C-mitotic Effect Research Articles

C-mitotic effect of aqueous extracts of Isodon coetsa (Buch.-Ham. ex D. Don) Kudo (Lamiaceae)

Isodon coetsa (Buch.-Ham. ex D. Don) Kudo (Lamiaceae) belongs to a genus typified for the presence of bioactive ent-kaurane diterpenoids and have been used in traditional Chinese folk medicine as antibacterial, antiinflammatory and antitumour agents for a long time. The aqueous leaf extracts were tested for cytotoxicity screening using Allium cepa assay due to its biological activity. Root tips were treated with different concentrations of the extract viz., 0.005 %, 0.01 %, 0.05 %, 0.1 % for varying time durations (½ h, 1 h, 2 h, 3 h). Increase in mitotic index was observed in all treatments. High frequency of aberrations viz., C-metaphase, polyploidy, cytostasis and vagrant chromosomes were observed in treated root tips, which revealed the C-mitotic effect in the extract. The plant extracts show severe as well as partial C-mitotic activity which was time and concentration independent. C-mitotic activity was found to be prominent during 1 h treatment at all concentrations which may be due to the cytostatic effect. Higher duration treatments at all concentration revealed a decrease probably due to the cytotoxic effect during prolonged exposure. Induction of polyploidy was observed at all treatments, which do not show any correlation either with concentration or time, probably due to the metabolic imbalance of the treated cells. Thus, it is concluded that Isodon coetsa leaf extracts possess C-mitotic activity which can be the effect of the presence of a colchicine like compound in it.

Risk evaluation of industrial wastewater on plants using onion(Allium cepa l) chromosome aberration assay

Rapid strides in industrialization coupled with incomplete sewage treatment system have led to indiscriminate discharge of potentially mutagenic wastewater into agricultural lands and water bodies. The response of Onion plant genetic material to the presence of potential cytotoxic and genotoxic substances in the environment was used to evaluate the toxicity of chemical effluent collected from a chemical industry in Enugu, Nigeria. The Allium test procedure was used. The concentrations of heavy metals (Cu, Co, Cd, Pb) were also determined. The data obtained showed that the mitotic index (MI) decreased as effluent concentration and treatment durations increased. Significant differences in MI at different concentrations and treatment durations were observed. Conversely, percentage of abnormal dividing cells significantly (P<0.05) increased as the concentration and treatment durations increased. Diverse chromosomal aberrations and cytotoxic problems were observed. These ranged from a mild C-mitotic effect at lower concentrations to vacuolated nucleues and even cytokinetic failure at higher concentrations. The adverse effects of this untreated wastewater on the ecosystem of which one of the most vulnerable are plants are discussed.

Influence of the Herbicide Stomp 330 on Morphogenetic Response of Triticale Callus Cultures. Cytological Evidences for Its Mutagenic Action.

Morphogenetic response of triticale callus cultures was studied after supplement of the herbicide Stomp 330 into the regeneration nutrient medium. It is found, that the regenerative ability is not affected at the lowest herbicide concentration. However, the higher doses reduce regenerative ability at different rates depending on the genotype. Rhizogenesis is completely suppressed. The necrotic calli increased and the fresh, unorganized growing calli reduced. Numerous abnormalities are observed in the treated tissues. Considerable part of the cells, especially those, situated in intensively proliferating zones are enlarged and vacuolized. Abnormalities as chromatin pycnosis and micronuclei are observed. The frequency of these abnormalities is proportional to the herbicide concentration. It is established, that Stomp 330 exerts strong C-mitotic effect in the callus cells, which is demonstrated by high frequency of polyploid cells.

Chromosome aberration assays in Pisum for the study of environmental mutagens.

