until now the results obtained indicate that there is no significant differences in the lipid peroxidation levels or in the induction levels od P4S0 of the exposed farmers as compared to the controls. For the urinary genotoxicity we could only find a slightly significant difference between exposed and controls when we used the YG I024 strain to test the urinary fraction after hydrolysis of the glucuronide conjugates excreted. As the farmers are exposed to mixtures of completely different compounds it would perhaps be worthy to increase the number of individuals studied to allow the consideration of subpopulations of exposed individuals to subgroups of compounds. Keyword(s}: pesticides; human biomonitoring
Ip XIII.921
In vitro Induction of micronuclei by benzo (a) pyrene on fish cell lines aod detection by now cytometry
P. Sanchez, C. Becerril, M. Carballo, M.J. Munoz, A. Castano. Diuision of Erwironmental Toxicology, ClSA-INIA, E-28130 Valdeolmos, Madrid, Spain; Toxicology Unit, CNAyN, 1. Carlos Ill, £-28220 Majadahonda, Madrid, Spain Increasing presence of genotoxic contaminants in aquatic environment can threat natural populations. This fact makes necessary to test aquatic genatoxic pollutants, in order to assess its environmental impact. In vivo test with fish, are expensive and large volume of samples is required, so for screening purposes, in vitro test can be used. As a consequence of its high chromosome number and its reduced size, micronuclei induction seems to be one of the best readily performed mutagenicity assay. In spite of this, the low frequency occurrence of micronuclei, makes its manual score tedious and time consuming. Flow cytometry, is an automatic technique that allow to perform a high number of samples in few minutes. In addition ofmicronuclei, cell distribution along the cellular cycle can be measured at the same time on the same sample. We have tested b(a)p on rainbow trout derived fish cell line (RTG-2) at O.OS, 0.1, O.S and I (/101 for 8, 24, 48 and 72 hours of exposure. O.OS (glml did no produce neither micronuclei increase nor cell cycle alterations at any of the tested exposure periods. Statistically significative differences in micronuclei increase was observed at: I (glmI after 8 h., at O.S (glmI after 24 h, and at 0.1 (g/ml after 48 h of exposure. Concerning to cell cycle distributions 0.1 (glml did no provoke statistically significative alterations, while I (glmI provoke a significative decrease of the portion of cells in GI phase of the cell cycle at any ofcxposure periods tested.
Ip XIII.931
SIIS
Analysis of BCL-Z translocation in buman TK6 Iymphoblastold cells following exposure to l,Z,3,4. dlepoxybutane
Hongwei Chen 1, Kathy G. Meyec2, Leslie Reci0 2 , Douglas A. Bell i • J C3-03. RD. Box 12233. National Institute of Environmental Health Sciences/National Institutes ofHealth, RTP, NC 27709, USA; 2 Chemical Industry I nstitute of Toxicology, 6 Davis Drive, RTP, NC 27709, USA 1,3-butadlene (BD) exposure has been reported to be associated with an increased risk of leukemia. BD is also a rodent carcinogen. 1,2,3,4diepoxybutane (DEB) is a genotoxic metabolites of 1,3-butadiene. A previous mutational spectrum study using human TK Iymphoblasts (TK6 cells) shows that a significant proportion of DEB genotoxicity is due to the induction of large deletions. In view of 1,3-butadiene's potential role in hematological malignancies, we hypothesized that 1,3-butadiene might induce bcl-2 translocation ID B lymphocyte cell cultures. In this study, we have used a PCR-based assay to determine the frequency of bcl-2 translocation in DEBexposed and control human cultures. TK6 cell cultures were exposed with DEB (4 uM x 24 hrs). DNA was extracted and purified from the celllysates following exposure. This PCR-based technique can detect bcl-2 translocation at a detection limit of I in 600,000 normal haploid genomes. In a total of 13 treated TK6 cell cultures, bcl-2 translocations were not detected in either control or DEB-treated cells. These results indicate that under our experimental conditions, DEB does not induce bcl-2 translocation in TK6 cells. Keyword(s}: 1,3-butadiene; Bcl-2 translocation; Mutagenesis
Ip XIII.941
Tbe use of genotoxlc endpoints to evaluate occupational exposure 10 antineoplastic drugs
