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.     

HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures

Criteria for scoring micronuclei and nucleoplasmic bridges in binucleated cells in the cytokinesis-block micronucleus assay for isolated human lymphocyte cultures are described in detail. Morphological characteristics of mononucleated cells, binucleated cells, and multinucleated cells as well as necrotic and apoptotic cells and nuclear buds are also described. These criteria are illustrated by a series of schematic diagrams as well as a comprehensive set of colour photographs that are of practical assistance during the scoring of slides. These scoring criteria, diagrams and photographs have been used in a HUman MicronNucleus (HUMN) project inter-laboratory slide-scoring exercise to evaluate the extent of variability that can be attributable to individual scorers and individual laboratories when measuring the frequency of micronuclei and nucleoplasmic bridges in binucleated cells as well as the nuclear division index. The results of the latter study are described in an accompanying paper. It is expected that these scoring criteria will assist in the development of a procedure for calibrating scorers and laboratories so that results from different laboratories for the cytokinesis-block micronucleus assay may be more comparable in the future.     

Evaluation of the general suitability of the rat for the micronucleus assay: the effect of cyclophosphamide in 14 strains.

To evaluate the general suitability of the rat for the micronucleus assay, we conducted the assay in males of 14 different strains, 13 inbred (ACI, BN, BUF, COP, DRH, F344, IS, LEW, RCS, SHR, WAG, WKYO, WTC) and 1 outbred (SD), using cyclophosphamide as the test chemical. Cyclophosphamide at 0 (vehicle), 5, 10, or 20mg/kg per day was administered orally twice, 24h apart, to five rats per dosage group. Bone marrow and peripheral blood were collected 24h after the second treatment.All 14 strains showed a positive response to cyclophosphamide, with slight differences in sensitivity. We concluded that the rat is suitable for the micronucleus assay regardless of strain.     

The quantitative analysis of micronucleus induction in human lymphocytes cultured in vitro (preconditions for making modified micronucleus assay)

The new modification of the method of micronucleus (MN) detection without cytochalasin-B is used in this paper. The code name of the method is called "method of micronucleus detection in mononucleated cells". The basis of this method is that it makes possible to analyze MN and chromosome aberrations (CA) at the same slides. To confirm the true supposition of the authors about correlation between MN quantity and chromosome/chromatid type aberrations so called "coefficient of transformation" was calculated and it was 7.9 +/- 0.41 for the chromosome type aberrations and over 67.2 +/- 30.2 for chromatid type aberrations. Mutagenic action of gamma-irradiation and 8-methoxypsoralen (8-MOP), activated with long-wave UV-light was estimated for the first cell cycle mitosis. When compared in straight experiments results of gamma-induced MN were received by two methods: the method of cytokinesis-block (commonly used) and by the suggested method, the "coefficient of transformation" of CA into MN was 3.6, when cytochalasin-B was used and 6.7 without using it. The total data give a possibility to make a new cytological micronucleus test for mutagens revealing. As we think the modified test is more simple, more reliable less laborious and less expensive.     

Feasibility of automating the micronucleus assay

The results of a feasibility study on the automation of the micronucleus assay in whole blood cultures of human lymphocytes are reported. The assay requires determination of the number of lymphocytes with micronuclei among the proliferating population. Using an in-house-assembled image analysis system, a prototype software package was developed that addressed two problems: micronuclei identification and discrimination of nonproliferating cells from proliferating lymphocytes (the only ones that can give rise to micronuclei). The results of manual verification of automated micronucleus scoring showed that 70% of all digitized micronuclei were extracted from the images and 90% of them were correctly classified and paired with a parent nucleus by an "affinity function". The discrimination between proliferating and nonproliferating cells was carried out by linear discriminant analysis of simple nuclear features extracted from Feulgen-stained cells. Among the Feulgen-stained nuclei that were identified by autoradiography as proliferating or not, 85% were correctly classified by a six-feature discriminant function.     

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.     

The micronucleus assay in exfoliated human cells: a mini-review of papers from the CIS

The critical analysis of the data concerning micronucleus assay in exfoliated epithelial cells presented by the investigators from the CIS is carried out. Twenty two articles are evaluated, and shortcomings of some of them are discussed. Presented results are compared whenever possible with literature data. The aim of the mini-review is a criticism of shortcomings of the papersforfurther improvement of the presentation of the data on micronucleus assay which will give the possibility to compare the results with the data presented by foreign investigators.     

Indirect mutagen assay in human lymphocyte in the presence of a metabolic activation system

Chromosome aberrations in cultured human lymphocytes were examined after exposures to various concentrations (from 1 X 10(-6) to 1 X 10(-3) mol X l-1) of cyclophosphamide (CP) in the presence or absence of a metabolic activation system (S9 mix). With metabolic activation, increases in the frequency of aberrant cells (AB. C.) produced by CP were significant and dose-dependent. At a concentration of 5 X 10(-4) mol X l-1, activated CP induced 29% AB. C. versus 6% AB. C. detected after exposures to CP without metabolic activation. The freshly prepared S9 mix did not virtually differ in its activation potency from the S9 mix stored for 3 weeks at -20 degrees C. CP preincubated for 100 min with S9 mix caused little or no increase in AB. C. frequency above the control level.     

