DNA damage in bone marrow cells of mouse males in vivo after exposure to the pheromone: Comet assay

It is shown by single-cell gel electrophoresis that the exposure of CBA mouse males with pheromone 2,5-dimethylpyrazine for 4 or 24 h increases DNA damage level in interphase nuclei of bone marrow cells. The results may reflect the work of a mechanism of formation of chromosomal aberrations and other mitotic disturbances, detected at the stage of ana-telophase, the frequency of which in dividing bone marrow cells increased after similar pheromonal exposure. The comparison of the damaged cell distribution types is proposed as an approach to analysis of comet assay data. The significance of the revealed effects on the immune system in the recipient organism is discussed.     

Action of two pyrazine-containing chemosignals on cells of bone marrow and testes in male house mouse Mus musculus L

Evolutionary conservative chemosignal 2,5-dimethylpyrazin that is pheromone in female mice has been shown to increase frequency of mitotic aberrations analyzed with aid of metaphasic and ana-telophasic analysis in bone marrow cells. Replacement of one of methyl radicals in the pheromone molecule by the carboxyl radical reveals specificity of action of the used derivative: the frequency of disturbances revealed only by the ana-telophasic analysis increases, whereas by the metaphasic analysis, no induction of disturbance is detected. In the sperm head abnormality test there is shown a rise of the anomalies by both compounds. Possible mechanisms of specific action of the tested substances on stability of genetic apparatus of the bone marrow dividing cells in the house mouse are discussed.     

The role of metabolic activation of promutagens in the genome destabilization under pheromonal stress in the house mouse (<Emphasis Type="Italic">Mus musculus</Emphasis>)

The hypothesis on a relationship between the high frequency of mitotic disturbances in bone marrow cells and the change in the activity of the S9 liver fraction containing promutagen-activating enzymes under olfactory stress in the house mouse Mus musculus has been tested. For this purpose, the effect of the pheromone 2,5-dimethylpyrazine on the frequency of mitotic disturbances in mouse bone marrow cells has been measured by the anaphase-telophase assay. In paralled, we compared the capacities of the S9 liver fractions from stressed and intact mice for activating the promutagen 2-aminofluorene in the Ames test utilizing Salmonella typhimurium. It has been demonstrated that the increased frequency of mitotic disturbances in bone marrow cells induced by the pheromonal stressor in male house mice is accompanied by an increased ability of the S9 liver fraction to activate the promutagen. The model system used in the study allowed the genetic consequences of the exposure to the olfactory stressor to be estimated and the possible mechanisms of genome destabilization to be assumed.     

The role of metabolic activation of promutagens in the genome destabilization under pheromonal stress in the house mouse (Mus musculus)

The hypothesis on a relationship between the high frequency of mitotic disturbances in bone marrow cells and the change in the activity of the S9 liver fraction containing promutagen-activating enzymes under olfactory stress in the house mouse Mus musculus has been tested. For this purpose, the effect of the pheromone 2,5-dimethylpyrazine on the frequency of mitotic disturbances in mouse bone marrow cells has been measured by the anaphase-telophase assay. The Ames test using Salmonella typhimurium has been employed to compare the capacities of the S9 liver fractions from stressed and intact mice for activating the promutagen 2-aminofluorene. It has been demonstrated that the increased frequency of mitotic disturbances in bone marrow cells induced by the pheromonal stressor in male house mice is accompanied by an increased promutagen-activating capacity of the S9 liver fraction. The model system used in the study allowed the genetic consequences of the exposure to the olfactory stressor to be estimated and the possible mechanisms of genome destabilization to be assumed.     

The balance hypothesis of the effect of socially important volatile chemosignals on reactivity of chromosome machinery of bone marrow dividing cells in the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis>

Volatile chemosignals released by female CBA mice are shown to affect the chromosome machinery of bone marrow cells in mature syngenic males in different ways depending on the experimental conditions. Chemosignals excreted by solitary adult females decrease the frequency of mitotic disturbances in bone marrow dividing cells of male recipients as compared with the spontaneous level in control animals. At the same time, 2,5-dimethylpyrazine, a pheromone released only by females caged at high densities, increases the frequency of mitotic disturbances. A preliminary 24-h treatment of males with chemosignals excreted by solitary females reduces the effect of a subsequent exposure to 2,5-dimethylpyrazine, however, the frequency of disturbances is still higher than that in the control. The simultaneous exposure to both chemosignals results in complete neutralization of the 2,5-dimethylpyrazine effect, and the frequency of mitotic disturbances does not differ from that observed after the exposure to solitary female chemosignals. It is hypothesized that the cytogenetic effects found in male recipients depend on the social housing conditions under which female chemosignal donors were kept.     

