Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation

The cytokinesis-block micronucleus technique was developed to overcome the kinetic problems inherent in the use of human lymphocytes for micronucleus assays. Using this technique the number of spontaneous micronuclei in lymphocytes from 42 individuals aged between 20 and 85 years was studied and was found to increase at a rate of 4.3% per year. Comparison with the results obtained with the conventional micronucleus assay confirmed that the conventional method markedly underestimates this age effect. The sensitivity of the cytokinesis-block method was determined by studying the effect of low-dose (less than 50 rad) X-irradiation. The results indicated that the dose-response was linear and a single in vitro exposure to 5 rad of X-rays could be unequivocally detected. We concluded that the cytokinesis-block micronucleus method is more sensitive and precise than the conventional micronucleus method and classical metaphase analysis, and that it will be of value for detecting chromosome damage induced in vivo by genotoxic agents.     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

重铬酸钾对蚕豆根尖细胞致畸效应的研究

以蚕豆根尖为材料,研究重铬酸钾对蚕豆根尖细胞的致畸效应。采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,以不同浓度的重铬酸钾为诱变剂,测定蚕豆根尖细胞的微核率和染色体畸变率。结果表明:重铬酸钾能诱发较高频率的微核率,即在一定浓度范围内,其微核率随重铬酸钾处理浓度的升高而增加,但高于一定浓度后反而呈下降趋势;不同浓度的重铬酸钾均使蚕豆根尖细胞有丝分裂指数增大;重铬酸钾还能诱导蚕豆根尖细胞产生较高频率的染色体畸变,且产生多种类型的染色体畸变。结论是重铬酸钾对蚕豆根尖细胞具有明显的致畸效应。     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

Spontaneous and mitomycin-C-induced micronuclei in human lymphocytes exposed to extremely low frequency pulsed magnetic fields

The cytokinesis block micronucleus method, a very sensitive cytogenetic assay, was used to ascertain the possible genotoxic effects of extremely low frequency pulsed magnetic fields in phytohemagglutinin-stimulated human lymphocytes cultures from 16 healthy donors. Four conditions were studied: i) lymphocytes not exposed to the field (control cultures); ii) lymphocytes exposed to the field; iii) lymphocytes treated with mitomycin-C and not exposed to the field; iv) lymphocytes treated with mitomycin-C and exposed to the field. Mitomycin-C-treated cultures were used as control for the micronucleus method, because it is known that mitomycin-C is a potent genotoxic agent, capable of inducing micronuclei. The frequency of micronuclei in field-exposed cultures was similar to the spontaneous frequency observed in control unexposed-cultures. Moreover, the exposure to pulsed magnetic fields did not affect the frequency of micronuclei induced by mitomycin-C, suggesting that, in the experimental conditions used, this kind of field neither affected the integrity of chromosomes nor interfered with the genotoxic activity of mitomycin-C.     

Genotype and the cryopreservation process affect the levels of aneuploidy and chromosome breakage in cultured human fibroblasts

Spontaneous micronucleus frequencies were measured in 11 human fibroblast strains, with early-passage cells that had never been frozen and with cells of comparable population doublings that had been cryopreserved in liquid nitrogen. The mean micronucleus frequency of the 11 strains increased from 14.0 +/- 0.7 to 20.4 +/- 1.8/1250 mononucleated cells (P = 0.002) after the freeze-thaw process. The nature of this increase in micronucleus frequency was examined using an immunodetection assay for the in situ identification of kinetochores in micronuclei. The increase in micronucleus frequency occurred primarily in the kinetochore-positive fraction, which is indicative of aneuploidy, but also by an increase in chromosome breakage in several strains. The findings were reproducible in repeat biopsies from two donors. Plating efficiencies of the 11 strains were studied during 1-9 and 10-20 population doublings from primary outgrowth, before freezing and again after freeze-thaw. The mean plating efficiency of frozen-thawed cells before nine doublings was significantly lower than that of cells of similar ages that had never been frozen (P = 0.004). The four strains that had a greater than 25% decrease in plating efficiency post freeze-thaw also had the highest aneuploidy index post freeze-thaw, suggesting that chromosomal imbalance contributes to the observed reduction in growth. We conclude that the genotype and culture manipulations of a fibroblast strain influence the outcome of the micronucleus assay.     

Collaborative validation study of the in vivo micronucleus test using mouse colonic epithelial cells

The in vivo micronucleus test using mouse colonic epithelial cells was evaluated as the 11th collaborative study organized by the Collaborative Study Group on the micronucleus test (CSGMT) with three model chemicals that were known to induce chromosome damage in mouse colonic cells. Five laboratories participated in this validation study. All three model chemicals, i.e. 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), N-methyl-N-nitrosourea (MNU), and mitomycin C (MMC), induced micronucleated colonic epithelial cells in a 4-day exposure protocol in all participating laboratories. We confirmed that the present single cell suspension method could be used to detect the model chemicals as micronucleus inducers in mouse colonic epithelial cells. Advantages of this method are that experiments are easy to perform and that intact cells can be analyzed. The present study suggested that the colon micronucleus assay proposed here is useful for mechanistic studies of colon carcinogenesis.     