The micronucleus assay in exfoliated human cells: A mini-review of papers from the CIS

A critical analysis of data from micronucleus assays in exfoliated epithelial cells presented by the investigators from the CIS is carried out. Twenty two articles are evaluated, and the shortcomings of several of them are discussed. These results are compared whenever possible with literature data. The aim of this mini-review is a criticism of the shortcomings of these papers in order to stimulate improvement of the presentation of micronucleus assay data, which will allow the comparison of these results with data presented by foreign investigators. Published in Russian in Tsitologiya i Genetika, 2007, Vol. 41, No. 2, pp. 56–66. The text was submitted by the authors in English.     

Screening for interindividual differences in radiosensitivity by means of the micronucleus assay in human lymphocytes

The response of unstimulated peripheral lymphocytes to a single dose of 3 Gy of 137Cs gamma rays was analysed in blood samples from 30 donors by a conventional micronucleus assay and from 14 donors by the cytokinesis-block (CB) method. Significant interindividual variations could be detected for the baseline levels and for induced levels of micronuclei. An age effect could be demonstrated with the conventional method for the number of spontaneous MN, but not with the CB method. The corresponding numerical estimate was 3.4 +/- 1.3% increase per year. No such increase was apparent for induced frequencies. Provided that cell proliferation kinetics is reliably taken into account the micronucleus assay could be helpful for diagnosing potential radiosensitive individuals.     

The micronucleus assay in Anodonta cygnea for the detection of drinking water mutagenicity

A micronucleus test in gill cells of the freshwater mussel Anodonta cygnea has been proposed for the detection of drinking water genotoxicity. Animals were exposed for 28 days to a drinking water sample and collected every week. Highly significant increases in spontaneous MN frequency were observed at each sampling, especially after 13 days of exposure. As positive control 2 doses of mytomicin C (MMC) were used (10(-8) and 10(-7) M). A second experiment was performed at a municipal waterworks in order to assess the role of water treatment processes in the production of mutagenic compounds. The most prevalent genotoxic effects were detected after chlorination (mean: 10.47% +/- 3.05, p less than 0.001).     

The micronucleus assay as a biological dosimeter in hospital workers exposed to low doses of ionizing radiation

The health risk associated with low levels of ionizing radiation is still a matter of debate. A number of factors, such as non-target effects, adaptive responses and low-dose hypersensitivity, affect the long-term outcome of low-dose exposures. Cytogenetic bio-dosimetry provides a measure of the absorbed dose, taking into account the individual radiation sensitivity. The aim of the present study is to evaluate the value of the micronucleus (MN) test as a bio-dosimeter in hospital workers exposed to low doses of ionizing radiation. Blood samples were obtained from 30 subjects selected among workers exposed to X- and gamma-radiation, and 30 controls matched for sex, age and smoking from the same hospital. Micronucleus frequencies were analyzed by use of the cytokinesis-block method. The MN frequency was compared among the groups considering the confounding factors and the length of employment. No increase in the number of bi-nucleated cells with MN (BNMN), but a significant increase in the number of mono-nucleated cells with micronuclei (MOMN) was observed in exposed subjects compared with the controls. The relationship between MN frequency and accumulated dose (mSv) was evaluated. The length of employment did not affect the extent of MN frequency, but an increase of BNMN and MOMN cells was observed based on the accumulated radiation dose. Our study shows the sensitivity of the MN test in the detection of cytogenetic effects of cumulative exposure levels, suggesting the potential usefulness of this assay in providing a biological index in medical surveillance programs.     

The use of bromodeoxyuridine labeling in the human lymphocyte HGPRT somatic mutation assay

The autoradiographic assay developed by Strauss and Albertini (1979) to quantitate human in vivo somatic mutation at the hypoxanthine guanine phosphoribosyl-transferase locus uses tritiated thymidine to identify mutant cells by their ability to pass through 'S' phase in the presence of 6-thioguanine. An alternative method, based on the incorporation of bromodeoxyuridine (BrdUrd) into the DNA of proliferative cells, followed by differential staining with the fluorescence-plus-Giemsa method, was used to identify 3 classes of lymphocyte nuclei: (a) small darkly stained nuclei, (b) large, reddish-colored nuclei with an apparent nucleolus, and (c) large, bluish-colored nuclei. By double labeling with BrdUrd and tritiated thymidine, it was determined that only the nuclei of the third class had incorporated BrdUrd. These results demonstrate that the technique used for sister-chromatid differentiation can be used to detect putative HGPRT mutants and to determine variant frequencies at the HGPRT locus.     

Studies on three structurally related phenylenediamines with the mouse micronucleus assay system.

Three structurally related compounds, 4-chloro-o-phenylenediamine (COP), 4-nitro-o-phenylenediamine (NOP) and p-phenylenediamine dihydrochloride (PPD), are used in fur dyes, inks and hair coloring formulations. COP has been reported to be carcinogenic in both rats and mice. NOP and PPD are non-carcinogens, but have consistently tested positive in short-term in vitro genotoxicity assays. Studies were undertaken to evaluate their genotoxicity with the in vivo mouse bone-marrow micronucleus assay. Five CD-1 male mice per dose were injected i.p. with the compounds and sacrificed at intervals of 24, 48 and 72 h. 2000 cells were scored per animal to determine the frequency of micronucleated-polychromatic erythrocytes (MPCE). COP induced significant dose-related increases in MPCE over the 3 doses tested at each of the sampling intervals. The peak response occurred at 24 h. No response was observed in animals treated with PPD or NOP.