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Urethane is readily detectable as an in vivo clastogen in the micronucleus test after oral administration to both rats and mice. Mice appear to be the more sensitive species. Positive results were obtained irrespective of the strain (CD1, CRH) or dosing regimen (single, triple-dose) used.
The in vivo mammalian micronucleus test can be conducted easily on peripheral blood samples since the maturation of erythrocytes involves the loss of the major nucleus. In addition, mature erythrocytes are relatively long-lived, so that the test potentially can detect genotoxic damage caused by chronic exposures. However, some species have spleens that remove micronuclei from the peripheral circulation, making such measurements problematical. This report summarises haematological and mutagenesis studies dealing with this subject and provides an anatomical interpretation of the phenomenon. Anatomical features can be used to identify those species in which micronuclei are removed by the spleen.
It has been reported that the sensitivity of the in vivo mouse bone marrow micronucleus test can be increased by inducing erythropoiesis with exogenous erythropoietin prior to treatment (Suzuki et al., 1989). In these studies we demonstrate that removing approximately 0.5 ml of blood from an adult male BDF1 mouse, another method for increasing the rate of erythropoiesis, synergistically increased the frequency of bone marrow micronucleated polychromatic erythrocytes induced by mitomycin C, with maximal enhancement occurring when the mutagen was given 24 h after bleeding. This enhancement response was also demonstrated for benzo[a]pyrene and dimethylnitrosamine but not for 2-acetylaminofluorene. These results indicate that bleeding mice prior to chemical treatment is a simple method for increasing the sensitivity of the micronucleus assay.
6 dosages of diethylstilbestrol-diphosphate (DES-dp), ranging from 0.01 to 500 mg per kg of body weight were compared to saline and phosphate buffered saline (negative controls) and two dosages of cyclophosphamide (positive control) in the micronucleus test with 115 ICR mice. DES-dp failed to generate a significant increase in micronucleated polychromatic erythrocytes over negative controls. Cyclophosphamide produced a dose-related increase in micronuclei similar to previously published reports. Iit was therefore determined that the micronucleus test did not detect the types of chromosomal changes known to be generated by DES-dp and DES.
To study possible clastogenic effects of 4-chloromethylbiphenyl (4CMB), a mouse micronucleus test was performed (as part of the UKEMS genetic toxicology trial). Mice were given 4CMB by intraperitoneal injection, once daily for 5 consecutive days. Although the range of dosages employed included highly toxic or lethal doses, the frequency of micronucleated polychromatic erythrocytes in the bone marrow was not significantly greater in 4CMB-treated groups, than in control-group animals treated with the Tween 80 vehicle alone. Thus, in this test, 4CMB did not induce chromosomal damage resulting in micronucleus formation, in bone-marrow erythrocytes.
Cytogenetic studies were performed in 22 patients treated with methotrexate (MTX). In some patients, metaphases from both bone-marrow cells and peripheral blood cells were studied. In the bone-marrow preparations an increased number of structural chromosomal aberrations was present, whereas abnormalities were not observed in the peripheral blood cells. An examination of the bone-marrow chromosomes must therefore be included in the study of the possible chromosome-breaking effect of chemical agents. The results obtained with the micronucleus test and chromosome studies were compared in 10 patients treated with MTX. The micronucleus test was more sensitive than the chromosome analysis as regards the clastogenic effect of MTX.
The purposes of the present study were the evaluation of the genotoxic effect of mixture of paraquat and linuron commercial formulations. the evaluation of the possible interaction between the two pesticides and the comparison of different statistical tests used for the data analysis. For this purposes the bone marrow micronucleus test was used. Dose levels were determined in the base of the LD50 of each component. According the experimental design, three dose levels of the mixture were administered at groups of five Wistar rats from each sex. Sampling time was 24 h for all groups plus 48 h for the highest dose and the positive control groups. In addition, the formulations were studied separately at two dose levels. Cyclophosphamide was used as positive control. For the statistical analysis of the results different parametric and nonparametric tests were used. The mixture of the two pesticides under the study conditions was not proven to be genotoxic. However, cytotoxicity and no signs of interaction were indicated. Minor declinations between the statistical tests used for data analysis observed.