Effect of the estrus cycle stage on sensitivity to pheromone 2,5-dimethylpyrazine in the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis>

Frequency of cytogenetic disturbances was estimated in mitotically dividing bone marrow cells of CBA strain female mice after 24-h long action of pheromone 2,5-dimethylpyrazine (2,5-DMP). The stage of estrus cycle of each animal was taken into acount at the moment of the end of the pheromone action. The analysis was performed using the anatelophase method that allows evaluating frequencies of various types of disturbances—bridges, fragments, delayed chromosomes. The spontaneous level of the mitotic disturbances revealed by the anatelophase method in animals of the control group amounts to 5.4%. Action of pheromone 2,5-dimethylpyrazine induced the mitosis disturbances detected in the dividing bone marrow cells at the anaphase-telophase stage in the females at the di-+ postestrus stage. The corresponding frequency of disturbances after the pheromone action was equal to 9.2%. In the female in estrus, the mitotic disturbance level amounted 6.7%, which not differed statistically significantly from control. It is suggested that differences in female mouse hormonal state at different estrus cycle stages affect sensitivity to olfactory signals. Mechanisms of the revealed effect and significance of the differences in sensitivity to pheromone for reproductive processes are discussed.     

Effect of the estrous cycle stage on sensitivity to pheromone 2,5-dimethylpyrazine in the house mouse Mus musculus

Frequency of cytogenetic disturbances was estimated in mitotically dividing bone marrow cells of CBA strain female mice after the 24-h long action of pheromone 2,5-dimethylpyrazine (2,5-DMP). The stage of the estrous cycle of each animal was taken into account at the moment of the end of the pheromone action. The analysis was performed using the anatelophase method that allows evaluating frequencies of various types of disturbances--bridges, fragments, delayed chromosomes. The spontaneous level of the mitotic disturbances revealed by the anatelophase method in animals of the control group amounts to 5.4 %. Action of pheromone 2,5-dimethylpyrasine induced the mitosis disturbances detected in the dividing bone marrow cells at the anaphase-telophase stage in the females at the di- + postestrus stage. The corresponding frequency of disturbances after the pheromone action was equal to 9.2%. In the female in estrus, the mitotic disturbance level amounted 6.7%, which did not differ statistically significantly from control. It is suggested that differences in the female mouse hormonal state at different estrous cycle stages affect sensitivity to olfactory signals. Mechanisms of the revealed effect and significance of the differences in sensitivity to pheromone for reproductive processes are discussed.     

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.     

Balance hypothesis of action of socially significant volatile chemosignals on reactivity of chromosome machinery of the bone marrow dividing cells in the house mouse Mus domesticus L

Volatile chemosignals released by female CBA laboratory mice have been shown to produce action of different direction, depending on conditions of performance of experiment, on chromosome machinery of bone marrow cells in syngenic adult males. Thus, chemosignals secreted into environment by isolated adult females decrease frequency of mitotic disturbances in bone marrow dividing cells in male recipients as compared with spontaneous level in control animals. At the same time, 2,5-dimethylpyrazine - pheronome released only by high density caged females - increases frequency of mitotic disturbances. Preliminary 24-h-long action of chemosignals of isolated females decreases effect of the subsequent action of 2,5-dimethylpyrazine, although the level of disturbances exceeds that in control animals. The simultaneous action of used chemosignals neutralizes completely the 2,5-dimethylpyrazine action, the frequency of mitotic disturbances being not different from that after chemosignals of isolated females. The hypothesis is put forward about dependence of the revealed cytogenetic effects in male recipients on zoosocial conditions of maintenance of female donors of chemocommunication signals.     

