R. Rieger

Genetic toxicology of ethylenediaminetetraacetic acid (EDTA).

EDTA and its salts have a number of applications in medicine and pharmacy. EDTA is used to remove calcium from the human body, and serves as an anticoagulant and as a detoxicant after poisoning by heavy metals. It is often used in analytical chemistry for complexometric titrations and many other purposes. Because the compound is of rather low toxicity, it is used as a food additive to bind metal ions. EDTA affects the inhibition of DNA synthesis in primary cultures of mammalian cells. This may be due to impairment of enzymes involved in DNA replication. Some early studies have shown that EDTA leads to morphological changes of chromatin and chromosome structure in plant and animal cells. These alterations consist of dispersion or swelling of chromosomes or a loss of interphase chromatin structure. For several test systems, a low chromosome-breaking activity of EDTA has been reported. A weak activity in the induction of gene mutations has also been observed. It is well established that EDTA influences chromosome breakage by mutagenic agents. In particular, when applied in combination with chemical mutagens, EDTA enhances mutagen-induced aberration frequencies. Furthermore, the chelating agent is able to increase the incidence of meiotic crossing-over. This has been demonstrated for many gene loci in Drosophila melanogaster, Chlamydomonas reinhardi, Neurospora crassa and Zea mays. EDTA interferes with DNA repair processes that take place after exposure to mutagens. In E. coli or Micrococcus radiodurans as well as in Chinese hamster cells, the fast repair component detectable after treatment with ionizing radiation or bleomycin is inhibited by EDTA. In plant cells exposed to gamma-rays, EDTA inhibits unscheduled DNA synthesis. The mechanism by which EDTA causes these effects remains poorly understood. The sequestering of metal ions by the chelating agent is obviously responsible for functional and structural alterations of the genetic material. Although EDTA produces a whole set of genetic effects it seems to be a harmless compound to man as far as genotoxicity is concerned. The data presently at hand, however, are not sufficient for a reliable risk assessment.

Distribution of heterochromatin in a reconstructed karyotype of Vicia faba as identified by banding- and DNA-late replication patterns

Abstract1) The distribution pattern of heterochromatin characterized by Giemsa-banding, Quinacrine-banding and DNA-late replication has been studied in a reconstructed karyotype of Vicia faba with all chromosome pairs interdistinguishable. 2) By means of two Giemsa-banding methods both an interstitial and a centromeric Giemsa-banding pattern are described. The former one comprehends 14 “marker” and 18 “additional” bands of lower but characteristic visualization frequencies. The centromeric Giemsa-banding pattern consists of 7 bands, located in the centromeric and in the secondary constrictions of the metaphase chromosomes. Chromosomes with banding patterns intermediate between the interstitial and the centromeric Giemsa-banding have also been observed. 3) Quinacrine-banding revealed 10–12 brightly fluorescent bands and 1–2 regions of dim fluorescence. Most Q-bands occupy chromosomal positions also characterized by interstitial Giemsa bands. 4) The DNA-late replication pattern, analyzed both by autoradiography and by FPG-technique, revealed 9 late replicating chromosome regions; all of these correspond positionally to the sites of interstitial Giemsa bands. 5) The results are discussed with respect to (a) the relationships between the banding- and the DNA-late replication pattern; (b) banding and heterochromatin characteristics; (c) the correlations between the distribution of chromatid aberrations and special types of heterochromatin. — The patterns of heterochromatin distribution found are in basic conformity with the corresponding patterns reported for the standard karyotype of Vicia faba. The heterochromatin type characterized by both Giemsabanding and late replication is characteristic of all those chromosome regions which after mutagen treatments show up as aberration hot spots. Positional correlations between interstitial Giemsa marker bands and chemically induced isochromatid breaks are indicative of preferential aberration clustering in heterochromatin/euchromatin junctions.