The effects of vincristine sulfate (VINC) on micronucleus induction were studied in 2 strains of mice (MS/Ae: CD-1) following intraperitoneal (i.p.) or oral administration (p.o.) of the chemical. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the full-scale micronucleus test was performed with a sampling time of 24 h at doses of 0.063, 0.125, 0.25 and 0.5 mg/kg (i.p.) and 1.25, 2.5, 5.0 and 10 mg/kg (p.o.). The maximum frequency of micronucleated polychromatic erythrocytes was 7.15% in MS/Ae mice and 4.98% in CD-1 mice at 5.0 mg/kg p.o. in both cases. The maximum frequencies by the i.p. route (9.93% in MS/Ae mice; 11.68% in CD-1 mice) occurred at 0.25 mg/kg and 0.125 mg/kg, respectively. Although the doses showing a positive response were different between the 2 routes, VINC induced micronuclei very efficiently at all doses tested by both administration routes in both strains.
The in vitro micronucleus test is available to demonstrate the clastogenic and aneugenic potencies of chemicals. This test is easier to evaluate than metaphase analysis and, although used routinely by some laboratories as a prescreening test for the determination of chromosomal mutation induction potency, it has not been recognised by regulatory authorities as a test to be included in the battery of assays for human risk assessment. This limitation is due to the lack of a fixed protocol, a limited data bank, particularly in the case of clastogens, and to the lack of sufficient robust interlaboratory validation studies. If we aim to recognize this test in the form of an OECD guideline and to introduce it as an alternative to in vitro metaphase analysis in cell culture, it is necessary to begin a collaborative study in order to determine the best protocol which is valid for the detection of all clastogens as well as to determine clearly the limits of the test. The use of this in vitro test as a prescreening assay could be very useful as it takes structural and numerical chromosomal abnormalities into account and is simpler to perform than in vitro metaphase analysis.
The mutagenic potential of gadobenate dimeglumine formulation (E7155) was studied by the micronucleus test in rats. Single intraperitoneal injection of E7155 to Sprague Dawley rats at the dose of 5295.2 mg/kg (5 mmol/kg) did not induce any statistically significant increase in the frequency of micronucleate cells in the bone marrow sampled after 18, 42 and 66 hr from time of administration.
In the light of the CEC aneuploidy programme eight known or suspected aneugens were evaluated with the human lymphocyte 'in vitro' micronucleus test using the cytochalasin-B technique. Only colchicine, chloral hydrate and hydroquinone induced dose-dependent increases in micronucleus frequencies. The other five chemicals (cadmium chloride, econazole, pyrimethamine, thiabendazole and thimerosal) were all negative, both with and without S9, and treated in G1 or G2 phase. These data are in good agreement with results from different 'in vivo' studies. However, discrepancies were found between the results from this study of human lymphocytes and those of other cultured cell types where econazole, cadmium chloride, pyrimethamine and thiabendazole induce a significant increase of micronuclei.
The micronucleus test using erythrocytes of Pleurodeles waltl larvae (Amphibia, Salamandridae) was used to detect the possible genotoxicity of four polycyclic aromatic hydrocarbons (naphthalene, anthracene, phenanthrene and benzo[a]pyrene), which represent a major fraction of crude oil. Larvae were reared in water containing the test compound and the levels of micronucleated red blood cells were compared with those found in larvae reared in control water. The results are compared with published data from other tests used to evaluate the clastogenic or mutagenic properties of compounds. The results obtained confirm that benzo[a]pyrene has a strong genotoxic potential, whereas the genotoxicity of naphthalene is weak; in contrast, anthracene and phenanthrene gave a negative response.
A comparative analysis between the in vivo comet assay and the in vivo micronucleus test (MNT) was carried out in three aquatic organisms suitable for genotoxicity monitoring, carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and clam (Spisula sachalinensis), using a direct-acting mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and an indirect mutagen, benzo[a]pyrene (B[a]P). By optimizing the conditions for cell isolation, gill and liver (or digestive glands) were selected as test tissues of the comet assay for MNNG and B[a]P. The MNT employed the erythrocytes (or hemocytes), the most universal cell type for the assay. The analysis of DNA strand breaks using the comet assay and the micronucleus frequencies using the MNT revealed dose- and time-dependent increases between animals exposed to several concentrations of mutagens. But the statistical significance (P<0.05) obtained was higher by the comet assay than by the MNT. When the time profiles of genotoxic signals resulting from B[a]P exposure to carp were plotted representatively, clear distinctions between all concentrations were made in the comet assay, but not in the MNT. The correlation index defined in this study also showed a higher correlation between concentration and signal in the comet assay than in the MNT. It is suggested that the standardization of the comet assay is necessary for its methodological evaluation and use as a genotoxicity biomarker. We conclude that the comet assay has an excellent suitability for aquatic genotoxicity monitoring because of its high and reliable sensitivity.
