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.     

Micronucleus test on gas station attendants

In the present study, the micronucleus test was applied in exfoliated cells of buccal mucosa to assess the mutagenicity risk associated with occupational exposure for gas station attendants. For each individual, 2000 exfoliated buccal cells were analyzed for micronucleus frequency. A highly significant difference was found between exposed and control groups. Likewise, a significant difference was found between these groups regarding the frequency of binucleated and broken egg cells. To determine whether smoking, alcohol habit, age, gender, or working time could exert any additional effect, we determined the frequency of micronuclei and binucleated and broken egg cells amongst exposed and control individuals. The results allowed us to conclude that the individuals studied belong to a risk group and should periodically undergo biological monitoring and appropriate care.     

Exposure to asbestos: correlation between blood levels of mesothelin and frequency of micronuclei in peripheral blood lymphocytes

Inhalation of asbestos, a mineral extensively used in a variety of applications, is strongly associated with malignant mesothelioma (MM), a fatal cancer of the pleura. Soluble mesothelin-related peptides (SMRP) are a promising biomarker suggested for the screening of MM in healthy asbestos-exposed subjects. In the present study a comparison of micronucleus (Mn) frequencies in peripheral blood lymphocytes (PBL) between 44 asbestos-exposed and 22 control individuals has been performed, and the correlation with serum SMRP has been examined. SMRP levels were found to be significantly higher in subjects exposed to asbestos and in their various subgroups than in controls. Concerning micronucleated lymphocytes, a statistically significant difference from controls was seen in the percentages of both micronucleated mononucleated lymphocytes (MnMNL) and micronucleated binucleated lymphocytes (MnBNL), but the difference was markedly higher for the percentage of micronucleated polynucleated lymphocytes (MnPNL). With respect to the correlation between the frequency of the three types of micronucleated lymphocytes and serum-SMRP values of asbestos-exposed subjects, it was statistically significant for MnMNL, but not for MnBNL and MnPNL.     

Binucleated HeLa cells are formed by cytokinesis failure in starvation and keep the potential of proliferation

Many cytological studies have reported that the numbers of binucleated cells were elevated in various tumors. However, binucleated cells are observed in not only malignant tumors but also normal tissues. Thus, the clinical significance of binucleated cells is controversial. Here we attempted to elucidate the characteristics of binucleated HeLa cells using time-lapse microscopy. To examine the frequency, viability, proliferation, and formation mechanism of binucleated cells, we grew HeLa cells on chamber slides and tissue culture dishes in DMEM supplemented with (10, 3, 1 and 0.5 % media) and without fetal bovine serum (0 % medium). The proliferation was evaluated by the medium improvement examination (cultured for 2 more days in 10% medium after culturing in 0% medium; starvation). In the 0 % medium, 150 binucleated cells were formed by cytokinesis failure. There were significantly more binucleated cells in the 0 % medium than in the 10, 3, 1 and 0.5 % media. About twice the number of binucleated cells underwent mitosis in the improvement examinations than in the serum-free examination. We found here that starvation induced the binucleation of HeLa cells and that some binucleated cells can reproduce. These findings might be helpful for understanding binucleated cells in tumors.     

Preferential occurrence of chromosome 21 malsegregation in peripheral blood lymphocytes of Alzheimer disease patients

To further investigate our finding of high levels of spontaneous aneuploidy in somatic cells of Alzheimer's disease (AD) patients (Migliore et al. 1997), we studied the molecular cytogenetics of eight patients with sporadic AD and six healthy controls of similar age. Cytochalasin B-blocked binucleated peripheral blood lymphocytes from the AD patients and unaffected controls were used to measure micronucleus induction or other aneuploidy events, such as the presence of malsegregation in interphase nuclei (representing chromosome loss and gain). Dual-color fluorescence in situ hybridization (FISH) with differential labeled DNA probes was applied. We used a probe specific for the centromeres of chromosomes 13 and 21 combined with a single cosmid for the Down's syndrome region (21q22.2) to obtain information on spontaneous chromosome loss and gain frequencies for both chromosomes (13 and 21). FISH data showed that AD lymphocytes had higher frequencies of chromosome loss (evaluated as fluorescently labeled micronuclei) for both chromosomes, as well as higher frequencies of aneuploid interphase nuclei, again involving both chromosomes, compared to control lymphocytes. However, aneuploidy for chromosome 21 was more frequent than for chromosome 13 in AD patients. This preferential occurrence of chromosome 21 in malsegregation in somatic cells of AD patients raises the hypothesis that mosaicism for trisomy of chromosome 21 could underlie the dementia phenotype in AD patients, as well as in elderly Down's syndrome patients.     

Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm)

To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30-50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method.     

Induction of micronuclei in V79 cells by fractions of roofing asphalt fume condensate.

More than 50,000 workers in the United States are exposed to roofing asphalt fumes that may pose genotoxic and potential carcinogenic hazards. The Type III roofing asphalt is most frequently used in roof-application. Results of our previous studies showed that fume condensates of Type III roofing asphalts induced micronuclei (MN) in vitro in cultured V79 cells and DNA adduct formation in vivo in rat lung cells. In this study, the genotoxicity of whole fume condensates (WFC) of Type III roofing asphalt and its five chemical fractions (A, B, C, D and E) was determined by the micronucleus assay using V79 cells. Linear regressions were determined for the dose response of MN frequencies and percent of binucleated and multinucleated cells (MTC) following the treatment. Results showed that the numbers of micronucleated cells in cultures treated with Type III roofing asphalt WFC and its fractions B, C, D and E were significantly higher than that in the control culture, and that the slopes of the linear regression line for fractions B and C were greater than those for the WFC and fractions D and E. A clear dose response of binucleated cells was also induced by the WFC and fractions B and C. These findings indicate that: (1) WFC and all fractions, except fraction A, induced MN formation in cultured V79 cells; (2) fractions B and C possess the highest genotoxic activity; (3) the roofing asphalt WFC contains chemicals or chemical classes that induce not only chromosomal aberrations but also binucleation in V79 cells.     

Frequency of micronuclei induced in peripheral lymphocytes by trimethyltin chloride

The clastogenicity of trimethyltin chloride was evaluated in human peripheral blood lymphocytes with micronucleus counts (MNC) as the endpoint. Two concentrations (0.5 micrograms and 1.0 microgram) of trimethyltin chloride were added to lymphocytes of healthy male and female subjects of different age groups, in mitogen-stimulated and serum-supplemented culture medium (RPMI 1640, Gibco) for 48 h at 37 degrees C. A significant increase in micronucleus counts was observed with both doses, which was more pronounced with the lower dose. ANOVA in male and female donors revealed significant differences between age groups (P less than 0.001), chemical concentrations (P less than 0.001) and for the interaction of these 2 factors (P less than 0.05 in females only). However, no regular increase or decrease in MNC frequencies was observed with the donor's age. Higher frequencies of MNC enhancement were observed in male individuals than in females.     

Cytogenetic biomonitoring of workers from laboratories of clinical analyses occupationally exposed to chemicals

A cytogenetic monitoring study was carried out on a group of workers in clinical analysis laboratories to investigate the risk of occupational exposure to chronic low levels of chemicals.Thirty-four clinical laboratories have been involved in the study. In these laboratories, toxicants and analytical procedures utilized have been characterized. The individual occupational exposure of workers was assessed by use of a questionnaire concerning the chemical substances utilized. About 300 different chemicals have been identified.Cytogenetic analyses (chromosomal aberration and micronucleus tests) were carried out on a strictly selected group of 50 workers enrolled from these laboratories and compared to 53 controls (healthy blood donors) matched for gender and age.The exposed group shows a significantly higher frequency of genetic damage than the control group. Both chromatid and chromosome aberration frequencies in workers appear significantly higher than in controls. Similarly, comparison between micronucleated cells rates of exposed and unexposed groups show significantly higher frequencies of binucleated cells with micronucleus (BNMN) and of total micronuclei (MN tot) in workers than in controls.     

Cytogenetic biomonitoring of workers from laboratories of clinical analyses occupationally exposed to chemicals

A cytogenetic monitoring study was carried out on a group of workers in clinical analysis laboratories to investigate the risk of occupational exposure to chronic low levels of chemicals.Thirty-four clinical laboratories have been involved in the study. In these laboratories, toxicants and analytical procedures utilized have been characterized. The individual occupational exposure of workers was assessed by use of a questionnaire concerning the chemical substances utilized. About 300 different chemicals have been identified.Cytogenetic analyses (chromosomal aberration and micronucleus tests) were carried out on a strictly selected group of 50 workers enrolled from these laboratories and compared to 53 controls (healthy blood donors) matched for gender and age.The exposed group shows a significantly higher frequency of genetic damage than the control group. Both chromatid and chromosome aberration frequencies in workers appear significantly higher than in controls. Similarly, comparison between micronucleated cells rates of exposed and unexposed groups show significantly higher frequencies of binucleated cells with micronucleus (BNMN) and of total micronuclei (MN tot) in workers than in controls.     

Study of the cytogenetic effects of occupational exposure to pesticides on sanitation workers in Belo Horizonte, Brazil

Sanitation workers handling pesticides in the control of disease vectors constitute an occupationally exposed population to genotoxic substances. The aim of the present study was to investigate the relation between the occupational exposure to various pesticides and the presence of cytogenetic damage. Fifty-nine men were selected (29 sanitation workers and 30 control individuals) with ages varying between 18-57 years who lived and worked in the same area in Belo Horizonte (Brazil). The following parameters were determined for all individuals using the cytokinesis-block micronucleus (MN) assay in peripheral blood lymphocytes: MN/1000 binucleated cells (BC), BC with MN (BCMN)/1000 BC, nucleoplasmic bridges (NB)/1000 BC, apoptotic and necrotic cells/500 cells and nuclear division index. The analysis of covariance showed significantly higher (p < 0.05) mean frequencies of MN (15.81 +/- 1.31 vs 4.71 +/- 0.42), BCMN (15.10 +/- 1.22 vs 4.62 +/- 0.44), NB (4.59 +/- 0.76 vs 1.00 +/- 0.34), and necrotic cells (12.07 +/- 1.45 vs 5.17 +/- 0.70) in the exposed group when compared to the control group. There was no significant difference in the apoptotic cell frequency between the two groups, while the nuclear division index was significantly lower (1.49 +/- 0.02 vs 1.61 +/- 0.02) in the control group. Neither the time of exposure nor the smoking or alcohol drinking habit influenced the cytogenetic parameters examined. According to these results, occupational exposure to pesticides induced genotoxic and cytotoxic effects in sanitation workers.     

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.     

Induction of micronuclei by alkaloids extracted from Senecio brasiliensis and stored for 23 years

In the present study, we report the results of an investigation on pyrrolizidine alkaloids extracted from Senecio brasiliensis (Sprengel) Less., which were stored for more than 23 years under variable conditions of temperature and humidity and exposed to light. Both the crude alkaloid (integerrimine+retrorsine+impurities) and pure integerrimine conserved the ability to induce acute toxicity in mice, leading to the death of the animals in less than 24h. The alkaloids also conserved the potential to induce significant increases in micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow compared to the negative control. The administration of alkaloids to lymphocyte cultures blocked with cytochalasin-B showed no significant increase in micronucleus frequency in binucleated cells, probably due to the lack of a metabolic activation mechanism. However, an antimitotic effect was observed.     

Use of rat primary lung cells for studying genotoxicity with the sister-chromatid exchange and micronucleus assays

Primary culture of lung cells from CD rats was established for pulmonary genotoxicity studies using two genetic endpoints, sister-chromatid exchange (SCE) and micronucleus formation (MN). In the cell isolation study, a combined enzyme separation of rat lungs with trypsin (1.3 mg/ml) plus collagenase (50 U/ml) gave the highest yield of viable and colony-forming cells. For the MN assay, the cytokinesis block induced by cytochalasin B (CYB) was employed to enumerate MN in binucleated (BN) cells. Treatment of primary lung cells with 2 micrograms CYB/ml for two days appeared to be optimal for scoring micronuclei in CYB-induced BN cells. By this procedure, mitomycin C (MMC), triethylenemelamine, and benzo[a]pyrene caused a dose-related increase in micronucleated BN cells in vitro without metabolic activation. In the SCE assay, maximum second-division metaphases were obtained after cells were incubated with bromodeoxyuridine for 48-54 h. After this incubation time, high frequencies of SCE induced by MMC and 3-methylcholanthrene after in vitro exposure (without S9 activation) or in vivo exposure were observed. The results indicate that rat primary lung cells can metabolize polycyclic aromatic hydrocarbons and that this lung cell system is potentially useful for the detection of pulmonary genotoxicants.     

Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

Frequency of radiation-induced micronuclei in neuronal cells does not correlate with clonogenic survival

It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2alpha, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2alpha, G28, G120 and G44; SF2 >/= 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of (60)Co gamma radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2 相似文献   

Micronucleus investigation of alcoholic patients with oral carcinomas

The micronucleus test (MN) is used as an indicator of genotoxic exposition, since it is associated with chromosome aberrations. An increased mutation rate in oral squamous cells, indicated by an increased MN frequency, is also related to the development of oral carcinomas. We evaluated the frequencies of MN and other metanucleated anomalies in the buccal squamous cells of 30 alcoholics with oral or oropharyngeal carcinomas, and compared them to a control group of abstinent health individuals. Microscopic examination was made of 2000 cells per individual from each of three distinct areas of the mouth: around the lesion (A), opposite to the lesion (B) and in the upper gingival-labial gutter (C); C was used as a control region because of low tumor frequency. There was a seven-fold increase in MN frequency in region B, a three-fold increase in region A and a two-fold, though nonsignificant, increase in C; indicating a gradient of frequencies towards carcinogenesis: C --> A --> B. Comparisons of frequencies of various types of metanucleated cells: binucleated, karyorrhexis (KR), karyolysis (KL) and broken egg (BE) in patients and controls showed, with few exceptions, highly significant differences. This gave us a better understanding of the dynamics of this squamous epithelium, supporting a more efficient biomonitor based on these various metanucleated anomalies: the repair index RI=(KL+KR)/(MN+BE). Also, the apparently contradictory results from regression analysis revealed that the MN frequency decreased with age and alcohol consumption, probably because of slow cell proliferation, and consequently led to a loss of homeostasis due to aging. In addition, in the analysis of nonparametric variables only one CAGE question was significant, confirming the effect of alcohol. In conclusion, the MN test and the repair index could be used for monitoring clinical evolution, by means of intra- and inter-individual cellular comparisons, in subjects with healed or surgically removed tumors or leukoplastic lesions, after chemo- or radiotherapeutic treatments.     

