Micronucleus Assay In Human Lymphocytes Research Articles

The micronucleus assay in human lymphocytes: screening for inter-individual variability and application to biomonitoring.

Micronuclei levels were assessed in cytokinesis-blocked lymphocytes of 200 male and female healthy donors not occupationally exposed to genotoxic risks and of 33 male industrial painters handling genotoxic substances. Frequency of micronucleated cells was 9.87 +/- 3.1 per 1000 in the control population and was shown to have a large inter-individual variability. The study of factors contributing to this variability showed that only smoking could affect micronucleated cell rate, inducing an increase of 25%, whereas age and sex had no effect. Among the industrial painters, frequency of micronucleated cells averaged 18.30 +/- 7.39 per 1000: the difference between the two populations studied was shown to be statistically significant by the Mann-Whitney rank sum test (one-sided U test) and indicated that exposed painters need preventive measures.

Cytogenetic damage induced in human lymphocytes by four vanadium compounds and micronucleus analysis by fluorescence in situ hybridization with a centromeric probe

The genotoxicity of four vanadium compounds, sodium metavanadate (NaVO 3), ammonium metavanadate (NH 4VO 3), sodium ortovanadate (Na 3VO 4) and vanadyl sulfate (SVO 5), was evaluated in human lymphocyte cultures using structural and numerical chromosome aberrations, micronuclei, sister-chromatid exchanges and satellite chromosome associations as endpoints. These compounds were not found to increase the frequency of structural chromosome aberrations whereas a significant increase in numerical aberrations, micronuclei and satellite associations was found. Since these results could have been related to a possible mechanism of the action of vanadium as a mitotic spindle poison, the fluorescence in situ hybridization (FISH) technique was applied to the human lymphocyte micronucleus assay, by means of an alphoid centromere-specific DNA probe. The four vanadium salts showed a micronucleus percentage with positive signal (presence of centromere and thus of whole chromosome(s)) that was always higher than 68% at all doses tested. That confirmed the aneuploidogenic potentiality of vanadium.

The cytokinesis-block micronucleus technique and its application to genotoxicity studies in human populations.

The development of the cytokinesis-block (CB) technique has made the human lymphocyte micronucleus assay (MN) a reliable and precise method for assessing chromosome damage. Recent studies in our laboratory have confirmed that this method is a sensitive indicator of in vivo radiation exposure in patients undergoing fractionated partial-body radiotherapy and rodents exposed to uniform whole-body irradiation, thus supporting the application of the cytokinesis-block micronucleus (CBMN) assay for biological dosimetry. To further define the use of this assay in biomonitoring, we have also undertaken extensive studies to determine the spontaneous level of MN in normal human populations and its relationship to various lifestyle factors. During the past year, we have also developed a new variation to the CBMN assay that enables the conversion of excision-repairable lesions to MN within one cell-cycle using cytosine arabinoside. With this method the slope of the in vitro dose-response curves was increased by a factor of 1.8 for X-rays, 10.3 for ultraviolet (254 nm) radiation, and approximately 40-fold for methylnitrosourea. Consequently, the CBMN assay can now be used not only to measure whole chromosome loss or chromosome breaks but also excision repair events. The versatility and simplicity of the CBMN assay together with new developments in automation should enable its successful application in monitoring exposed populations as well as identifying mutagen-sensitive individuals within a population.

The cytokinesis-block micronucleus technique: A detailed description of the method and its application to genotoxicity studies in human populations

The development of the cytokinesis-block (CB) technique has transformed the human-lymphocyte micronucleus assay (MN) into a reliable and precise method for assessing chromosome damage. Recent studies in our laboratory have confirmed that this method is a sensitive indicator of in vivo radiation exposure in (a) patients undergoing fractionated partial-body radiotherapy and (b) rodnets exposed to uniform whole-body irradiation, thus supporting the application of the cytokinesis-block micronucleus (CBMN) assay for biological dosimetry. To further define the use of this assay in biomonitoring we performed extensive studies to determine the spontaneous level of MN in normal human populations and its relationship to various life-style factors. We have also developed a new variation to the CBMN assay that permits the conversion of excision-repairable lesions to MN within one cell-cycle using cytosine arabinoside. With this method the slope of the in vitro dose-response curves was increased by a factor of 1.8 for X-rays, 10.3 for ultraviolet (UV, 254 nm) radiation and approximately 40-fold for methylnitrosourea. Consequently the CBMN assay can now be used to measure not only whole chromosome loss or chromosome breaks but also excision-repair events. The versatility and simplicity of the CBMN assay together with new developments in automation should ensure its successful application in monitoring exposed populations as well as in identifying mutagen-sensitive individuals within a population.

A collaborative study on the improvement of the micronucleus test in cultured human lymphocytes.

A collaborative study between two laboratories has been carried out in order to investigate the reproducibility of the cytokinesis-block micronucleus assay in human lymphocytes. A modified protocol to obtain good quality slides from whole blood cultures has been developed. The spontaneous frequencies of micronuclei in binucleated lymphocytes from five male subjects were evaluated in each laboratory at two cytochalasin-B concentrations (3 and 6 micrograms/ml). Our data show that 6 micrograms/ml cytochalasin-B was more effective than 3 micrograms/ml to arrest cytokinesis in all the donors analysed. The mean frequency of micronuclei in binucleated cells was significantly lower at 6 micrograms/ml compared to 3 micrograms/ml cytochalasin-B in both laboratories. The results obtained indicate a very high concordance between both sets of data supporting the validity of the proposed protocol.

