journal of health literacy by Meimei Lin

International Journal of Health Literacy Volume 27 Number 1 June 2014

Editorial Board Paras K Pokharel BP Koirala Institute of Health Sciences

Angela Yee Man LEUNG Hong Kong University

Reginal N M. Kamoga, Community Health And Information Network (CHAIN)

Arungazina ALTYN Muafikovna Kazakhstan School of Public Health

Hiroki SUGIMORI Daito Bunka University

Botagoz Turdaliyeva Gaukhar Baisunova Kazakh National Medical University

Phoenix Kit Han MO The Chinese University of Hong Kong

Hye-Kyeong KIM The Catholic University of Korea

Eti Rimawati Nurjanah Nj Dian Nuswantoro University

Bambang WISPRIYONO Universitas Indonesia

Vo Van THANG Faculty of Public Health, Hue University of Medicine and Pharmacy

De Alwis Seneviratne SUNITHA ROHINI University of Colombo

Ali Ghufron Mukti Gadjah Mada University Minh KhuĂŞ PHAM Haiphong University of Medicine and Pharmacy Nguyen THANH HUONG Hanoi School of Public Health Truong Quang Tien Hanoi School of Public Health Shahzad Ali Khan Health Services Academy Islamabad Tabina ZAMAN Health Promotion Sarhad Institute of Health Sciences Islamabad

Tin Tin SU University of Malaya Win Myint OO University of Medicine Carmen Tolabing University of the Philippines Hirono ISHIKAWA University of Tokyo Tae Wha LEE Yonsei University

International Journal of Health Literacy Volume 1 Number 1 June 2015

CONTENTS Promoting health literacy: a case study in the prevention of diarrhoeal disease from Bangladesh/Raquiba A. Jahan

Promoting health literacy: a case study in the prevention of diarrhoeal disease from Bangladesh/Raquiba A. Jahan

The HUMN and HUMN xL international collaboration projects on human micronucleus assays in lymphocytes and buccal cells—past, present and future/ Michael Fenech*, Nina Holland, Errol Zeiger, Wushou P. Chang, Sema Burgaz, Philip Thomas, Claudia Bolognesi, Siegfried Knasmueller, Micheline Kirsch-Volders and Stefano Bonassi Promoting health literacy: a case study in the prevention of diarrhoeal disease from Bangladesh/Raquiba A. Jahan The HUMN and HUMN xL international collaboration projects on human micronucleus assays in lymphocytes and buccal cells—past, present and future/ Michael Fenech*, Nina Holland, Errol Zeiger, Wushou P. Chang, Sema Burgaz, Philip Thomas, Claudia Bolognesi, Siegfried Knasmueller, Micheline Kirsch-Volders and Stefano Bonassi Promoting health literacy: a case study in the prevention of diarrhoeal disease from Bangladesh/Raquiba A. Jahan The HUMN and HUMN xL international collaboration projects on human micronucleus assays in lymphocytes and buccal cells—past, present and future/ Michael Fenech*, Nina Holland, Errol Zeiger, Wushou P. Chang, Sema Burgaz, Philip Thomas, Claudia Bolognesi, Siegfried Knasmueller, Micheline Kirsch-Volders and Stefano Bonassi

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Original Article (International) Journal of Health Literacy Vol 15 No 4 2014 Dec pp1-7

Promoting Health Literacy: A Case Study in the Prevention of Diarrhoeal Disease from Bangladesh Raquiba A. Jahan1, Raquiba A. Jahan2 1

Department of Public Health & Community Medicine, A27, University of Sydney, NSW, 2006 Australia, Project Coordinator, Health Sector/CARE-Bangladesh 2 Department of Public Health & Community Medicine A27, University of Sydney NSW, 2006 Australia

Key Words

ABSTRACT

Bangladesh, diarrhoea, health education, health literacy, Previous SectionNext Section

The relationship between health education and health literacy is examined through a case study of a project to prevent diarrhoea in rural Bangladesh by CARE-Bangladesh in 1993. Health professionals in Bangladesh typically define health education as a one-way approach of information dissemination. Achieving health literacy implies that health education not only relays information, but also enhances a person's ability to think about healthy behaviours, seek and use information, and motivates people to take action to improve health. This paper illustrates how a traditional health education program failed to bring expected changes in the community to improve health. It then describes the development and implementation of the SAFE (Sanitation and Family Education) project by CARE-Bangladesh. SAFE is a diarrhoea prevention program which illustrates the concept of health literacy as a step to improved health outcomes. Emphasis is given on the dynamic process of communication, community needs and participation, identification of problems related to socio-economic and environmental factors, and role of the field workers. It argues that health promotion interventions must respond to the local context and needs to be continually reviewed and revised as necessary. Lessons learned and achievements of SAFE are highlighted. The paper suggests that readers review whether their existing programs are doing enough to raise health literacy and improve health outcomes, or if they are merely increasing awareness of related issues.

ARTICLE INFO Article History

Received XXXXXX Accepted XXXXXXX

Corresponding Author

Raquiba A. Jahan Department of Public Health & Community Medicine, A27, University of Sydney, NSW, 2006 Australia, Project Coordinator, Health Sector/CAREBangladesh © SSNM&AIRITI PRESS DOI: xxxxxxxxxxx http://www2.cch.org.tw/7477 Copyright © 2014 Asian Health Literacy Association

Introduction: Health Education and Health Literacy in Bangladesh In Bangladesh ‘health education’ is a widely used term in preventive medicine directed to promote healthy lifestyles. Health professionals typically define health education as a one-way approach to information dissemination. Generally, they do not take into consideration the actual use of information to improve health by the individual receiving the information.

Over the last few years, health professionals in Bangladesh have begun to analyse the relationships between health, knowledge and environmental support to achieve a more sophisticated understanding of how to change lifestyles. This analysis fits more comfortably with the concept of ‘health literacy’ as an outcome of health education, going further than the simple dissemination of messages and enhancement of people's ability to think about healthy behaviours, to seek and use information, and motivates people to take action to improve health. Such distinctions help to clarify differences between educational programs

R. A. Jahan

designed to achieve the different levels of health literacy described by Nutbeam (Nutbeam, 2000). These levels distinguish between compliance with expert-prescribed behaviour (functional health literacy), self-management of problems in partnership with health professionals (interactive health literacy), and empowerment (critical health literacy).

