HUMN Project Workshop at ICEMHP Turkey 2007

HUMN project workshop on the buccal micronucleus assay

International Conference on Environmental Mutagens in Human Populations in Antalya Turkey in May 20-23 2007

The workshop will be on Monday 21st May 2007 from 20.30 to 22.30 .

There is no registration fee. Anyone who is interested can attend.

Aims of the Workshop:

1. Discuss current state of knowledge on the buccal micronucleus assay.
2. Identify important gaps of knowledge regarding theory, biology and methods.
3. Decide on the appropriate plan of action to resolve the key methodological and knowledge gap issues.
4. Explore possibility of pooling data bases to determine most important variables affecting the assay

The HUMN project (www.humn.org) is an international collaborative project founded in 1997

Program for HUMN Workshop Antalya Turkey

Chairpersons: Michael Fenech and Nina Holland

Michael Fenech (Australia): Overview of the achievements and goals of the HUMN project and the objectives of the Buccal MN Workshop. (5 min)

Nina Holland (USA): Biomonitoring of cytogenetic damage in children and young adults using the buccal MN assay.  (25 min)

Siegfried Knasmueller (Austria): Important effects of staining methods and scoring criteria on the observed result in the buccal MN assay (25 min)

Claudia Bolognesi (Italy): Demographic and exposure variables affecting buccal MN frequency in adults. (10 min)

Sema Burgaz (Turkey) (yet to be confirmed): Use of the buccal MN assay in genotoxin exposure studies in Turkey. (10 min)

Stefano Bonassi (Italy): The use of statistical and epidemiological methods to identify methodological , demographic and exposure variables affecting MN frequency and its association with cancer risk. (25 min)

Open discussion on prospects and plans for an international collaborative study to harmonise and standardise the use of the buccal MN assay worldwide. (20 min) 

Report on the HUMN Project workshop on the Buccal Micronucleus Assay – Antalya Turkey 2007

International Conference on Environmental Mutagens in Human Populations
Venue: Corinthia Hotel, Tekirova, Antalya, Turkey.
Date 21st May 2007.

Authors of the Report:
Michael Fenech*, Nina Holland, Siegfried Knasmueller, Sema Burgaz and Stefano Bonassi.

* corresponding author

The workshop was attended by 70 representatives from various laboratories, universities, private companies and government departments from around the world. A list of participants and their email addresses was collected during the meeting.

The aims of the workshop were to:

• Discuss current state of knowledge on the buccal micronucleus (MN) assay.

• Identify important gaps of knowledge regarding theory, biology and methods. 

• Decide on plan of action to resolve the key methodological and knowledge gap issues. 

• Explore pooling data bases to determine most important variables affecting the assay 

As part of the preparation for this workshop the HUMN Project coordinating group has prepared a review to define the current status of the buccal MN assay and identify important knowledge and technological gaps that need to be resolved to achieve the potential of this minimally invasive procedure.  This manuscript will be published as part of the proceedings of the conference.

The workshop opened with a brief presentation of the history and objectives of the HUMN project by Michael Fenech. He emphasised the need to consider that MN assays are evolving from the single MN biomarker approach into “cytome” assay systems of multiple DNA damage (including MN, aneuploidy), cytotoxicity (including cell death) and cell proliferation biomarkers. This has already been demonstrated for the cytokinesis-block MN assay in lymphocytes and is expected to be the case also for the buccal MN assay as in fact was already indicated in some of the key publications on this method.

Nina Holland gave a presentation on the use of the buccal MN assay for biomonitoring of cytogenetic damage in children and young adolescents. She emphasized that little is known about baseline and intra-individual variability in this age group. She presented the controversial results of available validation studies on staining methods for buccal cells and highlighted the need for HUMN-coordinated buccal cell MN validation study to address effects of collection, processing, staining and scoring in different laboratories. Results from the studies of air pollution, inflammatory bowel diseases and asthma from her laboratory were used to illustrate the usefulness of this non-invasive biomarker of cytogenetic damage in epithelial tissues for children’s studies. Proposed future directions of research in this area include association of MN in buccal cells with cancer and other diseases, correlation with blood MN data, and studies of diverse ethnic and age groups.
 
