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PubMed1987

Detection of mutagenicity in drinking water using a micronucleus test in newt larvae (Pleurodeles waltl).

We have previously described a micronucleus test using erythrocytes from larvae of the urodele amphibians Pleurodeles waltl (pleurodele) and Ambystoma mexicanum (axolotl). The test is based on a comparison of the levels of micronucleated erythrocytes in blood smears from larvae reared in water containing a clastogen, with the levels from larvae reared in purified water. Using larvae from the pleurodele, we have employed this test to evaluate mutagenic activity in drinking water. Groups of larvae were reared in tap water, while control groups were reared in tap water which had been filtered over sand and active carbon to remove micropollutants. Seven separate tests carried out between October 1985 and May 1986 all gave positive results of varying degree depending on the time of year. This test is therefore able to detect clastogens in normal drinking water. It could be used for quality control of drinking water during the various stages in the treatment of raw water without any requirement for prior extraction or concentration of micropollutants.

Animals↗
PubMed2005

Mutagenic effects of mercury pollution as revealed by micronucleus test on three Amazonian fish species.

Genotoxic effect of mercury pollution over Amazonian fish species was evaluated by using the micronucleus test (MNT). Distinct mean frequencies of micronuclei (MN) were observed in three trophically distinct characin fish species collected in two riverine environments in the Amazon Basin: the Madeira (polluted area) and the Solimões (unpolluted area) rivers. Mean frequencies of MN observed in Prochilodus nigricans (detritivore), Mylossoma duriventris (omnivore), and Hoplias malabaricus (piscivore) from the Madeira River were significantly higher compared to the frequencies from the same species from the Solimões River. In addition, mean frequencies of MN from piscivore species were almost fivefold higher than the detritivore and/or omnivore species. We conclude that MNT in fish erythrocytes may be useful for indicating genotoxity of mercury in Amazon rivers.

Animals↗
PubMed1993

Amphibian micronucleus test(s): a simple and reliable method for evaluating in vivo genotoxic effects of freshwater pollutants and radiations. Initial assessment.

A micronucleus test was developed using larvae from two urodele amphibians (Pleurodeles waltl and Ambystoma mexicanum) and an anuran (Xenopus laevis). The methods for maintenance of adults, egg laying, and rearing the larvae are described, and the conditions required for optimal response are given for each of these species. The tests are carried out during a period of intense erythropoiesis when red blood cells are actively dividing in circulating blood. The micronuclei are observed on blood smears. The genotoxic effects of X-rays were evaluated at 12 different doses over a range of 6-1200 rad. All doses, even the very low dose of 6 rad, gave positive results. The test substances were added to the water in which the larvae were reared, and the results obtained after treatment for 12 days and/or 8 days with 47 different chemical compounds are listed. Detailed results are given as the lowest concentration producing a positive response or the highest concentration producing a negative response. The reliability of the test system using the newt is now well established, while the tests using the other two amphibian species are still under evaluation. Integration of this test in a test battery for quality control of water would aid the evaluation of risks to human health, as well as the protection of aquatic ecosystems.

Ambystoma mexicanum↗
PubMed1995

Detection of germ cell mutagenicity of trophosphamide by the spermatid micronucleus test in the rat.

The effects of the antineoplastic drug trophosphamide (TP) on male rat germ cells were examined with the spermatid micronucleus test (SMNT). We used the microdissection technique in order to isolate stage I of the seminiferous epithelial cycle, where the cells that have just passed the meiotic divisions can be found. Micronuclei (MN) were scored at different time-points after TP treatment at dose levels of 25 and 50 mg/kg. An induction of MN was detected in cells exposed at preleptotene (18 and 19 days) and late pachytene (3 days), as well as at the diplotene-diakinesis stage (1 day). The dose-response for MN induction was linear at all time intervals studied, except for 18 days time point. The highest frequency of MN (5.20 +/- 0.57/1000 spermatids) could be found with the lower TP dose at 18 days, corresponding to exposed preleptotene spermatocytes and reflecting S-dependent clastogenicity. While a significant increase in MN could only be detected in exposed preleptotene spermatocytes with the lower TP dose, the higher dose level also induced MN significantly in late pachytene and the diplotene-diakinesis stage. DNA flow cytometry at 18 days showed cytotoxicity of TP to exposed primary spermatocytes at pachytene, but no cytotoxicity to the preleptotene spermatocytes that exhibited a significant MN induction. The results show that the SMNT using the stage-I-specific examination of the rat seminiferous epithelium can detect the germ cell mutagenicity of TP and gives further evidence of the usefulness of this technique in the testing of chemicals for genotoxic effects in male germ cells.

