The effects of silver ions and silver nanoparticles on cell division and expression of <Emphasis Type="Italic">cdc2</Emphasis> gene in <Emphasis Type="Italic">Allium cepa</Emphasis> root tips

The effects of silver nanoparticles (AgNPs), silver ions (Ag⁺), and polyvinylpyrrolidone (PVP) on mitosis and expression of a gene encoding cyclin-dependent kinase 2 (cdc2) in onion roots were compared. Three concentrations (5, 10, and 15 mg dm^-3) were employed in combination with three incubation times (3, 6, and 9 h). PVP enhanced mitotic index and cdc2 expression. Both silver forms decreased mitotic index and cdc2 expression. Genotoxicity of both silver forms were indicated by three major distinguishable classes of chromosome aberrations: spindle disturbances, clastogenic aberrations, and chromosome stickiness. Concerning Ag⁺ treatments, significant enhancements in occurrence of any chromosome aberration type was associated with significant decrease in mitotic index. On the other hand, disturbed spindle in AgNPs treatments was observed even in absence of significant reduction in mitotic index suggesting that AgNPs inhibit cellular events occurring during mitosis to proceed normally rather than starting of cell division.     

Cytogenetic effect of plant tissue culture medium with certain growth substances onAllium sativum L. meristem root tip cells

The effect of plant tissue culture medium with different concentrations and combinations of growth regulators (kinetin, indol-3-ylacetic acid, 2,4-dichlorophenoxyacetic acid) was evaluated on mitosis ofAllium sativum meristem root tip cells. Different combinations of growth regulators at low concentrations had no effect on induction of mitotic aberrations or inhibition of mitotic activity. Inhibition of mitotic activity, a tendency to chromosome stickiness and clumping and a slight increase in the frequency of mitotic aberrations were observed at higher concentrations. It may be proposed that plant tissue culture media have no direct effect on induction of mitotic aberrations in plant tissue culturesin vitro.     

Effects of Ultraviolet and X-Ray Radiation on in vitro Cultivated Cells of Haplopappus gracilis

A cell strain of Haplopappus gracills was used for investigations of the effects of UV (2537 A) and X-ray irradiation. Mitotic inhibition and killing after UV exposure were studied. A survival curve of UV treated and then plated cells is presented. The LD₅₀ seems to be about 2000 erg. mm^?2 under the experimental conditions used. All types of chromosome aberrations are induced by UV irradiation, but the frequency is relatively low at doses which do not completely inhibit cell division. A mutant strain of chromosome type is isolated after UV treatment and then plating. Mitotic inhibition and killing after X-ray treatment were studied. A survival curve is presented and the LD₅₀ under the culture conditions used seems to be about 2000 R. The frequency of chromosome aberrations induced by X-rays is highly increased by aeration during X-ray treatment which indicates that some degree of cell anoxia exists in a cell suspension. There arr indications that chromosome aherrations may not cause growth inhibition to such an extent as is usually believed.     

The effect of 5-azacytidine on the growth and thymidine kinase activity of coleoptile sections ofTriticum

The growth of coleoptile sections ofTriticum and its stimulation by indole-3-acetic acid (IAA) are inhibited by 5-azacytidine added into the cultivation medium. 50 per cent depression of the elongation was observed at 2×10^?3M 5-azacytidine concentration. Thymidine kinase activity in cell-free extracts prepared from coleoptile sections treated with 5-azacytidine, and caleulated per 10 mg of their wet weight, is increased while IAA administration resulted in its depression. The observed changes in thymidine kinase activity can be explained assuming the different uptake of water due to 5-azacytidine and IAA treatment.     

The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people

Peripheral blood lymphocytes from unirradiated control subjects and workers exposed within permitted limits to γ-radiation, have been examined for the incidence of dicentric and acentric chromosome aberrations. The results are compared with a review of data published elsewhere. Background levels show inter-laboratory variation and possible reasons for this are discussed. By combining the present data with those from the literature the spontaneous incidence of dicentric aberrations is approx. 0.55 × 10^?3 and for acentrics is 3.7 × 10^?3. In occupationally exposed subjects a significantly higher incidence of aberrations was found. When allowance was made for the turnover of lymphocytes for the period over which each man had worked with radiation a linear dose-effect relationship was apparent. The incidence of dicentrics was 2.22 ± 0.94 × 10^?4 rad^?1 and for all unstable aberrations 8.24 ± 2.8 × 10^?4 rad^?1. These are in reasonable agreement with dose-response data obtained in vitro.     

