Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

FP7

ECNIS2 Informe resumido

Project ID: 266198
Financiado con arreglo a: FP7-KBBE
País: Poland

Final Report Summary - ECNIS2 (ECNIS2: towards ECNIS Centre for Research and Education on Cancer, Environment and Food)

Executive Summary:

As a result of common effort of the ECNIS researchers a number of initiatives important for European integrative research on cancer, biomarkers, and food have been performed in the course of ECNIS2 project including, among others, so called “ECNIS added values”:
The MEC database has been created in ECNIS with the purpose of pooling data from different epidemiological studies on cancer and biomarkers thus providing increased statistical power to evaluate the influence of various environmental or other factors on cancer risk. In ECNIS2 we have added 3 studies: on global DNA methylation and, metabolomics in EPIC Italy and LUMA analyses. EPIC dataset has been enriched with additional biomarkers for the analysis of SES. New statistical approaches and methods have been developed and explored for analysis of false positive and false negative rates, graphical and statistical assessment of causal relationships; missing data and use of ecological data.
Three core facilities were established: chemical standards and reference materials, antibodies, and virtual core facility for analytical techniques. Effort was concentrated on the synthesis of 8 chemicals for characterisation of the antibodies produced by the antibody core facility. During ECNIS2 production of 5 antibodies have been completed, 2 are in final stage of preparation. As the ECNIS2 programme ended on 31 May 2013, the agreement concerning the ownership and supply of antibodies has been agreed. The list of recently available techniques in ECNIS was circulated among ECNIS2 and EIEC partners for comment, then revised, and uploaded on the ECNIS webpage. The 32P-postlabelling interlaboratory trials led to the production of a set of DNA standards and the development of a standardized protocol, use of which reduced interlaboratory variability in quantitation of DNA adducts. Modifications to the protocol reduced the amount of radioactivity used and shortened the assay time, representing a significant increase in potential throughput. Under the European Standards Committee on Urinary (DNA) Lesion Analysis (ESCULA) , in order to improve inter-assay agreement and also to investigate intra-individual variability, a large-scale study was undertaken with 26 laboratories involved. Analysis of 8-oxodGuo levels in urine samples from healthy subjects and cancer patients showed that the major difference in values originated from ELISA versus chromatographic techniques. The use of calibrants improved intra-technique agreement for chromatographic techniques to the extent that these methods can be used for pooling of results in large-scale studies. On-going in vitro work involves use of NUDT1 knock-out cell lines, and examination of 8-oxodGuo excretion and gene expression. A second round of 4 trials was performed under The European Comet Assay Validation Group (ECVAG) which included 14 laboratories. They considered the intra and interlaboratory variation, a set of reference conditions, and variation in DNA damage in different European countries. The conclusion is that at present it is not possible to promote a standard comet assay protocol that will be adopted by all laboratories. The most important recommendation from ECVAG is to use the same comet assay unit. The transformation of primary comet assay values to lesions/106 by calibration curves is the preferred way. ECNIS Repository is an electronic platform for storing and providing access to digital documents dealing with the matters of environmental hazards for cancer. Recent number of records is 524. ECNIS Repository has 2530 visitors with 16415 items and 2687 downloads. Education and training program was continued, containing i) training courses, a web-based training course, and a fellowship program. Four ECNIS2 fellowships were awarded.
In view of lack of funding for support of ECRECEF from EC after ECNIS2 completion it was decided the only potential way for durable integration and support for some key elements of ECNIS2 was through Polish national funding under The European Institute for Research on Environmental Cancer (EIEC), which has been included on a Polish Road Map for Research Infrastructure and has a form of centrally coordinated ten research institutions. Both ECNIS and EIEC members agreed to continue ECNIS ideas under EIEC umbrella. All the planned activities will be performed with close collaboration with ECNIS Network of Excellence. ECNIS will retain name as a well know “trademark” with its key elements within EIEC structure.

