Antagonism in the action of kinetin and actinomycin D on the cytogenetic effect of radiation

A study was made of the effect of kinetin when administered separately or in combination with actinomycin D on the chromosomal aberrations in Crepis capillaris L. irradiated by X-rays. Dry seeds were treated after irradiation or seeds soaked, prior to irradiation. Significant decrease in the frequency of chromosomal aberrations was shown when actinomycin D was administered separately and prior to kinetin. The latter used separately and prior to actinomycin D slightly increased the level of the aberrations, as compared with the control. Noticeable radioprotective effect was observed after treatment of seeds with kinetin, prior to irradiation.     

Chromosomal aberrations in Crepis capillaris cells detected by FISH

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after mutagenic treatment. It has simple karyotype: three pairs of morphologically distinct and relatively large chromosomes. The frequency of structural chromosome aberrations and micronuclei in root meristem cells has been used for evaluation of the genotoxicity of chemicals and environmental pollutants. The introduction of fluorescence in situ hybridization method allows more detailed detection and localization of chromosomal rearrangements not only in mitotic but also in interphase nuclei. We demonstrate a few examples of the detection of chromosomal aberrations using rDNA and telomeric sequences as probes for in situ hybridization to C. capillaris chromosomes.     

Undulating kinetics factors in radiation mutagenesis]

Air-dry Crepis capillaris seeds, soaked for 14 and 20 hours, were dried to the moisture content of 1,1% with the following gamma-irradiation at a dose of 1500 r. Irradiated seeds were stored in the state of identical moisture for 38 days. The drying of three groups of seeds to moisture content of 1,1% provided their identical initial radiosensitivity. In the course of the storage mutability of treated seeds significantly increased as compared with the yield of mutations in non-stored seeds, and revealed undulating kinetics that involved all types of arising chromosome and chromatid rearrangements. The character of undulating kinetics is due to a correlation between functional state of nucleus (early G1, late G1, S) and water content in seeds.     

The cytogenetic effect of chronic irradiation revealed using an analysis of the first two mitoses in the root meristem of germinating seeds of Crepis tectorum from the 30-kilometer zone of the accident at the Chernobyl Nuclear Power Station

Peculiarities of the appearance of C. tectorum rootlet in the course of seed germination have prompted the authors to use the method of continuous germination of seeds in 0.01% colchicine solution and to analyze chromosome aberrations in the first tetraploid cells derived from early dividing cells. Since it has been shown that cells of the embryo's root meristem of dormant Crepis tectorum seeds are at the G1 stage, the observed, by the method used, chromatid aberrations in the cells that early enter mitosis, are probably induced by the incorporated radioactive products at stages S and (or) G2 during seed germination.     

Critical periods of the mitotic cycle: influence of aminopterin and thymidine on production of chromosomal aberrations by radiation in Crepis capillaris.

The influence of aminopterin (AP), tritiated thymidine ([3H] TdR) and "cold" thymidine (TdR) on production of chromosomal aberrations in meristematic cells of Crepis capillaris irradiated in different stages of the mitotic cycle with 300 rad of 63Co gamma-rays was studied. All the chemical treatments increased most of all the frequency of aberrations induced during two "critical periods" localized before the stage of DNA synthesis (fixation 9 h after irradiation) and before that of mitosis (4 h). Treatments with TdR and [3H]TdR increased most of all the frequency of chromatid aberrations when irradiation was performed in G1, and the frequency of gaps when irradiated in G2. Treatment with AP increased the yield of different types of aberration more uniformly. The modifying effect of the chemicals tested appeared to be independent of replicative synthesis. The "critical periods" are suggested to be the stages when regular "proof reading" and correction of spontaneous errors takes place [9,13]. In addition to this regular mechanism, radiation induces an "emergency" mechanism of repair. AP inhibits the mechanism of regular repair; in addition TdR and [3H] TdR suppress the lateral spread of primary injuries across the chromosome.     

Mutagenic activity of antineoplastic agents under conditions of normal and artificial modification

Mutagenic effect of vincristine, vinblastine, thiophosphamide, sarcolysine and the supermutagen ethyleneimine was studied in Crepis capillaris germinating seeds at the G phase. Metabolic conditions, prolongation of cell cycle, specific activity of preparation as well as inhibition of DNA synthesis were found to modify the mutagenic process induced by antitumour agents.     

Mutagenic action of latex polymerization stabilizers in various test systems

The cytogenetic activity of latex polymerization stabilizers (monoethanolamine, triethanolamine and neozon-D) is investigated in three different test systems. It is shown that monoethanolamine and triethanolamine are weak inductors of chromosome breaks in the culture of human lymphocytes and in the Crepis capillaris seeds and induce low levels of gene mutations in the Ames systems. The third stabilizer--neozon-D manifests higher mutagenic activity and definite cytotoxic effect. Monoethanolamine and triethanolamine as to their weak mutagenic effect are recommended as preferable stabilizers to be used in the latex industry.     

