Effect of two pyrazine-containing chemosignals on cells of bone marrow and testes in male house mice (<Emphasis Type="Italic">Mus musculus</Emphasis> L.)

The evolutionarily conservative 2,5-dimethylpyrazine chemosignal, a pheromone released by female mice, has been shown to increase frequency of mitotic disturbances in bone marrow cells assessed by using metaphase and ana-telophase analyses. The substitution of methyl radical in the molecule of pheromone by carboxyl reveals specificity of the effect of the latter derivative: the frequency of disturbances revealed by the ana-telophase analysis alone increases, whereas no induction of disturbance is detected by the metaphase analysis. An increase of the anomalies induced by both compounds has been shown in a sperm head test. Possible mechanisms underlying specific action of the tested substances on stability of the genetic apparatus of bone marrow dividing cells in the house mouse 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.     

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.     

Prometaphase accumulation in murine bone marrow cells following in vivo treatment with alloxan

The aim of this study was to determine the effect of alloxan, an inhibitor of N-acetylglucosaminyl transferase that acts during the G2/M transition, on the course of mitosis in murine bone marrow cells. Mitotic cells from animals treated with different doses of alloxan were analyzed for the frequency of prometaphasic and metaphasic chromosomes based on their morphology and length. The results indicate that alloxan treatment substantially increases the frequency of prometaphase chromosomes. This suggests that N-acetylglucosaminyl transferase is also involved in the G2/M transition in bone marrow cells. Alloxan treatment also provides a method for obtaining large chromosomes for the analysis of chromosome bands, FISH and sister-chromatid exchanges.     

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.     

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.     

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.     

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.     

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 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.     

Sensitivity of bone marrow cells damaged by antitumor drugs to cytotoxic action of effectors of a normal spleen

Different cytogenetic effects in bone marrow cells induced by antitumor drugs with different mechanisms of action was studied. The treatment of adriablastine causes the appearance of chromatid deletion, vinblastine-polyploid cells, cyclophosphamide-chromosomal and chromatid aberration in mice. It was shown that bone marrow cells with cytogenetic damage have altered susceptibility to normal spleen cells-effectors cytotoxic action.     

Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle

Apoptosis and cell proliferation are accepted to be responsible for the maintenance of homeostasis in the hematopoietic system. Understanding of the mechanisms of action of the aminothiols and ionizing radiation on normal hematopoietic cells requires determination of the correlation between apoptotic cell death and cell cycle distribution. The effects of WR-2721 ((S)-2-/3-aminopropylamino/ethylphosphorothioic acid; Amifostine) and ⁶⁰Co gamma-rays on apoptosis and cell cycle progression in the mouse bone marrow were determined. Adult male Swiss mice were exposed to 6 Gy gamma-rays only, or pretreated with WR-2721, at a dose of 400 mg/kg body weight, 30 min before gamma-irradiation. The laser scanning cytometry APO-BRDU^TM assay based on simultaneous analysis of cellular DNA content and the in situ detection of DNA strand breaks was used to identify apoptotic cells and to reveal the cell cycle position of apoptotic and nonapoptotic cells. Temporary changes in the frequency of apoptotic cells with fluorescein isothiocyanate (FITC) labeling of DNA strand breaks, and all bone marrow cells including apoptotic and nonapoptotic ones, whose DNA stained with propidium iodide, were observed in the particular phases of the cell cycle throughout the 96-h period after WR-2721 application and gamma-irradiation. The cell cycle phase specificity of WR-2721 and ⁶⁰Co gamma-irradiation was shown in terms of induction of apoptosis in bone marrow cells. The patterns of alterations in the frequency of apoptotic cells and all bone marrow cells with respect to their cell cycle position were dependent on the agent(s) applied and the time interval after treatment of mice with WR-2721 and/or gamma-rays. A modulatory, suppressive action of WR-2721 on apoptosis induction and the cell cycle perturbation caused in normal cells of the mouse bone marrow by gamma-rays was found.     

Role of stromal microenvironment in the regulation of bone marrow hemopoiesis after curantyl administration

Role of the stromal microenvironment in regulation of bone marrow hemopoiesis at the administration of the thrombocyte disaggregant curantyl was studied by the method of heterotopic transplantation of the mice bone marrow. It is shown that the action of curantyl on hemopoiesis is realised through the stem stromal cells of the bone marrow. It is noted that the inhibitory action of the preparation on proliferation of osteogenic precursor-cells is followed by activation of bone resorption processes in regenerating ectopic hemopoietic organ. Under the action of curantyl at low bone marrow cellularity in the focus of heterotopic hemopoiesis and femur an increase of mitotic activity in hemopoietic elements is noted. It is revealed that a phenomenon of ineffective megakaryocytopoiesis with intramedullary destruction of megakaryocytes leads to the local excretion of the thrombocyte released growth factor (TRGF) which has a compensatory character.     

Participation of thyroid hormones in the regulation of the genetic activity of normal and transformed human cells]

It is shown that 125J-thyroid hormones are localized on the structures of the interphasic nucleus and metaphasic chromosomes of cultured fibroblasts of 8--10-day human embryos. At the same time, the labelled thyroid hormones, though localizing in the interphasic nuclei of HeLa cells, are not accepted, unlike normal cells, by their metaphasic chromosomes. It is suggested that during transformation the acceptor ares of the HeLa cells genome lost their property to bind their own receptor complexes with thyroid hormones.     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

Cytogenetic control over the safety of merthiolate

The capacity of merthiolate (also known as thimerosal), a mercury organic compound used as preservative in vaccines, to induce chromosomal aberrations was studied in vivo in experiments on mice. The metaphasic method of the registration of anomalies was used. In a dose of 3.3 micrograms/kg body weight merthiolate did not produce a damaging effect on the chromosomes of marrow, spleen and embryonal liver cells. Cyclophosphamide (in a dose of 25-50 micrograms/kg) used as positive control induced a significant rise in the frequency of chromosomal transformations.     

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.     

The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form

The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells. Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells. The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones. It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts. The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow). In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.     

Cytogenetic effects of 1,4-dihydropyridine in various test systems

Cytogenetic effect of 1,4-dihydropyridine was studied in different test-systems. The preparation is shown to decrease the level of complete sex-chromosome losses in Drosophila and chromosome aberration frequency in Allium fistulosum seedlings. The preparation does not affect spontaneous mutability of bone marrow cells in mice, high doses of the preparation have no mutagenic potential. Thus, 1,4-dihydropyridine shows antimutagenic activity reducing the chromosome mutation level in sex and somatic cells of eucaryotic organisms. Absence of the effect on mice chromosomes may testify to the specificity of 1,4-dihydropyridine action.     

Regulatory role of bone marrow in immunogenesis. III. Humoral factors stimulating and suppressing antibody formation produced by bone marrow cells in in vitro cultures

As revealed by the method of cultivation of bone marrow and spleen cells, separated by nucleopore membrane, in two-chamber bottles, the bone marrow cells were capable of producing humoral factor stimulating antibody genesis by the spleen cells. A direct contact of the bone marrow cells with the actively proliferating antigen-stimulated cells of the spleen led to production of a spleen humoral factor suppressing the antibody genesis by the spleen cells. The suppressive action of the bone marrow cells on the antibody genesis in the culture of the spleen cells was mediated through the suppression of the spleen cells proliferation; proliferation of the bone marrow cells is enhanced.