Hamster Bone Marrow Cells Research Articles

Coumarin protects 7,12-dimethylbenz(a)anthracene-induced genotoxicity in the bone marrow cells of golden Syrian hamsters

Aim : Aim of the present study was to evaluate the antigenotoxic effect of coumarin by measuring the frequencies of micronuclei and the degree of DNA damage in the bone marrow cells of hamster treated with 7,12-dimethylbenz(a)anthracene (DMBA). Materials and Methods: Genotoxicity was induced in experimental hamsters by single intraperitoneal injection of DMBA (30 mg/kg b.w.). The frequency of micronucleated polychromatic erythrocytes (MnPCEs) and DNA damage were assessed in the bone marrow cells of experimental hamsters. The status of lipid peroxidation, antioxidants and phase I and II detoxification agents were utilized as biochemical end points to assess the dose-dependent antigenotoxic potential of coumarin in DMBA-induced genotoxicity. Results: Increase in micronuclei frequency was accompanied by increase in tail length, percent of tail DNA, tail movement and olive tail movement in the bone marrow cells of hamsters treated with DMBA alone. A significant increase in the levels of thiobarbituric acid reactive substances, antioxidants and phase I and II detoxification agents were also noticed in hamsters treated with DMBA alone. Oral pretreatment of coumarin at a dose of 100 mg/kg b.w to hamsters treated with DMBA significantly decreased the frequency of MnPCEs and DNA damage in the bone marrow cells. Also, coumarin restored the status of biochemical variables in the plasma and liver of hamsters treated with DMBA. Conclusions: Present study demonstrated the antigenotoxic effect of coumarin in DMBA-induced genotoxicity. The antigenotoxic potential of coumarin is probably due to its antioxidant potential and modulating effect on detoxification cascade during DMBA-induced genotoxicity.

Anti-Clastogenic Effect of Berberine against DMBA-Induced Clastogenesis

Our aim was to evaluate the protective effect of berberine on 7,12-dimethylbenz[a]anthracene (DMBA)-induced chromosomal aberrations and micro-nucleated polychromatic erythrocytes (MnPCEs) frequency in bone marrow cells of golden Syrian hamsters. The anti-clastogenic effect of berberine (50 mg/kg b.w. p.o.) was also assessed by measuring the status of phase II detoxification enzymes and oxidative stress, as biochemical endpoints, during DMBA (30 mg/kg b.w. i.p.) induced clastogenesis. Marked chromosomal aberrations, increased MnPCEs frequency and enhanced status of lipid peroxidation, antioxidants and phase I and II detoxification enzymes were noticed in hamsters treated with DMBA alone. Oral pre-treatment with berberine for 5 days to DMBA-treated hamsters significantly reduced the frequency of MnPCEs and chromosomal abnormalities as well as reversed the status of lipid peroxidation, antioxidants and phase I and II detoxification enzymes. The present study thus suggests that berberine has potent anti-clastogenic potential against DMBA-induced clastogenesis, which is probably due to its anti-lipid peroxidative potential and effect on modulation of phase I and II detoxification cascade.

Chromosome breakage induced in vivo by a carcinogenic hydrocarbon in bone marrow cells of the Chinese hamster.

Chromosome breakage induced in vivo by a carcinogenic hydrocarbon in bone marrow cells of the Chinese hamster

In vivo method for sister chromatid exchanges in Chinese hamster foetal and bone marrow cells.

An in vivo BrdU labelling method for sister chromatid staining in Chinese hamster foetal and bone marrow cells is reported. The mitotic index in bone marrow cells was many times higher than that in foetal cells (2.7 to 11.3 and 0.1 to 2.1, respectively) whereas both tissues produced metaphases of high quality. Sister chromatid exchanges (SCE) in the foetal cells of 3 non-treated hamsters varied from 5.4 to 7.4, and in the bone marrow cells of 11 non-treated hamsters, from 2.9 to 5.4.

The anti-rat Thy-1.1 monoclonal antibody R2-12C5 recognizing rat, mouse, hamster, and mongolian gerbil Thy-1 antigen had no effect on IL-2 induced rat thymocyte proliferation

The reactivity of MAb R2-12C5 with various rat lymphoid cell populations was defined by immunofluorescence staining following FACScan analysis. R2-12C5 reacted with 94% of rat thymocytes; approximately 85% of Mongolian gerbil, AKR mouse, and hamster thymocytes; 35% to 45% of rat, Mongolian gerbil, AKR mouse, and hamster spleen cells; 30% to 60% of rat, Mongolian gerbil, AKR mouse, and hamster mesenteric lymph node cells; 76% of rat bone marrow cells; 55% to 60% of Mongolian gerbil and hamster bone marrow cells; and 92% of AKR mouse bone marrow cells. Furthermore, R2-12C5 antibody reacted with mouse Thy1.1 gene-transfected mouse B-cell lymphoma K46T13 cells.