From a literature survey, 117 chemicals are tabulated that have been assayed in 179 assays for their clastogenic effects in Pisum. Of the 117 chemicals that have been assayed, 65 are reported at giving a positive reaction (i.e. causing chromosome aberrations), 30 positive with a dose response, five borderline positive. Seventeen chemicals gave a negative response. Eighty-one percent of the chemicals gave a definite positive response. A c-mitotic effect was detected from treatment with 17 chemicals. In addition to the above tabulation of chemicals, 39 chemicals have been reported with an antimitotic effect. Thirteen assays have been recorded for five types of radiation, which with the exception of ultrasound reacted positively. The results of assays with 38 chemicals and/or radiations in combined treatments, as well as 15 chemicals and three types of radiations that induce somatic mutations are tabulated. The Pisum sativum (2n=14) bioassay has been shown to be a very good plant bioassay for assessing chromosome damage both in mitosis and meiosis for somatic mutations induced by chemicals, radiations, and environmental pollutants. For some chemicals, the Pisum assay is not as sensitive in assessing clastogenicity as the Allium assay, although this should be considered in relative terms. Pisum fulvum (2n=14) has been used in clastogenic studies also, but to a much lesser extent.

Colcemid and econazole do not show aneugenicity in male mouse germ cells.

Colcemid (COM) and econazole (EZ) were tested for induction of mitotic arrest and C-mitotic effects in mouse bone marrow cells and for meiotic delay and induction of hyperploidy in mouse spermatocytes after single injection. Doses of 1 and 3 mg/kg COM were used and bone marrow and spermatocytes were sampled at 2, 6, 10, 14 and 18 h. For EZ a dose of 120 mg/kg and intervals of 6, 10, 14 and 18 h were chosen. At 2 h after COM treatment of bone marrow cells the mitotic index and C-mitotic effect were highest, then both decreased from 6 to 18 h. At 18 h the mitotic indices decreased to or significantly below the control level at doses of 1 and 3 mg/kg respectively, whereas the corresponding frequencies of C-mitotic cells were still significantly higher than in the controls. EZ also induced mitotic arrest and C-mitotic effects in mouse bone marrow cells. In contrast to COM, the effects of EZ were highest at 18 h after treatment. COM and EZ caused disturbances in progression from the first to second meiotic division, however, after COM treatment the ratios of MMII to MMI were significantly below the control. In contrast, after EZ treatment the ratios were significantly higher than in the controls. Such differences may result from the different mechanisms by which COM and EZ act on cell cycle progression. COM and EZ did not induce non-disjunction under the experimental conditions. However, EZ induced structural chromosomal aberrations.

Genotoxic Effects of Marshal in Allium cepa L.

The aim of this study is to investigate the c-mitotic effect of Marshal in the root tip cells of Allium cepa L. and whether it can induce the formation of micronuclei and other mitotic abnormalities. The roots of A. cepa were treated with 5x10-6, 10-5, 5x10-5 and 10-4 (v/v) final concentrations of Marshal for 2, 4 and 6 hours. Marshal significantly decreased the mitotic index (MI) at all treatment times and concentrations. However, the ratio of c-mitosis was significantly increased especially in the groups treated with Marshal for 2 hours. The maximum increase was shown in the root tip cells of A. cepa treated with 5x10-5 (v/v) final concentrations of Marshal for 2 hours. The formation of micronuclei and the ratio of the other mitotic abnormalities were also increased.

Effects of lead on root growth, cell division, and nucleolus of Allium cepa

The effects of different concentrations of lead nitrate on root growth, cell division, chromosome morphology and nucleolus in root tip cells of Allium cepa were studied. The concentrations of lead nitrate (Pb(NO 3) 2) were 10 −7, 10 −6, 10 −5, 10 −4, 10 −3 and 10 −2 m. It was observed that lead reduces root growth and causes mitotic irregularities, including c-mitosis, anaphase bridges, and chromosome stickiness. Also, interphase cells with micronuclei, irregularly shaped nuclei and nuclei with decomposed nuclear material were noticed. The c-mitotic effect reached its maximum in the meristem at above 10 −4 m Pb, when practically almost all the anomalous dividing cells are of this type. After treatment with Pb, there were many similar silver-stained particulate materials scattered in the nucleus in short, rounded meristem cells and in long, oblong root cap cells.