Non-random intrachromosomal distribution of chromatid aberrations induced by X-rays, alkylating agents and ethanol in Vicia faba

Abstract A reconstructed karyotype of Vicia faba with all chromosomes individually distinguishable was treated with triethylene melamine (TEM), cytostasan (CYT) (a new benzimidazol nitrogen mustard), mitomycin C (MI), ethanol (EA) and X-rays. The distribution within chromosomes of induced chromatid abberations was non-random for all agents. The number of segments involved in aberration clustering corresponded to the number of sites representing constitutive heterochromatin, or the regions immediately adjacent to these, as evidenced by the position of Giemsa marker bands. Which of these potential regions of aberration clustering reacted with preferential involvement in aberrations was, in part at least, dependent upon the inducing agent used. It is argued that this may be due to differences in the base composition and/or molecular conformation of heterochromatic regions. Unexpectedly, the distribution pattern of chromatid aberrations induced by mitomycin C was found to be different from those after treatment with the alkylating agents TEM and cytostasan although mitomycin C is assumed to induce aberrations via alkylation. If mitomycin C-induced aberrations are indeed due to alkylation, this indicates that different alkylating agents do not necessarily result in identical patterns of abberation clustering. The other two alkylating agents and ethanol resulted in similar patterns of preferential distribution of abberations. X-Ray induced chromatid aberrations also showed a non-random intrachromosomal distribution, but the clustering was less pronounced than after treatment with the chemical agents.

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.

New Karyotypes of Vicia faba L.

Five new karyotypes of V. faba are presented and the types and positions of the structural changes combined in the construction of these new karyotypes are described. All their chromosomes are easily distinguishable by morphological criteria. These new chromosome complements are presently used to study the inter- and intrachromosomal distribution of induced chromatid aberrations and related problems.

Inhomologenpaarung und Meioseablauf bei haploiden Formen von Antirrhinum majus L.

Summary1.Meiosis of haploid forms ofAntirrhinum majus L. (n = 8) is characterized by an extremely high degree of pairing between inhomologous or partially homologous chromosomes. In extreme cases this pairing leads to the formation of four bivalents. Besides bivalents trivalents and more complex pairing configurations were found in pachytene. Fold-backs of whole chromosomes or of chromosome segments occurred in a considerable number of PMC. Pairing within the haploid chromosome set took place in most cases between inhomologous chromosome segments, but in later stages of first meiotic division it was possible to show the existence of at least one intrachromosomal and one interchromosomal duplication. The interchromosomal duplication causes preferential pairing of two specific chromosomes.2.In diakinesis and meta-anaphase I pairing — configurations consisting of two or more chromosomes were found. According to form and valence of these configurations it was possible to establish with certain reservations a minimal chiasma frequency of 0.8 per PMC. One part (40%) of chiasmata is probably formed between the segments of the interchromosomal duplication. The other 60% are either chiasmata between further duplications, which cannot be demonstrated cytologically, or chiasmata between inhomologous segments. The possibility is not excluded that part of these configurations are not held together by chiasmata, but are pseudoconfigurations caused by matrix stickiness.3.The various PMC-types are grouped according to the different combinations of uni-, bi- and multivalents found in them. 42,34% of the analyzed PMC contained 8 univalents, 39,82% one bivalent, 11,94% two bivalents, 2,14% three bivalents, 0,27% four bivalents. The last 3,49% of PMC were characterized by trivalents and a few higher associations.4.The abnormal second meiotic division was analyzed in order to find out the way of origin of the various types of sporades containing one to eight microspores. Dividing univalents in first meiotic division, the type of distribution of univalents to the poles, the formation of restitution nuclei and abnormal cytokinesis were found to be responsible for the development of the different types of sporades. The haploid plants were practically self-sterile but by pollination with diploid forms some progeny could be raised.5.Form, extent and consequences of inhomologous pairing are discussed. It is supposed that there is a general pairing tendency of unknown nature in meiotic prophase which will be saturated by preferential contact between homologous chromosomes. The existence of homologous partners is supposed to prevent pairing of inhomologous chromosomes. But if there are only inhomologous chromosomes within one nucleus the general pairing tendency in meiotic prophase leads to associations of inhomologous chromosomes. The existence of homologous segments within one set of mostly inhomologous chromosomes causes preferential pairing of these identical regions, but does not suppress inhomologous pairing.