Relationship between the duration and number of cell cycles after mutagenic exposure and the incidence of sister chromatid exchanges

The incidence of sister chromatid exchanges during the second and third mitoses in Jungar hamster bone marrow cells and human lymphocytes is assessed at different times after mutagenic exposure to thiophosphamide. In the control, the mean number of sister chromatid exchanges in bone marrow cells did not change during three consecutive cycles irrespective of the rate of cell proliferation. Thiophosphamide exposure resulted in replicative or nonreplicative repair of injuries inducing sister chromatid exchanges. About 50–70% of injuries are eliminated during a replicative (mitotic) cycle. Nonreplicative repair is intensive in proliferating bone marrow cells and weakly expressed in blood lymphocytes.

Clastogenic action of thiotepa on bone marrow cells of the Armenian hamster

Thiotepa (TT) induced chromosomal aberrations in the bone marrow cells of the Armenian hamster, Cricetulus migratorius, in a dose-dependent manner. The maximal clastogenic action was observed 24 h after treatment with thiotepa. Male and female Armenian hamsters were similarly sensitive to the clastogenic action of thiotepa. The lowest effective concentration of thiotepa for Armenian hamsters was 2.5 mg/kg b.w. (6.1% of the LD 50). The results obtained and analysis of the literature data have shown that Armenian hamsters are much more resistant to the clastogenic action of thiotepa than various other species of rodent. This resistance is not due to the metabolic activation rates of chemical mutagen in the liver of Armenian hamsters, since thiotepa is a direct-acting clastogen.

Bioaccumulative potential and toxicity of endosulfan insecticide to non-target animals

1. Endosulfan insecticide is a polychlorinated compound used for controlling a variety of insects; it is practically water-insoluble, but readily adheres to clay particles and persists in soil and water for several years. Its mode of action involves repetitive nerve-discharges positively correlated to increase in temperature. This compound is extremely toxic to most fish and can cause massive mortalities. In fish, it causes marked changes in Na and K concentrations, decrease in blood Ca 2+ and Mg levels and inhibits Na, K and Mg-dependent ATPase (in brain). 2. Bioaccumulation of endosulfan is reported for marine animals; however, freshwater animals (e.g. crayfish) accumulate it to some extent, but they lose the compound rapidly during depuration. Endosulfan is generally less toxic to aquatic invertebrates than fish. However, it causes decreases in adenylate energy charge, oxygen consumption, hemolymph amino acids, succinate dehydrogenase, heart-beat (mussel) and altered osmoregulation. 3. Generally, mammals are less susceptible to endosulfan's toxicity than aquatic animals. The majority of studies conducted on laboratory mammals can be summarized, (a) Neurotoxicity: male rats are more sensitive than females to endosulfan, which decreases brain and plasma acetyleholinesterase activity. Endosulfan I (a metabolite) causes a significant change in norepinephrine, 5-HT and GABA. (b) Renal toxicity: inhibition of MFOs activity was noticed in rats; other effects included changes in proximal convoluted tubules and necrosis of the tubular epithelium, (c) Hepatotoxicity: chemically-induced aminopyrine N-demethylase and aniline hydrolase were found in rat liver, and reduction in the glycogen level occurred, (d) Hematologic toxicity: endosulfan exposure resulted in a significant decrease in the erythrocyte glutathione reductase, hemoglobin amount, RBC number and mean corpuscular volume. 4. Respiratory toxicity: involved dyspnea, acute emphysema, cyanosis and hemorrhages in the interalveolar partitions of rat's lungs. 5. Biochemical: in rats, endosulfan caused increased glucose-6-phosphate dehydrogenase activity, blood glucose level, phospholipid contents of the microsomal and surfactant system, and profoundly induced the activity of alcohol dehydrogenase and cytosolic glutathione S-transferases. It also decreased significantly Na +, K + and Mg 2+ ATPases, plasma calcium level and alkaline phosphatase in the intestinal epithelium. 6. Immunologic toxicity: rat serum antibody titer to tetanus toxin, IgG, IgM and gammaglobulins were significantly reduced. 7. Reproductive toxicity: degenerative changes in the seminiferous epithelium, induction of the rate-limiting enzyme in testosterone production (3β-hydroxysteroid transferase and 17β-hydroxysteroid transferase), histological changes in reproductive organs, testicular atrophy and the occurrence of ovarian cysts were noticed in rat. Reduction in the weight of secondary sex organ was also observed. 8. Developmental, teratogenic and genotoxicity: this insecticide caused a significant increase in the fetal résorption of rats. Skeletal abnormalities included underweight fetuses, small 4th and 5th unossified sternabrae. No fetotoxic or teratogenic activity was found in rabbits; however, in chickens, egg-hatchability and sterility occurred due to antimitotic activity. Endosulfan caused significant chromosomal aberrations in mouse and hamster bone-marrow cells and damage to spermatozoa cells. In Drosophila, sex-linked recessive lethals were noticed. In mice, dominant lethal mutations occurred; increase in abnormal sperm and decrease in count occurred. 9. Carcinogenic toxicity: not reported to be carcinogenic in B6C3F mice or humans by any route of exposure. 10. Toxicity to humans: endosulfan exposure has exhibited epileptic effects, hyperactivity, irritability, tremors, convulsions and paralysis in humans. A suicidal attempt by a 20-year old male who ingested 30% endosulfan caused hypoxia, followed by recurrent aspiration pneumonia, episodes of tachycardiogenic shock which was preceded by tachycardia and hypertension.

