TY - JOUR A1 - Adam, W. A1 - Ahrweiler, M. A1 - Saha-Möller, C. R. A1 - Sauter, M. A1 - Schönberger, A. A1 - Epe, B. A1 - Müller, E. A1 - Schiffmann, D. A1 - Stopper, Helga A1 - Wild, D. T1 - Genotoxicity studies of benzofuran dioxetanes and epoxides with isolated DNA, bacteria and mammalian cells N2 - 1.2-Dioxetanes, very reactive and high energy molecules. are involved as labile intermediates in dioxygenase- activated aerobic metabolism and in physiological processes. Various toxico1ogica1 tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonucleasesensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, fonnamidopyrimidines). Pyrimidinedimersand sites ofbase loss (AP sites) which were probed by UV endonuclease and exonuclease 111 are minor lesions in this system. While the alky1-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA I 00. DNA adducts formed with an intermediary alkyJating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter. since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TAIOO and they form DNA adducts. as detected by the 212Ppostlabelling technique. Our results imply that the type of D NA darnage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA darnage by energy transfer. i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA. while radical darnage and alkylation prevail in the cellular system. KW - Toxikologie KW - 1 KW - 2-Dioxetane KW - Benzefuran dioxetane KW - Benzefuran epoxide KW - DNA damage KW - Mutagenicity KW - DNA adduct . Repair endonuclease KW - FPG protein Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63420 ER - TY - JOUR A1 - Epe, B. A1 - Harttig, U. A1 - Stopper, Helga A1 - Metzler, M. T1 - Covalent binding of reactive estrogen metabolites to microtubular protein as a possible mechanism of aneuploidy induction and neoplastic cell transformation N2 - Neoplastic cell transfonnation induced by estrogens and some other carcinogen& such as benzene appears to involve the induction of mitotic aneuploidy rather than DNA damage and point mutations. As metabolic activation may also play an important roJe in the mechanism of carcinogenesis of these nongenotoxic compounds, we have studied the Interaction of reactive quinone metabolites of various estrogens and of benzene with the major microtubular protein, tubulin, in a cell-free system. Covalent binding of the radioactively labeled metabolites to the a- and 13-subunit of tubulin was found to depend on the structure of the metabolite. When the adducted tubulins were tested in vitro for their ability to polymerize to microtubules, Inhibition of microtubule assembly was obsened in every case, although to varying extents. It is proposed that the fonnation of covalent tubulin adducts may impair the formation of mitotic spindies and thus contribute to chromosomal nondisjunction and aneuploidy induction. KW - Toxikologie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63478 ER - TY - JOUR A1 - Hintzsche, Henning A1 - Jastrow, Christian A1 - Kleine-Ostmann, Thomas A1 - Kärst, Uwe A1 - Schrader, Thorsten A1 - Stopper, Helga T1 - Terahertz electromagnetic fields (0.106 THz) do not induce manifest genomic damage in vitro N2 - Terahertz electromagnetic fields are non-ionizing electromagnetic fields in the frequency range from 0.1 to 10 THz. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along human exposure to these fields. Up to now, only a limited number of investigations on biological effects of terahertz electromagnetic fields have been performed. Therefore, research is strongly needed to enable reliable risk assessment. Cells were exposed for 2 h, 8 h, and 24 h with different power intensities ranging from 0.04 mW/cm2 to 2 mW/cm2, representing levels below, at, and above current safety limits. Genomic damage on the chromosomal level was measured as micronucleus formation. DNA strand breaks and alkali-labile sites were quantified with the comet assay. No DNA strand breaks or alkali-labile sites were observed as a consequence of exposure to terahertz electromagnetic fields in the comet assay. The fields did not cause chromosomal damage in the form of micronucleus induction. KW - Toxikologie Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76268 ER - TY - JOUR A1 - Holler, E. A1 - Fischer, H. A1 - Weber, C. A1 - Stopper, Helga A1 - Steger, H. A1 - Simek, H. T1 - A DNA polymerase with unusual properties from the slime mold Physarum polycephalum N2 - Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase cx