Analysis for Pheromone-Induced Cytogenetic Disturbances Depending on Major Urinary Proteins of Laboratory Mouse Males

Young male CBA/LacStoRap mice for 2 h were exposed to pheromones that are transferred by major urinary proteins of sexually mature males of the same strain. The treatment was conducted using either a complete set of the major urinary proteins typical of the CBA mice or incomplete set of protein fractions detected in some animals. The effect of pheromones was estimated 24 h after treatment by cytogenetic analysis for disturbances in dividing bone marrow cells at anaphase–telophase and in germ cells at metaphase I. The frequency of both mitotic and meiotic disturbances was significantly increased after exposure to pheromones associated with the complete spectrum of the major urinary proteins. Conversely, no cytogenetic effect was observed in the absence of particular protein fractions. Possible consequences of the pheromonal effect on the genomes of somatic and germ cells are discussed as well as the relationships between pheromones and the major urinary proteins.     

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.     

Antimutagenic effect of chemosignals from isolated female house mouse on male germ cells (Mus musculus L.)

The influence of chemosignals from isolated mature females of the CBA strain on level of spontaneous and radiation-induced meiotic disturbances in spermatocytes I of males of the same strain was studied. Using an ana-telophase method, 24-hour exposure of males to soiled bedding containing isolated females’ chemosignals was shown to lead to a significantly lower frequency of chromosomal aberrations and other meiotic disturbances in spermatocytes I as compared to males kept on clean bedding. The same effect of female chemosignals was found in the germ cells of irradiated males (4 Gr). The mechanisms and importance of the revealed antimutagenic effect of mouse female chemosignals on the male reproductive cells in the reproduction process are discussed.     

Metaphase arrest and delay in cell cycle kinetics of root apical meristems and mouse bone marrow cells treated with leaf aqueous extract of Clerodendrum viscosum Vent

Objectives

To study cell cycle delay and metaphase arresting activity of leaf aqueous extract of Clerodendrum viscosum Vent. (LAECV) in root apical meristems and mouse bone marrow cells.

Materials and methods

Cell cycle delay and metaphase arresting activities of LAECV were analysed, in root apical meristems of onion and wheat, and in mouse bone marrow cells, by scoring mitotic index, metaphase frequency and transition of cells from metaphase to anaphase. Colchicine was used as the standard metaphase arresting drug. Phytochemicals present in LAECV were detected and their phytotoxic activity was evaluated by analysing green‐gram (Vigna radiata) seedling's root growth retardation and branch root swelling phenomenon.

Results

LAECV treatment resulted in dose‐dependent root growth retardation of green‐gram seedling root length (P < 0.01) and half maximal growth inhibitory concentration (IC₅₀) of LAECV was 0.87 mg/ml at 144 h. In onion and wheat root meristem cells the mitotic index decreased, metaphase frequency increased and transition from metaphase to anaphase reduced. Experimentation with mouse bone marrow cells indicated that LAECV induced metaphase arrest (164.3% increase in arrested metaphases per 300 mg/kg body weight, over 2.5 h). Phytochemicals like carbohydrates, glycosides, saponins, terpenoids, triterpenoids, tannins and trace amounts of alkaloids were detected in LAECV.

Conclusion

It may be said that LAECV contains mitostatic and metaphase arresting components that are able to induce significant metaphase arrest in root apical meristems and also in mouse bone marrow cells.

     

Myelotoxicity of epirubicin]

Epirubicin (pharmorubicin, India), an antitumor antibiotic of the anthracycline group, was studied in regard to its effect on peripheral blood, bone marrow and lymphoid organs (the thymus and spleen) of CBA mice after its intraperitoneal administration in a single dose equal to the MIC (7.8 mg/kg) and in a course dose (1/5 of the MIC 5 times a day). The cytogenetic impairments induced by the cytostatic were estimated on metaphase plates with the bone marrow specimens and by counting the peripheral blood erythrocytes with micronuclei (the micronuclear test). It was shown that epirubicin induced cytogenetic disturbances in the hemopoietic cells within the first 72 hours. The antibiotic had a marked reversible effect on the erythroid population and lymphoid tissues and a moderate toxic action on the granulocyte population. The antibiotic did not affect thrombocytopoiesis. The single administrations had a more pronounced and prolonged myelotoxic and lymphotropic effect.     