Assessment of genotoxic effects by Lindane

The well known and previously widespread insecticide lindane has been re-assessed for DNA-damaging activity. A first group of investigations using standard in vitro and in vivo mutagenicity assays did not indicate any genotoxic effects of lindane at all. The assay systems used were for the induction of HPRT mutations and sister chromatid exchanges in CHO cells cultured in vitro, and for micronuclei induction in vivo in bone marrow cells of rats, hamsters and mice. Also, lindane was assessed for its potential to induce sister chromatid exchanges in vivo in the bone marrow of Chinese hamsters. These specific assay systems had not been used previously for elucidating the genotoxic effects of this compound, but they are basically similar to other standard mutagenicity assays in which lindane has been shown to be devoid of genotoxic activity. The second part of the investigations was directed at re-evaluating a previously reported positive effect of the compound in primary rat hepatocytes in vitro. We performed in vitro and in vivo studies with hepatocytes from the rat liver and used alkaline elution to detect DNA damage. However, we could not demonstrate that lindane induced genotoxicity, unless considerable concomitant cytotoxicity was apparent as well. Finally, since lindane can be ingested and inhaled by humans, we also measured the induction of DNA damage in local target organs of absorption using single cell microgelelectrophoresis (the comet assay). In these cases lindane was genotoxic in cells of the gastric and nasal mucosa in vitro and also in vivo following appropriate routes of application (oral and inhalational exposure).

Comparative investigation of cyclophosphamide action on bone marrow cells of the Armenian hamster and 4 other species of rodents

We studied the clastogenic action of cyclophosphamide (CP) on bone marrow cells of the Armenian hamster (AH), Cricetulus migratorius. CP induced a dose-dependent linear increase in aberrant cells. The maximal cytogenetic action was observed 12 h after CP treatment. Male and female AHs were similarly sensitive to the clastogenic action of CP. We compared CP clastogenicity at a dose of 25 mg/kg on bone marrow cells of AHs, mice, rats, guinea pigs and Chinese hamsters 24 h after treatment. We observed that this dose of CP induced only 2.8% aberrant cells in bone marrow of AHs, but 42.8%, 32.2%, 25% and 14.6% aberrant cells in bone marrow of guinea pigs, rats, mice and Chinese hamsters respectively. AHs are much more resistant to the metaphase-arresting action of colchicine than other species of rodents (e.g., the colchicine dose for AHs is 100-fold more than for rats). Thus AHs are the most resistant of all rodent species studied to the clastogenic action of CP.

The possible role of probiotics as dietary antimutagens

Possible antimutagenic actions of probiotics - mainly lactic acid bacteria - were examined using in vitro and in vivo test systems. In the Ames test with Salmonella typhimurium TA1538 beef extract and nitrosated beef extract were used as mutagens. L. casei showed high antimutagenic activity on mutagenicity induced by nitrosated beef extract only without S9 mix, whereas Omniflora (a lyophilized preparation of lactobacilli and E. coli and its cellfree culture broth exhibited antimutagenic action only on beef extract. The actions of probiotics were more homogeneous when living animais were used in the tests. Using busulfan as a mutagen both the chromosome aberration test (with Chinese hamster bone marrow cells) and the micronucleus test (with bone marrow cells of Chinese hamsters and mice) showed strong anticlastogenic action when L. casei, Omniflora or yoghurt (with living bifiobacteria) were given orally at the same time as the mutagen. Lactobacilli were effective also after i.p. injection. Cell-free culture broths had no or only weak antimutagenic effects. Mutagen-induced chromosome aberrations and micronuclei were reduced by up to 80% by the lactobacilli.