A direct demonstration of somatically paired heterochromatin of human chromosomes

Human lymphocyte cultures were treated with different concentrations of 5-azacytidine for various lengths of time. This cytosine analog induces very distinct undercondensation in the heterochromatin of chromosomes 1, 9, 15, 16, and Y if applied in low doses during the last hours of culture. These regions are further distinguished by their intense distamycin A/DAPI-staining and highly methylated DNA. In interphase nuclei, these heterochromatic regions are frequently somatically paired. These somatic pairings are preserved up to the metaphase stage in the 5-azacytidine-treated cultures and are thus susceptible to direct analysis. The specific effect of 5-azacytidine on the heterochromatin of these chromosomes, its conserving effect on somatic pairing, and some of the consequences of the somatic pairing on the development of human chromosome aberrations are discussed.     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

Evaluation of chemically induced cytogenetic lesions in rabbit oocytes: II. A comparison of streptonigrin effects on somatic and germ cells

The dose-response relationships for streptonigrin (NSC-45383)-induced chromosome aberrations in rabbit somatic cells are compared with dose-response data derived from the analysis of inherited structural chromosome abnormalities in preimplantation embryos from female rabbits treated with streptonigrin prior to mating. The incidence of inherited aberrations assessed in over 1000 karyotype preparations from 361 6-day blastocysts obtained from 55 female rabbits is used to derive a measure of the transmissible cytogenetic damage induced in the oocytes. The cytogenetic damage assessed in 2300 lymphoblast metaphases from 23 rabbits and 2750 marrow-derived metaphases from 27 rabbits which were collected and prepared for examination 6 h after the initiation of streptonigrin dosing are used to obtain estimates of the somatic cell insult. A uniform maximum likelihood analysis technique is applied individually to the 3 sets of data to derive the coefficients of the dose-response relationships. The resulting equations are Y = 0.6 ± 28.0 (×10^?5) + 8.2 ± 5.1 (×10^?4χ for inherited aberrations in 6-day blastocysts, Y = 9.7 ± 3.3 (×10^?3 + 1.9 ± (×10^?3)χ for bone-marrow cells, and Y = 2.8 ± 0.7 (×10^?2 + 4.8 ± 0.2 (×10^?3)χ for the lymphoblasts. In the somatic tissues Y is the percentage of cells with chromosome breakage, while in the blastocyst data Y is the percentage of 6-day blastocysts with consistent structural chromosome aberrations, and in all equations χ is the total streptonigrin dose in μg/kg.The study shows that streptonigrin injections in the range of 30–90 μg/kg when given to sexually mature female rabbits cause dose-dependent increases in chromosome aberrations in 2 types of somatic cells and in the incidence of inherited aberrations recovered in 6-day blastocysts. The coefficients of damage recovered in blastocysts versus damage recovered in somatic cells have the ratio of 1:2.3:5.8 (blastocysts: bone marrow: lymphoblasts). The results are discussed in terms of risk assessment and kinetics of aberration loss during meiosis and early embryonic development. The conclusion drawn from the study is that somatic cell cytogenetic damage is in some way predictive of damage incurred by oocytes which can be passed on to preimplantation embryos, at least for agents like streptonigrin.     

Mitotic chromosomal abnormalities and DNA polymorphism in the Nile tilapia (Oreochromis niloticus,Linnaeus, 1758) as a biomarker for water pollution by heavy metals

Mitotic chromosomal aberrations and DNA polymorphism (RAPD marker) were carried out on the Nile tilapia Oreochromis niloticus collected from five sites in Minia governorate, Egypt to test their applicability as biomonitors for heavy metal contaminants of water. The diploid chromosome number of O. niloticus population was 2 n = 44. Different types of chromosomal aberrations were recorded (e.g., deletion, ring, centromeric attenuation, end-to-end association, dicentric chromosome, stickiness chromosomes, endomitosis, fragments and chromatid gap). The chromosomal aberrations varied between O. niloticus population collected from five sites, and the most common type was ring (R) chromosomes. Samples obtained from Bahr Yousef and Irrigation drain exhibited the highest aberration frequency. The frequency of chromosomal aberration was positively correlated with the concentration of heavy metals where their concentration in the surface water of Irrigation drain and Bahr Yousef exceeded the limits defined by WHO as well as the concentration of Pb in muscles. The RAPD marker was also used to identify genetic variation among Nile tilapia samples collected from five different water sources. It created polymorphic and unique bands that can be used as genetic markers to track DNA variations. The dendrogram also revealed that exposure to heavy metal pollution causes gradual accumulation of variance, whereas areas subjected to environmental stress showed higher genetic variation and clustered together.     

