The micronucleus assay as a test for the detection of aneugenic activity

The aim of this work was to determine the usefulness of the micronucleus assay for the detection of aneugenic potential. Chemicals affecting microtubule assembly, i.e., colchicine, vinblastine sulfate and tubulazole, and chemicals affecting targets other than microtubuli, i.e., mitomycin C, cyclophosphamide and miconazole, and the clastogens azathioprine and procarbazine were administered once orally or intraperitoneally to male and female mice. Bone marrow preparations were made at 24, 48 and 72 h after dosing. All the clastogens and aneugens, except miconazole, yielded positive results in the micronucleus test. Measurements of the area of the micronuclei and their distribution clearly showed that the chemicals affecting microtubule assembly produced larger micronuclei than did the clastogens. The pattern of area distribution of the micronuclei found with cyclophosphamide and mitomycin C was between those found for the tubulin inhibitors and the clastogens. These findings indicate that the micronucleus test not only detects chemicals affecting microtubule assembly, but also can discriminate them from clastogens by measurements of the area of the micronuclei.     

Micronucleus test and bone-marrow chromosome analysis: A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations

The sensitivities of 2 cytogenetic tests, chromosome analysis and the micronucleus test, were compared by using mice exposed to the substances methyl methanesulfonate (MMS), mitomycin C (MC) and procarbazine (Natulan®). The lowest dose at which a significant effect could be observed in bone-marrow cells of mice was determined. Both test systems proved equally sensitive for MC and procarbazine. Doses as low as 0.16 mg of MC per kg and 3.12 mg of Natulan® per kg significantly increased both the aberration rates and the micronucleus rates above those of the controls. In contrast, after exposure to MMS, chromosomal aberrations were elevated above control levels at 5 mg/kg, and the micronucleus rate differed significantly from that of the controls after a dose of 10 mg/kg. With the present protocol and sample size one can conclude that the micronucleus test is generally comparable in sensitivity to the chromosome analysis. However, the MMS data indicate that there might be chemicals for which the resolution of the chromosome analysis is higher.

When the mutagens were given in 2 single i.p. injections separated by 24 h, the polychromatic erythrocytes were analyzed for the presence of micronuclei 6 or 24 h after the second injection. The double treatment did not increase the micronucleus rates above the single-treatment results at either sampling interval.     

Kinetochore analysis of micronuclei allows insights into the actions of colcemid and mitomycin C

We have induced micronuclei in two strains of diploid human fibroblasts with a known aneugen, colcemid, and a known clastogen, mitomycin C. Using immunofluorescence to detect the presence of kinetochores in micronuclei, we were able to demonstrate a 26.8-fold increase in fluorescence-positive micronuclei (aneuploidy) in colcemid-treated cells. However, colcemid also induced an increase in kinetochore-negative micronuclei. Our findings support previous reports that suggest colcemid may induce chromosome breakage in addition to its major aneugenic effect. The frequency of kinetochore-negative micronuclei (chromosome breakage) in mitomycin C-treated cells rose an average of 7.9-fold in the two test strains, a clear reflection of its clastogenic action. However, a 4-fold increase in the kinetochore-positive fraction was seen. We conclude that the fibroblast micronucleus assay, coupled with kinetochore immunofluorescence, provides a useful screening approach for genotoxic agents. The delineation of the precise mechanism by which an agent perturbs the rates of chromosomal breakage or lag may require more detailed analysis.     

Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test

In this work, we have measured both micronuclei and other nuclear abnormalities in renal erythrocytes from European minnow Phoxinus phoxinus and mollie Poecilia latipinna, with the aim to contribute to the standardisation of the micronucleus test for fish species. Intraperitoneal injections of colchicine (10 mg/kg), cyclophosphamide (40 mg/kg), or mitomycin C (20 mg/kg) for 24 h induced diverse nuclear abnormalities in minnow erythrocytes, therefore nuclear abnormalities should be added to micronuclei as genotoxicity indicators in fish micronucleus tests. The adequacy of administration protocols based on intraperitoneal injections has been evaluated by injecting saline solution to both species: single or double injections have not induced neither micronuclei nor other nuclear abnormalities in any case. Finally, the differential sensitivity of both species to toxic heavy metals was evaluated by exposing individuals of both species to different doses (0.17, 1.7, 2x1.7, and 3.4 mg/kg) of cadmium and mercury for 24 h; we concluded that the mollie is sensitive to both metals whereas the minnow is not sensitive to mercury.     

Chromosomal instability in a woman with infertility and two unaffected brothers: a new familial chromosomal breakage syndrome?