Animals↗
PubMed1993

In vitro micronucleus test of the cardioprotective agent stobadine. A genotoxicological study.

The genotoxic effect of stobadine (1) was studied in vitro using the micronucleus test. Hamster and human fibroblastoid cells were used. In hamster cells, the highest concentration of 1 (1.10(-3) mol/l) caused a significant elevatin in the number of micronuclei, while in human cells no positive response was found for either of the concentrations used. Stobadine had no genotoxic effect on human fibroblastoid cells.

Animals↗
PubMed1999

Genotoxicity of water samples from Dianchi lake detected by the Vicia faba micronucleus test.

Dianchi Lake covers about a 300 km2 area. Kunming city on the edge of the lake is surrounded by industrial establishments. Farm land surrounds the remaining areas of the lake. The lake water is polluted by the Kunming city municipal sewage from 3 million inhabitants, the industrial effluent and farm runoff. Water samples were collected from 12 sites along the shore of the lake during the dry (May) and rainy (August) seasons for genotoxicity testing with the Vicia micronucleus assay during the year 1995. Genotoxicity in terms of micronuclei (MCN) frequencies in the root tip cells of Vicia showed a consistent elevated frequency of MCN over the control sample at the 0.05-0.01 levels of significance in both seasons. The MCN frequencies of sites A (Daguanhe), B (Gaoqiao) and C (Xiyuan) were 3.5-4 times as high as the control values (5.25/1000 cells) in the dry season. In the rainy season, the MCN frequencies of water samples from most of the 12 sites were relatively lower than those of the dry season except sites J (Haikou), and K (Kunyang). The average MCN frequency of the dry season samples was 14.97 per 1000 cells and that of the rainy season samples was 12.24 per 1000 cells while the average control value was around 5.00/1000 cells.

Biological Assay↗
PubMed1999

Evaluation of 10 aliphatic halogenated hydrocarbons in the mouse bone marrow micronucleus test.

Ten halogenated aliphatic hydrocarbons (carbon tetrachloride, 1-chlorohexane, 2,3-dichlorobutane, 1,2-dichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, hexachloroethane, 1,1,2-trichloroethane, 1,2,3-trichloropropane and 1,1,3-trichloropropene), previously assayed in genetic assays in fungi, were evaluated in the mouse bone marrow micronucleus test in order to assess their genotoxicity in vivo. All chemicals were administered once i.p. at 40 and 70-80% of their respective LD50 to male and female CD-1 mice, 24 and 48 h before killing. All treatments produced evident clinical symptoms, but no marked depression of bone marrow proliferation. No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes over the control values were observed at any sampling time with any of the 10 halogenated hydrocarbons assayed. The comparison of the results obtained in this study with the findings provided by in vitro micronucleus assays on the same chemicals, reported by other authors, indicate that mouse bone marrow is weakly sensitive to the genotoxic effects induced by halogenated hydrocarbons in other test systems. This suggests that the role of such an assay in carcinogen screening may be questionable for this chemical class. An examination of mouse bone marrow micronucleus test results with the halogenated aliphatic hydrocarbons classified as carcinogens by IARC supports this conclusion.

Animals↗
PubMed1995

Clastogenic and aneugenic effects of three benzimidazole derivatives in the in vitro micronucleus test using human lymphocytes.

