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Results for “Micronucleus Tests”

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PubMed1996

Studies for a genotoxic potential of some endogenous and exogenous sex steroids. II. Communication: examination for the induction of cytogenetic damage using the chromosomal aberration assay on human lymphocytes in vitro and the mouse bone marrow micronucleus test in vivo.

The cytogenetic potential of 10 sex steroids (cyproterone acetate, drospirenone, gestodene, cyclodiol, cyclotriol, ethinylestradiol, atamestane, lilopristone, onapristone and propylmesterolone) with various medical indications was determined using the chromosomal aberration test in human lymphocytes in vitro and the mouse bone marrow micronucleus test in vivo. Nine of these sex steroids (gestodene was omitted) were investigated in the human lymphocyte assay and found to be negative with respect to the induction of chromosomal aberrations either with or without metabolic activation. In all assays the highest concentration evaluated was either clearly cytotoxic or, in case of noncytotoxicity, resulted in visible precipitates in the culture medium. Evaluation of the data from the mouse bone marrow micronucleus test indicated that the seven steroids (cyproterone acetate, drospirenone, gestodene, ethinylestradiol, atamestane, onapristone and propylmesterolone) investigated failed to induce enhanced frequencies of micronucleated polychromatic erythrocytes in male and female mice. The steroids were tested up to dose levels which induced signs of toxicity in the experimental animals or, in the case of non toxic compounds, the animals were treated up to the maximum recommended dose of 2 g/kg body weight. Evaluation of all data indicates that the investigated estrogens, progestins and other sex steroids had no genotoxic potential detectable with the chromosomal aberration assay on cultured human lymphocytes or the mouse bone marrow micronucleus test.

Animals↗

PubMed1984

Micronuclei in human bone-marrow cells: evaluation of the micronucleus test using human leukemia patients treated with antileukemic agents.

The relevance of the micronucleus test to human studies was investigated by using bone marrow from leukemic patients treated with antileukemic drugs. The median incidence of micronucleated erythrocytes and erythroblasts, respectively, increased from control values of 0.04 and 0.72% to 0.29 and 25.3% in leukemic cases; and the frequency of micronucleated erythroblasts was inevitably higher than the control value in cases that showed a higher frequency of micronucleated erythrocytes, but the reverse was not true. These results indicate that almost the same changes of micronucleus formation that are observed in the mouse micronucleus test are produced in human bone marrow by antileukemic drugs--mutagenic compounds--and, if the micronuclei were scored restrictively in erythroblasts, the application of the micronucleus test to human bone marrow would be reasonable.

Adolescent↗

PubMed2006

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.

Animals↗

PubMed1997

The chromosomal effect of birchen dust as determined by the micronucleus test.

In a wood processing factory, the measured air concentration of birchen dust was 1.26 +/- 0.41 mg/m3, and the micronucleus frequency of peripheral blood lymphocytes in 83 workers exposed to wood dust was 1.13 +/- 2.83/1000, which was significantly higher (P < 0.01) than that of control group (0.51 +/- 1.41/1000). The number of exposed workers with positive micronucleus test was 9.6%, which was higher than that of control group (4.5%), but the difference was not significant (P > 0.05). The micronucleus test in mice treated with water extracts of unsteamed and unbaked birchen dust showed that the micronucleus frequencies in all treated groups were significantly higher than that of control group (P < 0.01) and there was also a dose response correlation (r = 0.96, P < 0.0005). The results of steamed and baked birchen dust extracts were significantly lower than those of the unsteamed and unbaked ones at the same doses (P < 0.001). This suggests that when the birchen dust is steamed at the temperature of 100 degrees C for 24 h or baked at the temperature of 80 degrees C, its inducing effect in micronucleus test could be lowered.

Adult↗

PubMed1991

Micronucleus test in 2-cell embryos as a simple assay system for human sperm chromosome aberrations.