6-Methyl-5-Azacytidine-Synthesis,Conformational Properties and Biological Activity. A Comparison of Molecular Conformation with 5-Azacytidine

Abstract

The title compound was prepared by the isocyanate procedure and the tri-methylsilyl method. The measurement of ¹H NMR spectrum of 6-methyl-5-azacytidine (1) revealed a preference of γ^t (46%) rotamer around C(5′)-C(4′) bond, a predominance of N conformation of the ribose ring (K_eq 0.33) and a preference of syn conformation around the C-N glycosyl bond. An analogous measurement of 5-azacytidine has shown a preference of γ⁺ (60%) rotamer around the C(5′)-C(4′) bond, a predominance of N conformation of the ribose ring (K_eq 0.41) and a preference of anti conformation around the C-N glycosyl bond. 6-Methyl-5-azacytidine (1) inhibits the growth of bacteria E. coli to the extent of 85% at 4000 μM concentration and the growth of LoVo/L, a human colon carcinoma cell line, to the extent of 30% at 100 μM concentration but did not inhibit L1210 cells at ≤ 100 μM concentration. 6-Methyl-5-azacytidine (1) exhibited no in vitro antiviral activity at ≤ 1 μM concentration.

     

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.     

Aluminum Induces Chromosome Aberrations in Cells of Wheat Root Meristem

The yield and pattern of chromosome structure aberrations in wheat seedlings treated with aluminum nitrate and aluminum sulfate at various concentrations have been determined by the anaphase method. Aluminum has a genotoxic effect causing genome, chromatid, and chromosome aberrations in apical root meristem cells. The relationship between the total yield of structural mutations and the aluminum concentration follows a bell-shaped curve. The mutagenic activity of aluminum nitrate peaks at 10^–3mg/ml, which is twice as high as the permissible concentration limit (PCL) of aluminum in potable water. The maximum of the mutagenic activity of aluminum sulfate is observed at 5 × 10^–4mg/ml, i.e., one PCL. Tap water boiled for 2 h in an aluminum vessel has virtually no genotoxic effect on wheat cells.     

The type and time of occurrence of aminopterin-induced chromosome aberrations in cultured Potorous cells

Many inhibitors of DNA synthesis have been found to induce chromosome aberrations. Our kinetic studies indicate that treatment of cellswith 10^?7M aminopterin in the presence of 10^?4M glycine, 10^?4M hypoxanthine, and 10^?4M thymidine allows continued normal cell growth. Omission of thymidine, a treatment which is known to inhibit DNA synthesis while allowing RNA and protein synthesis to continue, leads to cessation of cell growth. Treament of Potorous cell cultures with aminopterin in the presence of hypoxanthine and glycine without thymidine led to the following observations: (1) only non-exchange chromatid aberrations were formed after aminopterin treatment; (2) the aberrations were induced only in cells treated during S, and the breaks were associated with the replicating region of the chromosome; (3) breaks were observed at the first metaphase after the beginning of treatment; and (4) thymidine could reverse the chromosome-breaking action of aminopterin. A model for the molecular mechanism is suggested.     

Cytogenetics of in vitro cultured somatic cells and regenerated plants of barley (Hordeum vulgare L.)

Summary Cytogenetic analysis of immature embryoderived calli and regenerated plants of barley has demonstrated high heterogeneity of callus cultures and significant differences in cytogenetic processes between different callus lines. Regenerated plants usually have a normal chromosome complement (2n=14). Tetraploid plaints occur with a frequency of 1%. No chromosome aberrations have been detected by Feulgen staining. The phenomenon of chromosome stickiness recorded from the 2nd day of culture was discovered in a majority of callus lines as well as the phenomena of chromatin hypercondensation and chromosome supercoiling. A possible contribution of cytogenetic and molecular processes to somaclonal variation is discussed.     

Effects of Copper on Root Growth, Cell Division, and Nucleolus of Zea Mays

The effects of different concentrations (10^–5 – 10^–2 M) of copper sulfate on root growth, cell division and nucleoli in root tip cells of Zea mays L. were investigated. 10^–5 M Cu stimulated root growth, but at higher concentrations (10^–4 – 10^–2 M) inhibited it. Cu had toxic effects on chromosomal morphology: c-mitosis, anaphase bridges, and chromosome stickiness were induced. Some nuclei had irregular shape and particles extruded from nucleoli to nuclei and finally from the nuclei into the cytoplasm.     

A comparison of the 8-hydroxydeoxyguanosine,chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes

We compared the mechanism of action of micronuclei (MN), unstable chromosome aberrations, and 8-hydroxydeoxyguanosine (8-OHdG) levels to evaluate the genotoxicity of methyl mercuric chloride (CH₃HgCl) and mercuric chloride (HgCl₂) in human peripheral lymphocytes. The chromosome aberrations in human peripheral lymphocytes exposed to various concentrations of CH₃HgCl or HgCl₂ increased in a concentration-dependent manner and were significantly higher than the control when the cells were incubated with 1 × 10⁻⁵ M (HgCl₂) or 2 × 10⁻⁶ M (CH₃HgCl). The increase in the incidence of micronucleated lymphocytes was significant among the exposed groups, being 2 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. CH₃HgCl was about 4-fold more potent than HgCl₂. We determined the 8-OHdG levels in human peripheral blood mononuclear cells(PBMC) and found that they were significantly higher in the exposed groups at 1 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. A detectable (p < 0.05) increase in the level of 8-OHdG was induced by CH₃HgCl at a concentration that was about 50% of the amount of HgCl₂ required to produce a similar response. The data confirmed the value of the MN and/or chromosome aberration assays for assessing of HgCl₂- and/or CH₃HgCl-induced genotoxicity, and indicated that they are about the same concentration as the 8-OHdG assay. The presence of genotoxic effects in peripheral blood lymphocytes exposed to the mercuric compounds indicated by the chromosome aberrations and the MN assays could be partly due either to the disturbance of the spindle mechanism, or to the elevated level of 8-OHdG brought by the generation of reactive oxygen species.     

The stage of chromosome duplication in the cell cycle as revealed by X-ray breakage and 3H-thymidine labeling

By means of combined experiments of X-irradiation and ³H-thymidine labeling of the chromosomes which are in the phase of synthesis, and the subsequent analysis at metaphase on the autoradiographs of the chromosomal damage induced during interphase, it was shown that in somatic cells from a quasi-diploid Chinese hamster line cultured in vitro the chromosomes change their response to radiation from single (chromosome type aberrations) to double (chromatid type aberrations) in late G₁. These results are interpreted to indicate that the chromosome splits into two chromatids in G₁, before DNA replication. — By extending the observations at the second metaphase after irradiation, it was also seen that cells irradiated while in G₂ or late S when they reach the second post-irradiation mitosis still exhibit, beside chromosome type aberrations, many chromatid exchanges, some of which are labeled. Two hypotheses are suggested to account for this unexpected reappearance of chromatid aberrations at the second post-irradiation division. The first hypothesis is that they arise from half-chromatid aberrations. The second hypothesis, which derives from a new interpretation of the mechanisms of production of chromosome aberrations recently forwarded by Evans, is that they arise from gaps or achromatic lesions which undergo, as the cells go through the next cycle, a two-step repair process culminating in the production of aberrations.This work was supported in part by grant No. RH-00304 from the Division of Radiological Health, Bureau of State Services, Public Health Service, U.S.A.     

Effects of alkylating agents on lymphocytes from controls and from patients with Fanconi's anemia

Summary The frequency of sister chromatid exchanges (SCE) and chromosome aberrations and the dynamics of cell division in peripheral blood lymphocytes of four patients with Fanconi's anemia were studied after in vitro exposure to alkylating agents TEPA and mitomycin.SCE frequency was significantly increased even after very low doses of mutagens, while chromosome aberrations were significantly increased only after high doses (0.160 g/ml mitomycin and 10^-5 M TEPA). The responses of Fanconi's anemia cells and control cells did not differ significantly. The increased frequency of both SCE and chromosome aberrations was accompanied by gradual delay of cell division, which was most conspicuous in cells from patients with Fanconi's anemia.     

Allium cepa as a biomonitor of ochratoxin A toxicity and genotoxicity

Ochratoxin A (OTA) is a toxin produced by Aspergillus and Penicillum moulds. Since OTA has not yet been evaluated in plant systems, this paper focused on describing the controversial effect OTA in an Allium root test model, which has known sensitivity to genotoxins and could be useful in toxin screening. Analyses of root growth and the root meristematic zone in response to OTA treatment were undertaken. The results show OTA toxicity to root growth at a concentration of 10 ug·ml^?1 associated with inhibition of proliferation activity. Cytological changes observed in the Allium chromosome aberrations assay, at a concentration of 5.0 ug·ml^?1, showed that OTA was able to induce genotoxicity at the chromosome level. These results indicate that plants cells (Allium cepa) are very sensitive to the mycotoxin OTA, as observed at the highest concentration. Under these conditions, OTA produced toxicity and cytogenetic injury. Evidence in vitro and in vivo indicates that OTA can induce damage at the DNA level.     

Induction of a high incidence of damage to the X chromosomes of Rattus (Mastomys) natalensis by base analogues,viruses, and carcinogens

Cultured embryonic cells from Rattus (Mastomys) natalensis were treated with the base analogues 5-bromodeoxyuridine and 2-aminopurine, the viruses herpes simplex virus and adenovirus type 12, and the carcinogens 7,12-dimethylbenz(a)anthracene and urethan. Treatment with these agents, except urethan, causes an increase in the incidence of chromosome aberrations. The induced aberrations are not randomly distributed among the chromosomes or within a chromosome. The X chromosome, especially its long arm, is more sensitive to these agents than is any other chromosomes in the complement. — A possible relationship among the high incidence of damage, the positive heteropycnosis, and the late replication in the X chromosome of R. natalensis is discussed.