Three benzimidazole compounds thiabendazole (TBZ), carbendazim (MBC) and mebendazole (MEB) were analysed with the in vitro cytochalasin-B micronucleus test on human lymphocytes. TBZ was tested in isolated lymphocyte cultures and MBC and MEB were tested in both isolated lymphocyte and whole blood cultures. TBZ was tested up to 300 microM with and without S9-mix. Although signs of toxicity, without S9, were observed by a decrease in the division index at 300 microM, an increase in the frequency of micronucleated binucleates was not found with or without S9. MBC and MEB induced a statistically significant concentration-dependent increase in the micronucleus frequency. The effective concentration range for MEB (0.3-1.5 microM) was ten times lower than for MBC (5-25 microM). By means of fluorescence in situ hybridization with a 30 nucleotide oligomer of the alpha centromeric regions, common for all chromosomes, on the induced micronuclei MBC and MEB were found to induce a significant increase of centromere positive micronuclei in a dose-dependent manner. MBC and MEB are poorly soluble in water and therefore have a low bioavailability in vivo. However, increased micronucleus frequencies were found in this in vitro micronucleus study at doses comparable to in vivo plasma levels in mice and should, therefore, not be neglected in the risk evaluation of those compounds.

Adult↗
PubMedDate not supplied

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides.

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.

Acridine Orange↗
PubMed2004

Examination of the potential genotoxicity of pure capsaicin in bacterial mutation, chromosome aberration, and rodent micronucleus tests.

There is widespread dietary exposure to capsaicin in the form of chili peppers, while capsaicin's analgesic qualities have led to increased use of a topical herbal remedy in various impure forms. Most recently, injection of pure capsaicin has been proposed as a means of relieving a variety of debilitating diseases, in which case tissues would receive relatively high and direct exposure. The purpose of the present study, where a series of standard assays were performed in accordance with the Organisation for Economic Cooperation and Development guidance, was to clarify earlier conflicting reports concerning potential genotoxicity of capsaicin prior to administering it to patients in an injectable form. The results confirm the absence of genotoxic activity of high-purity capsaicin in the bacterial mutation and chromosome aberration tests. In addition, no evidence of cytotoxicity or genotoxicity was seen in the rat bone marrow micronucleus test, where systemic exposure to pure capsaicin was achieved using the subcutaneous route and a rising dose toleration protocol. It is concluded that pure capsaicin is not active in the standard battery of genotoxicity assays recommended by the International Conference on Harmonisation for evaluation of new medicines; earlier reported in vitro genotoxic activity is probably associated with mutagenic impurities in commercial grades of the material.

Animals↗
PubMed1980

Studies on benzene mutagenesis. I. The micronucleus test.

The mutagenic action of benzene was studied by means of the micronucleus test performed on mice. A linear dose effect relationship was found for the percentage of micronucleated erythrocytes, against the benzene-dose logarithms. A significant dose effect correlation was found either after the standard 30-h experiments or after a prolonged 54-h one. A higher effect was found in the prolonged experiments, suggesting the induction of a delay in the cell cycle by benzene.

Animals↗
PubMed2005

Comparative study of the comet assay and the micronucleus test in amphibian larvae (Xenopus laevis) using benzo(a)pyrene, ethyl methanesulfonate, and methyl methanesulfonate: establishment of a positive control in the amphibian comet assay.

The present investigation explored the potential use of the comet assay (CA) as a genotoxicity test in the amphibian Xenopus laevis and compared it with the French standard micronucleus test (MNT). Benzo[a]pyrene (B[a]P), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS) were used as model compounds for assessing DNA damage. Damage levels were measured as DNA strand breaks after alkaline electrophoresis of nuclei isolated from larval amphibian erythrocytes using the CA in order to establish a positive control for further ecotoxicological investigations. The results led to the selection of MMS as a positive control on the basis of the higher sensitivity of Xenopus laevis to this compound. The CA and MNT were compared for their ability to detect DNA damage with the doses of chemical agents and exposure times applied. EMS and MMS were shown to increase micronucleus and DNA strand break formation in larval erythrocytes concurrently. However, B[a]P increased micronucleus formation but not that of DNA strand breaks. Time-dose experiments over 12 days of exposure suggest that the CA provides an earlier significant response to genotoxicants than does the MNT. In Xenopus the CA appears to be a sensitive and suitable method for detecting genotoxicity like that caused by EMS and MMS. It can be considered a genotoxicity-screening tool. The results for B[a]P show that both tests should be used in a complementary manner on Xenopus.