Traditional Health Education: Advantages and Limitations In general, health education in Bangladesh has relied upon the techniques of formal education under which most professionals have been trained. This model of education is basically a one-way, teacher-dominated delivery system. In some cases the approach used by the education worker to work with communities is not based on the principles of community development, i.e. respect, dialogue and negotiation with community members (Aubel et al., 1998). In this model, information is passed from the expert to the learner. Relatively few staff members are needed for this kind of approach and it is generally less costly in terms of time and money. The assumption in Bangladesh has been that people being told what they should do by health educators will then proceed to follow those instructions, resulting in improved health for the individual. This traditional method represents a generalized approach to promoting health which was designed for mass audience coverage. In other words, it is an approach which uses the same educational material for all audiences despite any cultural differences, or differences in norms, or beliefs that may exist within Bangladesh. As is often the case, educational materials are pre-packaged and the information describing healthy behaviours is standardized. One of the main problems with this model is that achievement is often reduced to meeting quantitative targets related to exposure to the message, or ability to recall facts. These targets indicate coverage (e.g. number of school children informed) but do not indicate the resulting behavioural changes, if any, that occurred (Jahan et al., 1995;Jahan et al., 1996). The fact that many recipients of health education in Bangladesh can recite the information they have received is often cited by health professionals as proof of program success, and there is an assumption that by having the information, individuals will then take steps to improve their health.

Health Literacy: Advantages and Limitations The operational or functional meaning of health literacy refers to the capacity of individuals to gain access to health information and use that information to improve health. Health literacy is an outcome to health education, and can be used as a reference point to dictate process and methods of communication used. A health education program called SAFE (Sanitation and Family Education), developed and implemented by CARE-Bangladesh, is described in the following section. This model is used to reflect upon the concept of health literacy. The model is based on recognition that each situation is different and requires methods that are appropriate to a particular community. The practical and sequential components of the model include the following (Bateman et al., 1993). • Community needs assessment through qualitative and quantitative studies. This task identifies the opportunities and constraints to behaviour change through health education. • Discussion on establishing the link between behaviour change and personal benefits such as health benefits or financial savings. • Acquisition of knowledge and skills through participation. • Development of locally appropriate solutions through joint partnerships with the community taking into consideration the local values, beliefs and practices. • C o n t i nu o u s a d a p t a t i o n o f ch a n g e s by t h e community. • Work on a series of small steps to behaviour change which are manageable, achievable and result in recognizable health benefits. Using this approach, facilitators, or field workers, serve as partners of community change rather than as teachers who impose pre-determined solutions. This method yields the best results when it employs participation of those affected. The participatory technique is consistent with the elements underlying the concept of ‘critical health literacy’. The essence of development is to empower people to take charge of their own health and to foster a spirit of self-reliance. Limitations of this approach include the need for well-trained facilitators with flexibility and accountability to the community people. It requires staff commitment to community participation, practical experience on both quantitative and qualitative research techniques, and the unique capability to

Prevention of diarrhoeal disease Bangladesh

continuously revise education strategies. In addition, it is a time-consuming approach that involves working with a community at a pace that is suitable to their ability in order to ensure the development of health literacy and subsequent sustainable healthy behaviour changes.

Case Study In Health Literacy— Diarrhoea Prevention In Bangladesh Problem Bangladesh is a small country of ~148 000 km2 with a population of ~120 million people. In Bangladesh, the leading causes of child death are related to diarrhoea, malnutrition, vaccine-preventable diseases and respiratory infections (Bangladesh Bureau of Statistics, 1996). Most women, particularly in rural areas, are largely confined to their homes and neighbourhoods. Like other developing countries in Asia, most women are illiterate, uninformed about nutrition or basic health and hygiene, and are without any independent income. As a result, her children suffer from chronic malnutrition and repeated episodes of preventable diseases such as diarrhoea. Diarrhoeal diseases are mainly caused by unsafe water, inadequate sanitation and poor hygiene which are common factors in shanty living conditions and densely populated countries such as Bangladesh. Faecal contamination of the environment usually results from the lack of, or non-use of, sanitation systems. Many people in Bangladesh use water polluted by faeces for a variety of purposes, and have improper sanitation practices that create multiple avenues for faecal– oral transmission of disease-causing organisms. Recent studies suggest that, although most Bangladeshis consider faeces a pollutant, few could make the association between faecal contamination and the transmission of organisms that cause diarrhoea (Zeitlyn and Islam, 1991).

Background A health initiative entitled Water Sanitation and Hygiene project (WASH) was developed by CAREInternational, Bangladesh as a relief effort after the devastating 1991 cyclone in Bangladesh. WASH focused on the repair of damaged water sources and the construction and supply of hygienic latrines. It also included a small hygiene education component that focused on drinking safe water, the installation and use of latrines, and hand washing to prevent and control diarrhoeal diseases. The intervention messages were selected from a standardized package

of education materials that were not field tested, and the implementation incorporated only one-way communication. In other words, the community was simply given information rather than participating in the identification of problems and development of solutions. Importance was given on the quantity of latrines and tubewells (hand pumps for safe water) that were installed rather than their use. An evaluation of WASH showed minimum changes in behaviour related to health and hygiene (Ahmed, 1992; Brahman et al., 1993). Findings from this evaluation revealed that very few children under the age of 6 years used latrines, hand-washing agents (soap, ash) were not found in very many households, and the rate of maintenance of latrines installed was poor. Community members continued to practice many behaviours which made them susceptible to a range of diarrhoeal diseases related to polluted water and poor sanitation. The evaluation of WASH recommended an intensive community-based health education program in project areas for success in community health improvement.

Program Implementation In 1993–1994 CARE-international, Bangladesh implemented an innovative community health education pilot project titled Sanitation and Family Education (SAFE) in Chittagong, the coastal area of south-east Bangladesh. This program was developed as a result of the evaluation performed by the WASH project (Brahman et al., 1993). Objectives of the SAFE project included the following. • The development and assessment of models for hygiene education outreach. • The development of effective hygiene education outreach methods and strategies to promote behaviour change. • The design and implementation of a behaviourbased monitoring and improvement system for the hygiene education program. In SAFE, two models of health education outreach were tested. The first model dealt with education sessions only with community men and women. It was developed on the traditional concept of dissemination of information leading to functional health literacy. In this model other community members, e.g. children, community leaders were not included as the participants and use of information received by the community members was not focused on. The second model was quite different from this traditional one. It had a wider approach that included separate education sessions for men, women, children (both in and out of school), and key opinion leaders in the community, and use of information 3

R. A. Jahan

for improvement of community health was the main focus. Education materials for adult men and women were the same in both models, but in the second model education materials were tailored towards different groups. The information disseminated in the second model was made easily understandable to different participants using poems, drawing pictures, stories and songs. This was more orientated to interactive or critical health literacy. Both the SAFE models used the following steps to achieve their goals: 1. assessment of community needs; 2. identification of achievable goals; 3. program implementation; 4. establishment and use of behaviour-based monitoring system; 5. adaptation of changes when necessary.

Assessment of Community Needs At the beginning of the SAFE program, hygiene problems were identified based on quantitative and qualitative research activities. For example, baseline surveys showed that many families living 20 min or more away from the safe water sources experienced less diarrhoea compared to those that were closer to the sources (Bateman et al., 1993). This surprising result required further examination. Observations in both groups of households revealed that, in general, closer families were more careless about water storage, and often drank water by cupping their hands to retrieve water from the source. Specific intervention messages were developed based on these assessments. The education workers were trained to be facilitators, rather than teachers, so that they were able to assist communities in assessing their situations and developing solutions. The role of the facilitators was to support the learning and discovery process by the communities themselves. For example, the communities reported that access to water sources was limited by distance and ownership. Focus group discussions indicated that community members did not have the economic means or influence to tackle these constraints directly, and so they simply used pond water which was unsafe to drink. Because the baseline survey results showed that no increased risk of diarrhoea was associated with using pond water for non-drinking purposes, it was feasible to recommend collecting, storing and using the tubewell water for drinking only and to use pond water for other purposes. The result of identifying needs with the communities themselves was an education program which was responsive to communities' needs and 4

priorities by accommodating local beliefs, norms and practices as much as possible. This approach was far more successful than one such as WASH which centred around an expert's perception of the communities' needs and what they should do.

Identification of Achievable Goals Those behaviours most closely linked to diarrhoea transmission and most amenable to change in the short term were focused on first. It was assumed that if these were successfully addressed, then there would be a high likelihood of an observable impact on health which would, in turn, motivate people to seek further improvements in their behaviour. Through several formal and informal discussions with community members, a number of realistic goals were set (Bateman et al., 1995a). For example, it seemed unrealistic to promote the idea of using tubewell water for all purposes, a standardized and ideal message, but realistic, was to: • promote drinking only tubewell water; • limit uses of pond water to decrease risk of diarrhoea; • promote careful collection and storage of safe water (keeping hands out of the water, using narrownecked storage containers and keeping containers covered). Similar, manageable goals were defined for other issues including latrine use, environmental cleanliness, hand washing, food hygiene and diarrhoea management (Laston et al., 1995; Bateman et al., 1995a;Bateman et al., 1995b).

Information Dissemination Several approaches to promote hygiene education were developed and tested jointly with the community (Bateman et al., 1995a). These included: • courtyard sessions—used in both models. The following techniques were used exclusively in the second model: • teashop sessions with general people; • children's education sessions (in and out of school); • sessions with key opinion leaders.

Courtyard Sessions

These were participatory and lively education sessions facilitated by field workers with mostly women, although men were also invited to attend. Participatory learning techniques and other interactive training methods were used. Participants themselves shared their experience of building and modifying the design of latrines suitable for the specific village situation. At the end of each session, participants

Prevention of diarrhoeal disease Bangladesh

described what they would do differently to improve their hygiene behaviour and community health overall.

Teashop Sessions

The community indicated that men played an important role in decision-making in rural Bangladesh. For example, they are responsible for the decisions to purchase and install hardware (latrines and tubewells). They also exercise considerable influence on the shaping of society norms which might affect the freedom of movement of field workers and the opportunities for women to gather in a place and discuss hygiene behaviour. Everyone agreed that it was vital to include men in the outreach program to ensure their support and co-operation. Although the courtyard sessions were used for men and women, in practice only two or three men ever attended the courtyard sessions. The sessions were being conducted during the day when men were busy working. Those who did come lost interest, assuming that these sessions were really only for women and they, themselves, did not need any health education. At this point, the community members suggested that education sessions should be conducted at public places like teashops and clubs where men gathered when they were not working. This was a highly successful initiative to help male community members see the relationships between expected hygiene practices and good health.

Childrenâ&#x20AC;&#x2122;s Education Sessions

Field workers conducted education sessions to encourage children to play interactive games which incorporated the link between good hygiene behaviours and improved health. The program was based on indigenous games, poems, stories identified from within the community and adapted to serve an educational purpose. They were tested and modified accordingly. The children recited rhymes and played games with educational messages, and they took pride in their ownership/involvement in developing program strategies. Sessions with key opinion/community leaders For wider dissemination of infor mation, key opinion leaders were identified by community members through focus group discussions and community mapping, the most common participatory rural appraisal (PRA of Robert Chambers) method. Community members identified key opinion leaders as those people in the community to whom they turned to for advice and guidance but were not necessarily official leaders. This group included a teashop owner, a schoolteacher, a hawker (saleswoman), a woman in the community who set bones when individuals

injured themselves, and a volunteer social worker. These key opinion leaders were not expected to be outreach workers but rather people to turn to for sound information and meaningful advice.

Behaviour-Based Monitoring System One of the impor tant and innovative components of the education model was the development and use of a behaviour-based monitoring system. This monitoring system included observation technique and identified what people actually did rather than what they reported they did. For example, regular observations of households and individuals, at pond, water pump or defecation sites, were carried out to examine actual hygiene practices. These observations were carried out by trusted individuals in the community and conducted in a discreet manner (Bateman et al., 1995a).

Continuous Adaptation of Implementation Strategies The approaches to hygiene education evolved slowly as communities themselves made suggestions and provided feedback. T hey par ticipated in monitoring data collection, analysing findings and developed feasible solutions. During project implementation the following qualitative techniques were used to refine inter ventions as and when necessary (Zeitlyn et al., 1994; Laston et al., 1995; Jahan et al., 1996).

Focus Group Discussions Focus group discussions with men and women in the community provided important input on interventions for further refinement. Community members offered practical sug gestions such as designing a fixed defecation site in the yard for young children who were scared of the darkness of the latrines. Women indicated that the air drying of hands after washing was not accepted in the community. They suggested a clean rag should be kept readily available in the household for hand drying (Zeitlyn et al., 1994). The members of households with latrines provided suggestions for further improvement of latrine design which were incorporated into the interventions and demonstrations.

Group discussions with children Group discussions with children provided effective feedback on the pictorial stories and games used in the education sessions. Those that conducted the discussions recommended appropriate revisions of educational methods and training materials such as 5

R. A. Jahan

children lead the games instead of the field workers, they recommended exclusion of risky games which involved running (hadudu) and inclusion of local indigenous games, to change colours of pictures of characters of the stories as suited.

Key Informant Interviews Unstructured interviews with the key opinion leaders provided input in refining messages and strategies. For example, the schoolteacher suggested that children could participate in health promotional activities during school and help maintain the school latrines. The social worker suggested that the practice of making traditional instant food with unsafe water should be addressed and changed.

Observations As described earlier, direct observations were made to determine actual hygiene practices during the intervention period to monitor effects of the project and determine changes or revisions that might be necessary for improvement of the program. Observations at safe and unsafe water sources indicated that community members still engaged in risky behaviours. The community recognized that the education sessions needed to be monitored frequently to ensure that the hygiene education emphasized the relationships between behaviour change and improved health. The use of these qualitative techniques provided rapid feedback to project personnel to allow for prompt revision of project activities. The education program was modified on a continual basis according to the discussions with and suggestions from a wide variety of community members.

Program Achievement In 1994–1995 an evaluation of the SAFE project was conducted in both model areas using quantitative survey and qualitative methods which showed that SAFE had a strong impact on diarrhoea in Bangladesh. For more detailed information regarding research design, sample selection and results on the SAFE program have been published elsewhere (Bateman et al., 1995a; Bateman et al., 1995b).

Conclusion The key lessons learned from the SAFE project are well documented. Based on experience from SAFE, key elements of health promotion programs should include working through different avenues at the community level (Bateman et al., 1995a) which include the following. • Identification and analysis of problems, and 6

definition of realistic and achievable solutions with specific outcomes. • Collection and analysis of information on existing beliefs, norms and practices of the community. • Interactive communication within the community leading to skills development and local ownership of the program. • Development, testing and refining of participatory extension methods and materials. • Development, testing and refinement of tailored education messages that target all members of a community including men, women and children. • Creation of a behaviour-based monitoring system to track the progress and revise strategies. SAFE is a practical example of a health education program directed towards the achievement of interactive and critical health literacy. SAFE demonstrates the importance of addressing many facets of a problem in order to improve health literacy within a community. It is important that a facilitator, rather than a teacher, work jointly with community members in order to address problems successfully and motivate community members to improve their understanding of certain health issues. SAFE shows that improving health literacy does not just involve a health educator telling a community what they can do to improve their health, but involving them in every step of the process so that they have a greater understanding of the problems they face and are empowered to take action to deal with them. Community involvement which results in empowerment of community members to take leadership roles is crucial to a successful program. Goals of the program must be clearly defined and directed towards specific outcomes which will lead to improved health literacy. Lastly, program flexibility and a willingness to change is key to developing a sustainable program that is successful in improving health. SAFE model is being replicated in many projects within CARE-International, Bangladesh and other CARE missions around the world. To enhance the capacity of health promotion activities of local non-government organizations (NGOs), CAREInternational, Bangladesh, initiated a 5-year (1996– 2000) training-oriented partnership program with NGOs. The concept of health literacy to improve health has proven to be successful in the SAFE model area, Bangladesh, and most importantly, men, women and children in these areas are leading healthier lifestyles as a result.

Prevention of diarrhoeal disease Bangladesh

References [1] Ahmed, S. (1992) Report on Evaluation of WASH Project. Unpublished document, CAREBangladesh. [2] Aubel, J., Jahan, R. and Hena, A. (1998) Report on the Mid-term Evaluation. SAFER project, CARE-Bangladesh. [3] Bangladesh Bureau of Statistics (1996) Progotir Pathey: Achieving the Mid Decade Goal for Children in Bangladesh. [4] Bateman, O. M., Jahan, R., Brahman, S., Zeitlyn, S. and Laston, S. L. (1995a) Prevention of Diarrhoea Through Improving Hygiene Behaviours. CARE-ICDDR,B joint publication, Dhaka, Bangladesh. [5] Bateman, O. M., Jahan, R., Brahman, S., Zeitlyn, S. and Laston, S. L. (1995b) Report on the Monitoring and Improvement System. SAFE Pilot Project, CARE-Bangladesh. [6] Bateman, O. M., Zeitlyn, S., Jahan, R. and Brahman S. (1993) Report on the Baseline Survey. SAFE Pilot Project, CARE-Bangladesh. [7] Brahman, S., Jahan, R. and Ritchie, N. (1993) Concept Paper: Sanitation and Family Education Pilot Project. Unpublished document, CARE-Bangladesh.

[8] Jahan, R., Bateman, O. M., Brahman, S., Laston, S. L., Zeitlyn, S., Jupp, D. and Durandin, F. (1996) Hygiene behaviour change and community participation. Learning for Health, 8, 10–14. [9] Jahan, R., Jupp, D., Durandin, F., Bateman, O. M., Brahman, S. and Laston, S. L. (1995) ‘Don’t just say it, do it’: issues for consideration when planning for behaviour change in hygiene education programs. SAFE Pilot Project, CAREBangladesh. [10] Laston, S. L., Brahman, S., Jahan, R. and Bateman, O. M. (1995) Final Report on the Qualitative Assessments. SAFE Pilot Project, CAREBangladesh. [11] Nutbeam, D. (2000) Health literacy as a public health goal: a challenge for contemporary health education and communication strategies into the 21st Century. Health Promotion International, 15, 259–267. [12] Zeitlyn, S., Brahman, S., Bateman, O. M. and Jahan, R. (1994) Report on the Qualitative Assessments. SAFE Pilot Project, CARE-Bangladesh. [13] Zeitlyn, S. and Islam, F. (1991) The use of soap and water in two Bangladeshi communities: implications for the transmission of diarrhoea. Reviews of Infectious Diseases, 13, S259–S264.

Original Article (International) Journal of Health Literacy Vol 15 No. 4 2014 Dec pp1-7

The HUMN and HUMN xL International Collaboration Projects on Human Micronucleus Assays in Lymphocytes and Buccal Cells—Past, Present and Future Michael Fenech*, Nina Holland1, Errol Zeiger2, Wushou P. Chang3, Sema Burgaz4, Philip Thomas, Claudia Bolognesi5, Siegfried Knasmueller6, Micheline Kirsch-Volders7 and Stefano Bonassi8 1

Department of Nutritional Genomics and DNA Damage Diagnostics, Commonwealth Scientiﬁc and Industrial Research Organisation Food and Nutritional Sciences, Gate 13 Kintore Avenue, PO Box 10041, Adelaide BC, South Australia 5000, Australia 2 School of Public Health, University of California, Berkeley, CA 94720-7360, USA 3 Errol Zeiger Consulting, 800 Indian Springs Road, Chapel Hill, NC 27514, USA 4 College of Public Health and Nutrition, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan 5 Department of Toxicology, Faculty of Pharmacy, Gazi University, 06330, Hipodrom, Ankara, Turkey 6 Environmental Carcinogenesis Unit, National Institute for Research on Cancer, Genoa, Italy 7 Institute of Cancer Research, Medical University of Vienna, Vienna, Austria 8 Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium and Unit of Clinical and Molecular Epidemiology, Istituto do Ricovero e Cura a Carattere Scientiﬁco San Raffaele Pisana, Via di Val Cannuta, 247 00166 Rome, Italy * To whom correspondence should be addressed. CSIRO Food and Nutritional Sciences, PO Box 10041, Adelaide BC, South Australia 5000, Australia.

ARTICLE INFO

ABSTRACT

Article History

The International Human Micronucleus (HUMN) Project (www.humn. org) was founded in 1997 to coordinate worldwide research efforts aimed at using micronucleus (MN) assays to study DNA damage in human populations. The central aims were to (i) collect databases on baseline MN frequencies and associated methodological, demographic, genetic and exposure variables, (ii) determine those variables that affect MN frequency, (iii) establish standardised protocols for performing assays so that data comparisons can be made more reliably across laboratories and countries and (iv) evaluate the association of MN frequency with disease outcomes both cross-sectionally and prospectively. In the first 10 years of the HUMN project, all of these objectives were achieved successfully for the MN assay using the cytokinesis-block micronucleus (CBMN) assay in human peripheral blood lymphocytes and the findings were published in a series of papers that are among the most highly cited in the field. The CBMN protocol and scoring criteria are now standardised; the effect of age, gender and smoking status have been defined, and it was shown prospectively using a database of almost 7000 subjects that an increased MN frequency in lymphocytes predicts cancer risk. More recently in 2007, the HUMN coordinating group decided to launch an equivalent project focussed on the human MN assay in buccal epithelial

Received on June 14, 2010; revised on July 24, 2010; accepted on July 26, 2010

Corresponding Author

Michael Fenech Department of Nutritional Genomics and DNA Damage Diagnostics, Commonwealth Scientiﬁ c and Industrial Research Organisation Food and Nutritional Sciences, Gate 13 Kintore Avenue, PO Box 10041, Adelaide BC, South Australia 5000, Australia Tel: þ618 83038880; Fax: þ618 83038899; Email: michael.fenech@csiro.au © SNM&AIRITI PRESS DOI: xxxxxxxxxxx http://www2.cch.org.tw/7477 Copyright © 2014 Asian Health Literacy Association

M. Fenech et al .

cells because it provides a complementary method for measuring MN in a tissue that is easily accessible and does not require tissue culture. This new international project is now known as the human MN assay in exfoliated cells (HUMN xL ). At present, a database for > 5000 subjects worldwide has been established for the HUMN xL project. The interlaboratory slide-scoring exercise for the HUMN xL project is at an advanced stage of planning and the analyses of data for methodological, demographic, genetic, lifestyle and exposure variables are at a final stage of completion. Future activities will be aimed at (i) defining the genetic variables that affect MN frequencies, (ii) validation of the various automated scoring systems based on image analysis, flow cytometry and laser scanning cytometry, (iii) standardisation of protocols for scoring micronuclei (MNi) in cells from other tissues, e.g. erythrocyte and nasal cells and (iv) prospective association studies with pregnancy complications, developmental defects, childhood cancers, cardiovascular disease and neurodegenerative diseases.

The Origins of the Humn Project In the 1980s, several exciting developments with regards to MN assays in humans were starting to emerge, which included the development of improved methods for scoring MNi in lymphocytes, buccal cells and erythrocytes. The cytokinesis-block micronucleus (CBMN) technique, which was first described in the mid-1980s (1,2), significantly improved the reliability of micronucleus (MN) measurements in lymphocytes and led to its exponential adoption by numerous laboratories worldwide and has since been the most widely used method for studying micronuclei (MNi) in human populations. For this reason, it became increasingly evident that the possibility of measuring DNA damage in human populations worldwide was feasible and that there was, therefore, a need for an internationally coordinated effort to achieve this goal reliably and sustainably. The precise point in time of the origin of the HUMN project is difficult to define but the idea crystallised during a chance meeting and discussion about these ideas between Angelo Abbondandolo and M.F. during the international conference ‘Chromosome Segregation and Aneuploidy’ in Sorrento, Italy, April 24–29, 1995. During the discussion, Angelo recommended that S.B. would be an ideal partner to explore this project concept. After a year of email correspondence, S.B. and M.F. decided to launch the HUMN project at the Seventh Inter national Conference of Environmental Mutagenesis in Toulouse, France, September 7–12, 1997. They were overwhelmed by the great interest in the HUMN project, which had as its primary aims the collection of data worldwide to determine the main variables affecting lymphocyte MN frequencies, the establishment of scoring criteria 2

for this assay, the performance of an inter- and intralaboratory slide-scoring exercise to validate established scoring criteria and deter mine reproducibility of scoring across and within laboratories and a prospective study to test the hypothesis that MN frequency in lymphocytes predicts cancer risk. A coordinating group of founding members was set up, which consisted of M.F., S.B., N.H., W.P.C. and E.Z. to execute and oversee the various aspects of the project [the HUMN coordinating group in 2007 consisted of, in alphabetical order, S.B., W.P.C., M.F.(Chair), N.H., M.K.-V. and E.Z.]. Because of their expertise with the buccal MN assays, S.K., S.B., C.B. and P.T. were included in 2009 to form the HUMN xL coordinating group. Through the efforts of the coordinating group and numerous international collaborators, all the objectives were met successfully within the following 10 years and the resulting publications are among the most cited in the MN ﬁeld (3–8). The results of these studies showed that inter- and intralaboratory variation in slide scoring, age, gender and smoking status are important factors affecting observed MN frequency in human lymphocytes. Furthermore, the prospective study did in fact show that a mid- or high-tertile level of MN frequency predicted an increased cancer risk several years later (8). The ﬁndings from these studies are explained in more detail below.

The HUMN Project Manifesto The ﬁrst publication from the HUMN project was a ‘manifesto’ that explained the rationale and objectives of the project (3). The HUMN project was organised to collect data on MN frequencies in different human populations and different cell types.

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The test procedures considered by this project were assays using human lymphocytes (cytokinesis-block method), exfoliated epithelial cells, as well as other cell types. Data (including descriptions of the populations monitored, detailed test protocols and test results) were voluntarily submitted by a large number of laboratories throughout the world and entered into an unified database. The information was used to: (i) determine the extent of variation of ‘normal’ values for different laboratories and the influence of other factors potentially affecting baseline MN frequency, e.g. age, gender and lifestyle, (ii) provide information on the effect of experimental protocol variations on MN frequency measurements, (iii) design and test optimal protocols for the different cell types and (iv) determine the extent to which MN frequency is a valid biomarker of ageing and risk for diseases such as cancer. It was evident at the time that the lymphocyte CBMN assay was the most widely used and robust method for measuring MN in human populations; for this reason, the initial HUMN project focus during the first 10 years was on this assay.

International lymphocyte CBMN assay database and analysis of methodological and demographic variables affecting MN frequency In early 1997, an invitation letter was sent by the HUMN project steering committee to 130 laboratories that had published studies on the MN test in human lymphocytes using the CBMN assay. The letter included a questionnaire for some basic information concerning the data available in each laboratory. The committee received 42 responses and based on that information, a so-called ‘information package’ was prepared and sent to the interested laboratories. All of these laboratories had used the lymphocyte CBMN assay in their investigations. A more sophisticated questionnaire requesting detailed information about the laboratory protocol, scoring criteria, individual data of subjects in the study and references of the published papers was sent with this information package. The identities of the individual participants were not identiﬁed by the laboratories. An Excel ﬁle, to be used as a template for submitting original data to the coordinating center (Italian National Research Centre on Cancer, Genoa), was also sent. A total of 25 databases were received from laboratories in 16 countries, mostly in Europe but also in Asia, America, Australia and New Zealand. This resulted in a database on information for nearly 7000 subjects for whom MN frequency in

lymphocytes was determined and the outcomes of the analyses of these data with respect to the effect of laboratory protocol, scoring criteria and host factors were published in 2001 (4). Substantial 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 foetal calf serum and in the culture method. Furthermore, discrepancies in criteria for scoring MNi were also evident. Evaluation of the data showed that the overall median MN frequency in non-exposed (i.e. normal) subjects was 6.5 per thousand and the interquartile range was between 3 and 12 per thousand. MN frequencies increases monotonically with age in both genders, with the steepest increase after 30 years of age (Figure 1). A more pronounced effect of age in females was conﬁrmed by different female:male MN frequency ratios in age-classes ,40 years of age [1.08; 95% conﬁdence interval (CI): 1.03–1.14] and in those over this age (1.23; 95% CI: 1.18–1.28). On average, females were shown to have a 19% higher level of MN frequency (95% CI: 14–24%) compared to males. The best random-effects model, which included genotoxicant exposure factors, host factors, methods and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods (4).

Outcomes of a Slide-Scoring

Fig. 1. Effect of age and gender on MN frequency in peripheral lymphocytes determined using data from the HUMN project. Note: FRs are adjusted by laboratory and exposure to genotoxic agents. All age-classes after 30 years were signiﬁcantly higher than the reference class (0–9). For more details refer to (4).

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Exercise Using Standardized Slide-Scoring Criteria for Mni and Nucleoplasmic Bridges in Lymphocytes One of the objectives of the HUMN project is to identify the methodological variables that have an important impact on the scored MN frequencies or micronucleated (MNed) cell frequencies measured in human lymphocytes using the CBMN assay. In the previous study discussed above (4), we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria (5), an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved, including some laboratories with more than one scorer (6). The results of this study showed that even under these optimised conditions, there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories correctly ranked the MNed cell frequencies in cells from cultures that were un-irradiated or exposed to 1 or 2 Gy of gamma rays. The study also showed that the intra-scorer median coefﬁcient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2 Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model, it was estimated that radiation dose explained 67% of the variance, while the staining method, cell sample, laboratory and covariances explained 0.6, 0.3, 6.5 and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. Nucleoplasmic bridges (NPBs) were also estimated by the laboratories, however, inexperience in the use of this biomarker of chromosome rearrangement was reﬂected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies (3–6) clearly indicate that even after standardising culture and scoring conditions, it is necessary to calibrate scorers and laboratories if MN, MNed cell and NPB frequencies are to be reliably compared among laboratories and among populations. The calibration procedure involves using a standard set of slides of known MN, MNed and NPB frequency that each scorer has to score and for whom deviation from the real value is recorded. This variation ratio could then be used to correct data to the real value 4

depending on the scorer. These slides can also be used to qualify laboratories for performing the test. Standard protocol and scoring criteria for the CBMN assay with peripheral blood lymphocytes The slides for the inter-laboratory slide-scoring exercise were prepared using the standard laboratory protocol for the isolated lymphocyte CBMN assay established by Michael Fenech’s laboratory in Australia. This protocol for isolated lymphocytes as well as that for whole blood cultures was described in detail and published in 2007 (9). It also includes the scoring criteria used in the slide scoring comparison exercise and is the recommended method for performing the CBMN assay in human peripheral blood lymphocytes. The detailed scoring criteria used in the slide-scoring exercise study described above were also published separately (5). Criteria for scoring (i) MNi, NPBs and nuclear buds in binucleated cells, (ii) mononucleated cells, binucleated cells and multinucleated cells and (iii) necrotic and apoptotic cells are described in these papers. These different end points and scoring 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. It is expected that these scoring criteria will assist in the development of a procedure for calibrating scorers and laboratories performing the lymphocyte CBMN assay so that results from different laboratories for the CBMN assay may be more comparable in the future. It is also important to note that this detailed CBMN assay protocol in lymphocytes, which was originally developed for in vivo biomonitoring in humans, also served as the basis for the validation of the lymphocyte CBMN assay for genotoxicity testing in vitro, which is now incorporated in the OECD guidelines that were established for this purpose (10). As a consequence, we have for the ﬁrst time the possibility to use the same lymphocyte CBMN assay standard to measure the genetic impacts of a physical or chemical agent both in vitro and in vivo. This will allow cross-validation and testing of the predictivity of the lymphocyte CBMN in vitro test with respect to ex vivo MN induction in lymphocytes of human populations exposed to physical or chemical agents that were tested in vitro with the same test.

A Definitive Study on the Effect of Smoking on MN in Peripheral Blood Lymphocytes The effect of tobacco smoking on the frequency of MNi in human lymphocytes has been the object of many population studies. In most reports, the results

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were unexpectedly negative, and in some instances, smokers had lower frequencies of MN than nonsmokers. An analysis of the pooled 24 databases from the HUMN international collaborative project was performed with the aim of understanding the impact of smoking habits on MN frequency (7). The complete database included 5710 subjects, with 3501 non-smokers, 1409 current-smokers and 800 formersmokers, in studies of exposure to occupational and environmental genotoxicants. The analysis of the combined databases confirmed the small decrease of MN frequencies in current-smokers [frequency ratio (FR) 5 0.97, 95% CI:0.93–1.01] and in former-smokers (FR 5 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. However, a typical J-shaped curve was observed for the smokers who were not exposed to genotoxic agents with a significant increase in MN frequency occurring in individuals smoking ≧ 30 cigarettes/day (FR 5 1.59, 95% CI:1.35–1.88). This analysis confirmed that smokers generally do not experience an overall increase in MN frequency. However, when the interaction with occupational exposure was taken into account, heavy smokers were the only group showing a significant increase in genotoxic damage as measured by the CBMN assay in lymphocytes. Based on these results, some general recommendations for the design of bio-monitoring 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 subgroup of heavy smokers ( ≧ 30 cigarettes/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 always be tested. Since this study was published, it was shown that spontaneous and cigarette smoke nitrosamine-induced frequencies of MNi, NPBs and nuclear buds in lymphocytes of smokers who develop lung cancer are higher than those of matched smoker controls who do not develop lung cancer (11,12). These results suggest that DNA damage biomarkers in the CBMN assay are only likely be substantially elevated as a result of smoking in those with abnormally high susceptibility to carcinogenic effects of genotoxicants contained in cigarette smoke. Whether the elevated MN frequency in smokers with lung cancer is only due to increased susceptibility to cigarette smoke genotoxicants or also due to the presence of cancer itself will not be known

until comparisons are also made for MN frequency between non-smoker and smoker lung cancer cases.

The Risk for Cancer in the HUMN Project Cohorts is Predicted by Mn Frequency in Peripheral Blood The ultimate objective of the HUMN project studies was to test the hypothesis that an elevated MN frequency in human tissues is predictive of cancer risk (8). This critical validation step is essential to justify the use of such techniques in human biomonitoring studies in populations, which are suspected to be at a higher cancer risk due to inappropriate environmental or diet and lifestyle exposures that may be genotoxic and to test whether preventive strategies could actually reduce DNA damage. Therefore, although much theoretical evidence has been accumulated supporting the causal role of MN induction in cancer development, prospective cohort studies are needed to validate MN as a cancer risk biomarker. A total of 6718 subjects from 10 countries, screened in 20 laboratories for MN frequency between 1980 and 2002 in ad hoc studies or routine cytogenetic surveillance, were selected from the database of the HUMN international collaborative project and followed up for cancer incidence or mortality. To standardise for the inter-laboratory variability, the subjects were classiﬁed according to the percentiles of MN distribution within each laboratory as low, medium or high frequency. A signiﬁcant increase of all cancers incidence was found for subjects in the groups with medium (RR 5 1.84; 95% CI: 1.28–2.66) and high MN frequency (RR 5 1.53; 95% CI: 1.04–2.25) (8). The same groups also showed a decreased cancer-free survival, i.e. P 5 0.001 and P 5 0.025, respectively. This association was present in all national cohorts and for all major cancer sites, especially urogenital (RR 5 2.80; 95% CI: 1.17–6.73) and gastrointestinal cancers (RR 5 1.74; 95% CI: 1.01–4.71). The results from this study provide evidence that MN frequency in peripheral blood lymphocytes is a predictive biomarker of cancer risk within a population of healthy subjects. Other studies have since been published showing that a higher MN frequency in lymphocytes measured using the CBMN assay is associated prospectively with increased pregnancy complications (13) and cardiovascular disease mortality (14,15). The success of the HUMN project with the peripheral blood lymphocyte CBMN assay resulted in further collaborations on 5

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pooled analyses of related databases to test the impact of genotype (16–18) although this is not discussed here as it is the subject of another review in this special issue (19). Furthermore, it brought to attention the need to achieve similar advances with the MN assay in buccal cells in which there is an increasing interest because of their ease of collection, their proximity to food and air-borne genotoxicants and relevance to carcinogenesis in epithelial tissues.

The HUMN xL Project on MN Frequencies in Human Buccal Cells The MN assay in exfoliated buccal cells is a minimally invasive and potentially useful method for monitoring genetic damage in humans. Although this assay has been used since the 1980s to demonstrate cytogenetic effects of environmental and occupational exposures, lifestyle factors, dietary deficiencies and different diseases, important knowledge gaps remain about the characteristics of MNi and other nuclear abnormalities, the basic biology explaining the appearance of various cell types in buccal mucosa samples and effects of diverse staining procedures and scoring criteria in laboratories around the world and their relationship to disease states and outcomes (20). To address these uncertainties, the HUMN project coordinating group initiated a new international validation project for the buccal cell MN assay similar to the project previously performed using human lymphocytes (as described above) (21–23). To distinguish it from the lymphocyte project, this project was given the acronym HUMN xL , i.e. human MN assay in exfoliated buccal cells. The planned research will explore sources of variability in the assay (e.g. between laboratories and scorers, as well as inter- and intra-individual differences in subjects) and resolve key technical issues, such as the method of buccal cell collection and staining, optimal criteria for classification of normal and degenerated cells and for scoring MNi and other abnormalities. The harmonization and standardisation of the buccal MN assay will allow a more reliable comparison of the data among human populations and laboratories, evaluation of the assay’s perfor mance and consolidation of its worldwide use for biomonitoring of DNA damage. To further consolidate this initiative, the first HUMN xL project workshop on the Buccal MN Assay was held at the International Conference on Environmental Mutagens in Human Populations in Antalya, Turkey in 2007 (22). It was attended by 70 representatives from various laboratories, universities, 6

private companies and government departments from around the world. The aims of the workshop were to (i) discuss current state of knowledge on the buccal MN assay, (ii) identify important gaps of knowledge regarding theory, biology and methods, (iii) decide on a plan of action to resolve the key methodological and knowledge gap issues and (iv) explore the possibility of the pooling of databases to determine the most important variables affecting the assay. It was agreed at the workshop that four activities should be initiated as soon as possible, namely (i) a method for collection of databases from different laboratories, (ii) writing of a protocol based on the most commonly used and best-validated methods, (iii) development of slide scoring procedures and (iv) an inter-laboratory slidescoring exercise, in this order. A follow-up workshop was held at the 10th International Conference on Environmental Mutagens in Florence in 2009 in which progress on these activities was reported. The following has been achieved so far. 1. A systematic review on the current status and knowledge gaps regarding the buccal MN assay was completed and published (20). The review concluded that although many studies have consistently shown a statistically significant increases in the buccal cell MN frequency in human populations exposed to genotoxic agents or a decrease as a result of vitamin supplementation or chemopreventive measures, the magnitudes of the changes are usually relatively small. Different confounding factors influencing the MN frequency in peripheral lymphocytes, such as gender, age and lifestyle habits have been considered for the buccal cell MN assay. However, the majority of studies failed to demonstrate any influence of age or sex, although only a few studies have investigated a broad age range. The low baseline MN frequency in buccal cells may amplify the statistical problems in the scoring but at the same time provide a low background level against which genotoxic effects may be observed more readily. In this context, the review considered that it was mandatory to standardise the buccal cell MN assay protocol, including the scoring criteria and that an inter-laboratory calibration exercise be organised to validate the protocol. This standardised method would enable collection of data from different laboratories and different countries, which are more comparable and minimize uncertainty due to methodological variables and scoring criteria. In addition, the development of automated scoring systems is encouraged as a critical advancement for high-throughput and statistically powerful analysis.

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2. A detailed protocol for buccal cell collection, slide preparation and scoring of cells based on the best available methods was in fact prepared and published (24). This protocol describes one of the current established methods for buccal cell collection using a small-headed toothbrush, the generation of a single-cell suspension, slide preparation using cytocentrifugation, fixation and staining using Feulgen and Light Green for both bright field and fluorescence microscopic analysis. Also, the scoring criteria for MNi and other nuclear anomalies are described in detail. The protocol in its current form takes ~4 h to complete from the time of buccal cell collection to the generation of stained slides for microscopic analysis. 3. A survey of the current use and practice of the buccal MN assay was completed as an initial step in the HUMN xL project initiative (23). An invitation to join the HUMN xL project was sent out together with a questionnaire to all laboratories that have published on the buccal MN assay. Overall, 188 messages were delivered and 58 laboratories from 25 countries agreed to participate (43 indicating intention to contribute buccal MN data). The questionnaire was designed to collect methodological information regarding the laboratory’s performance of the assay and to assess the extent and type of epidemiological data that are routinely collected. The results provided an overview of the most commonly used methods for buccal cell collection and preparation, slide preparation, staining, scoring criteria and an evaluation of epidemiological data, including demographics, genetic background, gender, health status, occupation, exposure, lifestyle and dietary habits. According to this survey, a potential database of up to 15 000 subjects may be available for future pooled analyses. A number of protocol discrepancies emerged, implying that standardisation of the method is a major priority. The results of this survey will (i) identify technical and epidemiological key variables that impact on buccal MN frequency in human populations, (ii) drive the design of future intra- and inter-laboratory validation studies and (iii) determine the role of buccal MN frequency and other biomarkers, in monitoring genomic damage and predicting cancer and other degenerative diseases. 4. At the time of writing this paper, 29 databases were received from laboratories located in 16 countries and the combined HUMN xL database currently contains results for 5157 subjects. This database is being analysed to determine the association

of buccal MN frequency with methodological, exposure, diet, lifestyle and demographic variables. 5. A systematic review of recent literature on the buccal MN assay was carried out to provide a state-of-the-art evaluation of how critical topics such as control for confounding, sample size and statistical power, number of cells scored, end point selection and statistical modelling are considered (25). In addition, a meta-analysis was performed on the combined database to estimate the impact of most common confounders on MN frequency and to provide a baseline value and range of MN frequency in the control population. (23,25). Age, gender and smoking habit were the most commonly studied confounders being reported in 98.4, 85.7 and 90.5% of databases, respectively. Univariate statistics were estimated in most studies while multivariate statistical analysis was applied only in the 47.6% of studies. The mean baseline MN frequency in controls was 1.10/1000 cells (95% CI: 0.70–1.72), and the relative increment in subjects exposed to genotoxic agents or affected by disease correlated with similar observations in lymphocytes (R 2 5 0.74). A minimum sample number of 4000 cells is recommended to reduce the variability of the MN mean estimates, in contrast to the current practice of scoring only 2000 cells (in 81% of submitted databases). The authors of this review recommended the use of Poisson or Negative Binomial statistical models when .2000 cells are scored and that studies scoring smaller numbers of cells should consider the use of statistical models taking into account the excess of zeros, e.g. the Zero Inﬂated Poisson (ZIP) models. Further more, the review (25) concluded that the quality of papers published on the buccal MN assay can be substantially improved, with better consideration of basic issues such as power analysis, control for confounding, choice of the statistical model and the number of cells to be scored.

Future Challenges Although the HUMN and HUMN xL projects have been largely successful in advancing our knowledge of the use of MN assays to study DNA damage in humans, there are still several challenges to be met. The following is a list of these challenges, the sequence of which is not necessarily related to priority. 7

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(a) MN assays in other tissues such as erythrocytes, nasal epithelium and hair root cells have been shown previously to be of relevance to human studies in specific scenarios (e.g. erythrocytes in micronutrient deficiency and nasal epithelium in air pollution) (26,27). Protocols for these systems have been reported but these have not been standardised or validated with respect to variables that may be expected to affect baseline frequencies and in terms of prospective association with disease outcomes. A focus on MN assays in these other tissues will eventually occur once the HUMN xL inter-laboratory slide-scoring exercise is completed. The latter exercise is due to commence late in 2010 and those interested in participating should contact the corresponding author of this paper. (b) Up to now, only the lymphocyte MN assay has been tested for its sensitivity to the impact of diet and lifestyle factors and the studies reported to date are sparse. A greater effort with large welldefined populations is required to replicate these initial studies and identify those diet and lifestyle patterns that associate with reduced or increased MN frequencies. (c) Although several studies have reported on the impact on MN frequency of common singlenucleotide polymorphisms (SNPs) in candidate genes involved in metabolic pathways that may directly or indirectly affect genome stability, most of these studies were underpowered (19). Furthermore, it is important that genome-wide association studies using random SNP analysis be performed with large cohorts to identify unknown genes that may strongly affect MN frequency or to verify those that have been identified as being determinants of this biomarker when mutated. (d) An important development in MN assays is the adoption of the cytome approach that not only scores MN but also captures other nuclear abnormalities such as nuclear buds and NPBs as well as capturing frequencies of necrotic and apoptotic cells as well as the proportion of cells undergoing cell division (9,24). In the cytome approach, even MN in non-divided cells may need to be considered due to alternative mechanisms of MN formation such as nuclear budding or in the case of lymphocytes due to preexisting MN expressed in aberrant in vivo nuclear divisions (9,28). The comprehensive micronucleus cytome approach is increasingly being adopted as it enables all major nuclear anomalies and cytotoxicity events to be captured simultaneously. 8

It will take considerable effort to validate the other biomarkers in the cytome system but this will become necessary if the addition of these indices improves association with genotoxicant exposures and/or disease outcomes as has been suggested by a recent study on the association between the CBMN cytome assay biomarkers in lymphocytes and lung cancer (11,12). (e) During the past decade, great advances have occurred that have enabled automated scoring of MNi as well as high-content analysis of MNi and nuclei using molecular probes that provide additional information on DNA damage mechanism (e.g. centromere and telomere detection in MNi and nuclei). Procedures and reagents for these methods require standardisation particularly if they are to be used on a routine basis for MN analyses. (f) Larger and/or longer studies are required to verify the results of previous studies concerning the association of MN frequency with pregnancy complications, cancer and cardio-vascular disease (8,13–15). Prospective studies of MN in cells from the umbilical cord (at birth) with respect to association with cancers in childhood and later in life are as yet uninvestigated and deserve attention given that the carcinogenic risk from DNA damage early in life may be of great significance. Associations with other diseases such as Alzheimer’s disease, Parkinson’s disease and diabetes have been reported in cross-sectional studies (29,30) but prospective data are required for ultimate validation of the MN biomarker as a predictor of these degenerative diseases. (g) Future studies should also explore the relationship of MN expression with chang es in DNA methylation and the associated transcriptome, metabolome and proteome profiles to unravel the underlying molecular mechanisms that correlate with this DNA damage biomarker. This ‘omic’ data could provide valuable information on the likely origin of MN when the exposure profile is unknown or difficult to ascertain. In conclusion, the activities of the HUMN and HUMN xL projects have transformed the approach to MN assay validation, standardised the performance of the assay and scoring procedures and facilitated the wide-spread adoption of this valuable technology as a tool for investigating the most fundamental pathology of the human condition, i.e. damage to the human genome. The ultimate goal is to see the validated MN assays becoming a routine diagnostic in the new disease prevention paradigms and

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strategies required for this new millennium based on personalised prevention of DNA damage.

Acknowledgements We would like to thank all the scientists and laboratories that have contributed to the various activities of the HUMN and HUMN xL ; they are listed or indicated in the publications associated with this paper and/or appear as co-authors of this paper. Also recognised are the thousands of volunteers who have donated their time and tissue samples to enable MN frequency data to be collected. Finally numerous funding bodies, from different coutries, have supported this research and all are acknowledged in the HUMN and HUMN xL publications. Conﬂict of interest statement: None declared.

References [1] Fenech, M. and Morley, A. A. (1985) Measurement of micronuclei in lymphocytes. Mutat. Res., 147, 29–36. [2] Fenech, M. and Morley, A. A. (1986) Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat. Res., 161, 193–198. [3] Fenech, M., Holland, N., Chang, W. P., Zeiger, E. and Bonassi, S. (1999) The HUman MicroNucleus Project—An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans. Mutat. Res., 428, 271–283. [4] Bonassi, S., Fenech, M., Lando, C. et al. (2001) 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. Environ. Mol. Mutagen., 37, 31–45. [5] Fenech, M., Chang, W. P., Kirsch-Volders, M., Holland, N., Bonassi, S. and Zeiger, E. (2003) HUman MicronNucleus project. HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat. Res., 534, 65–75. [6] Fenech, M., Bonassi, S., Turner, J. et al. (2003) HUman MicroNucleus project. Intra- and interlaboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project.

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