Repeated chromosomal analysis of peripheral blood lymphocytes and skin fibroblasts from a woman referred for amenorrhoea, streak gonads, hyperthyroidism, adiposity and elevated α-fetoprotein levels but no other manifestations of known chromosomal breakage syndromes demonstrated an increased spontaneous chromosomal breakage rate (ISCBR). Chromatid and chromosomal breaks were more numerous than sporadic rearrangements and dicentric chromosomes. Exposure of the cells to mitomycin C, diepoxybutane, X-rays or UV irradiation induced an increase in chromosomal and chromatid abnormalities over that in controls. A micronucleus assay demonstrated an increase in the incidence of formation of micronuclei and the population doubling time of the fibroblasts of the proposita was delayed. Chromosomal analysis was performed on lymphocytes of the parents and of five sibs of the proposita. Two brothers had chromosomal abnormalities identical to those of the patient and elevated α-fetoprotein levels, however, without any clinical abnormalities. The parents were affected by only a moderate ISCBR whereas two brothers and one sister were chromosomally normal. The clinical, chromosomal and biochemical findings in this family represent a novel chromosomal instability syndrome. Received: 30 October 1996 / Accepted: 27 March 1997     

Detection of micronuclei in peripheral blood of mitomycin C-treated mice using supravital staining with acridine orange.

The induction of micronuclei in peripheral blood from mitomycin C (MMC)-treated mice was examined using a supravital acridine orange staining method. Male ICR mice were intraperitoneally given MMC at a single dose of 0.25, 0.5, 1, or 2 mg/kg. Blood was sampled from the tail 24, 48, 72, and 96 h after treatment, and the frequency of micronucleated reticulocytes (MNRETs) was examined. The induction of MNRETs peaked at 48 h after treatment with MMC; there was a clear, dose-related increase in MNRETs. In a multiple-treatment study, mice were treated with 4 consecutive daily injections of MMC at a dose of 0.13, 0.25, 0.5, or 1 mg/kg. The frequency of MNRETs increased markedly 24 h after the second treatment as compared with the first treatment, and did not change significantly until 24 h after the fourth treatment. The frequency of MNRETs decreased to approximately control values 96 h after the last treatment. In addition, a slight but statistically significant increase in the number of micronucleated normochromatic erythrocytes in peripheral blood was detected by means of Giemsa staining 7 days after the last treatment. These results confirm the usefulness of the supravital acridine orange staining method to evaluate micronucleus induction in mouse peripheral blood.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Cytotoxic and genotoxic effects of sodium hypochlorite on human peripheral lymphocytes in vitro

Chlorination is widely used method in the disinfection of drinking and utility water worldwide. In this study, cytotoxic and genotoxic effects of sodium hypochlorite were investigated by the cytokinesis-block micronucleus assay and chromosomal aberration analysis on human peripheral lymphocytes in vitro. A significant increase in chromosomal aberration frequency was observed in all treatments of NaOCl (0.030, 0.065, 0.100, 0.25, 0.5, 1, 2, 4 μg/mL) at 24 and 48 h compared with the negative control and mitomycin C (MMC, 0.3 μg/mL), which was used as a positive control. NaOCl significantly increased the frequency of micronuclei in a dose dependent manner. The results showed that there was a significant correlation between NaOCl concentration and chromosomal aberration, micronuclei frequency, necrotic cells, apoptotic cells and binucleated cells.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 μl of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS · JEMS).Male CD-1 mice were intraperitoneally injected once with 12.5–100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored.The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Development and validation of an in vitro micronucleus assay platform in TK6 cells

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.     

Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-dioxane in the bone marrow and liver of young CD-1 mice

1,4-Dioxane, a widely used industrial chemical and rodent hepatocarcinogen, has produced mixed, largely negative results in the mouse erythrocyte micronucleus assay. In contrast, a recent report has indicated that 1,4-dioxane induces micronuclei in mouse hepatocytes following in vivo treatment. The objective of this study was to confirm these earlier results and identify the origin of the induced micronuclei. Following an initial range-finding study, mice were administered 1,4-dioxane by gavage at doses ranging from 1500 to 3500 mg/kg. The test animals were also implanted with BrdU-releasing osmotic pumps to allow cell proliferation to be measured in the liver and to increase the sensitivity of the hepatocyte assay. Upon sacrifice, the frequency of micronuclei in the bone marrow erythrocytes and in the proliferating BrdU-labeled hepatocytes was determined. Significant dose-related increases in micronuclei were seen in both the liver and the bone-marrow with significant increases being detected at all the tested doses in the bone marrow and at the 2500 and 3500 mg/kg doses in the liver. Using CREST staining or pancentromeric FISH to determine the origin of the induced micronuclei, it was determined that 80-90% of the micronuclei in both tissues originated from chromosomal breakage. Small increases in centromere-containing micronuclei were also seen in the hepatocytes. Decreases in hepatocyte proliferation as well as in the ratio of bone marrow PCE:NCE were also observed. Based on these results, we conclude that at high doses: (i) dioxane exerts genotoxic effects in both the mouse bone marrow and liver; (ii) the induced micronuclei are formed primarily from chromosomal breakage; and (iii) dioxane can interfere with cell proliferation in both the liver and bone marrow.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Formation of micronuclei in erythrocytes of the fathead minnow (Pimephales promelas) after acute treatment with mitomycin C or cyclophosphamide