Animals↗
PubMed2001

Anticlastogenicity of two derivatives of 1,4-dihydroisonicotinic acid in mouse micronucleus test.

Effects of two derivatives of 1,4-dihydroisonicotinic acid (1,4-DHINA) against the monofunctional alkylating agent ethyl methanesulfonate (EMS) were studied in the micronucleus test in (CBA x C57Bl/6(j)) mice. Adult males and pregnant females were treated with an antimutagen (i.p.) and 12h later they were exposed to EMS (i.p.). The frequencies of micronucleated (MN) polychromatic erythrocytes (PCEs) in mouse bone marrow and foetal liver were analysed 6, 12, 18, 24, 30, 36, 48 or 24, 48 and 72 h after the mutagen injection. In adults, the maximum number of MNPCEs was observed 36 or 24h after the EMS administration. In foetuses, which were treated in a maternal organism, such peak was found at 24h. Pre-treatment of mice with the antimutagens 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (DHP) and glutapyrone (GP) decreased the yield of MNPCEs in male bone marrow. Having been observed at a peak of MN induction, the anticlastogenic effect of DHP (1/10 LD(50) or 340 mg/kg) reached 30%. DHP at the doses of 0.5-1mM/kg did not affect the EMS-induced frequency of MNPCEs in bone marrow, whereas GP inhibited it at the similar millimolar concentrations. Simultaneously with maternal bone marrow, foetal liver cells were analysed for MNs in the transplacental test. The anticlastogenic effect of DHP (1/10 LD(50)) was found to be more prolonged and higher in females than in males and to average 50%, but this antimutagen was not efficient in foetuses. Both antimutagens did not change the polychromatic/normochromatic erythrocyte (PCE/NCE) ratio as compared with EMS action. Results presented indicate a peak of EMS-induced micronucleated cells in mouse bone marrow 24 or 36 h and in foetal liver 24h after animal treatment. Two 1,4-DHINA derivatives exhibited anticlastogenic activity in adults, but not in foetuses.

Animals↗
PubMed1983

The micronucleus test with mouse spleen cells.

The results of this study show that the micronucleus test can be carried out with mouse spleen cells as well as with cells from bone marrow. Polychromatic erythrocytes occurred in the spleen at a frequency of about 9% of the whole spleen cells compared with about 13% in the bone marrow. 3 test compounds were used to compare the frequency of micronuclei in cells from the 2 tissues. Mitomycin C and cyclophosphamide induced micronucleated polychromatic erythrocytes in both spleen and bone marrow. Fosfomycin, an antibiotic having a broad spectrum of antimicrobial activities, did not induce micronucleated erythrocytes in either organ.

Animals↗
PubMed1994

The spermatid micronucleus test with the dissection technique detects the germ cell mutagenicity of acrylamide in rat meiotic cells.

