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Modulation of restriction enzyme-induced chromosome damage by electroporated T4 DNA ligase
Abstracts/Mutation Research360 (1996)201-300 studied on five test systems. There was in bacteria and yeast a direct relationship with increased toxic effects as a function of the elongation of the alkyl chain of the alkoxy substituents. However, no structure-toxicity relationship was found after their application on plants and Drosophila. All anaesthetics were non-mutagenic to S. typhimurium. Pentyloxy and heptyloxy derivatives increased rates of genetic changes in S. cerevisiae, mainly point mutations at the isoleucine locus. Pentyloxy and heptyloxy derivatives slightly increased the frequency of chromosome aberrations in V. faba root-tip meristems. No chlorophyll mutations were detected after the treatment of H. vulgare with pentyloxy, hexyloxy and heptyloxy derivatives. No sex-linked recessive lethals only aneuploids were induced in D. melanogaster males. Results obtained showed that test compounds did not manifest structure-genotoxicity relationships in V. faba assay and D. melanogaster assays. 2-17
Aphidicolin-induced fragile sites in Chinese hamster cells. Relationship with telomere-related sequences
219
ascribed to FS, since they are evolutionarily conserved: 51 FS are shared by man, chimpanzee and gorilla. Moreover, FS have been reported in Drosophila, while non-random chromosomal aberrations have been shown in other animals such as rat, Persian voles, Chinese hamster and domestic pig. In this work we present evidence for the presence of aphidicolin-induced FS in hamster chromosomes, using Chinese hamster embryonal fibroblast (CHEF) cells in two passage, p18 and p33, during spontaneous progression to tumorigenicity and in 835T2 a tumor derived cell line. A significant excess of breakage has been detected at chromosomal bands lq22, 3cen, 3p21 and Xq21 in CHEFpl8, CHEFp33 and 835T2 cells, whereas 3q31 band is expressed only in CHEFp33. Each band, except 3q31, is involved in structural chromosomal rearrangements reported by other investigators. Experiments carried out by FISH technique with a telomeric probe showed the presence of interstitial telomeric arrays in almost all the chromosomes, prevalently located at centromeric level. The association between FS and telomeric level, sequences will be specifically investigated after Gbanding on hybridized metaphases. 2-18
A. Musio, I. Sbrana, G. Rainaldi a; Dipartimento di Scienze dell'Ambiente e del Territorio, Universith di Pisa, Via S. Giuseppe 22, Pisa, Italy, a Istituto di Mutagenesi e Differerenziamento C.N.R., Via Svezia, 2 Pisa, Italy The genetic and molecular basis for chromosomal fragility at common fragile sites (FS) is not known, nor is anything definitively known the mechanism of their expression. Since telomere sequences are prone to recombination, breakage and fragility, a feature peculiar to FS, it has been proposed that some FS could correspond to interstitial telomere repeats. The location of telomere-related sequences by FISH on human chromosome 2 in the ql 1-q14 region, where two FS map, supports this hypothesis; however, no direct evidence of the association between FS and telomere sequences has been yet reported. Whatever the causative aspect of DNA structure or function may be, a biological meaning can be
Modulation of restriction enzyme-induced chromosome damage by electroporated T4 DNA ligase Trinidad Ortiz, Joaquin Pifiero, Felipe Cortrs; Department of Cell Biology, Faculty of Biology, University of Seville, Spain The possible modulation by T4 DNA ligase of the DNA double-strand breaks produced by restriction endonucleases in living mammalian cells was studied. Four restriction endonucleases, two producing blunt-ended double-strand breaks ( DraI and ScaI) and two producing cohesive ended DNA doublestrand breaks (EcoRI and BamHI) were tested. An electroporation method was used for the introduction of T4 DNA ligase and the restriction endonucleases into cultured Chinese hamster ovary
220
Abstracts / Mutation Research 360 (1996) 201-300
(CHO) cells, an corresponding pairs of every restriction enzyme and ligase were introduced together 22 h later (including 3 h in colcemid), the cells were fixed and frequencies and distribution of chromosomal aberrations were studied. The pattern BrdU labelling showed that more than 95% of cells were in their first mito after electroporation with either the restriction enzyme alone or the combination restriction enzyme + ligase. Our results showed that T4 DNA ligase was able to reduce the aberration yields produced by the four restriction endonucleases tested. This decrease was dependent on the mode of break end structure, i.e., lower for restriction endonucleases with blunt-end cutting than when a restriction enzyme inducing cohesive ends was tested. One interesting observation was that, in spite of the lower frequency of aberrati when T4 DNA ligase was present, the relative proportion of deletion and exchange aberrations was maintained to a great extent. In our opinion, this feature deserves further discussion. In the case of deletions, the operation of a mechanism of a rapid ligation should result in a restitution of the original structure. Since also exchange-type aberrations were lowered by T4 DNA ligase, this seems to support the existence of an error-free ligation, avoiding the misrejoining with other double-strand break present in either the same chromosome (intrachange) or another chromosome (interchange), 2-19