Perturbation of cell division by acrylamide in vitro and in vivo

Exposure of V79 Chinese hamster cells to acrylamide (AA) caused a concentration-dependent increase in the incidence of spindle disturbances. A c-mitotic effect with the appearance of C-metaphases, a mitotic block and the concomitant disappearance of ana-telophase figures, was observed after 6 h of treatment with concentrations ranging from 0.01 to 1.0 mg/ml of AA. Intraperitoneal injection of male mice with the highest tolerated dose of 120 mg/kg of AA showed no mitotic arrest in bone marrow cells. However, 1 h and 3 h after treatment the frequencies of cells with highly condensed and separated chromatids was reduced indicating an effect on mitotic progression. In spermatocytes of mice AA caused a meiotic delay from 2 h to 22 h after treatment determined by a reduced ratio of second/first meiotic divisions. The meiotic delay was predominantly due to a prolongation of interkinesis. The present results show that AA causes disturbances of cell division in vitro and in vivo. They suggest that AA might induce aneuploidy in mammalian cells in vitro by interfering with proper functioning of the spindle similar to the effect of colchicine. In vivo, particularly in spermatocytes, the progression of cell division was altered by AA. It cannot be explained simply by an effect on spindle function however, this alteration may also cause errors in chromosome segregation.

Effects of TPA on mitosis of V79 Chinese hamster cells. Reduced precision of chromosome distribution and modification of entrance into and exit out of mitosis.

Protein kinase C (PKC) was activated in V79 Chinese hamster cells with 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Within 30 min soluble activity decreased concomitant with a 10-fold increase of particulate activity. The latter was still elevated after 3 h but was back to control levels after 24 h of treatment; by then soluble activity was lost. The frequency of mitotic cells with signs of abnormal spindle function increased within 15 min and reached a plateau after 45-60 min which lasted throughout the 4 h treatment. The c-mitotic effect was delayed and significantly lower when 10 nM TPA was combined with 50 microM of the protein kinase inhibitor H7. The frequency of disturbed mitotic cells decreased after 24 h of treatment but remained significantly higher than in non-treated cells. Change of medium and addition of new TPA caused a slight but significant further increase. It is suggested that PKC takes part in eliciting the c-mitotic effect of TPA. However, the sustained effect coincident with down-regulation points to significant alterations of the level or the activity of an as yet unidentified ultimate elicitor. TPA also caused a transient block in the G2 phase which was ameliorated by H7 and which could not be detected at all in TPA-pretreated cells (24 h) given new TPA. This suggests that PKC takes part in eliciting the G2/M block as well but the mechanism is different from the one(s) behind the c-mitotic effect. V79 cells were found to exit from mitosis in the presence of 0.2 microgram/ml of colcemid but TPA-pretreated cells showed a decreased exit rate. There was no sign of hampered exit among cells going into mitosis soon after the G2 block was reversed, which implies that the spontaneous reversal of the block does not involve rapid down-regulation of PKC.

Bombesin impairs spindle function in mitotic V79 Chinese hamster cells by a receptor-dependent mechanism

Bombesin belongs to a family of peptides acting as local hormones with roles in growth regulation, neural function and secretion. Upon binding to its receptor bombesin primarily elicits an increase of inositolphosphates and diacylglycerol, events leading to increased [Ca 2+] i and activation of protein kinase C. When asynchronously growing V79 Chinese hamster cells were treated with bombesin in the 10 −9−10 −7 M concentration range their content of inositolphosphates increased and so did the frequency of mitotic cells with abnormal chromosomal arrangements (c-mitoses). Both effects were abolished by simultaneous addition of the synthetic peptide antagonist D-Arg 1,D-Phe 5,D-Trpu 9-Leu 11-substance P that binds to certain bombesin receptors. These results demonstrate that the V79 cells most probably have receptors for bombesin and that the weak but significant c-mitotic effect is mediated by such receptors.

Mitotic spindle damage induced by 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes

A 3-h exposure of V79 Chinese hamster cells with the sensory irritant 2-chlorobenzylidene malonitrile (CS) caused apolar mitoses in a dose-dependent manner. With a preparation and staining technique that allows for the visualization of the spindle apparatus and the chromosomes it was found that unlike in Colcemid-induced c-metaphases residual spindle fibers or microtubule material were present in the majority of CS-induced c-metaphases. The observation suggests different mechanisms for the induction of the c-mitotic effect by the two spindle poisons.