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides with triethylenemelamine.

A micronucleus assay using mouse peripheral blood and supravital staining with acridine orange (AO) was validated by two laboratories on triethylenemelamine-treated mice. Dose- and time-dependent increases in micronucleated peripheral reticulocytes were observed. This new method can be used as an alternative to the conventional bone marrow micronucleus assay.     

Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay

Silver nanoparticles (AgNPs) have antimicrobial properties, which have contributed to their widespread use in consumer products. A current issue regarding nanomaterials is the extent to which existing genotoxicity assays are useful for evaluating the risks associated with their use. In this study, the genotoxicity of 5 nm AgNPs was assessed using two standard genotoxicity assays, the Salmonella reverse mutation assay (Ames test) and the in vitro micronucleus assay. Using the preincubation version of the Ames assay, Salmonella strains TA102, TA100, TA1537, TA98, and TA1535 were treated with 0.15-76.8 μg/plate of the AgNPs. Toxicity limited the doses that could be assayed to 2.4-38.4 μg/plate; no increases in mutant frequency over the vehicle control were found for the concentrations that could be assayed. Human lymphoblastoid TK6 cells were treated with 10-30 μg/ml AgNPs, and additional cells were treated with water and 0.73 gy X-rays as vehicle and positive controls. Micronucleus frequency was increased by the AgNP treatment in a dose-dependent manner. At a concentration of 30 μg/ml (with 45.4% relative population doubling), AgNPs induced a significant, 3.17-fold increase with a net increase of 1.60% in micronucleus frequency over the vehicle control, a weak positive response by our criteria. These results demonstrate that the 5 nm AgNP are genotoxic in TK6 cells. Also, the data suggest that the in vitro micronucleus assay may be more appropriate than the Ames test for evaluating the genotoxicity of the AgNPs.     

Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report of the 5th collaborative study by CSGMT/JEMS · MMS

The main goal of the Collaborative Study Group for the Micronucleus Test (CSGMT) was to validate a new method for the micronucleus test, recently introduced by Hayashi et al. (1990), using mouse peripheral blood cells stained supravitally with acridine orange (AO). The micronucleus tests were performed on CD-1 mice using 23 chemicals with various modes of action. As a rule, one chemical was studied by two participants. Peripheral blood sampled from the same animal was examined 0, 24, 48, and 72 h (or longer) after treatment. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on observation of 1000 reticulocytes per mouse.All chemicals induced MNRETs dose-dependently. Interlaboratory differences in the induction of MNRETs were in an acceptable range for most chemicals tested. Although differences were observed with some chemicals, there were no discrepancies in qualitative judgment. Most chemicals gave the greatest response 48 h after treatment, which was less variable than in the bone marrow assay (greatest response, 24–48 h). These results suggest that the peripheral blood assay using the AO supravital staining technique generates reproducible and reliable data to evaluate the clastogenicity of chemicals. This makes the peripheral blood micronucleus assay an attractive alternative to the conventional bone marrow assay.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

Studying the genotoxic effects induced by two kinds of bentonite particles on human B lymphoblast cells in vitro

The aim of the present study was to evaluate the genotoxic effects induced by native and active bentonite particles (BPs) on human B lymphoblast cells using comet assay and cytokinesis-block micronucleus (CBMN) assay in vitro. The cells were exposed to BPs at the concentrations of 30, 60, 120 and 240μg/ml for 24, 48 and 72h, respectively. The quartz contents of native and active BPs were 6.80±0.20 and 6.50±0.10%, respectively. Gypsum and DQ-12 quartz served as negative and positive controls. The results of comet assay showed that DNA damage induced by native and active BPs was significantly higher than that induced by gypsum control (P<0.05 or <0.01), and increased with exposure concentration and duration. When the cells were exposed to BPs at the doses of 120 and 240μg/ml for 72h, DNA damage induced by active BPs and native BPs was significantly higher than that induced by DQ-12 quartz (P<0.01), and DNA damage induced by active BPs enhanced significantly, as compared with native BPs (P<0.01). The results of CBMN assay demonstrated that both native BPs and active BPs could induce significant micronuclei, as compared with gypsum control (P<0.05 or <0.01). However, there was no significant difference of micronucleus frequency (MNF) among native BPs, active BPs and DQ-12 quartz. The water-soluble fractions from two kinds of BPs did not induce significant DNA damage and micronuclei. These findings indicated that the genotoxicity induced by active BPs and native BPs could be detected in comet assay and CBMN assay in vitro, the insoluble particle fractions from BPs may play a main role in the genotoxic effects induced by BPs.     

