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P XIV C.4 Analysis of mutagenic effect of low frequency electromagnetic fields by chromosome painting
S136
S-XIV: New mutagenicity tests and their evaluation
Ip XIV c.21
Immunonuorescence analysis of Diazepam-Induced mitotic apparatus abnormalitIes and chromosome loss In Chinese hamster cells
Massimo Izzo', Antonio Antoccia1 , Francesca Degrassi", Caterina Tanzarella 1 . / CentroGeneticaEooluzionisttca CNR. c/o Dip. Genetica Bioi.
Maleealare. Universita "La Sapienza", P. Ie A. Moro 5. 00185 Ramo. Italy; 2Dip. Biologia, "Ramo Tre", V.le Marconi 446. 00154 Roma, Italy Indirect immunofluorescent staining was performed with antibodies directed against mitotic apparatus proteins to study the mechanisms leading to Diazepam (DZ)-induced chromosomal malsegregation. After a 3 h treatment with 80 ug/ml of DZ, the mitotic index of hamster cells was strongly increased by the presence of "contracted" metaphases. The use of an anti a-tubulin antibody confirmed that "contracted" metaphase. were provided with monopolar spindles. Staining with anti-dynein antibody suggested that chromosomes arranged in monopolar spindles were attached to short fibers originating from spindle pole. Interestingly, nearly fifthy percent of DZinduced monopolar mitosis showed a punctate staining of centrosome when evaluated with g-tubulin antibody. This observation seemed to indicate that DZ interfered with the correct assembly of centrosomes by inhibiting the recruitment of g-tubulin to the centrosome. No qualitative differences were observed between untreated and treated cells after actin detection. After 15-30 min of recovery from DZ, nearly twenty-percent anaphases were abnormal, consisting mainly of multipolars and lagging chromosomes. This observation is consistent with the high frequency of CREST-positive MN scored in 3 h cytochalasin-blocked binucleated cells after removal of DZ. Our data show that DZ IS a powerful inducer of chromosome loss through a mechanism related to interference in the correct assembly of centrosomes. Keyword(s): Diazepam; Centrosomes; aneuploidy
Ip XIV c.31
Analysis of radiation-Induced chromosome aberrations In Chinese hamster splenocytes by FISH uslng chromosome-specific DNA libraries
M. Grigorova, Y. Xiao, A.T. Natarajan. Dept.RadiationGenetics & Chemical Mutagenesis, Leiden University. Leiden, The Netherlands Application of the fluorescent in situ hybridization (FISH) technique using chromosome specific DNA libraries for assessing radiation-induced chromosome damage has facilitated the detection of different types of translocations, In human lymphocytes, the frequencies of radiation induced translocations are higher than dicentrics. However, in mouse splenocytes the frequencies of X-ray induced translocations and dicentrics are similar. This difference between the two species has been attributed to their karyotypic features. A suitable model to study the influence of the karyotype on the induction of chromosome aberrations is the Chinese hamster, since its karyotype, similar to human, is composed of both metacentric and acrocentric chromosomes. We have estimated the frequencies of chromosome aberrations induced by different doses of 200 kV X-rays (0.75, 1.5, 3.0 Oy) and I Mev fast neutrons (0.25, 0.5, 1.0 Gy) in splenocytes from Chinese hamster by bicolour FISH using DNA libraries specific for chromosomes # 2, 3, X and 8. Only first division metaphases were analysed. The results indicate that both X-rays and neutrons induce more translocations than dicentrics, the ratio between the two types of exchanges being 1.5 and 2 respectively. The RBE of the neutrons for induction of exchanges was found to be around 3. Among the chromosomes studied, an increased involvement of chromosome #8 in exchange aberrations, about 2-3 times more than expected on the basis of its DNA content was found. The high content of interstitial telomeric sequences in chromosome #8 may be responsible for the observed increased sensitivity of this chromosome. Keyword(s): Radiation; exchanges; heterogeneity
Ip XIV
CAl
Analysis of mutagenic effect of low frequency electromagnetic fields by chromosome painting
Jose M. Garcia-Sagredo, Concepcion Villalon, Isabel Vallcorba, Carmen Sanchez-Hombre, Teresa Ferro, Carlos San Roman. Medical Genetics
Department. University Hospital Ramony Cajal, Madrid, Spain The experimental evidence on genetic effects with 50-60 Hz electromagnetic fields (EMF) indicate contradictory results; however, the majority of the reports failed to demonstrate adverse effects. Lymphocytes from normal donors, were in vitro exposed to a 50 Hz EMF at a flux density of 300, 115, and 28 I1Tdunng the time of cultures for cytogenetic analysis (72 h). After harvesting, microscope slides were hybridized with whole-chromosome DNA-libranes for chromosomes #1 and #2. Under fluorescence microscope, stable chromosome involving chromosomes I and 2 were easily detected. Our results shows 0.16% stable aberrations in controls, and 0.38%, 0.46%, 0.45% in exposed cultures under 28, 115 and 300 ILT respectively. (0.59 in controls, 1.41, 1.69, 1.63% in cell equivalents). No significant differences between chromosome abnormalities scored in control cultures and chromosome abnormalities scored in exposed cultures were detected. These result. show that a low-frequency SO Hz EMF at intensity levels similar to high intensities in human environmental exposures, have no cytogenetic effects in human lymphocytes "in vitro" at the detectable level of FISH analysis. Acknowledgment: This work was made by grant (96/1593) from FISS. Keyword(s): EMF SO Hz; FISH; Chromosome painting
