The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxicity of the 2-furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene (2-betaNF) has been evaluated in cultured human peripheral blood lymphocytes at concentrations ranging from 0.5 to 15microg/ml. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were used and scored as indicators of genetic damage. To asses the role of the metabolism mediated by the enzymes present in the S9 mix, over the possible genotoxic potential of the test agent, the cultures for MN and SCE demonstrations were treated for 3h in presence and in absence of rat liver microsomal fraction. The results indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of the presence/absence of metabolic fraction. Nevertheless, a slight increase in the SCE frequency was observed in those cultures treated without the S9 mix; although this slight increase disappeared in the experiments carried out with the microsomal fraction. In addition, cytotoxic/cytostatic effects of (2-betaNF) were observed mainly in the cultures treated without the S9 fraction.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed eventhough the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.     

Effect of p53 haploinsufficiency on melphalan-induced genotoxic effects in mouse bone marrow and peripheral blood

Mice heterozygous for a p53 null mutation develop tumours induced by genotoxic carcinogens with a shorter latency than wild type mice and have been proposed as an alternate animal model for carcinogenicity testing. Some literature data suggest that p53+/- mice might also be more sensitive to the short-term effects of genotoxic agents and manifest a haploinsufficiency phenotype that could contribute to the higher tumour susceptibility. We have compared the induction of micronuclei in bone marrow and blood of p53+/- and p53+/+ isogenic mice after treatment with a single or multiple doses of melphalan (MLP), a crosslinking genotoxic carcinogen. We have also characterized the mechanism of micronucleus induction with CREST staining of kinetochore proteins to distinguish between chromosome break- and chromosome loss-induced micronuclei. Significant increases of micronucleated bone marrow polychromatic erythrocytes and blood reticulocytes were induced under all MLP exposure conditions. The frequency of micronucleated blood erythrocytes increased linearly with duration of exposure. Micronuclei were essentially a consequence of chromosome break events. After a single MLP dose, a significant reduction of the frequency of polychromatic erythrocytes in bone marrow of p53+/+ animals suggested the induction of cytotoxicity/cell cycle delay. This effect was not observed in p53+/- mice. We believe this finding to provide some evidence of a haploinsufficiency phenotype in the modulation of cell cycle/apoptotic pathways mediated by the p53 protein. In bone marrow of wild type mice, an increased effect of multiple MLP doses was detected over that of a single administration, whereas, in p53+/- mice, no differential effect was found of different exposure durations. Possibly, the probability of micronucleus formation increased under chronic exposure because of increased cell division in response to peripheral anemia and a reduction of p53 protein level had a small effect on cell cycle modulation and on such indirect mechanism of micronucleus induction. However, pairwise comparisons between the frequencies of cells with micronuclei in wild type and p53+/- mice under all exposure conditions did not show statistically significant differences, suggesting that the observed effects of p53 haploinsufficiency were weak and temporary and a higher/faster induction of irreversible chromosome damage could not account for the increased susceptibility of p53+/- mice to MLP-induced tumours.     

The in vivo genotoxic effects of sodium metabisulphite in bone marrow cells of rats

This study is designed to investigate the genotoxic effect of sodium metabisulphite (SMB), which is used as an antimicrobial substance in foods on bone marrow cells of rats. Four different concentrations of SMB (250, 500, 750 and 1000 mg/kg body weight) were given rats (Rattus norvegicus var. albinos) for 6, 12 and 24 hours treatment period by intraperitoneal (IP) and gavage (GV) administrations. In this study, we found that intraperitoneal implement of SMB generally more effective increasing the percentage of abnormal cells and CA/cell in all concentrations and treatment period. In addition, mitotic index (MI) data of intraperitoneal injection are lower than gavage. It can be concluded that potential genotoxic effects of SMB by IP injection is higher than GV injection.     

The in vivo genotoxic effects of sodium metabisulfite in bone marrow cells of rats

This study is designed to investigate the genotoxic effect of sodium metabisulphite (SMB), which is used as an antimicrobial substance in foods on bone marrow cells of rats. Four different concentrations of SMB (250, 500, 750 and 1000 mg/kg body weight) were given rats (Rattus norvegicus var. albinos) for 6, 12 and 24 h treatment period by intraperitoneal (IP) and gavage (GV) administrations. In this study, we found that intraperitoneal implement of SMB generally more effectively increases the percentage of abnormal cells and CA/cell in all concentrations and treatment period. In addition, mitotic index (MI) data of intraperitoneal injection are lower than gavage. It can be concluded that potential genotoxic effects of SMB by IP injection are higher than GV injection. The text was submitted by the authors in English.     

The effects of colcemid on mouse bone marrow.

Following Colcemid administration, mitoses accumulate preferentially in the subendosteal region of the bone marrow of the mouse. This finding suggests that the most rapidly proliferating cells are localized to the subendosteal region, and complements previous radioautographic studies which have demonstrated a corresponding labelling gradient in the marrow. Quantitative estimates of cell cycle time by the stathmokinetic method were precluded by the presence of significant Colcemid induced interphase cell loss. Colcemid also affected cell differentiation in the marrow. Following Colcemid administration there was a fall in mature granulocytes in the marrow, and a concommitant rise in marrow megakaryocytes.     

