Review
The HUman MicroNucleus project on eXfoLiated buccal cells (HUMNXL): The role of life-style, host factors, occupational exposures, health status, and assay protocol

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Abstract

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMNXL collaborative study. The HUMNXL project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52–1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p < 0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40 cig/day, FR = 1.37; 95% CI 1.03–.82) and decreased with daily fruit consumption (FR = 0.68; 95% CI 0.50–0.91). The results of the HUMNXL project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency.

Introduction

The number of publications referring to the human buccal micronucleus (MN) cytome assay (BMCyt) has greatly increased in the last decade [1]. This growing interest may be explained by several factors, including its relative technical simplicity and the variety of complementary toxicological endpoints evaluated. These include micronuclei (MNi, a biomarker of chromosome breakage or loss occurring as a result of chromosome fragments or whole chromosome lagging during mitosis), and other nuclear anomalies such as nuclear buds (indicative of gene amplification), binucleated cells (caused by cytokinesis-failure or arrest), and various forms of cell death measured as condensed chromatin, karyorrhectic, pyknotic or karyolitic cells as well as the frequency of basal and fully differentiated cells [2]. Furthermore, the minimal invasiveness of cell collection, their ease of storage and slide preparation makes the BMCyt the preferred choice for molecular epidemiology studies. In addition, the evidence linking MN frequency in peripheral lymphocytes of healthy individuals with the risk of cancer [3], cardiovascular diseases [4], and neurodegenerative disorders [5] lends further support to the use of the BMCyt in biomonitoring genetic damage in humans.

The mechanism of MN formation in buccal exfoliated cells is consistent with the model proposed for lymphocytes. MNi are small extranuclear bodies formed by chromosome fragments or whole chromosomes lagging behind at anaphase during nuclear division that are not incorporated into resulting daughter nuclei [1]. The MN assay in a rapidly dividing tissue, such as the buccal mucosa, allows the assessment of DNA damage without the need for an ex vivo cell replication step. Another advantage of this minimally invasive approach is that it can be used without establishing cell culture, a technique normally associated with classical metaphase analyses, and also with interphase analysis, such as in the cytokinesis block method in binucleated lymphocytes. After exposure it takes 2–3 weeks till MN can be detected in exfoliated cells [6], [7]. This feature allows detecting only the effect of recent acute or chronic exposures, and the efficiency of this approach in detecting toxic effects in other regions of the body is often discussed [1]. More detail about the formation of MNi in buccal exfoliated cells, including determinants and technical features, can be found in a recent comprehensive review [1], while a detailed protocol of the assay has been published by the HUMNXL project consortium [2].

The collection of exfoliated cells from the inner wall of the cheek is minimally invasive, and therefore this assay is well suited for large biomonitoring studies, especially in pediatric populations. Other advantages are the specificity for detecting the effects of exposure to inhaled or ingested genotoxic agents, and the easy storing of samples – before and after processing – in fixative solution or as fixed slides at room temperature. In addition, the strong correlation of MN frequency in buccal exfoliated cells with MN frequency in lymphocytes [8] (Fig. 1) implies that (i) systemic genotoxic effects within the blood stream may also impact on and be detectable in buccal cells and (ii) findings on exposure and genetic variables affecting MN frequency in lymphocytes may potentially also apply to some degree to buccal cells, including the association of MN with cancer risk [3].

In order to have widespread acceptance and credibility in human population studies the BMCyt requires standardization of the protocol, of scoring criteria, and a better knowledge of critical features affecting the assay outcomes, including the definition of the spontaneous MN frequency. These limitations can be best addressed by international collaborative studies which enable the creation of a network of laboratories, and as a consequence the assembly of large databases which allows a more detailed analysis of the assay's performance and study of the biological/clinical events associated with this biomarker. Based on the experience of the HUman MicroNucleus (HUMN) project which was initially focused on the cytokinesis-block MN cytome assay in peripheral blood lymphocytes (www.HUMN.org; [9]), an international collaborative study on MN frequency and other nuclear anomalies in buccal exfoliated cells was launched at the Conference on Environmental Mutagens in Human Populations, held in Antalya, Turkey in 2007 [10], [11]. This new project was designated HUMNXL (‘XL’ referring to eXfoLiated cells), and was specifically designed to (i) identify technical variables that affect the measurement of buccal MN frequency in human populations, (ii) identify life-style variables affecting this frequency, (iii) identify protocol variables that affect the recovery and scoring of MN, (iv) use this information to design intra- and inter-laboratory validation studies of the method and scoring criteria, and (v) determine the role, if any, of buccal MN and other nuclear alterations in monitoring genomic damage and the prediction of cancer and other degenerative diseases.

The first activity for the HUMNXL project was to perform a state-of-the-art, worldwide survey among laboratories that reported using the buccal MN exfoliated cell assay or that were interested in starting the assay for future studies. Overall, 58 laboratories from 25 countries initially agreed to participate in a collaborative survey. This exercise provided valuable information about the most widely used technical procedures, the extent and the quality of epidemiological data, and enabled the HUMNXL steering committee1 to identify a large network of interested laboratories [12].

In the report presented here as the second step of the project, a database of more than 5000 subjects with data for buccal MN and other nuclear anomalies has been compiled and analyzed, using data from 30 laboratories worldwide with experience in the use of the assay. The pooled dataset was analyzed to assess the impact of host factors, occupation, life-style, health status, and technical features of the protocol on the occurrence of MN in exfoliated buccal cells. In addition, the study was designed to provide a set of reference range values of spontaneous buccal cell MN frequency, for each age and gender group, against which future studies may be calibrated and/or compared.

Section snippets

Subjects and laboratories

All laboratories that participated in the initial HUMNXL state-of-the-art survey [12], and claimed direct experience with the buccal MN assay including publication of their data in peer-reviewed journals, were invited to contribute databases of their original studies to the HUMNXL coordinating center. An information package was sent to all laboratories that accepted the invitation. The package included (i) a detailed questionnaire requesting information about laboratory protocol, scoring

Effects of methodological aspects of the protocol

A summary of the various procedures used by the laboratories, and their contributions to the MN frequencies is reported in Table 3. With respect to cell collection, the most commonly used methods, a toothbrush or tongue depressor, produce similar MN frequencies [2.00‰ vs. 2.63‰; FR (frequency ratio) = 0.91 (0.29–2.86)]. The MN frequency from the only laboratory that used a metal spatula are much higher, i.e., 5.47‰, while the use of the Cytobrush, apparently resulted in a lower MN frequency

Discussion

The results of this survey – which involved a representative number of laboratories using the buccal MN assay in population studies – provided a unique opportunity to understand the technical, biological and epidemiological features of the BMCyt. Several parameters were investigated, including the spontaneous occurrence of cells with chromosomal damage in normal and degenerated buccal cells, the variability associated with these spontaneous values, the influence of life-style and genotoxic

Conclusions

The large number of laboratories that volunteered to take part in this project, and the data that were submitted, confirmed the feasibility of international collaborative studies and their important role as a valuable tool for biomarker validation. The HUMNXL study addressed a number of technical, mechanistic, and epidemiological items concerning the human buccal MN assay, providing reference frequency values and creating the basis for a more detailed validation effort. Original evidence has

Conflict of interest

None.

Acknowledgements

We are extremely grateful to the many scientists, their laboratory associates and staff, as well as the volunteers who have contributed their buccal cell samples for enabling these important buccal micronucleus assay data to be collected and compiled. Financial support to the project was given by grants funded by Associazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry of Health, Fondazione Buzzi Onlus, Casale, Italy; Channel 7, Adelaide, Australia.

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