on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzymewas 30-40% pure and form HS2 enzyme 60% with regard toprotein contents of the preparations. Form HS2 enzymewas generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptidein a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as weil as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase ß by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis. KW - Toxikologie Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63501 ER - TY - JOUR A1 - Kirchner, S. A1 - Stopper, Helga A1 - Papp, T. A1 - Eckert, I. A1 - Yoo, H. J. A1 - Vig, B. K. A1 - Schiffmann, D. T1 - Cytogenetic changes in primary, immortalized and malignant mammalian cells N2 - Some chromosomes in transformed rat cells and somatic cell hybrids fail to display the presence of kinetochore proteins as detected by antikinetochore antibodies. Suchchromosomes (K- Chromosomes) may constitute a novel mechanism for the genesis of aneuploidy. Wehave analyzed primary~ immortalized and malignant marnmalian cells for the presence of kinetochore proteins and micronuclei. Our resuJts suggest a correlation of the K- chromosome and micronucleus frequency with the variability in chromosome number. Upon in situ hybridization with the minor satellite and alpha satellite sequences some Kchromosomes showed a signal. This indicates that the observed lack of kinetocbores is not necessarily due to a lack of centromeric DNA. We conclude that dislocated K- chromosomes may become incorporated into micronuclei which are prone to loss. Such events would be associated with the generation of aneuploidy. KW - Toxikologie KW - Micronuclei KW - Kinetochore KW - Chromosome distribution Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63439 ER - TY - JOUR A1 - Stopper, Helga A1 - Eckert, I. A1 - Schiffmann, D. A1 - Spencer, D. L. A1 - Caspary, W. J. T1 - Is micronucleus induction by aneugens an early event leading to mutagenesis? N2 - This study was designed to investigate a previously unidentified potential mechanism for mutation induction as well as to clarify a biological comequence of micronucleus formation. We compared the induction of micronuclei with mutation inductioo as measured by trißuorothymidine (TFI') resistance in mouse L5178Y cells using four aneugens: colcemid, diethylstilbestrol, griseofulvin and vioblastine. AU four compounds induced micronuclei which appeared in the first cell cycle after treatment. More than 85% of the micronuclei induced by each compound stained positive for the presence of kinetochores implying that the micronuclei contained wbole cbromosomes. However, these same compounds were unable to induce TFf resistance under tbree different treatment regimes. We concluded that tbese compounds, under conditions where tbey induce primarily kinetochore positive micronuclel, were not able to induce mutations. Thus, the induction of micronuclei containing wbole chromosomes barborlog a select.able gene is not an early event leadlog to mutations in these cells. KW - Toxikologie Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63390 ER - TY - JOUR A1 - Stopper, Helga A1 - Jones, H. A1 - Zimmermann, U. T1 - Large scale transfection of mouse L-cells by electropermeabilization N2 - Mouse L-cells were transfected by electropenneabilization using the selectable plasmid pSV2-neo which confers resistance to G-418 (Geneticin). 1be DNA concentration used was 1 l'gfml, the field strength was 10 kV fcm, the duration of the pulse was S ~s. Transfeetion yield was optimal at a temperature of 4°C when using a time in between consecutive pulses of 1 minute compared to shorter (of the order of seoonds) or Ionger (3 minutes) time intervals. A more detailed study of the relationship between the number of pulses applied (up to 10) and transfection yield showed it to be almost linear in this range at 4 o C. The yield of transfectants in response to 10 pulses was up to 1000 per 106 cells (using 3.3 pg DNA per cell). The inßuence of the growth phase of the cells on the transfection yield and I or the subpopulation of the mouse L--ceU line used was shown. Furthennore the clone yield depended on the DNA per ceU ratio within a very small range. KW - Toxikologie KW - Electrical breakdown KW - Electropermeabilization KW - Volume distribution KW - Gene transfer KW - Transfection KW - (Mouse L-cell) Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63497 ER - TY - JOUR A1 - Stopper, Helga A1 - Kirchner, S. A1 - Schiffmann, D. A1 - Poot, M. T1 - Cell cycle disturbance in relation