Genotoxicity studies on the organophosphorus insecticide chloracetophone

Chloracetophone ( O, O-dimethyl-2,2,2-trichloro-1-(chloroacetoxy)phosphonate), a new insecticide of the organophosphorus group of pesticides, was tested for genotoxicity in a variety of systems with different genetic end-points and varying parameters. The test systems included 2 microbial systems, Salmonella and Aspergillus for point mutations and mitotic segregation, respectively, and human lymphocyte cultures and mammalian bone marrow cells (from rats and hamsters treated acutely and subacutely) for chromosomal aberrations and micronuclei. Chloracetophone was negative in Aspergillus at concentrations of 1–500 μg/ml, in human lymphocyte cultures at concentrations of 2.5–40 μg/ml, in rats at doses of 420–21 mg/kg b.w. and in hamsters at doses of 210–42 mg/kg b.w. for chromosomal aberrations. It did not cause any increase of micronuclei in human lymphocytes and rat bone marrow cells but did cause a significant increase in hamster bone marrow cells. Chloracetophone induced base-pair substitutions in strain TA100 of Salmonella with and without metabolic activation at a concentration range of 2000 6000 μg/plate.

Re-examination of potassium sorbate and sodium sorbate for possible genotoxic potential

Potassium sorbate and sodium sorbate were investigated for possible genotoxic actions using the Salmonella/mammalian-microsome test, HGPRT and sister chromatid exchange (SCE) test with Chinese hamster ovary cells, the micronucleus test on bone marrow cells of mice and Chinese hamsters, and the chromosome aberration and SCE test on Chinese hamsters. In all the in vitro tests no signs of genotoxicity were detected. Whereas no in vivo mutagenicity of potassium sorbate and sodium sorbate with freshly prepared aqueous solutions and with stored potassium sorbate was found, investigations with stored sodium sorbate revealed weak clastogenic activity by increased chromosome aberrations and elevated numbers of micronuclei at doses of 200 mg/kg body weight, but no induction of SCEs.

Genotoxic evaluation of chronic fluoride exposure: sister-chromatid exchange study

As part of a continuing investigation, this study was conducted to examine the genotoxic effects of chronic exposure to sodium fluoride (NaF) in drinking water on the frequency of sister-chromatid exchange (SCE) in the bone-marrow cells of male Chinese hamsters. Animals at about 3 weeks of age were randomly assigned to 6 groups, each with at least 3 hamsters, and were maintained on a low fluoride diet (< 0.2 ppm F) throughout the experiment. At 4 weeks of age, the animals in groups I–V began to receive drinking water containing fluoride at concentrations of 0, 1, 10, 50 and 75 ppm, respectively. Group VI was treated with cyclophosphamide and served as the positive control. The animals were sacrificed at 24 weeks of age by cervical dislocation. The humeri and plasma were analyzed for fluoride content, which was found to increase with the increase in fluoride concentration in drinking water. Slides of chromosomes from bone-marrow cells were prepared and blindly examined for the frequency of SCE. The mean scores of SCE for the hamsters receiving drinking water containing F concentrations up to 75 ppm for 21 weeks ranged from 4.28 to 6.28 per cell, and were not significantly different from those of the negative controls (4.60–5.44/cell). The results indicated that chronic fluoride exposure had no effect on the frequency of SCE in Chinese hamster bone-marrow cells under the conditions of the present investigation.

Sister-chromatid exchanges induced by triethylenemelamine: in vivo and in vivo/in vitro studies in mouse and Chinese hamster bone marrow and spleen cells

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15–405 μg/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 μg/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.

Chromosomal abnormalities and sister-chromatid exchange in bone marrow cells of mice and Chinese hamsters after inhalation and intraperitoneal administration: II. Cyclophosphamide

The genotoxic effects of cyclophosphamide (CPP), a human and animal carcinogen requiring metabolic activation, were studied in bone marrow cells of mice and Chinese hamsters, analyzing chromosome abnormalities (CA) and sister-chromatid exchange (SCE) after a 2-h inhalation or a single intraperitoneal administration. In order to compare the genotoxicity after the different routes of administration in the dose range of 10–110 mg CPP/kg body weight, the systemic dose obtained by inhalation was calculated from blood concentrations and the inhalation duration after an analysis of the CPP blood kinetics. In NMRI mice the frequency of bone marrow cells with chromosome abnormalities was higher after aerosol exposure than after intraperitoneal administration of comparable CPP doses. In Chinese hamsters the CA frequency was similar with both exposure routes. Inhaled CPP was found to induce a higher frequency of CA and SCE in the bone marrow cells of mice compared to those of Chinese hamsters. The findings suggest that for genotoxins requiring metabolic activation species differences exist with respect to the influence of the route of entry and the sensitivity of bone marrow cells.