The use of micronucleus assay in rat hepatocyte primary cultures for determining the genotoxicity of organophosphorous insecticides E. Piatti, L. Marabini, E. Chiesara; Department of Pharmacology, Chemoteraphy and medical Toxicology 'E. Trabucchi', University of Milan, Via Vanvitelli 32, 20129 Milan, Italy The potential hazard of pesticides must be carefully considered, due to their wide utilization and persistence in the environment. Teratogenic, carcinogenic
and genotoxic effects of some classes of pesticides are well known (IARC, 1991); however, the results of genotoxicity tests in mammalian cells or in vivo studies are conflicting. The micronucleus (MN) test in rat hepatocyte primary cultures was used in order to evaluate the in vitro genotoxic effect of dimethoate (DIM) azinphosmethyl (AZN) and diazinon (DIA). In addition the MN test was also performed at a ratio of 10:6:4, respectively, this being the mixture frequently found in foodstuffs by residual analysis. Rat hepatocytes provided a metabolization of compounds more similar to that of an in vivo situation, without requirement of complementary biotransforming enzymes. This approach also allows the study of experimental doses at concentrations which would be limited in vivo by animal toxicity. In fact, one of the main problems with the organophosphorous compounds (OPP) is that they are extremely toxic and consequent only a limited range of doses can be studied. Rat hepatocytes were stimulated to proliferate in vitro by epidermal growth factor (EGF) and were treated for 48 h with the test substances. The doses were chosen on the basis of cytotoxicity tests such as trypan blue exclusion, mitotic index inhibition and neutral red inclusion as well as protein content. The results showed that AZN was the most to doses higher than 25 Ixg/ml) and DIM the least toxic compound (at doses higher than 500 ~ g / m l ) . None of the three compounds showed a genotoxic effect when tested separately. However, a statistically significant increase in MN frequency was seen when the three OPP were combined at the lowest doses tested for the single compounds (5 + 3 + 2 p,g/ml of DIM, AZN, DIA, respectively). A further analysis with lower doses for the mixture confirmed a clear genotoxic effect even at one third of the lower dose. It is possible that, in the particular combination tested here, one of the OPP in detoxification of the other(s) by blocking some enzymatic activity as suggested by Gilot et al. (Mutation Res., 1983, 117, 139-148). In order to understanding this phenomena better, the genotoxic effects of the OPP mixture were also evaluated in cultured hepatocytes isolated from phenobarbital-induced animals. Preliminary data show a frequency in the OPP-mixture treated cells similar to that of the negative controls.
Aphidicolin-induced fragile sites in Chinese hamster cells. Relationship with telomere-related sequences
219
ascribed to FS, since they are evolutionarily conserved: 51 FS are shared by man, chimpanzee and gorilla. Moreover, FS have been reported in Drosophila, while non-random chromosomal aberrations have been shown in other animals such as rat, Persian voles, Chinese hamster and domestic pig. In this work we present evidence for the presence of aphidicolin-induced FS in hamster chromosomes, using Chinese hamster embryonal fibroblast (CHEF) cells in two passage, p18 and p33, during spontaneous progression to tumorigenicity and in 835T2 a tumor derived cell line. A significant excess of breakage has been detected at chromosomal bands lq22, 3cen, 3p21 and Xq21 in CHEFpl8, CHEFp33 and 835T2 cells, whereas 3q31 band is expressed only in CHEFp33. Each band, except 3q31, is involved in structural chromosomal rearrangements reported by other investigators. Experiments carried out by FISH technique with a telomeric probe showed the presence of interstitial telomeric arrays in almost all the chromosomes, prevalently located at centromeric level. The association between FS and telomeric level, sequences will be specifically investigated after Gbanding on hybridized metaphases. 2-18
A. Musio, I. Sbrana, G. Rainaldi a; Dipartimento di Scienze dell'Ambiente e del Territorio, Universith di Pisa, Via S. Giuseppe 22, Pisa, Italy, a Istituto di Mutagenesi e Differerenziamento C.N.R., Via Svezia, 2 Pisa, Italy The genetic and molecular basis for chromosomal fragility at common fragile sites (FS) is not known, nor is anything definitively known the mechanism of their expression. Since telomere sequences are prone to recombination, breakage and fragility, a feature peculiar to FS, it has been proposed that some FS could correspond to interstitial telomere repeats. The location of telomere-related sequences by FISH on human chromosome 2 in the ql 1-q14 region, where two FS map, supports this hypothesis; however, no direct evidence of the association between FS and telomere sequences has been yet reported. Whatever the causative aspect of DNA structure or function may be, a biological meaning can be