2-Chlorobenzylidene malonitrile (CS) causes spindle disturbances in V79 Chinese hamster cells

Exposure of V79 Chinese hamster cells to 2-chlorobenzylidene malonitrile (CS), a chemical used as a sensory irritant for riot control, caused a concentration-dependent increase in the incidence of spindle disturbances. A C-mitotic effect with the appearance of C-metaphases, a metaphase block and the concomitant disappearance of ana-telophase figures were observed after a 3-h treatment. The results indicate that CS might induce aneuploidy in mammalian cells by interacting with the mitotic apparatus.

Spindle disturbances in mammalian cells IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism

The glutathione-specific agents diamide, diethyl maleate and 1-chloro-2, 4-dinitrobenzene were found to induce a low frequency of c-mitosis (15%) at non-toxic concentrations concomitant with a 30–40% decrease of non-protein sulfhydryls. The frequency of c-mitosis did not increase further with increased concentrations until non-protein sulfhydryl levels were obtained suggesting depletion of reduced glutathione. The observed shape of the concentration-response curve for c-mitosis is particular to these 3 agents and caffeine among 22 different compounds being tested under comparable conditions. This suggests a similar mechanism of action and from what is known about caffeine this mechanism probably involves an impaired control of cytoplasmic free Ca 2+. It is speculated that this impairment with the glutathione-specific agents is primarily due to depletion of a particular pool of reduced glutathione. Tertiary butylhydroperoxide which is a substrate for glutathione peroxidase(s) also causes c-mitosis when there is no significant decrease of non-protein sulfhydryls. The c-mitotic response was found to be biphasic with maintained control levels at an intermediate concentration. The humps in the concentration-response curve for c-mitosis appeared coincident with a mitogenic response (G 1 → S). Since the latter type of effect most probably is Ca 2+ dependent and since the spindle is sensitive to Ca 2+ it is tentatively suggested that the c-mitotic effect of tertiary butylhydroperoxide is due to an increase of cytoplasmic Ca 2+. Measurements performed imply that an increase of glutathione disulfide (diamide) is more inhibitory to uptake and incorporation of thymidine than a decrease of reduced glutathione per se (diethyl maleate). This difference is probably due to secondary effects on pertinent protein sulfhydryls with diamide, one possible target being the ribonucleotide reductase.

Induction of somatic mutations in species of Allium. I. Effect of dES on the somatic chromosomes of Allium cepa.

The effect of diethyl sulphate (dES), a known mutagen, on the chromosomes of Allium cepa has been studied. Three different concentrations of this alkylating agent were tried. While 30 mM concentration has been shown to induce various chromosomal anomalies, 20 mM concentration does not interfere much with the mitotic cycle. 25 mM concentration of this mutagen has c-mitotic effect on the chromosomes of Allium cepa. However, all the three concentrations lower the mitotic index.

Some aspects on the organization of microfilaments and microtubules in relation to nondisjunction.

One possible mechanism behind nondisjunction is a malfunctioning spindle. A defect or lack of spindle is a criterion for c-mitosis. In chemical mutagenesis research the c-mitotic effect is a well known cytological phenomemon, which can be induced by many different compounds. Pioneer work was performed during the 1940's by Ostergren and Levan, and their results and conclusions are briefly discussed. Since colchicine can induce c-mitosis and c-meiosis, by definition, and nondisjunction, a correlation between these phenomena is logical. The general importance of the spindle protein tubulin is considered and some new data from the cell biology literature on spindle formation and function as well as chromosome structure are briefly summarized. This knowledge can be used to correlate cytological and biochemical parameters among which c-mitosis and changes in sulfhydryl group metabolism after chemical treatment are the most obvious ones.

Cytological effects of paper mills effluents on somatic cells of Allium cepa.