Thomas H. Connor", Ellen S. Bakec2. J Unioersity of Texas Health Science Center, Houston, Texas, USA; 2National Aeronautics and Space Administration, Houston, Texas, USA Genotoxic endpoints have been employed to ascertain whether occupational exposure to antineoplastic drugs poses a health risk to health care workers who are involved with their admixing, administration and disposal. These have included urine mutagenicity, SCEs, chromosomal aberrations, micronuclei induction, HPRT mutations and DNA damage. We reviewed S3 reports published since 1979 that examined a genotoxic endpoint as an indicator of occupational exposure in 78 separate tests. Based on the authors' evaluations, twenty-eight tests for urine mutagenicity demonstrated 13 positive effects and IS negative effects. Twenty tests for SCE resulted in 6 positive, 12 negative and 2 equivocal outcomes. Nineteen tests for chromosomal aberrations resulted in I I positive, 7 negative and I equivocal outcomes. Seven tests for micronuclei produced 4 positive, 2 negative and I equivocal outcome. Three tests for HPRT mutations resulted in I of each outcome. The single study that examined DNA damage produced a positive outcome. Four studies that evaluated exposure before and after changes in handling practice noted a reduction in exposure following the changes. Few attempts have been made that have correlated environmental exposure with a biological endpoint. One study which examined urinary excretion of a specific drug and chromosomal aberrations did not observe any correlation between the two. Thus, these methods appear to be unsuitable for routine monitoring of exposure. All are nonspecific and subject to many confounders and may not be sensitive enough to detect routine occupational exposure. Keyword(s}: Antineoplastic agents; Occupational exposure
Ip XIII.91!
Blomonltorlng studies on an buman population exposed to pesticides
Helena Borba, Margarida Monteiro, Maria Jose Proenca, Jose Rueff. Department of Genetics, Faculty of Medical Sciences UNL, Rua da Junqueira, 96, 1300 Llsboa, Portugal We are studying a population of farmers occupationally exposed to pesticides. Our aim is to help validate and optimise the use of a combination of methods applied to human populations exposed to possible genotoxic compounds. The biomarkers studied until now were: · the urinary genotoxicity using the Ames test (TA98 and YGI024); different urinary fractions were prepared using the XAD-2 extraction procedure previously or after hydrolysis of the glucuronide conjugates excreted. · an indicator of lipid peroxidation - using the erytrocitary and urinary MDA-TBA reactive products formed measured by the conventional spectrophotometric method and by an HPLC analytical technique. · an indicator ofthe induction levels of P4S0 - evaluated using the inhibition of f3-glucuronidase by the urinary excreted d-glucaric acid hydrolised in the corresponding lactones. From the 31 farmers who applied pesticides and 16 controls studied until now the results obtained indicate that there is no significant differences in the lipid peroxidation levels or in the induction levels od P4S0 of the exposed farmers as compared to the controls. For the urinary genotoxicity we could only find a slightly significant difference between exposed and controls when we used the YG I024 strain to test the urinary fraction after hydrolysis of the glucuronide conjugates excreted. As the farmers are exposed to mixtures of completely different compounds it would perhaps be worthy to increase the number of individuals studied to allow the consideration of subpopulations of exposed individuals to subgroups of compounds. Keyword(s}: pesticides; human biomonitoring
Ip XIII.921
In vitro Induction of micronuclei by benzo (a) pyrene on fish cell lines aod detection by now cytometry
P. Sanchez, C. Becerril, M. Carballo, M.J. Munoz, A. Castano. Diuision of Erwironmental Toxicology, ClSA-INIA, E-28130 Valdeolmos, Madrid, Spain; Toxicology Unit, CNAyN, 1. Carlos Ill, £-28220 Majadahonda, Madrid, Spain Increasing presence of genotoxic contaminants in aquatic environment can threat natural populations. This fact makes necessary to test aquatic genatoxic pollutants, in order to assess its environmental impact. In vivo test with fish, are expensive and large volume of samples is required, so for screening purposes, in vitro test can be used. As a consequence of its high chromosome number and its reduced size, micronuclei induction seems to be one of the best readily performed mutagenicity assay. In spite of this, the low frequency occurrence of micronuclei, makes its manual score tedious and time consuming. Flow cytometry, is an automatic technique that allow to perform a high number of samples in few minutes. In addition ofmicronuclei, cell distribution along the cellular cycle can be measured at the same time on the same sample. We have tested b(a)p on rainbow trout derived fish cell line (RTG-2) at O.OS, 0.1, O.S and I (/101 for 8, 24, 48 and 72 hours of exposure. O.OS (glml did no produce neither micronuclei increase nor cell cycle alterations at any of the tested exposure periods. Statistically significative differences in micronuclei increase was observed at: I (glmI after 8 h., at O.S (glmI after 24 h, and at 0.1 (g/ml after 48 h of exposure. Concerning to cell cycle distributions 0.1 (glml did no provoke statistically significative alterations, while I (glmI provoke a significative decrease of the portion of cells in GI phase of the cell cycle at any ofcxposure periods tested.