A micronucleus test method to assess radiation-induced chromosomal damage in human spermatozoa is described, and its efficiency examined by comparison with that of sperm chromosome analysis. Human spermatozoa were exposed in vitro to 1.11 and 2.13 Gy of 137Cs gamma-rays at a dose rate of 1.36 Gy/min. After interspecific in vitro fertilization of irradiated spermatozoa with zona-free hamster oocytes, a total of 193 monospermic eggs were examined with the micronucleus test at the 2-cell stage, and a total of 304 male pronuclear chromosome plates were analyzed according to our established method. The incidence of 2-cell embryos with micronuclei coincided well with the incidence of spermatozoa with chromosomal breaks and fragments (51.6% vs. 50.3% in the 1.11-Gy group and 82.7% vs. 79.3% in the 2.13-Gy group). A similar correlation was also found between the number of micronuclei per embryo and the number of breaks and fragments per spermatozoon (0.85 vs. 0.88 and 1.50 vs. 1.45 in the 2 dose groups, respectively). These results indicate that our micronucleus test is useful as a simple and rapid method for assessing the clastogenic effects of various environmental mutagens on human sperm chromosomes.

Animals↗

PubMed1983

An application of Acridine Orange fluorescent staining to the micronucleus test.

Acridine Orange fluorescent staining was applied to the micronucleus test in mice and rats. Micronuclei emitted bright green fluorescence and were easily distinguished from micronucleus-like inclusions or contaminants. In rat bone-marrow cells, micronuclei with green fluorescence could be easily distinguished from granules accidentally dispersed from broken mast cells, which showed bright red fluorescence. Therefore, it is recommended that the Acridine Orange staining method be used to provide more reliable data in the micronucleus test.

Acridine Orange↗

PubMed1989

The automated bone marrow micronucleus test.

A new technology is presented which offers high-quality slides enabling the fully automated scoring of large quantities of erythrocytic cells for micronuclei by computerized image analysis. The techniques are applicable to bone marrow specimens as well as to peripheral blood obtained from various species of laboratory animals as well as from man. The key steps leading to this improved slide quality are the total removal of nucleated hematopoietic cells and the production of 'flat' cells by cytocentrifugation on polylysine-coated slides. The new procedures also allow the quantitative elimination of artifact-producing leukocytic granules from the rat bone marrow, even for the Fischer-344 strain, thus making the rat micronucleus test an attractive system for routine purposes in genetic toxicology. In addition, the proportion of immature erythrocytes can, if desired, be increased to more than 90% by using a Percoll step-gradient. This greatly facilitates the peripheral blood micronucleus test in laboratory animals as well as in (splenectomized) humans. First results, using peripheral blood from 2 rat strains, indicate that the immature erythrocyte population is very useful for micronucleus analysis, which encourages the development of a rat peripheral blood micronucleus test. This is an interesting application because it allows repeated testing in the same animals, resulting in fewer rats being needed, as no separate control groups are necessary. A further advantage is the possibility of concomitantly using rats from an ongoing toxicological study for micronucleus testing. The present results demonstrate that the new methodology is a valuable tool for improved micronucleus testing. Possible consequences in the field of genetic toxicology are discussed.

Animals↗

PubMedDate not supplied

Effects of benzene in a micronucleus test on peripheral blood utilizing acridine orange-coated slides.

A micronucleus test was conducted on the peripheral blood of mice and rats utilizing acridine orange-coated slides (AO-coated method) after oral administration of benzene. Blood was sampled at 0, 24, 48, and 72 h after administration of benzene at doses of 500, 1000, and 2000 mg/kg in both mice and rats. The highest occurrence of micronucleated reticulocytes (MNRETs) was observed at 48 h after administration in both species. Species differences was found in the frequency of MNRETs, with the number being lower in rats than in mice. The present results indicate that the micronucleus test can easily detect chromosome aberrations in peripheral blood induced by benzene administration in both mice and rats. Furthermore, the AO-coated method used in this study was simpler to perform and allowed for easier detection of the effect than the conventional method.

Acridine Orange↗

PubMed1987

Analysis of the results of the micronucleus test in patients presenting upper digestive tract cancers and in non-cancerous subjects.