Chromosomal abnormalities and sister-chromatid exchange in bone marrow cells of mice and Chinese hamsters after inhalation and intraperitoneal administration: I. diepoxybutane

Diepoxybutane (DEB), a direct-acting animal carcinogen, was found to increase the frequency of structural chromosomal abnormalities (CA) and sister-chromatid exchange (SCE) in bone marrow cells of mice and Chinese hamsters, when inhaled from an aerosol during a 2-h head-only exposure or administered as a single intraperitoneal injection. For the purpose of comparing the genotoxicity in the 2 species, both after inhalation and intraperitoneal administration, the systemic DEB dose obtained by inhalation was determined on the basis of blood concentrations and inhalation duration after the investigation of the blood kinetics. The bone marrow cells of male and female NMRI mice were found to be more sensitive than those of Chinese hamsters to the genotoxic activity of DEB.

Preparation of Chinese hamster bone marrow and spleen primary cell cultures for sister chromatid exchange and chromosomal aberration studies

Procedures for preparing and culturing Chinese hamster bone marrow and spleen cells for cytogenetic studies are described. Animals are killed by cervical dislocation, then the bone marrow is flushed from femora and tibia with Ham's F12 medium into centrifuge tubes. Bone marrow cells are collected by low-speed centrifugation (285 ×g). Approximately one million cells are cultured in a 30-ml Falcon flask with 5 ml complete medium containing 3.95 ml Ham's F12 medium, 1.0 ml fetal bovine serum (FBS), and 0.05 ml penicillin-streptomycin (5000 U/ml and 5000 µg/ml stock). Spleens are obtained aseptically, transferred to centrifuge tubes containing 2 ml of RPMI 1640 and smashed with a sterile spatula. The debris is removed, cells are collected by low-speed centrifugation (285 ×g), and washed three times with phosphate buffered saline supplemented with 2% heat inactivated FBS. Approximately one million cells are suspended in a 30-ml Falcon flask with 5 ml complete medium containing 3.70 ml RPMI 1640. 1.0 ml heat inactivated FBS, 0.05 ml penicillin-streptomycin, 0.05 ml of 200 mMl-glutamine solution, 1 × 10−5M 2-mercaptoethanol, and 0.20 ml lipopolysaccharide. For sister chromatid exchange analysis, 20 µM of 5-bromo-2′ -deoxyuridine is also added to the culture medium for 24 to 28 h for bone marrow cells and 36 to 40 h for spleen cells. These culture systems can also be used for chromosomal aberration studies.

The anticlastogenic potential of fatty acid methyl esters

To test for possible anticlastogenic effects of fatty acids, the methyl esters of fatty acids — short-chain to long-chain — were examined on busulfan in Chinese hamster bone-marrow cells using the chromosome aberration test. When the experimental animals were treated with fatty acid esters and the mutagen, the chromosome-breaking actions of busulfan were not modulated by the short-chain fatty acids, but the fatty acids from lauric acid (C 12) up to nonadecanoic acid (C 19) reduced the rate of aberrant metaphases from 9.4 to about 3% at doses of 100 mg/kg and less. Other chemical properties of the fatty acids (saturated or not, number of double bonds, even- or odd-numbered) had no influence on the anticlastogenic effects. The only exceptions to this rule were arachidonic acid, which had no effect, and γ-linolenic acid, which had no consistent effect on the action of busulfan.

Induction of chromosomal aberrations in the Syrian hamster by insecticides tested in vivo.

The insecticides demeton, dimetoat, dichlorovos, endosulfan, trichlorofon, carbaryl, lindane, methoxychlor, propoxur and malathion were examined for their ability to induce chromosomal aberrations in the bone marrow cells of the Syrian hamster (Mesocricetus auratus) treated in vivo. Mutagenicity of commercial preparations was examined at four doses: LD50; 1/2, 1/5 and 1/10 LD50. The positive control was an IP injection of cyclophosphamide to hamsters at a dose of 40 mg/kg body wt. Demeton, dichlorovos and endosulfan gave positive results. Malathion, dimethoate and the mixture of methoxychlor and propoxur were weakly clastogenic; at some doses these compounds induced statistically significant increases in the number of aberrations. Trichlorfon and the mixture of carbaryl and lindane were negative in this test.