Despite the widespread use of the fathead minnow in ecotoxicology, there have been relatively few studies on genotoxicity biomarkers in this small, warm-water fish species. Consequently, we investigated the effect of two known genotoxins, mitomycin C and cyclophosphamide, on micronucleus induction in spleen and peripheral blood erythrocytes of this species. Initially, 96-h experiments after intra-peritoneal (i.p.) injections of mitomycin C and cyclophosphamide were undertaken to determine the maximum tolerated dose (MTD). From these studies, MTDs of 10 and 400 mg/kg, respectively, were obtained: doses that were higher than those reported for other fish species. Next, an assessment of micronucleus induction at 1, 2, 4, 8 and 14 days after injection was undertaken for each compound at the MTD. Mitomycin C at 10 mg/kg significantly induced micronuclei in erythrocytes from the spleen, but not from the peripheral blood, at 8 and 14 days. In addition, the overall levels of micronuclei observed were lower than most previously published data from other fish species. In contrast to mitomycin C, treatment with 400 mg/kg cyclophosphamide failed to significantly induce micronuclei in erythrocytes from any of the tissues employed, in contrast to previous reports of significant induction in other species. The reasons for the apparent relative insensitivity of the fathead minnow to these clastogens, with respect to both MTDs and micronucleus induction, are not clear. The fathead minnow, however, has previously been described as relatively insensitive compared to other fish species with respect to selected carcinogens and cytochrome P450 inducers; the latter suggesting that the lack of a significant induction following cyclophosphamide exposure may be due to low metabolic activation in vivo. Consequently, further clarifying work is required to delineate the response shown, considering the extensive use of this species in ecotoxicology research and regulatory testing.     

SO2衍生物诱发蚕豆根尖细胞微核和后期异常的研究

研究SO₂体内衍生物--亚硫酸钠和亚硫酸氢钠混合液(3:1 mmol·L^-1/mmol·L^-1)诱发蚕豆根尖细胞微核和后期异常的效应。结果表明:SO₂衍生物处理可诱发蚕豆根尖间期细胞微核和核芽,使分裂后期出现多种染色体异常,如断片、桥以及滞后染色体等。异常细胞中以微核细胞和染色体断裂细胞居多。在一定浓度范围内,细胞异常率与处理液浓度之间表现正的线性相关。这些研究结果表明,蚕豆根尖间期微核和后期染色体异常有可能用作检测SO₂污染的生物剂量计。     

The effect of hyperthermia on micronucleus induction by mutagens in mice

We administered mitomycin C (0.5 mg/kg) intraperitoneally to hyperthermic-treated mice and examined the effect of hyperthermia on micronucleus induction. Hyperthermia enhanced micronucleus induction. The timing of chemical administration relative to the start of hyperthermic treatment (37 degrees C ambient temperature) influenced micronucleus frequency, and the effect was greatest 2 h after the start of hyperthermic treatment. But the hyperthermic treatment did not change the time course of micronucleus induction. In addition, we investigated the effect of hyperthermia on micronucleus induction by chemicals with different modes of action, i.e., alkylating agents (mitomycin C at 0.1-0.5 mg/kg, cyclophosphamide at 1.25-10 mg/kg), a spindle poison (colchicine at 0.05-1.0 mg/kg), and an antimetabolite (5-fluorouracil at 2.5-50 mg/kg). Hyperthermia enhanced only the clastogenicity of alkylating agents.     

Measurement of micronuclei in lymphocytes

The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.     

Sampling times in micronucleus testing.

A series of micronucleus inducers were evaluated in the mouse bone marrow micronucleus test to determine if a 72-h sampling time enhances the sensitivity for detecting genotoxic agents. Male and female Swiss albino mice were dosed once with 7,12- dimethylbenz[a]anthracene, 6-mercaptopurine, benzo[a]pyrene, benzene, cyclophosphamide, 2-acetylaminofluorene, tubulazole, or mitomycin C. According to the EEC and OECD guidelines, the mice were killed at 24, 48 and 72 h after dosing. All test compounds induced an increase in the number of micronucleated polychromatic erythrocytes at 24 and/or 48 h. From the results obtained, it was evident that the 72-h sampling time does not enhance the sensitivity of the micronucleus test. The present data show that for screening purposes two sampling times at 24 and 48 h are sufficient to detect clastogens as well as aneugens. Although quantitative differences were found in sensitivity to micronucleus inducers between male and female mice, no qualitative differences were observed between the two sexes.     

The cytonucleus test in the rat: a combined metaphase and micronucleus assay

The suitability of the rat as a species choice for the micronucleus assay and the possibility of combining both metaphase and micronucleus analysis using one set of animals were investigated. Cyclophosphamide, trenimon, vinblastine sulphate and dimethyl benzanthracene were used to optimise the study design and experimental procedures. The sample times ranged from 12 to 72 h after a single dose of the compound. A maximal response in both micronucleus and chromosome aberration induction was observed 24 h after dosing. This sample time is recommended as the single sample time for the screening of all classes of compound. Using the optimal conditions, a number of different mutagens/carcinogens were then analysed for the induction of both micronuclei and chromosome aberrations in the same animal. The compounds selected were mitomycin C, methotrexate, 5-fluorouracil, hexamethyl phosphoramide, benzo[a]pyrene, benzidine and diaminoterphenyl. The results show that the rat is a responsive test species and that it is possible to combine both metaphase and micronucleus analysis in the same animal.