Evaluation of genotoxic effects in human leukocytes after in vitro exposure to 1950 MHz UMTS radiofrequency field

In the present study the third generation wireless technology of the Universal Mobile Telecommunication System (UMTS) signal was investigated for the induction of genotoxic effects in human leukocytes. Peripheral blood from six healthy donors was used and, for each donor, intermittent exposures (6 min RF on, 2 h RF off) at the frequency of 1950 MHz were conducted at a specific absorption rate of 2.2 W/kg. The exposures were performed in a transverse electro magnetic (TEM) cell hosted in an incubator under strictly controlled conditions of temperature and dosimetry. Following long duration intermittent RF exposures (from 24 to 68 h) in different stages of the cell cycle, micronucleus formation was evaluated by applying the cytokinesis block micronucleus assay, which also provides information on cell division kinetics. Primary DNA damage (strand breaks/alkali labile sites) was also investigated following 24 h of intermittent RF exposures, by applying the alkaline single cell gel electrophoresis (SCG)/comet assay. Positive controls were included by treating cell cultures with Mitomycin-C and methylmethanesulfonate for micronucleus and comet assays, respectively. The results obtained indicate that intermittent exposures of human lymphocytes in different stages of cell cycle do not induce either an increase in micronucleated cells, or change in cell cycle kinetics; moreover, 24 h intermittent exposures also fail to affect DNA structure of human leukocytes soon after the exposures, likely indicating that repairable DNA damage was not induced.     

Y Chromosome aneuploidy,micronuclei, kinetochores and aging in men

This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant mcrease in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.     

Individual responsiveness to induction of micronuclei in human lymphocytes after exposure in vitro to 1800-MHz microwave radiation

The widespread application of microwaves is of great concern in view of possible consequences for human health. Many in vitro studies have been carried out to detect possible effects on DNA and chromatin structure following exposure to microwave radiation. The aim of this study is to assess the capability of microwaves, at different power densities and exposure times, to induce genotoxic effects as evaluated by the in vitro micronucleus (MN) assay on peripheral blood lymphocytes from nine different healthy donors, and to investigate also the possible inter-individual response variability. Whole blood samples were exposed for 60, 120 and 180 min to continuous microwave radiation with a frequency of 1800 MHz and power densities of 5, 10 and 20 mW/cm(2). Reproducibility was tested by repeating the experiment 3 months later. Multivariate analysis showed that lymphocyte proliferation indices were significantly different among donors (p<0.004) and between experiments (p<0.01), whereas the applied power density and the exposure time did not have any effect on them. Both spontaneous and induced MN frequencies varied in a highly significant way among donors (p<0.009) and between experiments (p<0.002), and a statistically significant increase of MN, although rather low, was observed dependent on exposure time (p=0.0004) and applied power density (p=0.0166). A considerable decrease in spontaneous and induced MN frequencies was measured in the second experiment. The results show that microwaves are able to induce MN in short-time exposures to medium power density fields. Our data analysis highlights a wide inter-individual variability in the response, which was confirmed to be a characteristic reproducible trait by means of the second experiment.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane.

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF1 mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

Genotoxic effect and nitrative DNA damage in HepG2 cells exposed to aristolochic acid

Aristolochic acid (AA), extensively used as a traditional herbal medicine, was withdrawn from the market in the last century because it was found to be a potent carcinogen in humans and animals. The aim of this study was to evaluate the genotoxic effect of AA and obtain further insight into whether the nitrative DNA damage can be induced by reactive nitrogen species (RNS), including nitric oxide (NO) and its derivative peroxynitrite (ONOO(-)) using human hepatoma HepG2 cells. To identify the genotoxic effect, the comet assay and micronucleus test (MNT) were performed. In the comet assay, 25-200microM of AA caused a significant increase of DNA migration in a dose-dependent manner. A significant increase of the frequency of micronuclei was found in the range between 12.5 and 50microM in the MNT. The results showed that AA caused DNA and chromosome damages. To elucidate the nitrative DNA damage mechanism, the level of nitrite and 8-hydroxydeoxyguanosine (8-OHdG), which can be generated by ONOO(-), were monitored with the 2,3-diaminonaphthalene (DAN) assay and immunoperoxidase staining, respectively. The results showed that AA causes a significant increase in the levels of NO and formation of 8-OHdG at concentrations >/=50microM. This observation supports the assumption that AA could exert genotoxicity probably via NO and its derivatives at higher concentrations in HepG2 cells.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF₁ mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

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.     

Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique.

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.