A micronucleus test was performed on 75 subjects of whom 38 presented with cancer of the upper digestive tract and 37 were free of disease; the absence of cancerous or pre-cancerous lesions in this latter group was confirmed by endoscopy and vital staining. The daily levels of alcohol and tobacco consumption of the 75 subjects were determined by precise questioning: 78% of the non-cancerous subjects smoked less than 10 g of tobacco per day whereas 79% of the cancer patients smoked 10 g or more daily. The alcohol intake of 78% of the non-cancerous subjects and 63% of the cancer patients was less than 101 ml per day. Only 10% of the cancer patients had combined daily intake levels corresponding to the threshold of sensitivity of the micronucleus test as defined by previous studies. The mean frequency of micronucleated buccal cells was 0.26% in the cancer patients and 0.13% in the non-cancerous subjects. All non-cancerous patients presented a negative test. Only 5% of the cancer patients presented a micronucleated cell frequency above 1% and could thus be considered as positive. It thus appears that the micronucleus test was not significantly positive in our population of 38 cancer patients.

Adult↗

PubMed1989

Effect of route of administration in the micronucleus test with potassium bromate.

The effect of intraperitoneal injection (i.p.) versus oral gavage administration (p.o.) of potassium bromate was examined using the micronucleus test in 2 strains of male mice (MS/Ae and CD-1). First, a small acute toxicity test and a pilot micronucleus experiment were performed to determine the appropriate dose range and sampling time for the full-scale micronucleus test. The full-scale test was carried out using doses of 18.8, 37.5, 75, and 150 mg/kg in the i.p. test and of 37.5, 75, 150, and 300 mg/kg in the p.o. test. The sampling time was 24 h for both mouse strains. Potassium bromate induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently by both routes of administration in both mouse strains. No distinct difference in route of administration was observed in the test with MS/Ae mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.

Administration, Oral↗

PubMed2001

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Animals↗

PubMedDate not supplied

Effects of multiple dosing of phenacetin in the micronucleus test.

As a part of the international cooperative study to identify the most sensitive regimen in the micronucleus test, phenacetin was given i.p. to male CD-1 mice at doses of 37.5, 75, 150, 300, 400, and 600 mg/kg once, twice, thrice or four times and the bone marrow cells were harvested 24 h after the final dosing. Positive responses were seen at 600 mg/kg after single and triple dosing and at 400 and 600 mg/kg after double dosing. No dose level gave a positive response after quadruple dosing. A repeated-dosing effect was detected at double and triple dosing. Although triple dosing gave the highest magnitude of micronuclei at 600 mg/kg, double dosing showed a sufficient sensitivity and was more convenient from the viewpoint of selecting a suitable test dose and carrying out the micronucleus test.

Animals↗

PubMed2000

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage.

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.

Acridine Orange↗

PubMed1989

Micronucleus test with 2-acetylaminofluorene by intraperitoneal injection and oral administration.

The effect of route of administration on the outcome of the mouse micronucleus test was evaluated in 2 laboratories by administering 2-acetylaminofluorene (2-AAF) by intraperitoneal injection (i.p.) and oral gavage (p.o.) to 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus test, the full-scale experiment was performed with a 24-h sampling time at doses ranging from 75 to 600 mg/kg by both routes. The results indicated that 2-AAF induced micronucleated polychromatic erythrocytes (MNPCEs) at all doses tested by both routes. In the MS/Ae strain, higher doses were required by p.o. than by i.p. to reach a similar level of MNPCE incidence. On the other hand, similar responses were recorded by both administration routes with CD-1 mice. Since the LD50 for the p.o. route was higher than that for the i.p. route in both strains, the route-related difference with MS/Ae mice became small when the comparison between i.p. and p.o. was made on the basis of the LD50. Thus both i.p. and p.o. routes are acceptable in the micronucleus test of this chemical.

2-Acetylaminofluorene↗

PubMed2000

Evaluation of the effect of steviol on chromosomal damage using micronucleus test in three laboratory animal species.