Intra-chromosomal distribution patterns of mutagen-induced SCEs and chromatid aberrations in reconstructed karyotypes of Vicia faba

Abstract The FPG (fluorescent plus Giemsa) technique was used in a study of the frequency and the inter- and intra-chromosomal distribution patterns of SCEs after incorporation of BrdUrd into the chromosomal DNA as well as after additional mutagen treatment in two reconstructed karyotypes of Vicia faba . The kartotypes used orginated from combining of different translocations and pericentric inversions (each homozygously) and allowed the exact morphological phological identification of each of the 6 chromosome pairs. Treatment with ethanol (EA), maleic hydrazide (MH) and mitomycin C (MMC) resulted in a strong increase of SCE frequency. With one exception (the nucleolus-organizing secondary constriction) the inter- and intra-chromosomal distribution of SCEs was length proportional, independently of whether or not additional mutagen treatment was performed. This indicates absemce of coincidence between the intra-chromosomal distribution patterns of mutagen-induced SCEs an chromatid aberrations for the following reasons: (1) chromatid aberrations have been shown to be non-randomly and, in part at least, mutagen-specifically distributed; (2) chromosome segments, which were found to be differently involved in chromatid aberrations when differently positiones in the two karyotypes, were equally involved in mitomycin-C-induced SCEs. The results are discussed with respect to possible connections between the mechanisms leading to SCEs and to chromatid aberrations.

Chromosomenaberrationen bei Vicia paba und Ascitestumoren der Maus nach Einwirkung von N-nitroso-N-Methylharnstoff

SummaryN-nitroso-N-methylurea was tested for its ability to induce chromosomal aberrations in root-tip meristems of Vicia faba and in two ascites tumour strains (Ehrlich mouse ascites carcinoma and S2-sarcoma) of the mouse. In all cases the agent was found to be active (Table 1,8). The effectivity of the agent in aberration induction was strikingly different in the two tumour strains and possible reasons for that are discussed.In Vicia the radiomimetic activity of nitrosomethylurea was studied in more detail. The compound had a delayed effect (Table 2) and the aberrations induced were exclusively of the chromatid type. They were preferentially localized in the heterochromatic segments of the chromosomes and unevenly (not length proportional) distributed between the chromosomes. The distribution-ratio of aberrations between the short (5 pairs) and long chromosomes (one pair) was in favour of the short ones and dependent on concentration and duration of treatment (Table 1,2). The distribution of isolocus breaks and chromatid translocations between cells was in accordance with expectation on the basis of a Poisson-distribution (Table 3) and the effectivity of nitrosomethylurea was dependent on the temperature of the treatment solution (Table 4). The agent was almost inactive in the absence of oxygen and its activity was greatly reduced when the meristems were pretreated with the enzymeinhibitor sodium azide. Pretreatments with 2,4-dinitrophenol, KCN, hydroxylamine and chloromycetin did not affect significantly the rate of induced aberrations (Table 5). Pretreatment with the chelator EDTA sensitized to the radiomimetic effects of nitrosomethylurea (Table 6). Breaks induced by the compound stayed open for approximately 4–8 hours (Table 7).The experimental results are discussed and the tentative conclusion as to the mode of action of nitrosomethylurea is, that the aberrations become induced by a breakdown product of the agent the formation of which is dependent on the presence of oxygen and an enzyme containing heavy metal. Probably this decomposition product is not an alkylating agent.