Project Context and Objectives:
According to World Cancer Report, 2008 (IARC) there were an estimated 12.4 million new cancer cases and 7.6 million cancer deaths worldwide in 2008. With an estimated 2.9 million new cases and 1.7 millions of deaths each year, cancer remains a major public health problem in Europe. Epidemiological studies show that the environment, including nutrition, is a major determinant of cancer risk in the general population. Human exposure to environmental carcinogens usually involves low levels of relatively weak carcinogens, frequently found as complex mixtures, and occurs over a prolonged period of time, making exposure assessment a major problem. To explore the risk associated with these exposures, there has been a rapid increase worldwide in the use of biomarkers to monitor exposure and to determine the resultant biological effects, together with studies of human susceptibility factors. Such information is essential for us to understand the reasons for the large variations in the incidence of major cancers that occurs across European countries and is a prerequisite for any future major epidemiological studies to validate hypotheses to explain these variations. Also rapidly increasing worldwide is the study of food components which may modulate the effects of environmental carcinogens, and biomarkers are playing a major role in assessing the effectiveness of chemoprevention measures using these food constituents.
Substantial progress has already been made in improving the sensitivity, throughput and applicability of biomarkers of exposure, biological effect and susceptibility, but the promise for improving the health of the public using these tools is far from being realized. One of the reasons for this is that the methodology is complex and specialized, and the expertise is only available in a limited number of laboratories. As a result, most biomarker methods have not been properly validated, either analytically or in the field with respect to human health effects.

Achievements of ECNIS – base of ECNIS2
The original ECNIS project brought together some of the best European research groups from 25 institutions of 13 countries active in the area of environmental cancer and its modulation by nutrition and genetic makeup. Through works carried out during five and a half years, a vivid and mature network of strongly collaborating participants, using harmonized, integrated resources, data bases, procedures and quality standards for common use as a base for performing coordinated joined research activities was established. One of the primary objectives of the network had been to integrate the research efforts and to facilitate exchange of the knowledge and expertise. The ECNIS network has formed an extraordinary multidisciplinary forum for exchange of knowledge and expertise among almost 200 participating researchers. The Network is unique in the range of methodologies involved and the state of the art technology available in the laboratories of the ECNIS partners. None of the existing individual laboratories could provide such a level of multidisciplinary capabilities and research methods.

To accomplish its scientific aims ECNIS had focused on the field of biomarkers and their application in population studies as well as on the studies of mechanisms of gene-environment and gene-food interactions in carcinogenesis. ECNIS funds supported scientific events including conferences, symposia and workshops – during five years; altogether as many as 63 workshops were organized with almost 3 000 attendees, besides conferences with support or satellite ECNIS events. As many as 181 joint publications acknowledging ECNIS were published. Furthermore, ECNIS researchers performed in-depth evaluations of the state of science relevant to cancer etiology and have formulated corresponding proposals for future research. The conclusions of such deliberations are published in the series "ECNIS Reports" and disseminated in the form of workshops directed at a broad range of stakeholders.

The ECNIS2 project aimed at continuing the integrative and coordinative works initiated during ECNIS network with an ultimate goal of transforming network into a virtual centre, the European Centre for Research and Education on Cancer, Environment and Food
(ECRECEF).

The foundations for the development of the ECRECEF centre were developed within ECNIS
by bringing together researchers from different disciplines (epidemiology, chemical analysis, genetics, molecular biology, nutrition, exposure assessment, risk assessment, harmonization and standardization of methodologies and analytical techniques). The initiatives undertaken within ECNIS were continued during ECNIS2 and focused on the main pillars, so called “ECNIS added values”, including: Molecular Epidemiology and Cancer" (MEC) database, core facilities for: standardized chemicals, antibodies and analytical methodology, European Standards Committee on Urinary DNA Lesion Analysis (ESCULA), and European Comet Assay Validation Group (ECVAG), ECNIS Repository, and training and educational program. Research coordination, education and dissemination will be key outputs, together with the formulation of strategy for transformation of ECNIS into the Centre (ECRECEF). Continued support for the development validation and application in population studies of biomarkers, improved data bases, and core facilities were additional important components. The intention was to prepare realistic, costed plans for the use for these facilities and the implementation of validated biomarker techniques in European cancer molecular epidemiology studies.

Project objectives:
The specific ECNIS2 aims include:
• Strengthening the integration of the research within Europe devoted to cancer biomarkers validation, standardization and use in the population based studies
• Enhancing the interaction between research networks active in the area of environmental cancer etiology and prevention
• Integration of the research on biomarkers reflecting dietary exposure and mechanisms of carcinogenic and anticarcinogenic (protective) effects of food components on carcinogenesis
• Comprehensive education and training in the field of molecular epidemiology
• Providing management structure for coordination of the network activities
and finally
• Creation of ECNIS Center for Research and Education on Food, Environment and Cancer

The activities of as many as 21 ECNIS2 partner institutions from 13 countries (incl. 4 SMEs) was organized within the framework of nine workpackages; five Infrastructural (WP1-WP5), three Thematic (WP6-WP8) and Managerial –WP9.