Ip XIV c.sl
Development of arm-specific painting probes for Chinese hamster chromosomes and their use In the detecllon of complex chromosome rearrangements Induced by bleomycin
Y. Xiao l , F. Darroudi', S. Siml 2 , A.T. Natarajan'; / MGC. Department of Radiation Genetics and Chemical Mutagenesis. Leiden University. Wassenaarseweg 72. 2JJJ AL. Leulen, The Netherlands; 21stituto di Mutagenesi e Differenziamento, C.N.R.. Pisa, Italy The technique of fluorescence in situ hybridization (FISH) using chromosome-specitic probes allows the detection of simple as well as complex chromosome rearrangements. However, chromosome-specific probes also have their limitations. For example, some structural chromosome alterations (such as pericentric inversion) can not be detected by these probes. Recently, we have generated arm-specific painting probes for Chinese hamster chromosomes by means of microdissection with subsequent DOPPCR. The specificity of these probes was examined by two-color FISH. Using the arm specific probes, we have studied the chromosome aberrations induced by Bleomycin (radiormmetic agent) in Chinese hamster primary embryonic cells. The results obtained so far demonstrate that arm-specific painting probes are very useful to study bleomycin-mduced chromosome rearrangements, in particular, complex aberrations, intra-chromosome exchanges (such as pericentric inversions) and centric rings. Keyword(s): FISH; Chinese hamster arm-specific probes; Bleomycin
Ip XIV c.61
Analysis of the Induction and the penlstence of Xray Induced chromosome aberrations In mouse bone marrow cells using ftuorescence In sltu hybrIdization
Y. Xiao", F. Darroudi', M. Grigorova', A.T. Natarajan'. /MGC, Department of Radiation Genetics and Chemical Mutagenesis. Leiden University. Wassenaarseweg 72. 2JJJ AL. Leiden, The Netherlands We have developed mouse chromosome-specific DNA hbraries for chromosomes #1, 2, 6, 8, 13, IS, X and Y. In combination with fluorescence in situ hybridization (FISH) technique, these probes were employed to study the induction and the persistence of X-ray (I and 3 Gy) induced chromosome
S-XIV: New mutagenicity tests and their evaluation
Ip XIV c.21
Immunonuorescence analysis of Diazepam-Induced mitotic apparatus abnormalitIes and chromosome loss In Chinese hamster cells
Massimo Izzo', Antonio Antoccia1 , Francesca Degrassi", Caterina Tanzarella 1 . / CentroGeneticaEooluzionisttca CNR. c/o Dip. Genetica Bioi.
Maleealare. Universita "La Sapienza", P. Ie A. Moro 5. 00185 Ramo. Italy; 2Dip. Biologia, "Ramo Tre", V.le Marconi 446. 00154 Roma, Italy Indirect immunofluorescent staining was performed with antibodies directed against mitotic apparatus proteins to study the mechanisms leading to Diazepam (DZ)-induced chromosomal malsegregation. After a 3 h treatment with 80 ug/ml of DZ, the mitotic index of hamster cells was strongly increased by the presence of "contracted" metaphases. The use of an anti a-tubulin antibody confirmed that "contracted" metaphase. were provided with monopolar spindles. Staining with anti-dynein antibody suggested that chromosomes arranged in monopolar spindles were attached to short fibers originating from spindle pole. Interestingly, nearly fifthy percent of DZinduced monopolar mitosis showed a punctate staining of centrosome when evaluated with g-tubulin antibody. This observation seemed to indicate that DZ interfered with the correct assembly of centrosomes by inhibiting the recruitment of g-tubulin to the centrosome. No qualitative differences were observed between untreated and treated cells after actin detection. After 15-30 min of recovery from DZ, nearly twenty-percent anaphases were abnormal, consisting mainly of multipolars and lagging chromosomes. This observation is consistent with the high frequency of CREST-positive MN scored in 3 h cytochalasin-blocked binucleated cells after removal of DZ. Our data show that DZ IS a powerful inducer of chromosome loss through a mechanism related to interference in the correct assembly of centrosomes. Keyword(s): Diazepam; Centrosomes; aneuploidy
Ip XIV c.31
Analysis of radiation-Induced chromosome aberrations In Chinese hamster splenocytes by FISH uslng chromosome-specific DNA libraries