On the induction of segment extension and chromatid structural changes in Vicia faba chromosomes after treatment with 5-azacytidine and 5-azadeoxycytidine

Abstract When root tips of Vicia faba were treated with 5-azacytidine (5-ACR) or 5-azadeoxycytidine (5-ACD) local uncoiling (segment extension) of specific chromosome segments and chromatid aberrations was produced. The peak of cells with segment extension was observed 2–8 h after treatment (non-delayed effect); cells with chromatid aberrations appeared after recovery times of more than 20 h (delayed effect). Colchicine labelling of cells after 5-ACR treatment showed that chromatid aberrations were present in 4n cells, i.e. , they became induced during the interphase preceding the second mitosis. 5-ACR post-treatment after X-raying increased the frequency of X-ray-induced chromatid aberrations, and pretreatment was without influence on the aberration yield induced by X-rays alone. The preliminary conclusion drawn from the experimental results is that segment extension is due to the inhibition of RNA and, concomitantly, protein synthesis or the failure of complex formation between DNA and specific proteins. The incorporation of the analogues into DNA is presumed to be responsible for the induction of chromatid aberrations.

Die Auslösung von Chromosomenaberrationen beiVicia faba durch chemische Agenzien

ZusammenfassungDiese Arbeit soll einen Überblick über die Induktion von Chromosomenaberrationen durch chemische Agenzien beiVicia faba geben. Zuerst werden einige Angaben zur Charakterisierung des Untersuchungsobjektes und der eingesetzten Methoden gemacht (S. 160). Im zweiten Teil wird der Versuch unternommen, eine möglichst vollständige Übersicht über sämtliche chemische Substanzen zu geben, die beiVicia faba auf ihr Vermögen, Chromosomenmutationen zu induzieren, überprüft worden sind. Die publizierten Befunde konnten durch das Entgegenkommen von A. Loveless (London), B. A. Kihlman (Uppsala), N. S. Cohn (Athens, Ohio) und D. Davidson (St. Andrews, Schottland) durch bisher unveröffentlichtes Material ergänzt werden. Im letzten Teil der Arbeit werden Angaben über kombinierte Einwirkung verschiedener Agenzien (S. 167), über die inter- und intrachromosomale Aberrationsverteilung (S. 170), über biochemische, die Aberrationsauslösung beeinflussende Faktoren (S. 174) und über einige Befunde und Probleme im Zusammenhang mit dem Wirkungsmechanismus der Radiomimetika gemacht (S. 181).SummaryThis paper is to give a survey of the work on the induction of chromosome aberrations by chemicals inVicia faba. Firstly some data characterizing object and methods are given (p. 213). In the second part it is tried to give a list as complete as possible of all chemicals tested for radiomimetic activity inVicia faba independent of their being effective or ineffective (p. 219). Published data are supplied by unpublished ones generously given by Drs. A. Loveless (London), B. A. Kihlman (Uppsala), N. S. Cohn (Athens, Ohio) and D. Davidson (St. Andrews, Scotland). The last part of the paper is concerned with the effects of combined treatment ofVicia faba with different agents (p. 256), with the distribution of aberrations between and within chromosomes (p. 259), with biochemical factors tested for influences on the radiomimetic activity of various compounds (p. 265) and with data and problems of the mode of action of some radiomimetic substances (p. 272).Краткое содержаниеДается обзор работ по индукции хромосомных перестроек химическими соединениями уVicia faba L. Сначала приводятся некоторые данные для характеристики объекта исследования и применяемых методов (стр. 213). Во второй части авторы пытаются дать возможно более полный список химических веществ, которые были испробованы на их способность вызывать уV. faba хромосомные мутации. Приводимый здесь материал, благодаря коллегиальной помощи А. Лавлеса (Лондон), Б. А. Кильмана (Упсала), Н. С. Кона (Этенс, Огайо) и Д. Дэвидсона (Ст. Андруз, Щотландия), мог быть значительно пополнен неопубликованными данными. В последней части работы даются сведения о комбинированном действии различных веществ (стр. 256), о распределеннии перестроек между хромосомами и внутри хромосом (стр. 259), о биохимических факторах, влияющих на индукцию аберраций (стр. 265) и о некоторых проблемах механизма действия радиомиметических веществ (стр. 272).