Internal and external consultation with the scientific community and other stakeholders through a series of internal meetings and two workshops satellite to relevant conferences was organized within WP1 (ECNIS Centre establishment) in order to establish the optimal organizational formula for the transformation of existing ECNIS infrastructure into
ECRECEF. However, in view of lack of funding for support of ECRECEF from EC after the formal completion of ECNIS2, the only potential way for durable integration and support for some key elements of ECNIS2 was identified in the context of Polish national funding, which foresees strong international collaboration. The basis for this activity has been created in the form of a European Institute for Research on Environmental Cancer (EIEC), which has been included in the Polish Road Map for Research Infrastructure (Polish governmental initiative, cofinanced by EU Structural Fund). The European Institute of Environmental Cancer has a form of centrally coordinated network facilitating integration of 10 institutes, clinics and laboratories with a high degree of specialisation to perfect/ameliorate the integration of activities towards biomarker-based research on environment, food, genetics and cancer. The specific objectives of the EIEC is in full agreement with ECNIS objectives. Both ECNIS and EIEC members agreed to continue to pursue the objectives of ECNIS under the umbrella of EIEC. The commitments were expressed during the ECNIS2 General Assembly. All the planned activities will be performed with close collaboration with ECNIS Network of Excellence. ECNIS will retain name as a well know “trademark” with its key elements within EIEC structure.

The organizational work within ECNIS2 WP1 was run in parallel with other WPs, dedicated to the most relevant infrastructural and thematic activities continuing from ECNIS. Activities on validation, standardization and quality control measures for biomarkers continued within WP2 (Validation and Standardization) towards the development of protocols and quality controls for the use of biomarkers in studies of environmental cancer elaborated. Inventories of resources and facilities developed within ECNIS have been maintained. The efforts continued to further consolidate and extend the Molecular Epidemiology of Cancer (MEC) data base with more pooled analyses on suitable biomarkers in MEC performed (WP3). Development of the ECNIS2 website, preparation of newsletters, brochure, update of the ECNIS Repository and organization of workshop for policy makers and researchers was done within WP4 (Spreading of Knowledge) to foster communication and interaction between researchers and other stakeholders in Europe and beyond. WP5 (Education and training) comprised training courses, a web-based training course, and a fellowship program. A database of “Research teams, networks and projects on Molecular epidemiology of carcinogenesis” has been created within WP6 (Biomarkers use for cancer prevention) and made available on the ECNIS website. Interactions between research teams, networks and project was promoted via setting up of targeted working groups (to explore important and emerging issues), and organization of corresponding workshops. Within WP7 (Dietary exposure and cancer risk as modified by genetic polymorphisms) inter-laboratory comparisons of genotyping methods was continued and research on genetic polymorphisms related to the metabolism of different micronutrients, and exposures to toxic food components (e.g PAHs, cadmium,acrylamides) and cancer risk continuously reviewed with the aim of formulation of new dietary advice, and future research strategy. Links to other consortia and projects have been further developed. Training activities organized within ECNIS2 complemented with material specific for ethics and communication developed in the framework of WP8 (Ethics, Communication and Gender). A help-desk as a tool for advice and support for transnational research in the specific research domain of food, environment and cancer and implying the use of human biomarkers has been expanded. Integration and coordination of consortium activities in the process of the transformation from ECNIS Network of Excellence into the ECNIS Centre for Research and Education on Cancer , Environment and Food/EIEC was ensured by effective managerial workpackage (WP9 - Management).
Project Results:
The main objective of the ECNIS2 project was to facilitate the effective coordination of European research in the area of environmental cancer molecular epidemiology by laying the foundations for the creation of an ECNIS Center for Research and Education on Food, Environment and Cancer. In the context of this objective, and as a result of common efforts of the ECNIS researchers, a number of initiatives important for European integrative research on cancer, biomarkers, and food have been performed in the course of ECNIS2 project, including, among others, so called “ECNIS added values”:
1. Molecular Epidemiology and Cancer" (MEC) database,
2. Core facilities
3. European Standards Committee on Urinary DNA Lesion Analysis (ESCULA).
4. European Comet Assay Validation Group (ECVAG)
5. ECNIS Repository
6. Training instruments