Cytotoxic and genotoxic effects of Br-containing oxaphosphole on Allium cepa L. root tip cells and mouse bone marrow cells

The continuous production and release of chemicals into the environment has led to the need to assess their genotoxicity. Numerous organophosphorus compounds with different structures have been synthesized in recent years, and several oxaphosphole derivatives are known to possess biological activity. Such chemical compounds may influence proliferating cells and cause disturbances of the genetic material. In this study, we examined the cytotoxicity and genotoxicity of 4-bromo-N,N-diethyl-5,5-dimethyl-2,5-dihydro-1,2-oxaphosphol-2-amine 2-oxide (Br-oxph). In A. cepa cells, Br-oxph (10(-9) M, 10 (-6) M and 10 (-3) M) reduced the mitotic index 48 h after treatment with the two highest concentrations, with no significant effect at earlier intervals. Mitotic cells showed abnormalities 24 h and 48 h after treatment with the two lowest concentrations but there were no consistent changes in interphase cells. Bone marrow cells from mice treated with Br-oxph (2.82 x 10 (-3) μg/kg) also showed a reduced mitotic index after 48 h and a greater percentage of cells with aberrations (principally chromatid and isochromatid breaks). These findings indicate the cytotoxicity and genotoxicity of Br-oxph in the two systems studied.     

Clastogenic effects of acrylamide in mouse bone marrow cells

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.     

Cytogenetic effects of ribavirin on mouse bone marrow

The micronucleus test and mitotic chromosome analysis were used to study the in vivo mutagenic activity of ribavirin on bone marrow cells of Swiss albino mice. To determine the incidence of micronuclei, mice were injected i.p. twice, at an interval of 24 h. with the drug at doses of 20, 100 and 200 mg/kg. Animals were killed 6 h after the second dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. Ribavirin significantly (P less than 0.05) induced micronuclei in polychromatic erythrocytes at all doses. A study was conducted to investigate the cytogenetic effect of the drug on mitotic chromosomes. Ribavirin at 200 mg/kg/day was administered to mice for 3 and 5 days. Repeated treatment with the high dose of ribavirin produced a highly significant (P less than 0.02) increase in abnormal metaphase spreads. The results indicate that ribavirin is mutagenic to bone marrow cells of mice as evaluated by the micronucleus test and by chromosome analysis.     

Genotoxic effects of deltamethrin in the mouse bone marrow micronucleus assay

The genotoxicity of deltamethrin was studied in Swiss albino male mice (five animals/group) using the bone marrow micronucleus assay. Deltamethrin (two i.p. injections, 30 h and 6 h before sample collection) was found to induce micronuclei at 162.5 and 300.0 mg/kg body weight. A lower dose (32.5 mg/kg body weight) failed to induce a significant increase in micronuclei over the control level.     

Cytogenetic effects of dacarbazine on mouse bone marrow cells in vivo

The mutagenicity of dacarbazine was assayed in an in vivo test utilizing mouse bone marrow cells. The dose rates used in the experiments were computed according to the standard surface area of the mouse and were proportional to the human dose rate. These were 0.27, 0.44 and 0.60 mg/30 g body weight, each given twice daily at an interval of not less than 6 h. The duration of drug treatment was 24, 48 and 72 h. This alkylating agent proved to be mitodepressive and produced a 3-fold reduction in the mitotic index. The drug also induced chromosome anomalies mainly in the form of chromatid gaps and breaks. These anomalies were proportional to dose rate and duration of drug treatment.     

The effects of age, sex and diet on the clastogenic action of cyclophosphamide in mouse bone marrow

7-week-old and 12-week-old mice of both sexes received either a control or protein-deficient diet for 3 weeks. Afterwards, they were given a single dose of cyclophosphamide (0.5 mg/10 g b.wt.) before being sacrificed. The relationship between age and the clastogenic action of cyclophosphamide can be observed in the bone marrow cells of male mice but not in those of female mice. 12-week-old males on a 75% protein-deficient diet have a lower frequency of cells with cyclophosphamide-induced chromosome aberrations than has the control group. On the contrary, 7-week-old males and females, and 12-week-old females, show that protein-deficient diets act synergistically with the clastogenic action of cyclophosphamide. These results are discussed taking the metabolism of the drug into account. Animal age also plays a role in the formation of chromosome rearrangements; this type of aberration is significantly more frequent in younger animals of both sexes than in older ones exposed to the drug.     

Clastogenic effects of cesium chloride on mouse bone marrow cells in vivo

Clastogenic effects of cesium chloride (CsCl) on mouse bone marrow cells in vivo following oral administration were studied after 24 h. The incidence of chromosome aberrations increased linearly with increasing concentrations of the chemical from 1/20th to 1/5th of the LD50. The frequency of cell division was also enhanced by the lower doses but higher doses were mitostatic. This report is the first on the clastogenicity of cesium on animals.     