Chemosignals from isolated females have antimutagenic effect in dividing the cells of bone marrow from male mice of the CBA strain

A level of X-ray induced mitotic disturbances in the cells of the bone marrow of male mice was studied under the modifying influence of chemosignals from isolated adult female mice of the CBA strain. It has been shown that the frequency of chromosomal aberrations in irradiated (4 Gr) males after exposing them for 24 hours on bedding soiled with female chemosignals is lower than in irradiated males in cages with clean bedding. The mechanisms and importance of the antimutagenic effect of female house mouse chemosignals are discussed.     

Chromosome mutations in marshland and dry valley populations of Abies sibirica Ledeb

Results of chromosome mutation investigation in Abies sibirica populations growing in fen and dry valley in Western Siberia are presented. The frequency of chromosome aberrations and mitotic irregularities is higher in dry valley population of the fir, than in its fen population. However, the spectrum of abnormalities registered in metaphase and ana-telophase cells of the fir from fen is broader, compared to that from dry valley. These features may be due to the fir genome reorganization in different environmental conditions, and indicate the plant reaction to stress factors.     

Human cells in suspension. 1 Human lymphoid and granulopoietic cells in primary and secondary cultures: effects of in vitro induction and prolonged methanol acetic acid fixation on metaphase chromosome structure.

Modifications of Hungerford's method (1965) for production of chromosomal slides from human lymphoid cells in culture have been developed. Modified in vitro induction of banding and uncoiling has been used to produce chromosomal slides from human neoplastic cells of granulopoietic origin. The chromosomes are well spread and appear either long, thin and segmented or uncoiled. It is suggested that it is the combined action of the prolonged fixation used, and the in vitro induction, which leads to the observed structural alteration of the chromosomes. A method for increasing the yield of metaphase cells when working with bone marrow has been developed on the basis of culturing the granulopoietic cells in medium containing colony stimulating factor (CSF). Comparative analysis of metaphases from primary and secondary cultures of bone marrow cells showed that the culturing conditions for the secondary cultures do not induce chromosome abnormalities in the cells during the growth period.     

Chromosomal abnormalities and spleenocyte production in laboratory mouse males after exposure to stress chemosignals

Activity of antibody producing spleenocytes and chromosome stability in bone marrow cells from laboratory mouse males of CBA strain after exposure to different chemosignals excreted by stressed or irradiated syngeneic donors was studied. It has been shown that the exposure of the recipient males to volatiles from donor males (stressed by swimming) decreases quantity of antibody-producing cells in 1, 3 and 10 days after the treatment. The same exposure increased the chromosome aberrations level in dividing bone marrow cells from CBA recipients in 1 day after the treatment. Similar changes were observed in 24 h after exposure to volatiles of irradiated donors or to synthetic mouse pheromone, 2,5-dimethylpyrazine. Possible mechanisms of chemosignals effect on the immune system are discussed.     

Possible mechanisms for occurrence of chromosomal restructuring. Interchromosomal contacts during metaphase and their role in chromosomal restructuring

The influence of colchicine-hypotonic treatment on interchromosomal aberrations at metaphase was studied in bone marrow cells of BALB mice irradiated by X-rays within the dose range from 0.25 to 1.50 Gy. In was found that after 30 min treatment with 0.002% colchicine of cells dividing 10 h following irradiation, the frequency both of chromosomal exchanges and interchromosomal contacts decrease about 3.5 times, the amount of chromosomal breaks increasing. It is calculated from the data of this experiment that two breaks induced by irradiation, which were scored at the same K metaphase as independent ones, appeared to be associated with each other at high frequency through exchange in the absence of colchicine or hypotonic treatment. It is assumed that regions of interchromosomal contacts at native metaphase are the most radiation-sensitive zones of the genome preferentially involved in chromosomal aberrations of X-irradiated cells.     

Effects of “Pheromone-Like” pyrazine-containing compounds on stability of genetic apparatus in bone marrow cells of the male house mouse <Emphasis Type="Italic">Mus musculus</Emphasis> L.

There was studied effect of female house mouse pheromone 2,5-dimethylpyrasine and other pyrazine-containing substances on the genetic apparatus stability of dividing bone marrow cells of male mice of the strain CBA. Differences in action of the used chemosignals are revealed. Role of the method of action on induction of analyzed mitotic aberrations is shown. Spectrum of the aberrations is analyzed. Dependence of cytogenetic activity of the used substances on their structural peculiarities and significance of the revealed effects are discussed.