Laying the foundations of an ECNIS Center for Research and Education on Food, Environment and Cancer
In view of the lack of funding for support of ECRECEF from EC after the formal completion of ECNIS2, the only potential way for durable integration and support for some key elements of ECNIS2 was identified in the context of Polish national funding, which foresees strong international collaboration. The basis for this activity has been created in the form of a European Institute for Research on Environmental Cancer (EIEC), which has been included in the Polish Road Map for Research Infrastructure (Polish governmental initiative, cofinanced by EU Structural Funds). The European Institute of Environmental Cancer has the form of a centrally coordinated network facilitating integration of 10 institutes, clinics and laboratories with a high degree of specialisation to perfect/ameliorate the integration of activities towards biomarker-based research on environment, food, genetics and cancer. The specific objectives of the EIEC is in full agreement with ECNIS objectives. Both ECNIS and EIEC members agreed to continue to pursue the objectives of ECNIS under the umbrella of EIEC. The commitments were expressed during the ECNIS2 General Assembly. All the planned activities will be performed with close collaboration with ECNIS Network of Excellence. ECNIS will retain name as a well know “trademark” with its key elements within EIEC structure.

In addition to the above, work was conducted on the charting of European activities in the area of environmental cancer research and on the formulation of future research strategy in the area of molecular epidemiology of environmental cancer. In relation to the first objective, a database of “Research teams, networks and projects on Molecular epidemiology of carcinogenesis” was constructed and specific contact was established with ongoing relevant European initiatives as well as with relevant stakeholders, including the organisation of a stakeholders’ workshop titled «Cancer and the Environment: European research & preventive policy» (Krakow, October 2012; see Dissemination Report). As regards the formulation of research strategy, the main conclusions emerging out of the consultations which were carried out were that areas of major importance, which were considered likely to have major impact in European research in the middle-to-long term, at the same time requiring broad support, were a) the creation of new, continent-wide prospective cohorts and biobanks specifically focused on questions relevant to the study of environmental causes of cancer, and b) the exploitation of the power of new technologies of holistic biological analysis in the context of population-based studies. Both of these research foci take note of and provide support for the development of the newly emerging concept of the exposome which promises to revolutionize exposure assessment and the study of the environmental determinants of chronic diseases including cancer. In fact the support of exposome-related research is a third focus identified in these discussions and was widely recognised as one for which efforts should be made to promote its adoption and incorporation into the activities of FP7 and beyond. In this context various opportunities were taken to inform policy makers at EC level of the importance attached by the environmental cancer research community to the formulation of a research strategy based on the exposome and also to improve the potential of the ECNIS partners to become active, as a multidisciplinary collection of research teams, in relevant research.
As regards the other two foci identified as important for development a coordinated research policy (new biobanks and exploitation of new technologies), a symposium dedicated to the topic ("Design of future molecular epidemiology studies and new biomarkers") was organised by ECNIS in collaboration with the Molecular Epidemiology group of the UK Environmental Mutagen Society in London in December 2012.