M. Grigorova, Y. Xiao, A.T. Natarajan. Dept.RadiationGenetics & Chemical Mutagenesis, Leiden University. Leiden, The Netherlands Application of the fluorescent in situ hybridization (FISH) technique using chromosome specific DNA libraries for assessing radiation-induced chromosome damage has facilitated the detection of different types of translocations, In human lymphocytes, the frequencies of radiation induced translocations are higher than dicentrics. However, in mouse splenocytes the frequencies of X-ray induced translocations and dicentrics are similar. This difference between the two species has been attributed to their karyotypic features. A suitable model to study the influence of the karyotype on the induction of chromosome aberrations is the Chinese hamster, since its karyotype, similar to human, is composed of both metacentric and acrocentric chromosomes. We have estimated the frequencies of chromosome aberrations induced by different doses of 200 kV X-rays (0.75, 1.5, 3.0 Oy) and I Mev fast neutrons (0.25, 0.5, 1.0 Gy) in splenocytes from Chinese hamster by bicolour FISH using DNA libraries specific for chromosomes # 2, 3, X and 8. Only first division metaphases were analysed. The results indicate that both X-rays and neutrons induce more translocations than dicentrics, the ratio between the two types of exchanges being 1.5 and 2 respectively. The RBE of the neutrons for induction of exchanges was found to be around 3. Among the chromosomes studied, an increased involvement of chromosome #8 in exchange aberrations, about 2-3 times more than expected on the basis of its DNA content was found. The high content of interstitial telomeric sequences in chromosome #8 may be responsible for the observed increased sensitivity of this chromosome. Keyword(s): Radiation; exchanges; heterogeneity
Ip XIV
CAl
Analysis of mutagenic effect of low frequency electromagnetic fields by chromosome painting
Jose M. Garcia-Sagredo, Concepcion Villalon, Isabel Vallcorba, Carmen Sanchez-Hombre, Teresa Ferro, Carlos San Roman. Medical Genetics
Department. University Hospital Ramony Cajal, Madrid, Spain The experimental evidence on genetic effects with 50-60 Hz electromagnetic fields (EMF) indicate contradictory results; however, the majority of the reports failed to demonstrate adverse effects. Lymphocytes from normal donors, were in vitro exposed to a 50 Hz EMF at a flux density of 300, 115, and 28 I1Tdunng the time of cultures for cytogenetic analysis (72 h). After harvesting, microscope slides were hybridized with whole-chromosome DNA-libranes for chromosomes #1 and #2. Under fluorescence microscope, stable chromosome involving chromosomes I and 2 were easily detected. Our results shows 0.16% stable aberrations in controls, and 0.38%, 0.46%, 0.45% in exposed cultures under 28, 115 and 300 ILT respectively. (0.59 in controls, 1.41, 1.69, 1.63% in cell equivalents). No significant differences between chromosome abnormalities scored in control cultures and chromosome abnormalities scored in exposed cultures were detected. These result. show that a low-frequency SO Hz EMF at intensity levels similar to high intensities in human environmental exposures, have no cytogenetic effects in human lymphocytes "in vitro" at the detectable level of FISH analysis. Acknowledgment: This work was made by grant (96/1593) from FISS. Keyword(s): EMF SO Hz; FISH; Chromosome painting
Ip XIV c.sl
Development of arm-specific painting probes for Chinese hamster chromosomes and their use In the detecllon of complex chromosome rearrangements Induced by bleomycin
Y. Xiao l , F. Darroudi', S. Siml 2 , A.T. Natarajan'; / MGC. Department of Radiation Genetics and Chemical Mutagenesis. Leiden University. Wassenaarseweg 72. 2JJJ AL. Leulen, The Netherlands; 21stituto di Mutagenesi e Differenziamento, C.N.R.. Pisa, Italy The technique of fluorescence in situ hybridization (FISH) using chromosome-specitic probes allows the detection of simple as well as complex chromosome rearrangements. However, chromosome-specific probes also have their limitations. For example, some structural chromosome alterations (such as pericentric inversion) can not be detected by these probes. Recently, we have generated arm-specific painting probes for Chinese hamster chromosomes by means of microdissection with subsequent DOPPCR. The specificity of these probes was examined by two-color FISH. Using the arm specific probes, we have studied the chromosome aberrations induced by Bleomycin (radiormmetic agent) in Chinese hamster primary embryonic cells. The results obtained so far demonstrate that arm-specific painting probes are very useful to study bleomycin-mduced chromosome rearrangements, in particular, complex aberrations, intra-chromosome exchanges (such as pericentric inversions) and centric rings. Keyword(s): FISH; Chinese hamster arm-specific probes; Bleomycin
Ip XIV c.61
Analysis of the Induction and the penlstence of Xray Induced chromosome aberrations In mouse bone marrow cells using ftuorescence In sltu hybrIdization
Y. Xiao", F. Darroudi', M. Grigorova', A.T. Natarajan'. /MGC, Department of Radiation Genetics and Chemical Mutagenesis. Leiden University. Wassenaarseweg 72. 2JJJ AL. Leiden, The Netherlands We have developed mouse chromosome-specific DNA hbraries for chromosomes #1, 2, 6, 8, 13, IS, X and Y. In combination with fluorescence in situ hybridization (FISH) technique, these probes were employed to study the induction and the persistence of X-ray (I and 3 Gy) induced chromosome