The antimutagenic activity Angelica archangelica L. water and alcohol extracts thio-tepa against mutagenicity was investigated by the micronucleus test in mouse bone marrow and peripheral blood cells. The reduction of thio-tepa mutagenic activity was more prominent when the extracts were injected 2-hours before thio-tepa treatment as it could be seen at the simultaneous treatment. The observed reduction of micronucleus frequencies was up to 77%. No genotoxic effects of Angelica extracts had been seen at the concentrations 50-100 mg/kg.
The mutagenic activity of vastak and durs ban pesticides was studied by the micronucleus test in mouse bone marrow. The frequency of micronuclei in polychromatic erythrocytes was tested at 24, 36 and 42 h after oral administration of 50% LD50 dose of vastak (14 mg/kg) and durs ban (30.5 mg/kg). Significantly different increase in micronucleated polychromatic erythrocytes was established at 24, 36 and 48 h after vastak administration, and at 24 and 36 h after durs ban treatment. Doses of 25% LD50 for both pesticides showed no mutagenic activity, as judged by the induction of micronuclei in polychromatic erythrocytes.
The genotoxic potential of the pyrethroid flumethrin was evaluated by using the combined protocol of metaphase analysis and micronucleus test in vivo in mouse bone marrow. The dermal route was tested in a single treatment and the intraperitoneal (i.p.) route in a single and a multiple treatment. Flumethrin showed a cytotoxic effect on both myelopoiesis and erythropoiesis, as evidenced by a reduction in the mitotic index and in polychromatic erythrocyte values. An increase in the frequency of gaps after the dermal exposure and of breaks only at the highest dose tested in the i.p. treatment indicates a weak clastogenic potential of the compound. A significant increase in the frequency of micronucleated polychromatic erythrocytes was observed after single and multiple i.p. treatments. In the latter, the induction of micronuclei was highly significant but not accompanied by an increase in breaks. This may indicate that the clastogenic effect might not account by itself for the induction of micronuclei, which could also have arisen from an aneugenic potential of flumethrin.
The micronucleus test with peripheral blood using acridine orange-coated slides was validated in male CD-1 mice treated once with N-ethyl-N-nitrosourea (ENU) at doses of 6.25, 12.5, 25.0, and 50.0 mg/kg body weight. Peripheral blood preparations were made 0, 24, 48, and 72 h after treatment. The frequencies of micronucleated peripheral reticulocytes in the ENU-treated groups increased dose-dependently, peaking at 48 h after treatment. The results indicate that the method used in the present study can be an alternative to the method using bone marrow polychromatic erythrocytes.
The usefulness of the micronucleus assay using mouse peripheral blood erythrocytes and acridine orange (AO)-coated slides was evaluated with methyl methanesulfonate (MMS). The micronucleus test was carried out at doses ranging from 20 to 80 mg/kg body weight in CD-1 mice by intraperitoneal injection. Peripheral blood cells were examined from 0 to 72 h after treatment at 12- or 24-h intervals. Bone marrow cells from other mice treated with 80 mg/kg MMS were also sampled at the same times. The frequency of micronucleated reticulocytes (MNRETs) increased dose-dependently at every sampling time except 72 h, and the maximum frequency of MNRETs was observed at about 36 h after treatment. Micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow after a dose of 80 mg/kg were significantly induced at 12 h to 36 h, and the maximum frequency of MNPCEs was observed at 24 h after treatment. The induction of MNRETs was delayed by about 12 h compared to that of MNPCEs in bone marrow, and the maximum frequencies of MNRETs were lower than those of MNPCEs, but the induction of MNRETs by MMS was significant and dose-dependent. It is concluded, therefore, that bone marrow cells could be replaced by peripheral blood cells as material for the micronucleus assay using AO-coated slides.