As a part of the development and validation of the spermatid micronucleus test (SMNT) in the project 'Detection of Germ Cell Mutagens' sponsored by the CEC we studied the mutagenicity of acrylamide (AA) and mitomycin C (MMC). Of two alternative techniques, we used the 'dissection technique' based on microdissection of seminiferous tubules offering a narrow window for evaluation of cell stage sensitivity, and including DNA-specific staining and scoring. AA given as a single injection of 50 or 100 mg/kg did not significantly increase MN frequencies. When a subchronic treatment (4 x 50 mg/kg) was given, a significant increase over background was observed 18 and 19 days after the last injection, indicating genotoxic activity in preleptotene spermatocytes and late spermatogonial stages. MMC given as single injections of 0.5 or 1.0 mg/kg increased MN frequencies significantly 17, 18, 19 and 20 days after treatment as a result of clastogenicity in S phase cells. DNA flow cytometry did not show cytotoxicity of AA to preleptotene spermatocytes, but a small decrease in the numbers of stem cells. If spindle disturbances are caused by AA, as suggested, they were not detectable by induction of spermatid MN in vivo 1 or 3 days after treatment or by treatment with AA of cultured segments of seminiferous tubules undergoing meiotic divisions in vitro. In conclusion, the SMNT with the dissection technique is able to show the germ cell clastogenicity of AA and MMC. AA was observed to have a much weaker MN inducing potency than MMC.

Acrylamide↗
PubMed1990

Acute cytogenetic effects of tyramine and MTCAs on mouse bone marrow cells in vivo by the micronucleus test.

We studied the acute cytogenetic effects of tyramine and MTCAs--precursors of the mutagen present in soy sauce--on mouse bone marrow cells in vivo by the micronucleus test. The incidence of MNPCE in bone marrow cells gradually increased and reached a maximum level 24 h after intraperitoneal injection of tyramine or MTCAs and decreased within 36 h. A dose-dependent increase in MNPCE was clearly observed for both compounds. Compared to the values for the untreated control, significant positive results were obtained with 0.5 mmole tyramine/kg (68.5 mg/kg) and with 0.1 mmole MTCAs/kg (23 mg/kg) 24 h after intraperitoneal administrations. Micronuclei were significantly induced but no severe reduction in the ratio of PCEs/NCEs was observed.

Animals↗
PubMed2005

Mercury chloride genotoxicity in rats following oral exposure, evaluated by comet assay and micronucleus test.

Mercury is a toxic element which is easily absorbed after ingestion or inhalation and deposited mainly in the kidney. The aim of this study was to evaluate the effects of mercury chloride in rats. Female rats, aged 14 weeks, were receiving mercury chloride in oral doses of 0.068, 0.136, and 0.272 mg kg(-1) body weight (b.wt.) for five consecutive days. Three days after the last dose, the animals were killed. The liver and the kidney were dissected and mercury measured using vapour generation atomic absorption spectrometry. The results show a significant increase in mercury mass fraction in the kidney after two higher doses of mercury chloride, while liver mercury burden showed a significant increase only after the highest dose. Blood samples were analysed using the comet assay and supravitally acridine orange stained micronucleus test. Tail length, tail moment and micronucleus frequency were significantly higher in the treated rats than in control rats, regardless of the dose of mercury chloride, while the difference between the treated groups for both comet and micronucleus parameters was not statistically significant.

Administration, Oral↗
PubMed1987

Lack of genotoxic effects of fluoride in the mouse bone-marrow micronucleus test.

The purpose of this investigation was to examine the potential genotoxic influence of sodium fluoride (NaF) on mammalian cells by means of a mouse bone-marrow micronucleus test. Mice of genotype B6C3F1 were obtained at about eight weeks of age and maintained on a low-fluoride diet (< 0.2 ppm F) and distilled water ad libitum throughout the experiment. At approximately 12 weeks of age, the animals were randomly assigned to seven groups, with multiple sampling schedules. The animals were intubated with various doses of NaF, ranging from 0.1 mg/kg to the Maximum Tolerable Dose (MTD), with sampling at 30, 48, and 72 hours after treatment. Negative (distilled water) and positive (cyclophosphamide) controls were included. Coded slides of femur marrow cells were prepared and examined, without identification as to treatments, for the frequency of micronucleated polychromatic erythrocytes (MN-PCE). The humera were analyzed for fluoride for monitoring of the absorption of fluoride following oral intubation. The results indicated that doses of NaF up to the MTD did not significantly increase the frequencies of MN-PCE when compared with the negative controls, although the bone fluoride content increased as the dose of NaF was increased.

Analysis of Variance↗