Factors distinguishing couples at risk for nondisjunction

Micronucleus frequencies were determined on 24 young parents of trisomic infants, 21 individuals with recurrent unexplained abortions, and 42 control individuals with proven reproductive success. In addition to measurements of spontaneous micronucleus frequencies, mitomycin C induced frequencies were determined at two doses (2.5 and 5.0 ng/mL). Using the 95% confidence limits established from control data as an arbitrary upper limit, 16 of 24 parents of trisomics and 5 of 21 recurrent aborters were detected by the micronucleus assay to above this cutoff. The effects of sex, age, pregnancy status, and a variety of environmental exposures were studied by comparing the micronucleus frequencies of the exposed and unexposed populations. The data suggested that vitamins were associated with a lower micronucleus frequency and tea drinking with an increased micronucleus frequency in parents of trisomics, an effect not seen in controls. These results suggest that a biologic basis for nondisjunction may be associated with elevation in spontaneous and induced micronuclei. In addition, tea and vitamins may modulate micronucleus frequencies in parents of trisomics who appear to be more sensitive to these influences than the controls.     

Cytogenetic monitoring of fishermen with environmental mercury exposure

Due to high mercury levels in many Mediterranean aquatic organisms, people who live in this area and consume large amounts of seafood are exposed to a toxicological hazard. A group of 51 fishermen exposed to mercury through eating contaminated seafood from the northern Tyrrhenian Sea underwent cytogenetic monitoring. This work is part of a research project consisting of the evaluation of micronuclei (MN), chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in peripheral blood lymphocytes. Here we present data on mercury levels in blood and on micronucleus frequencies in peripheral blood lymphocytes of fishermen. The range of mercury concentrations in blood was 10.08–304.11 ng/g fresh weight, the average was 88.97±54.09 ng/g. Micronucleus frequency was defined with at least 2000 binucleated cells scored for each person; the average was 8.74 ± 2.56 expressed on 1000 binucleated cells. A statistical correlation was found between MN frequency and total mercury concentration in blood (p = 0.00041, r = 0.674), as well as between MN frequency and age (p = 0.017). No other parameters taken into account correlated with MN frequency.     

A comparative biomonitoring study of populations residing in regions with low and high risk of lung cancer using the chromosome aberration and the micronucleus tests

Chromosome aberration (CA) and micronucleus (MN) tests were performed in peripheral blood lymphocytes from people residing in two districts of Chiang Mai, Thailand, a high-risk area, Saraphi (n=107), where the lung cancer incidence is three-fold higher than in a low-risk area, Chom Thong (n=118). The percentage of cells with CAs was significantly lower in the Saraphi population than in the Chom Thong population (0.47+/-0.91 versus 1.04+/-1.18, P=0.0001) as was the percentage of CAs (0.49+/-0.91 versus 1.08+/-1.21, P<0.0001) and the mitotic indices (1.25+/-0.44 versus 1.33+/-0.33, P=0.025). The frequency of MN in binucleated (BN) cells, however, was significantly higher in the Saraphi population (12.01+/-3.57 versus 9.99+/-3.11, P<0.0001) as was the percentage of BN cells with MN (1.14+/-0.31 versus 0.93+/-0.23, P<0.0001). There was no difference in the nuclear division indices (1.49+/-0.07 versus 1.47+/-0.11, P=0.1759) between the two populations. With regard to the effect of confounding factors, it was found that cigarette smoking influenced both CA and MN frequencies, and that the chewing of fermented tea leaves or betel nuts affected CA and sex affected MN frequencies. An increasing of CA and MN frequencies were seen in smokers and chewers over non-smokers and non-chewers, with CA frequencies being higher in Chom Thong smokers and chewers and MN frequency being higher in Saraphi smokers. However, pesticide exposure and alcohol consumption had no impact on CA and MN frequencies. Due to the conflicting results obtained in the two tests, we cannot make a clear statement regarding the potential effects of the environmental exposures in the two study populations.