Modulation of restriction enzyme-induced chromosome damage by electroporated T4 DNA ligase Trinidad Ortiz, Joaquin Pifiero, Felipe Cortrs; Department of Cell Biology, Faculty of Biology, University of Seville, Spain The possible modulation by T4 DNA ligase of the DNA double-strand breaks produced by restriction endonucleases in living mammalian cells was studied. Four restriction endonucleases, two producing blunt-ended double-strand breaks ( DraI and ScaI) and two producing cohesive ended DNA doublestrand breaks (EcoRI and BamHI) were tested. An electroporation method was used for the introduction of T4 DNA ligase and the restriction endonucleases into cultured Chinese hamster ovary
220
Abstracts / Mutation Research 360 (1996) 201-300
(CHO) cells, an corresponding pairs of every restriction enzyme and ligase were introduced together 22 h later (including 3 h in colcemid), the cells were fixed and frequencies and distribution of chromosomal aberrations were studied. The pattern BrdU labelling showed that more than 95% of cells were in their first mito after electroporation with either the restriction enzyme alone or the combination restriction enzyme + ligase. Our results showed that T4 DNA ligase was able to reduce the aberration yields produced by the four restriction endonucleases tested. This decrease was dependent on the mode of break end structure, i.e., lower for restriction endonucleases with blunt-end cutting than when a restriction enzyme inducing cohesive ends was tested. One interesting observation was that, in spite of the lower frequency of aberrati when T4 DNA ligase was present, the relative proportion of deletion and exchange aberrations was maintained to a great extent. In our opinion, this feature deserves further discussion. In the case of deletions, the operation of a mechanism of a rapid ligation should result in a restitution of the original structure. Since also exchange-type aberrations were lowered by T4 DNA ligase, this seems to support the existence of an error-free ligation, avoiding the misrejoining with other double-strand break present in either the same chromosome (intrachange) or another chromosome (interchange), 2-19
The use of micronucleus assay in rat hepatocyte primary cultures for determining the genotoxicity of organophosphorous insecticides E. Piatti, L. Marabini, E. Chiesara; Department of Pharmacology, Chemoteraphy and medical Toxicology 'E. Trabucchi', University of Milan, Via Vanvitelli 32, 20129 Milan, Italy The potential hazard of pesticides must be carefully considered, due to their wide utilization and persistence in the environment. Teratogenic, carcinogenic
and genotoxic effects of some classes of pesticides are well known (IARC, 1991); however, the results of genotoxicity tests in mammalian cells or in vivo studies are conflicting. The micronucleus (MN) test in rat hepatocyte primary cultures was used in order to evaluate the in vitro genotoxic effect of dimethoate (DIM) azinphosmethyl (AZN) and diazinon (DIA). In addition the MN test was also performed at a ratio of 10:6:4, respectively, this being the mixture frequently found in foodstuffs by residual analysis. Rat hepatocytes provided a metabolization of compounds more similar to that of an in vivo situation, without requirement of complementary biotransforming enzymes. This approach also allows the study of experimental doses at concentrations which would be limited in vivo by animal toxicity. In fact, one of the main problems with the organophosphorous compounds (OPP) is that they are extremely toxic and consequent only a limited range of doses can be studied. Rat hepatocytes were stimulated to proliferate in vitro by epidermal growth factor (EGF) and were treated for 48 h with the test substances. The doses were chosen on the basis of cytotoxicity tests such as trypan blue exclusion, mitotic index inhibition and neutral red inclusion as well as protein content. The results showed that AZN was the most to doses higher than 25 Ixg/ml) and DIM the least toxic compound (at doses higher than 500 ~ g / m l ) . None of the three compounds showed a genotoxic effect when tested separately. However, a statistically significant increase in MN frequency was seen when the three OPP were combined at the lowest doses tested for the single compounds (5 + 3 + 2 p,g/ml of DIM, AZN, DIA, respectively). A further analysis with lower doses for the mixture confirmed a clear genotoxic effect even at one third of the lower dose. It is possible that, in the particular combination tested here, one of the OPP in detoxification of the other(s) by blocking some enzymatic activity as suggested by Gilot et al. (Mutation Res., 1983, 117, 139-148). In order to understanding this phenomena better, the genotoxic effects of the OPP mixture were also evaluated in cultured hepatocytes isolated from phenobarbital-induced animals. Preliminary data show a frequency in the OPP-mixture treated cells similar to that of the negative controls.