Ip XIII.931
SIIS
Analysis of BCL-Z translocation in buman TK6 Iymphoblastold cells following exposure to l,Z,3,4. dlepoxybutane
Hongwei Chen 1, Kathy G. Meyec2, Leslie Reci0 2 , Douglas A. Bell i • J C3-03. RD. Box 12233. National Institute of Environmental Health Sciences/National Institutes ofHealth, RTP, NC 27709, USA; 2 Chemical Industry I nstitute of Toxicology, 6 Davis Drive, RTP, NC 27709, USA 1,3-butadlene (BD) exposure has been reported to be associated with an increased risk of leukemia. BD is also a rodent carcinogen. 1,2,3,4diepoxybutane (DEB) is a genotoxic metabolites of 1,3-butadiene. A previous mutational spectrum study using human TK Iymphoblasts (TK6 cells) shows that a significant proportion of DEB genotoxicity is due to the induction of large deletions. In view of 1,3-butadiene's potential role in hematological malignancies, we hypothesized that 1,3-butadiene might induce bcl-2 translocation ID B lymphocyte cell cultures. In this study, we have used a PCR-based assay to determine the frequency of bcl-2 translocation in DEBexposed and control human cultures. TK6 cell cultures were exposed with DEB (4 uM x 24 hrs). DNA was extracted and purified from the celllysates following exposure. This PCR-based technique can detect bcl-2 translocation at a detection limit of I in 600,000 normal haploid genomes. In a total of 13 treated TK6 cell cultures, bcl-2 translocations were not detected in either control or DEB-treated cells. These results indicate that under our experimental conditions, DEB does not induce bcl-2 translocation in TK6 cells. Keyword(s}: 1,3-butadiene; Bcl-2 translocation; Mutagenesis
Ip XIII.941
Tbe use of genotoxlc endpoints to evaluate occupational exposure 10 antineoplastic drugs
Thomas H. Connor", Ellen S. Bakec2. J Unioersity of Texas Health Science Center, Houston, Texas, USA; 2National Aeronautics and Space Administration, Houston, Texas, USA Genotoxic endpoints have been employed to ascertain whether occupational exposure to antineoplastic drugs poses a health risk to health care workers who are involved with their admixing, administration and disposal. These have included urine mutagenicity, SCEs, chromosomal aberrations, micronuclei induction, HPRT mutations and DNA damage. We reviewed S3 reports published since 1979 that examined a genotoxic endpoint as an indicator of occupational exposure in 78 separate tests. Based on the authors' evaluations, twenty-eight tests for urine mutagenicity demonstrated 13 positive effects and IS negative effects. Twenty tests for SCE resulted in 6 positive, 12 negative and 2 equivocal outcomes. Nineteen tests for chromosomal aberrations resulted in I I positive, 7 negative and I equivocal outcomes. Seven tests for micronuclei produced 4 positive, 2 negative and I equivocal outcome. Three tests for HPRT mutations resulted in I of each outcome. The single study that examined DNA damage produced a positive outcome. Four studies that evaluated exposure before and after changes in handling practice noted a reduction in exposure following the changes. Few attempts have been made that have correlated environmental exposure with a biological endpoint. One study which examined urinary excretion of a specific drug and chromosomal aberrations did not observe any correlation between the two. Thus, these methods appear to be unsuitable for routine monitoring of exposure. All are nonspecific and subject to many confounders and may not be sensitive enough to detect routine occupational exposure. Keyword(s}: Antineoplastic agents; Occupational exposure