Cytogenetic activity of various organophosphorus insecticides

The action of certain insecticides belonging to the group of organophosphorous compounds (chlorophos, metaphos, etaphos, phosphamid, phosalone, carbophos, selecron) has been studied by the test of chromosome aberrations in Crepis capillaris cells. It is shown that these insecticides possess mutagenic activity in all phases of the cell cycle and are mutagens of the undelayed action.     

Interaction between chemical mutagens with a delayed effect and metabolites of seeds

Summary A study was made of chromosome aberrations in Crepis capillaris seedlings, induced by the reaction products of chemical mutagens with seed metabolites. Interaction between ethylenimine and seed metabolites of some plants of the family Compositae (C. capillaris, Taraxacum officinale, Pyrethrum carneum, Helianthus annuus) has been found to lead to the formation of highly active secondary mutagens whose action remains similar to that of ethylenimine, although the effect of ethylenimine is enhanced dozens of times. The substances responsible for this enhancement effect are contained in the fruit coating of the seed. The metabolites of seeds of other plants studied (Triticum vulgare, Hordeum vulgare, Fagopyrum esculentum) enhanced the effect of ethylenimine only 1.5–2.0 times. Unlike ethylenimine, the effect of its derivatives (thioTEP and phosphazine) and of ethyl methanesulphonate, HN2 and maleic hydrazide is not enhanced after their interaction with metabolites of compositae plant seeds. Experiments with HN2 revealed an almost complete inactivation of the mutagenic action of NH2 by metabolites of C. capillaris seeds. The observed modification of the mutagenic action of ethylenimine and NH2 after successive treatment of seedlings with mutagens and metabolites of seeds points to the preservation of the mutagen in the cell. It is concluded that when chemical mutagens act on the cells, chromosome aberrations are induced not only by the chemical agent itself, but also by its reaction with cell metabolites.     

Interaction between chemical mutagens with a delayed effect and metabolites of seeds

Summary A study was made of the cytogenetic effect of mutagens with a delayed effect (ethylenimine and ethyl methanesulphonate) on Crepis capillaris seeds. The effect was found to depend on the physiological condition of the seeds. In seeds not subjected to prolonged storage, where only chromatid aberrations were occurring spontaneously, mutagens also induced chromatid aberrations only. If, however, because of physiological changes in the seeds (e.g. upon prolonged storage or when seeds were kept at an elevated temperature and humidity) a large number of chromosome-type aberrations appeared, they also appeared when the seeds were acted upon by mutagens with a delayed effect. The action of such mutagens was also found to depend on spontaneous mutation in seeds with different rates of germination. Special experiments showed that the interaction of ethylenimine with the metabolites of seeds in vitro leads to the formation of secondary active mutagens differing from ethylenimine in the nature of their action. The induction of chromosome-type aberrations by treating seeds with alkylating compounds may be due to the action of secondary mutagens.     

Transformed roots of Crepis capillaris--a [corrected] sensitive system for the evaluation of the clastogenicity of abiotic agents

The presence of a large number of pollutants, including mutagenic agents in the environment is a problem of a major concern. Rapid progress in plant biotechnology, especially in the development of cell transformation methods, including the production of transformed roots -- 'hairy roots' -- has opened new possibilities to use transformed root cultures in plant bioassays for the evaluation mutagenic effects of different agents. We have used Crepis capillaris hairy roots for evaluation of cytogenetic effects of mutagenic treatment. Effects of maleic acid hydrazide (MH) and X-ray treatment were analysed in chromosomal aberration, sister chromatid exchange (SCE) and TUNEL tests. Comparison of cytogenetic effects in hairy roots and roots of seedlings showed a much higher sensitivity of hairy roots, which makes them convenient material for monitoring DNA damage after mutagenic treatment.     

The genetic activity of an alkaline solution of formaldehyde

Toxicity and genetic activity of disinfectant consisting of 1.5% paraform and 0.5% NaOH were studied. It was found that the preparation caused a weak mutagenic effect on Crepis capillaris and Chlorella roots, but had no effect in the Ames test and gonadal cells of mammals. The studies of mutagenic effect of the preparation on somatic cells of white mice show limits of the cytogenetic effect at the level of 3.5 g/kg (1/10 LD50).     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Cytogenetic activity of the herbicide sodium trichloroacetate

It is established that sodium trichloracetate does not induce chromosomal aberrations in bone marrow cells of mice, does not modify mutagenic effect induced by rubomicin, but increases chromosome aberration frequency in the culture of human peripheral lymphocytes and in germs of Crepis tectorum and Allium cepa seeds.     

Plant tissue culture as a model system for mutagenicity testing of chemicals

Two karyotypically stabilized callus strains of Crepis capillaris (2n = 7 and 2n greater than or equal to 12, respectively) were employed to illustrate the utility of plant tissue culture method for screening and analysis of cytogenetic effects of chemicals following long-term treatment. The cytogenetic analysis of callus cells revealed significant differences in the toxic and mutagenic effects of chemicals under study (2,4-D, MH, NMU and kinetin). A certain dose-response relationship (though not necessarily linear) was observed. The maximum cytogenetic activity of chemicals was associated with certain intermediate concentrations (4.5 X 10(-5) M-9 X 10(-5) M), whereas a further increase in the dose either gave rise to the opposite effects (i.e. decrease in the ana- and telo-phase aberration rates and increase in the modal karyotype frequency) or the aberration rate remained unchanged.     

Modifying effect of dioxane on the cytogenetic activity of N-nitroso-N-methylurea

Dry seeds of Crepis capillaris L. were treated with N-nitroso-N-methyl urea (NMU) and with dioxane (DO) an organic solvent, at pH 7.0 and pH 5.7. The treatment with NMU only was used as a positive control. Three concentrations of NMU were applied. The cytogenetic activity of NMU was found to considerably decrease at two values of pH, while NMU was solved in DO. The relationship between different types of chromosome aberrations remained unchanged in this case.     

Mechanism of the chromosome damaging effect of ftorafur]

Chromosome damage induced by three antineoplastic drugs -- ftuorafur (Ft), 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FUdR) hase been studied in Djungarian hamsters cell line after 24 hours exposition with these agents before the fixation. Ft at a dose of 10 micron/ml induced aberrations in 56.7% of metaphases. 60.0% of aberrant metaphases were obtained in experiments with 0.1 micron/ml of 5 FU. FUdr at the same dose induced 24.0% of aberrant metaphases. The high frequency of chromatid breaks and gaps was typical for the mutagenic action of these fluorinated pyrimidines. The addition of Ft or 5-FU to the cell cultures 1--12 hours before fixation did not produce any significant chrosome damage, while further exposition with the drugs for 15--24 hours caused breaks in more than 50% of metaphases. Thymidine at a concentration of 1.0 micron/ml suppressed the chromosome breaking effect of Ft and 5-FU. The results obtained are in accordance with the idea that fluorodeoxyruidinemonophosphate is the ultimate mutagen for both Ft and 5-FU and that the aberrations observed are due to the lack of thymidine nucelotides caused by the former agents while DNA replication.     

Mechanism of action of antimutagens. I. ionol modification of aberrations induced by different factors]

The capacity of decreasing the frequency of chromosome aberrations, spontaneous and induced by ionizing radiation (gamma-rays) and alkylating compounds (ethylene imine) was established to be inherent in ionole (2,6-di-tretbutyl-4-methylphenole). Crepis capillaris was used as the experimental material. Ionole was particularly efficient with respect to aberrations induced by ethylene imine. The decrease of the frequency of spontaneous and induced aberrations did not affect their spectrum.     

Mechanism of action of antimutagens. II. Relationship between the antimutagenic effect of ionol and the stage of the mitotic cycle of Crepis capillaris L.]

When a synchronous population of Crepis capillaris L. cells was used as the experimental material, the repair of spontaneous and induced aberrations by antimutagens was established to take place at the G1 stage. A definite period within this stage when the repair proceeds is discovered.     

Influence of physiological conditions on DNA repair and mutagenesis in higher plants

In barley ( Hordeum vulgare L.) and grass pea ( Lathyrus sativus L.), caffeine, an inhibitor of DNA repair activity, and Na₂ethylenediaminetetraacetate, an inhibitor of DNA-endonucleases, sharply decreased the excision repair of pyrimidine dimers induced in DNA by ultraviolet irradiation. An inhibitor of RNases, diethylpyro-carbonate, did not inhibit the process of excision, and in one experiment it even enhanced excision. Caffeine markedly increased the frequency of mutations and inhibited the growth of seedlings after UV-radiation. Such enhancement was greater with the higher UV fluence. Results of chemical inhibition were further confirmed by the suppression of repair by low temperatures: the frequency of chromatid aberrations induced with propyl methanesulfonate was increased more than 3 times and chromatid aberrations 1.5 times. Evidence for participation of repair enzymes in the modification of mutation processes was also obtained in the experiments which combined γ-irradiation and treatment with propyl methanesulfonate. Conditions favouring repair activity caused a drastic reduction in the frequency of aberrations, whereas with conditions preventing enzyme function the mutation frequency increased. In one of the experiments of this series we were able to demonstrate, with identical mutagenic treatment, that by changing post-mutagen conditions (wetting and drying of seeds, storage after mutagenic treatment) it was possible to alter the mutation frequency and to obtain below-additive, additive and synergistic mutational response.