Molecular Epidemiology and Cancer" (MEC) database
Within the original ECNIS project a special effort had been made to facilitate the optimal utilization of already existing knowledge and data, through the construction of the "Molecular Epidemiology and Cancer" (MEC) database. The MEC database was created in ECNIS with the purpose of pooling data from different epidemiological studies on cancer and biomarkers thus providing increased statistical power to evaluate the influence of various environmental or other factors on cancer risk. MEC allowed to (a) conduct validation studies, e.g. on the degree of laboratory error and intra-individual variability in biomarker levels, including determinants of such variability; (b) develop updated tools (e.g. bayesian methods) for pooled analyses of biomarkers; (c) study the relationship between some external exposures (e.g. dietary variables, air pollution) and selected outcomes such as adducts; (d) study the relationship between biomarkers and hard outcomes such as cancer. To this end we have created a database that encompasses epidemiological studies from all over Europe and includes biomarkers belonging to the following categories: (a) adducts (protein and DNA); (b) micronuclei, SCE, chromosome aberrations; (c) proteomics; (e) metabolomics; (e) transcriptomics. MEC stores, organizes and links exposure, genomic, biomarker, phenotypic and disease status information. A document manager (DMS) is used to archive original datasets with author level access control. A bibliographic tool (set up on a per project criterion) was also created to integrate data with study information and references. A set of statistical tools (R code developed for meta-analyses and integrated in the web application, and SAS scripts for data pooling and regression (externally run)) were created and embedded within the MEC.
The original content of MEC included 17 molecular epidemiology datasets from 15 European contributors, including data for the following biomarkers measured in humans: adducts (32P post labelling, etheno adducts), 8-OH-dG, protein adducts, lipid peroxidation products, mutations (eg HPRT, KRAS), chromosome aberrations and SCE, transcriptomics and proteomics (limited), serum cotinine, ascorbic acid and other vitamins, fatty acids, genetic data (SNP): metabolic polymorphisms, DNA repair, cell-cycle genes, Environgenomarker epigenetics – LINE1 methylation. In ECNIS2 we have added the following studies: Global DNA methylation in EPIC Italy (857 subjects), Metabolomics in EPIC Italy (colon cancer, 388 subjects), LUMA analyses from EPIC (376 subjects). The EPIC dataset has been enriched with additional biomarkers for the analysis of SES. As regards the incorporation of further omic data, steps have been taken for additional omic data to be added, eg. metabolomic data from the Envirogenomarkers project as well as epigenomic data from the HuGeF colon and breast cancer studies.
The analysis of pooled biomarker data poses a number of challenges to be addressed during the pooling project, and the statistical analysis of the data. The main challenge is biomarker inter-laboratory variability. Often, large differences in biomarker measurements observable in pooled databases are due (at least partly) not to real differences between subjects, but to analytical procedures within laboratories. To overcome this problem we have developed statistical approaches that have been applied to our pooled analyses and will be applied to further analysis of new pooled data sets. Statistical methods have been developed and explored for analysis of false positive and false negative rates, graphical and statistical assessment of causal relationships; missing data and use of ecological data. Special emphasis was given to the development of Bayesian statistical tools for meta- and pooled analyses.
During ECNIS2 particular emphasis was placed on the statistical analysis of methylation data, genome wide in particular, using generalised linear models coupled with ad-hoc multiple testing and/or FDR correction strategies. The inclusion of random effects in the model enables to assess and correct for a potential batch effect, and therefore eases the compilation of data obtained from different experimental conditions. Bayesian variables selection approaches is also used (e.g. Evolutionary Stochastic Search; ESS++). This considers predictors simultaneously, and hence implicitly corrects for multiple testing. These approaches typically seek for the best combination of predictors (here the per locus methylation fraction  or m coefficient) to predict the outcome of interest (disease status or exposure levels), which, in turn, can be complex (e.g. multidimensional response). To accommodate to case/control data, and based on the recent Multilevel Inference for SNP Association study model, the multiple linear regression model on which the ESS++ is currently based, has been generalized. Although ESS has been shown to provide parsimonious results, a particular attention is paid to minimize the number of false positive discoveries. In that respect, a cross-validation procedure (using train/test split) is carried out. Furthermore, initial guesses of the MCMC algorithm built-in in ESS are chosen from a preliminary association study based on the Bayesian False Discovery Probability measure (BFDP), a stochastic alternative to the q-value which also accounts for the costs of false discovery and non-discovery.
These procedures have been applied in the analyses described below, focusing in particular on the relationship between several foods/nutrients and the levels of selected biomarkers:
The impact of dietary intakes of food groups, nutrients, and patterns on genomic DNA methylation in PBLs of healthy individuals was investigated based on the data and blood samples collected from the European Prospective Investigation into Cancer and Nutrition (EPIC) were used. We investigated 376 healthy women with dietary data collected.through FFQ, anthropometric measures, lifestyle, and environmental exposures. Global methylation levels in PBLs in the blood of subjects collected at recruitment was analyzed using LUminometric Methylation Assay (LUMA). We have shown that even though independent intakes of food groups and nutrients were not associated with percent global methylation, intakes of nutrients that impact on the one carbon metabolism influence DNA methylation. Having below median intakes of folate and the B vitamins, coupled with above median alcohol consumption increase the risk of hypomethylation which is a common feature in tumour cells and may be implicated in carcinogenesis. Adequate intake of the one carbon metabolism related nutrients and moderate alcohol consumption may prevent hypomethylation.
The HuGEF colon cancer dataset was used to investigate the association between metabolite features and risk of colon cancer. Metabolomics data from 194 incident colon cancer cases and 194 matched controls (age [+/- 5 years] -participants of the EPIC Italy cohort were analysed. Metabolic profiling was performed on blood samples taken at recruitment. One hundred and thirty four metabolite features were derived from this cohort. The number of significant associations and the top ten hits for each variable were determined. The overlap in selected significant metabolites between variables was calculated, and the metabolites that appeared in the top ten hits most frequently, identified.
The association of several indicators of socioeconomic status (SES) over the life-course with DNA methylation of candidate genes was analyzed using the Italian component of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Candidate genes have been selected based on a literature review of the papers published on socioeconomic status and gene regulation patterns (in humans and primates). Low SES was found to consistently predict low methylation of pro-inflammatory genes. Its noteworthy that 6 out of 10 probes that were significantly associated with household's highest occupational position (5/8 after correction for multiple comparisons) belong to the NFATC1 gene, one of the three genes whose expression was more strongly associated with social rank in macaques. We also highlighted IL8RB, while the study in macaques found an association with IL1A. Associations are also described in our study between SES and AHRR and IGF-2 methylation, but the pattern is not consistent across the gene probes. This was the first study to assess DNA methylation patterns in relation to SES in such a large population (N=857), and to use indicators of SES spanning through the life-course.