The cytological effects of paper mills effluents on somatic cells of Allium cepa were presented. The cytological effect decreased by decreasing the concentration of the effluents. Disturbed metaphase, anaphase and telophase, occurrence of binucleate and tetranucleate cells comprised the most dominant types of abnormalities. Clumping of chromosomes, contracted chromosomes, pleiomorphic and micronuclei formation, cell vacuolation and polyploid cells are found less frequently. The c-mitotic effect and other abnormalities may be due to the action of alkalies, acids and bleaching agents present in the paper mill effluents.

Chromosome aberrations in plants as a monitoring system.

The potential of higher plants as a first-tier assay system for detecting chemical mutagens is evaluated. The use of plant tissue (primarily root tips and pollen mother cells) for studying the induction of chromosomal aberrations is one of the oldest, simplest, most reliable, and inexpensive methods available. Specific types of abnormalities have been induced by different classes of pesticides. Chromosome clumping, contraction, stickiness, paling, fragmentation, dissolution, chromosome and chromatid bridges, C-mitosis, and endoploidy have been reported in the literature. Examples of cytogenetic studies with pesticides demonstrating the usefulness of higher plants as a monitoring system are reviewed. Pesticides which cause chromosome aberrations in plant cells also produce chromosome aberrations in cultured animal cells. Frequently, the aberrations are identical. For example, studies have shown that compounds which have a C-mitotic effect on plant cells have the same effect on animal cells. It is recommended that plant systems be accepted as a first-tier assay system for the detection of possible genetic damage by environmental chemicals.

Cytogenetic effects of styrene and styrene oxide

Styrene and styrene oxide induce various cytogenetic effects, similar in both human lymphocytes in vitro and onion root-tip cells in vivo. Styrene appears to cause chromosome breakage in both systems, and in Allium it shows a strong c-mitotic effect. Styrene oxide, on the other hand, seems to destroy the tertiary folding of the chromatin. Cytotoxicity of styrene oxide is very high (complete mitotic inhibition occurs on 0.03% v/v) in human lymphocytes, whereas, in Allium, styrene is slightly more toxic than styrene oxide. Styrene glycol, a further metabolite of styrene oxide, does not cause mitotic inhibition.

Cytogenetic effects of styrene and styrene oxide on human lymphocytes and Allium cepa.

Styrene and styrene oxide induce cytogenetic effects already at very low concentrations (0.01% v/v or even less); the effects are similar in both in vitro human lymphocytes and in vivo onion root tip cells (Allium cepa L.). It is characteristic that styrene treatment is more potent in causing chromosome breakage in both systems. In Allium styrene induced inhibition of mitotic spindle action as revealed by a strong c-mitotic effect. Also the number of micronuclei and nuclear bridges increased in both test systems, especially after styrene oxide treatment. Furthermore, the metaphase chromosome morphology in the cells treated with styrene oxide was strongly affected. In both systems, chromosome destruction was observed, or else the chromosome material was decondensed and resulted in a characteristic fuzzy appearance of Allium chromosomes or a banded appearance of human lymphocyte chromosomes. A specific effect of styrene oxide on the chromosomal proteins is thus suggested. The data obtained from the autoradiographic studies with Allium support the idea that [7--3H] styrene oxide binds irreversibly to the cytoplasmic and nuclear macromolecules.

Maytansine-Induced Mitotic Arrest in Human Lymphoblasts

Maytansine, a novel ansa macrolide (1), has potent anti-tumor and antimitotic activity (2, 3). It blocks cell cycle traverse in mitosis with resultant accumulation of metaphase cells (4). Inhibition of brain tubulin polymerization in vitro by maytansine has also been reported (3). The C-mitotic effect of this drug is similar to that of the well known Vinca- alkaloids, vinblastine and vincristine. This study was carried out to examine the effects of maytansine on the cell cycle traverse and the fine struc- I ture of human lymphoblasts.Log-phase cultures of CCRF-CEM human lymphoblasts were exposed to maytansine concentrations from 10-6 M to 10-10 M for 18 hrs. Aliquots of cells were removed for cell cycle analysis by flow microfluorometry (FMF) (5) and also processed for transmission electron microscopy (TEM). FMF analysis of cells treated with 10-8 M maytansine showed a reduction in the number of G1 cells and a corresponding build-up of cells with G2/M DNA content.