The cytokinesis-block micronucleus cytome assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Cytostatic effects are measured via the proportion of mono-, bi- and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios. Further information regarding mechanisms leading to MNi, NPBs and NBUDs formation is obtained using centromere and/or telomere probes. The assay is being applied successfully for biomonitoring of in vivo genotoxin exposure, in vitro genotoxicity testing and in diverse research fields such as nutrigenomics and pharmacogenomics as well as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. The procedure can take up to 5 days to complete.

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Cytokinesis-block micronucleus cytome assay

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.

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

Chromosomal changes: induction, detection methods and applicability in human biomonitoring.

The effect of tobacco smoking on the frequency of micronuclei (MN) in human lymphocytes has been the object of many population studies. In most reports, the results were unexpectedly negative, and in many instances smokers had lower frequencies of MN than non-smokers. A pooled re-analysis of 24 databases from the HUMN international collaborative project has been performed with the aim of understanding the impact of smoking habits on MN frequency. The complete database included 5710 subjects, with 3501 non-smokers, 1409 current smokers, and 800 former smokers, among subjects in occupational and environmental surveys. The overall result of the re-analysis confirmed the small decrease of MN frequencies in current smokers (frequency ratio (FR)=0.97, 95% confidence interval (CI)=0.93–1.01) and in former smokers (FR=0.96, 95% CI=0.91–1.01), when compared to non-smokers. MN frequency was not influenced by the number of cigarettes smoked per day among subjects occupationally exposed to genotoxic agents, whereas a typical U-shaped curve is observed for non-exposed smokers, showing a significant increase of MN frequency in individuals smoking 30 cigarettes or more per day (FR=1.59, 95% CI=1.35–1.88). This analysis confirmed that smokers do not experience an overall increase in MN frequency, although when the interaction with occupational exposure is taken into account, heavy smokers were the only group showing a significant increase in genotoxic damage as measured by the micronucleus assay in lymphocytes. From these results some general recommendations for the design of biomonitoring studies involving smokers can be formulated. Quantitative data about smoking habit should always be collected because, in the absence of such data, the simple comparison of smokers versus non-smokers could be misleading. The sub-group of heavy smokers (≥30 cigarettes per day) should be specifically evaluated whenever it is large enough to satisfy statistical requirements. The presence of an interaction between smoking habit and occupational exposure to genotoxic agents should be always tested.

Effect of smoking habit on the frequency of micronuclei in human lymphocytes: results from the human micronucleus project

Associazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of Health, Fondazione Buzzi Onlus, Casale, Italy

The human micronucleus project on exfoLiated buccal cells (HUMNXL): the role of life-style, host factors, occupational exposures, health status, and assay protocol

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of "normal" values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin-B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5 per thousand and the interquartile range was between 3 and 12 per thousand. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14-24%). Statistical analyses were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.

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HUman MicroNucleus Project: International database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei

Ludmilla Mikhalevich

Papers

HUman MicroNucleus Project: International database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei