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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

The effect of serotonin on the hematopoietic stem cells of the bone marrow]

Haemopoietic stem cells content and proliferative activity were studied in the bone marrow of female F1 (CBA x C57Bl6) mice after single (50 mg/kg) and chronic (0.5 mg/kg daily for 7 days) serotonin (S) injections. It is shown that 9-day and 12-day COEs contents in the bone marrow of experimental mice has been increasing for 24 h after single S injection. After chronic S injections twofold increase of 12-day COEs is observed without any increasing of 9-day COEs. Total myelokaryocyte number is increased too. The study of proliferative status by in vitro incubation of bone marrow cells with ARA-C has shown that the numbers of 9-day and 12-day COEs in S-phase have increased both after single and chronic S injections. Possible mechanisms of stimulating effect of S on bone marrow stem cells are discussed.     

Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells

The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis. Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by [3H]thymidine labeling and radioautography. In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains. The turnover of small lymphocytes also increased. These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC. In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects. The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production. Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.     

Comparative Investigation of Mesenchymal Stem Cells Isolated from the Bone Marrow and Fetal Liver of Mouse and Rat

We studied the properties of cells forming fibroblast colonies from the bone marrow and fetal liver of mouse and rat. Bone marrow and fetal liver cells formed colonies in vitro including fibroblasts as well as a considerable proportion of macrophages. The colonies formed from bone marrow and hepatic cells of rat differed from the murine ones by a higher proportion of fibroblasts. Most colonies derived from the bone marrow of both mouse and rat included a fraction of cells expressing alkaline phosphatase, and hence, capable of osteogenic differentiation; the colonies derived from the fetal liver included low proportions of such cells. The cell layers derived from the colony-forming fibroblasts of both bone marrow and fetal liver of mouse maintained hematopoiesis in the peritoneal cavity of irradiated mice, which indicated that these progenitor cells can form hematopoietic microenvironment.     

Cycloheximide genotoxicity in in vitro and in vivo test systems

The aim of this study was to investigate if there was any genotoxic effect produced by the antibiotic cycloheximide, widely used as a fungicide in agriculture as well as in everyday laboratory practice. The battery of test systems included the bacterium Salmonella typhimurium (strains TA98 and TA100), the yeast Saccharomyces cerevisiae (D7), Allium cepa somatic cells and mouse bone marrow cells. This combination of test systems enabled us to establish possible effects caused by cycloheximide at different levels of the genome and to indicate a possible mechanism of action. The results obtained in experiments showed that cycloheximide did not induce frameshift or base-pair substitution mutations in S. typhimurium regardless of metabolic activation. In S. cerevisiae cycloheximide had only toxic effects but no increase of mitotic gene conversion was noticed under the conditions of the experiment. However, in A. cepa somatic cells as well as in mouse bone marrow cells cycloheximide showed its activity causing different genetic damages, e.g., chromosome breaks, mitotic disturbances and nuclear abnormalities.     

Effect of an erythrocytic chalone on the postsynthetic period of the erythroblastic mitotic cell cycle in mouse bone marrow

The effect of the erythrocytic chalone on the beginning, middle and end of the G2-period of the mitotic cycle has been studied in the erythroblastic cells of the mouse bone marrow. The end of S(-) and the beginning of G2(-) periods have been demonstrated to be the most sensitive to the effect in question. The erythrocytic chalone inhibits mitotic activity by decreasing the amount of prophases and inhibits incorporation of radioactive glycine into the dividing erythroblastic cells. The mechanism inhibiting their mitotic activity is discussed.     

Investigation of genotoxic and cytotoxic effects of micro- and nanosized titanium dioxide in six organs of mice in vivo

Titanium dioxide is manufactured worldwide in large quantities for use in a wide range of applications including as food additives, in cosmetics and pigments for coloring ingested and externally applied drugs. Although TiO(2) is chemically inert it can cause negative health effects, such as lung cancer in rats. However, the mechanisms involved in TiO(2)-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. In the present research genotoxicity and carcinogenicity of titanium dioxide were studied in a mouse model. We treated CBAB6F1 mice by oral gavage with titanium dioxide particles (microsized, TDM, 160nm; nanosized, TDN, 33nm) in doses of 40, 200 and 1000mg/kg bw, daily for seven days. Genotoxic effects were analyzed in the cells of brain, liver and bone marrow by means of the Comet assay and in the cells of bone marrow, forestomach, colon and testis with a poly-organ karyological assay (analysis of micronuclei, nuclear protrusions, atypical nuclei, multinucleated cells, mitotic and/or apoptotic index). TDM induced DNA-damage and micronuclei in bone-marrow cells and TDN induced DNA-damage in the cells of bone marrow and liver. TDM and TDN increased the mitotic index in forestomach and colon epithelia, the frequency of spermatids with two and more nuclei, and apoptosis in forestomach (only TDN) and testis. This is one of the first poly-organ studies of TDM- and TDN-induced genotoxicity in vivo in mice. These effects are caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the increasing use of TiO(2) nanoparticles, these findings indicate a potential health hazard associated with exposure to TiO(2) particles.     

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.     

Bone-marrow-derived cells cultured in serum-free medium reduce liver fibrosis and improve liver function in carbon-tetrachloride-treated cirrhotic mice

We have previously developed autologous bone marrow cell infusion (ABMi) therapy for liver cirrhosis patients. One problem associated with ABMi therapy is that general anesthesia is required to obtain 400 ml bone marrow fluid from liver cirrhosis patients. However, many patients with decompensated cirrhosis do not meet the criteria, because of decreased liver function or an increased bleeding tendency. To overcome these issues, our aim is to derive liver repair cells from small amounts of autologous bone marrow aspirates obtained under local anesthesia and to use these cells in liver cirrhosis patients. Here, we conducted, by using a mouse model, basic research aimed at achieving novel liver regeneration therapy. We cultured bone marrow cells aspirated from the femurs of C57 BL/6 Tg14 (act-EGFP) OsbY01 mice (green fluoresent protein [GFP]-transgenic mice). After 14 days of culture with serum-free medium (good manufacturing practice grade), the obtained spindle-shaped GFP-positive cells were injected (1×10⁴ cells) via the caudal vein into mice with carbon tetrachloride (CCl₄)-induced cirrhosis. Numerous cultured macrophages and some mesenchymal stem cells repopulated the cirrhotic liver. The results showed that serum albumin, liver fibrosis and liver function were significantly improved in the group treated with cultured bone marrow cells (P<0.01). Moreover, matrix metalloproteinase-9 expression was increased in the liver (P<0.01). Thus, infusion of bone-marrow-derived cultured cells improved liver function and liver fibrosis in mice with CCl₄-induced cirrhosis.     

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.     

Analysis for pheromone-induced cytogenetic disturbances depending on major urinary proteins of male laboratory mice

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 line. 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.     

Sister chromatid exchange in murine alveolar macrophages,regenerating liver and bone marrow cells — a simultaneous multicellular in vivo assay

Differential labeling of sister chromatids was achieved simultaneously in murine alveolar macrophages, regenerating liver, and bone marrow cells of partially hepatectomized mice as well as in alveolar macrophages and bone marrow cells of nonhepatectomized mice. The mean frequency of SCE/cell ±S.D. and the percentage of second division cells for each cell type were determined. No significant differences in mean frequencies of SCE/cell were observed among the cell types or between hepatectomized (alveolar macrophages –3.6±2.2, bone marrow –3.4±2.2; regenerating liver –3.6±2.4) and nonhepatectomized (alveolar macrophages —3.4 ±1.9; bone marrow —2.9±1.8). Although the percentage of second division cells was dependent upon cell type, no significant differences were apparent between hepatectomized (alveolar macrophages —57±8%; bone marrow —37±6%; regenerating liver –65±6%) and nonhepatectomized mice (alveolar macrophages –53±6%; bone marrow –36±4%). Comparisons between BrdU treated and nontreated nonhepatectomized mice revealed no significant alteration in mitotic yields.