Conversion of excision‐repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes

The human lymphocyte micronucleus (MN) assay is relatively insensitive to genotoxic agents that predominantly induce excision-repairable lesions such as adducts and abasic sites. In this study we have explored the possibility of using cytosine arabinoside (ARA) to convert excision-repairable DNA lesions to micronuclei (MN) within one cell cycle. The system consisted of human lymphocytes as target cells, the cytokinesis-block (CB) method for identifying cells that had completed one nuclear division only, and X-rays, methylnitrosourea (MNU), and ultraviolet light (UV) as mutagens. With each mutagen we have observed significant increments in induced MN in the cultures that had also been treated with ARA during G1. The slope of the dose-response curves for induction of MN was increased by a factor of approximately 1.8 for X-rays and 10.3 for UV and significant MNU induction of MN was only achieved in the cultures treated with ARA. Furthermore, a 24-hr gap between mutagen exposure and the start of the assay did not abolish the increased sensitivity in the cultures treated with ARA. These observations suggested that the combined ARA and cytokinesis-block micronucleus (CBMN) method may enhance the detection of exposure to genotoxic agents that predominantly induce excision-repairable lesions.

Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5–6 μg/ml for the last 28 h) was 9–21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 μg/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5–12 μg/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (≥ 3 poles) divisions which had a high frequency of anaphase aberrations (39–53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided ≥ 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.

Human lymphocyte micronucleus assay by the anti-BrdU method

Human lymphocyte micronucleus assay by the anti-BrdU method

Evaluation of twelve potential aneuploidogenic chemicals by the in vitro human lymphocyte micronucleus assay

Within the framework of the EEC Project on the validation of tests for genomic mutations, 12 chemicals were submitted to the micronucleus assay using in vitro cultured human lymphocytes to assess their potential aneuploidogenic activity; the results will be compared with those of other in vivo and in vitro short-term genotoxicity assays developed in different laboratories. Micronuclei were evaluated in binucleate cells, after continuous treatment for 48 or 72 hr. Five compounds gave clear positive results, showing a dose-effect relationship (acetaldehyde, cadmium chloride, colchicine, griseofulvin, vinblastine); three compounds gave positive results without a clear dose-effect correlation (chloral hydrate, hydroquinone, thiomersal) and four compounds were found to be negative (diazepam, econazole, pyrimethamine, thiabendazole).

The human lymphocyte micronucleus assay: a comparison between whole-blood and separated-lymphocyte cultures

The induction of micronuclei (MN) by vincristine, mitomycin C and cyclophosphamide was compared in purified lymphocytes and in whole-blood cultures. With both assays, cytokinesis was blocked by cytochalasin B and MN were only scored in binucleate cells. The data suggest that whole-blood cultures may be considered a better experimental condition for the detection of MN induced by chemicals in vitro.

The human lymphocyte micronucleus assay: Response of cord blood lymphocytes to γ-irradiation and bleomycin

The induction of micronuclei in human cord blood lymphocytes by treatment with γ-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 μg/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.

The suitability of the human lymphocyte micronucleus assay system for biological dosimetry

Human whole blood was irradiated with 220 keV X-rays at doses of 0–4.0 Gy. After incubation periods of 48, 60, 72, 84 and 96 h, lymphocytes were prepared without colcemid pretreatment according to 2 different methods, and micronuclei were scored. The crucial point of lymphocyte preparation was found to be the osmotic pressure of the hypotonic solution. Only a method that preserves the cytoplasm of lymphoblasts is suitable for a correct association of micronuclei with the main nucleus. Similar as for structural chromosome changes, now their intercellular distribution can be analysed. This is necessary for the derivation of appropriate statistical weights which have to be used for more reliable regression analyses. For 48 h, the data can be described by the linear model, for 84 and 96 h, by the linear-quadratic model. For 60 and 72 h no such definite conclusions can be drawn. For calibration purposes a standardized culture time cannot be recommended. Because the background frequency is high, the lymphocyte micronucleus assay system is not sensitive enough to detect a significant increase in the incidence of micronuclei after exposure to low doses (< 0.3 Gy).

Establishment of stochastic significance in Ames tests and its relevance to genetical significance

Mangifera indica L. extract (Vimang) consists of a defined mixture of components (polyphenols, terpenoids, steroids, fatty acids and microelements). It contains a variety of polyphenols, phenolic esters, flavan-3-ols and a xanthone (mangiferin), as main component. This extract has antioxidant action, antitumor and immunemodulatory effects proved in experimental models in both in vitro and in vivo assays. The present study was performed to investigate the genotoxicity potential activity of Vimang assessed through different tests: Ames, Comet and micronucleus assays. Positive and negative controls were included in each experimental series. Histidine requiring mutants of Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 strains for point-mutation tests and in vitro micronucleus assay in primary human lymphocytes with and without metabolic activation were performed. In addition, genotoxic effects were evaluated on blood peripheral lymphocytes of NMRI mice of both sexes, which were treated during 2 days with intraperitoneal doses of M. indica L. extract (50–150 mg/kg). The observed results permitted to affirm that Vimang (200–5000 μg/plate) did not increase the frequency of reverse mutations in the Ames test in presence or not of metabolic activation. Results of Comet assay showed that the extract did not induce single strand breaks or alkali-labile sites on blood peripheral lymphocytes of treated animals compared with controls. On the other hand, the results of the micronucleus studies (in vitro and in vivo) showed Vimang induces cytotoxic activity, determined as cell viability or PCE/NCE ratio, but neither increased the frequency of micronucleated binucleate cells in culture of human lymphocytes nor in mice bone marrow cells under our experimental conditions. The positive control chemicals included in each experiment induced the expected changes. The present results indicate that M. indica L. extract showed evidences of light cytotoxic activity but did not induce a mutagenic or genotoxic effects in the battery of assays used.