Siegfried Knasmüeller described the results of investigations concerning the impact of different staining procedures on the results of MN assays with exfoliated buccal cells. They investigated MN formation in oral mucosa cells from heavy smokers and non-smokers with DNA specific stains (Feulgen, acridine orange, DAPI) and non specific stains (Giemsa, May Grünwald). While no increase of MN frequencies in smokers was detectable with any of the DNA-specific stains, significant induction of MN (3-5 fold over the control level) was detectable in the smokers group relative to non-smokers with the non-specific ones. Furthermore, correlation analyses showed that the MN frequencies in Giemsa-stained slides correlated with nuclear aberrations such as karyorrhexis, karyolysis, condensed chromatin and binucleates while no such correlations were detected with Feulgen. These findings indicate that these nuclear anomalies as well as keratin bodies which are formed in cells of epithelial origin as a consequence of acute toxic effects may be misinterpreted as MN with non specific stains and lead to false positive results.    

Sema Burgaz provided an overview on the use of the buccal MN assay in genotoxin exposure studies in Turkey. She explained that her laboratory adopted the Feulgen/Fast green staining and scoring criteria of Tolbert and colleagues in all of her studies. In her presentation, she indicated that efficiency of detecting chromosomal damage due to occupational exposure to antineoplastic drugs was lower for MN in buccal cells as compared to that of MN in lymphocytes. Regarding the PAH exposure, she said that significant induction of MN in buccal cells from taxi drivers and traffic policemen were found and same effect was also observed by chromosome aberration rates in lymphocytes of same subjects. She provided data indicating that site-specific differences in MN were available for Maras powder, a kind of smokeless tobacco, and also suggested that buccal cells as well as nasal cells may be important targets of formaldehyde-induced genotoxic effects as measured by MN frequency. She finally emphasized that her studies were mainly based on chronic exposure situations and MN frequency in buccal cells seems to be informative for assessment of cytogenetic damage. She also stressed that background MN levels in her studies as well as other studies in Turkey  varied by a factor of ~3  and that  harmonization of the MN assay in buccal cells is needed for appropriate data comparison across laboratories.

In his presentation Stefano Bonassi focussed on the use of epidemiological methods to identify variables affecting buccal MN frequency and its association with cancer risk. He indicated that a project similar to that performed by HUMN for peripheral blood lymphocytes is being planned on base-line MN frequency in buccal cells with the aim of collecting information from laboratories that have published their studies in reputable peer-reviewed journals. Information relating to factors such as demographic, methodological, life-style, dietary, genetic and occupational exposure variables will be particularly important. The ultimate aim will also be to test whether MN frequency in buccal cells is predictive of cancer risk or other pathologies in the aerodigestive tract as well as other sites in the body. He also suggested that this new project focussed on buccal e(X)foliated cells should be called the HUMN-X project to distinguish it for the original HUMN project on peripheral blood.     

After these presentations there was an open discussion on priorities, prospects and plans for a collaborative study to improve and standardise buccal MN assay. There was an energetic discussion on various aspects of the assay reflecting the urgency and importance of resolving methodological issues to enable a harmonised application of this important method. Some of the notable suggestions from the audience include the following:

• A coordinated multi-centre approach is needed that is properly funded to compare the effect of staining methods (particularly DNA-specific stains) and base-line frequencies together with collection of DNA to investigate the impact of genotype as well as other biomarkers such as adducts.
• Large data bases are already available in various centres that could be contributed to the HUMN-X project. Some of these databases may vary in staining method and scoring criteria and could therefore potentially be used to identify key methodological variables that affect the measurements with this assay.
• Several participants emphasised the need to standardise the buccal cell collection, cell washing and slide preparation and staining methods as these are thought to be major variables between laboratories.
• It was generally acknowledged that there are considerable discrepancies among laboratories with regards to the scoring method for micronuclei depending on whether they are scored only in non-degenerate and degenerate cells or in basal cells or differentiated cells. Furthermore the various classifications of dead/dying cells (e.g. condensed chromatin, karyorrhectic, pyknotic and karyolitic) were not interpreted in the same way amongst laboratories. It was evident that there is a strong need for the scoring criteria to be clearly defined in detail as has been done for the cytokinesis-block micronucleus assay. It was suggested that a collection of photomicrographs of the various cell types scored in buccal MN assays in different laboratories should be initiated.
• A recurring issue that was raised is whether MN frequency in buccal cells and lymphocytes are correlated with each other. This issue needs to be resolved because if these biomarkers are not strongly and positively correlated with each other it becomes necessary to use both systems to comprehensively measure genome damage.
• In addition it was noted that for studies involving exposure to inhaled genotoxins (e.g. formaldehyde, diesel fumes etc.) it may be more appropriate to also sample nasal epithelial cells. Therefore a recommendation was also made to include nasal epithelial cells when the HUMN-X project initiates its efforts to define the methodology for the buccal MN assay.
• There was general agreement that as a minimum the main demographic, exposure, genetic and life-style variables that impact on the buccal MN assay should be properly defined when analysing contributed data bases.
• Practical issues relating to an inter-laboratory slide scoring exercise were actively discussed. An important issue was the collection of a positive control sample (i.e. high MN frequency) given that such samples are only possible form cancer patients undergoing radiotherapy in the head and neck region. Another aspect was that demands on participating laboratories had to be limited to a level that was acceptable and practical in the absence of external funding.
• It was agreed that a research proposal be put together and submitted for funding to NIH or other similar funding bodies and that such a proposal could be used by participating laboratories to seek local funds to enable/facilitate their participation.

It was agreed that two phases could be initiated within the next twelve months as follows:

1. A protocol for collection of data bases is to be developed and once this is ratified data base collection can commence. This aspect will be coordinated primarily through the efforts of Dr Stefano Bonassi at the Italian National Cancer Institute.
2. A detailed method paper is to be written based on the most commonly used protocols including procedures of cell collection, cell preparation, slide preparation, slide staining, scoring criteria and scoring method (i.e. number of cells scored and statistical power). The scoring criteria section would include detailed line diagrams of the various cell types scored in the buccal MN “cytome” assay together with clear photomicrographs of the various cell types stained using Feulgen/Light Green (under light and fluorescence microscopy) and DAPI staining (fluorescence only). Writing of the method paper will be led by Dr Michael Fenech (CSIRO Human Nutrition, Australia) with the assistance of Dr Nina Holland (University of California, Berkeley, USA), Dr Claudia Bolognesi (Italian National Cancer Institute) and Dr Siegfried Knasmueller (University of Vienna). The draft method paper will also be open to review by other experts prior to submission for publication in Nature Protocols. The method paper will be a joint publication of the HUMN Project and those providing input will be appropriately acknowledged.

The inter-laboratory slide scoring comparison cannot commence until the scoring criteria paper is finalised and published. In the interim plans for the inter-laboratory slide-scoring exercise will commence and a working group for this purpose will be set up and led by Dr Nina Holland with assistance from Dr Siegfried Knasmueller, Dr Claudia Bolognesi, Dr Micheline Kirsch-Volders, Dr Sema Burgaz, Dr Michael Fenech and Dr Stefano Bonassi. Given the importance of automation it will also be necessary to involve companies developing automated image cytometry systems at an early stage and to include such systems in the slide scoring exercise.

Acknowledgements:
The HUMN project coordinating group is thankful to the organisers of the ICEM conference in Antalya, Turkey for providing the venue and facilities for the meeting and to the participants at the workshop for their insightful contributions during the discussions.  Errol Zeiger is acknowledged for his assistance with reviewing the report.