Ultradian biorhythms in mouse bone marrow

Ultradian oscillations in the number of karyocytes isolated from the femoral bone of intact ACR mice have been demonstrated. The periodicity of oscillations did not depend on the season or the site of mice breeding. The bone marrow also showed ultradian oscillations in relative and absolute amount of lymphoid, myeloid and mitotic cells. It is postulated that differentiation and migration of bone marrow cells might have ultradian biorhythms.     

The effect of agent dose and treatment time on the intercellular distribution of sister-chromatid exchanges induced by genotoxic agents in mouse bone marrow cells in vivo

Using two methods of bromodeoxyuridine (BrdUrd) administration and three genotoxic chemicals, the effects of dose and treatment time on the intercellular distribution of sister-chromatid exchanges (SCE) in the bone marrow of male B6C3F1 mice were evaluated. The dispersion of SCE among solvent control mice infused intravenously with BrdUrd or implanted subcutaneously with a BrdUrd tablet partially coated with paraffin was largely consistent with a Poisson model. Intraperitoneal treatment with cyclophosphamide (CP; solvent = phosphate-buffered saline), 7,12-dimethylbenzanthracene (DMBA; solvent = corn oil) and, in mice infused with BrdUrd, mitomycin C (MMC; solvent = phosphate-buffered saline) induced a significant increase in SCE, the distribution of which was not distributed as a Poisson. For CP and MMC, the increase in dispersion was dose-dependent and independent of treatment time (-1, +1 or +8 h in relation to the start of the BrdUrd treatment). The lack of a treatment time effect suggests that there were no significant differences among treatment times in the distribution of the reactive forms of these two chemicals, no variation in cell-stage sensitivity, and no cellular toxicity to modulate the response. For DMBA, the increased dispersion of induced SCE depended on treatment time and was not simply related to dose. The increase in dispersion was agent-specific; at equal levels of SCE induction, the distribution of SCE in mice treated with DMBA exhibited greater dispersion than SCE in mice treated with either CP or MMC. These differences between DMBA and CP/MMC are probably due to DMBA's slower absorption/distribution kinetics, its requirement for metabolic activation to genotoxic metabolites and its extended half-life. These data suggest that analyzing the distribution of SCE, in addition to mean frequency, is a useful method for evaluating agent specific patterns in SCE induction.     

Potential protective effects of melatonin on bone marrow of rats exposed to cytotoxic drugs

Myelosuppression is the most serious, dose limiting, toxicity of cytotoxic drugs. Efforts to protect the bone marrow have been only variably successful, and no agreement exists on how to approach this problem. Melatonin, the major hormonal product of the pineal gland, is supposed to have both chemoprotective and myelostimulatory effects. This experimental study was carried out to test these two effects on the bone marrow of rats, daily intraperitoneally injected with 100 microg melatonin. Injection of 10 mg aracytin for 10 days produced a significant (P < 0.01) decrease in red blood cells count (RBCs), total leucocytic count, as well as platelets count. When melatonin was injected along with aracytin, it would significantly increase (P < 0.05) RBC count and (P < 0.01) blood platelet count. Injection of melatonin after aracytin treatment would significantly increase (P < 0.01) RBC, total leucocytic and platelet counts in comparison with rats treated with aracytin only. The effects of melatonin were more clear in rats treated with it after aracytin injection than those treated with melatonin and aracytin at the same time. Furthermore, it was found that aracytin produced a significant (P < 0.01) decrease in serum total proteins, albumin, and significantly increased the (P < 0.01) albumin/globulin ratio. Melatonin injection would significantly increase (P < 0.01) total protein, globulin, and significantly decrease (P < 0.01) the albumin/glubulin ratio when injected either with aracytin or after aracytin treatment. These results indicate that melatonin protects bone marrow, lymphoid tissues from damaging effect of cytotoxic drugs, as well as stimulating the suppressed bone marrow.     

Simulation study of the effects of multiple treatments in the mouse bone marrow micronucleus test

The effect of multiple treatment with chemicals in the micronucleus test was evaluated by simulation involving an estimation of the additive accumulation of micronucleated polychromatic erythrocytes (MNPCEs) on the basis of time-response data available on single treatments with mitomycin C, 1-beta-D-arabinofuranosylcytosine, 6-mercaptopurine, and methotrexate. The frequency of MNPCEs calculated for different multiple treatment regimens by the model could predict the effects observed in real experiments. On the other hand, the effect of multiple treatments on bone marrow depression, expressed as a decrease in the frequency of polychromatic erythrocytes, was exponential according to both the simulation and actual data. These results suggest that although increasing the number of treatments may additively enhance the MNPCE response obtained with some agents it may, in the case of bone marrow-toxic chemicals and doses, make micronucleus analysis more time-consuming and even impossible due to the exponential decrease of analyzable cells, especially in the case of manual scoring.