to micronucleus formation induced by the carcinogenic estrogen diethylstilbestrol N2 - In addition to its tumor-promoting activity in honnone-receptive tissue, the carcinogenic estrogen diethylstilbestrol (DES) has been found to induce cell transformation, aneuploidy and micronucleus formation in mammalian cells. The majority of these micronuclei contained whole chromosomes and were fonned during mitosis. Here a possible relationship between a disturbance in cell cycle progression and micronucleus fonnation is investigated by exposing Syrian hamster embryo (SHE) cells to DES. Continuous bromodeoxyuridine labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry was employed for analysis of cell cycle transit and related to the time course of micronucleus formation. Treatment of SHE cells with DES resulted in delayed and impaired cell activation (exit from the GO/G 1 phase), impaired S-phase transit and, mainly, G2-phase traverse. Cells forming micronuclei, on the other hand, were predominantly in G2 phase during DES treatment. These results suggest that impairment of Sand G2 transit may involve a process ultimately leading to micronucleus formation. KW - Toxikologie KW - Flow cytometry KW - Micronucleus formation KW - Diethylstilbestrol KW - Hoechst 33258 dye KW - Bromodeoxyuridine labeling KW - continuous Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82250 ER - TY - JOUR A1 - Stopper, Helga A1 - Körber, C. A1 - Schiffmann, D. A1 - Caspary, W. J. T1 - Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells N2 - 5-Azacytidine was originally developed to treat human myelogenous leukemia. However, interest in this compound has expanded because of reports of its ability to affect cell differentiation and to alter eukaryotic gene expression. In an ongoing attempt to understand the biochemical effects of this compound, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. In L5178Y mouse cells, 5-azacytidine induced micronuclei at concentrations at which we and others have already reported its mutagenicity at the tk locus. Using CREST staining and C-banding studies, we showed that the induced micronuclei contained mostly chromosomal fragments although some may have contained whole chromosomes. By incorporating BrdU into the DNA of SHE cells, we determined that micronuclei were induced only when the compound was added while the cells were in S phase. Microscopically visible effects due to 5-azacytidine treatment were not observed until anaphase of the mitosis following treatment or thereafter. 5-Azacytidine did not induce micronuclei via interference with formation of the metaphase chromosome arrangement in mitosis, a common mechanism leading to aneuploidy. SupravitalUV microscopy revealed that chromatid bridges were observed in anaphase and, in some cases, were sustained into interphase. In the first mitosis after 5-azacytidine treatment we observed that many cells were unable to perform anaphase separation. All of these observations indicate that 5-azacytidine is predominantly a clastogen through its incorporation into DNA. KW - Toxikologie KW - Micronuclei KW - L5178Y cells KW - 5-Azacytidine KW - Berenil KW - DES KW - Ethionine KW - Mitosis Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63411 ER - TY - JOUR A1 - Stopper, Helga A1 - Körber, C. A1 - Spencer, D. L. A1 - Kirchner, S. A1 - Caspary, W.J. A1 - Schiffmann, D. T1 - An investigation of micronucleus and mutation induction by oxazepam in mammalian cells N2 - Tbe benzodiazepines are a class of d.rugs that are widely used in the treatment of various psychiatric disorders. One member of um ~' oxazepam, is also a common metabolite of sevmd other benzod.iazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is incol:lsb1ent, we investigated the oxazepam-induced fonnation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and LS178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all tbree ceU llnes. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were fonned in the first mitosis after treatment. Kinetochore staining (CREST -antiserum) revealed the presence of kinetochores in -SO% of the micronuclei in aU tbree ceU types. ThJs resu1t was further confinned by in situ bybridization in LS178Y cells and indicates tbe presence of wbole Chromosomes or centric fragments as weU as acentric fragments in the oxazepam-induced micronuclei. The LS178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used. KW - Toxikologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-63404 ER -