The chromosomal damage activity of steviol, a product of enzymatic alteration of stevioside, a natural non-caloric sweetener was reevaluated by using a bone marrow micronucleus test in both male and female hamsters, rats and mice. The micronucleus test is used widely as a rapid and efficient alternative in chromosome analysis for detecting in vivo cytogenetic damage. Steviol at the dose of 4 g/kg body weight for hamsters and 8 g/kg body weight for rats and mice showed no effect on the frequencies of micronucleus formation in bone marrow erythrocytes of both male and female hamsters, rats and mice. Moreover, there was also no apparent change in the PCEs:NCEs (polychromatic erythrocytes:normochromatic erythrocytes) ratio of the male animals of all three treated species at 24, 30, 48 and 72 hour intervals. However, steviol at the given dose can cause significant reduction of PCEs to NCEs ratio of the female hamsters at 72 hours and female rats and mice at 48 and 72 hours after receiving steviol orally. From these results, it could be proposed that steviol at the given dose to the treated animals produced adverse metabolites and these metabolites could reach the bone marrow, the target organ for micronucleus test. These metabolites also exhibited a slightly cytotoxic effect but not clastogenic effect to the bone marrow erythrocytes.

Administration, Oral↗

PubMed1995

Evaluation of hydroquinone and chloral hydrate on the in vitro micronucleus test on isolated lymphocytes.

The cytochalasin B micronucleus test was performed in human peripheral lymphocyte cultures to assay the ability of hydroquinone and chloral hydrate to induce micronuclei, in the presence or absence of an exogenous metabolic activation system. Cultures and readings were performed in duplicate. No significant damage was found after treatment with chloral hydrate in this test system, whereas hydroquinone induced a clear positive response in one culture in the presence of metabolic activation during the G1 phase. Isolated lymphocytes used as a test system provide information about the test compound itself without interference by blood components. Comparison of the two readers' data showed few marked discrepancies in the number of micronuclei recorded in binucleated cells. Strict criteria for data analysis are therefore necessary to avoid intra-assay or operator variability.

Adult↗

PubMed1997

In vivo genotoxicity of selected herbicides in the mouse bone-marrow micronucleus test.

The herbicides alachlor, atrazine, terbuthylazine, gluphosinate-ammonium, isoproturon, pendimethaline and trifluralin were tested for genotoxicity in the mouse bone-marrow micronucleus test (MNT). Both atrazine and trifluraline caused a significant increase in the number of micronuclei at doses of 1,400 mg/kg body weight in female mice only. Alachlor, terbuthylazine, gluphosinate-ammonium, isoproturon and pendimethaline did not have any genotoxic effect in the mouse bone-marrow micronucleus test in either female or male animals.

Animals↗

PubMed1992

Mouse bone marrow micronucleus test using flow cytometry.

At present, the micronucleus test is the most popular short-term assay for the in vivo detection of clastogens or spindle poisons. As micronuclei are rare events, it has been argued that the conventional microscopical analysis based on 500-2000 cells per animal does not provide enough sensitivity. In addition, the manual scoring of micronuclei is time-consuming and tedious. As an attempt to solve these problems, an automated method for the analysis of micronuclei in mouse bone marrow erythrocytes was developed using flow cytometry. Femoral bone marrow cells, from mice treated with known in vivo clastogens [mitomycin C (MMC): 0.5, 1 or 2 mg/kg body weight intraperitoneally; benzene: 1000, 2000 or 4000 mg/kg body weight orally] or vehicles for the clastogens (physiological saline, olive oil; for control animals), were fixed with 1% glutaraldehyde, suspended in 70% ethanol for storing, stained with 4',6-diamidino-2-phenylindole (0.5 microgram/ml) and analyzed (50,000 cells per sample) in an EPICS V flow cytometer using a Coherent 5W UV laser at 150-200 mV. The frequency of micronucleated erythrocytes (MNEs) was calculated from the cytometer, by a computer program involving model fitting to normal Gaussian distribution. Correlations between MNE frequencies obtained by manual microscopic observations (1000-2000 cells per animal) and by the flow cytometric measurements were good for both group results (means of 4-5 mice; linear correlation coefficient r = 0.89-0.998) and individual data (r = 0.82-0.96). Repeated experiments with MMC showed good reproducibility for the flow cytometric scoring of micronuclei.

Algorithms↗