Thanks to work conducted within the original ECNIS project and subsequently within ECNIS2, the Molecular Epidemiology of Cancer (MEC) database has been further expanded and is developing into a powerful tool for the study of environmental cancer. An additional advantage of MEC is that, in addition to storing data derived from molecular epidemiology studies, it also incorporates tools for the analysis of these data, including the conduct of meta-analyses. Thus it became apparent that the incorporation of omics data emerging from population-based environmental health studies, which are currently relatively limited but are anticipated to increase substantially in the coming few years, along with the development of analysis tools targeted at omics data, would give MEC a unique character and help establish it as valuable resource in environmental carcinogenesis research. Furthermore, an increasing number of European environmental health research projects have been initiated which have produced omic data from human populations exposed to different environmental, dietary and lifestyle conditions, including recently initiated projects related to the exposome. This underlines the need for a central repository where such data can be collected and integrated with other kinds of data coming from traditional epidemiology (e.g. questionnaire-based dietary data) as well as molecular epidemiology data such as those already present in MEC.


Core facilities
The development of core facilities was considered to be an important part of the work of ECNIS from the earliest days of the network. Such facilities were intended to provide access to technologies that were too expensive for individual groups or required particular expertise and to provide specialist materials that were not available commercially or of an acceptable or consistent standard. After extensive discussion, three core facilities were established:
1. chemical standards and reference material, overseen by Albrecht Seidel, Biochemical Institute for Environmental Carcinogens (BIU);
2. antibodies, overseen by Marcus Cooke, University of Leicester; and a
3. virtual core facility for analytical techniques, overseen by Peter Farmer, University of Leicester.
1. Chemical standards
After ECNIS funding ended in April 2010, effort was concentrated on the synthesis of chemicals for characterisation of the antibodies produced by the antibody core facility (see below). Work continued on synthesis during ECNIS2, focussing on the first two chemicals because of the long synthetic route necessary to prepare the requisite building blocks. The chemicals available as of May 2013 are shown in Table 1.

After consultation between the partners it was decided to undertake a final prioritisation exercise to encompass Partners’ needs for chemical standards as well as haptens/antibodies because of changes in the techniques used in current projects, such as the increasing use of mass spectrometry rather than 32P-postlabelling. The questionnaire was circulated to members of the European Institute of Environmental Cancer (EIEC) as well as ECNIS2 partners so that future needs could be accommodated. The greatest interest was in (±)-anti-B[a]PDE-trans-N2-dG-3′-monophosphate, mixture of diastereomers, and [(±)-anti-B[c]PhDE-trans-N6-dA-3′-monophosphate, mixture of diastereomers, predominantly isotopically labelled for use in mass spectrometry. Of the chemicals that have already been synthesised, O6-carboxymethyl-2′-deoxyguanosine (Ca2+ salt) was most preferred.

2. Anti-DNA adduct antibodies
Partners were principally interested in using antibodies for determination of modifications to DNA and potential immunoaffinity columns for clean-up of extracellular matrices prior to analysis. The priority list of antibodies that was drawn up after consultation reflected these uses. After revision, the resultant list comprised:
1. 1,N6-Etheno-2'-deoxyadenosine
2. N2-Ethyl-2'-deoxyguanosine
3. N2-(2'-Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
4. 5,6-Dihydroxy-5,6-dihydrothymine
5. 8-Oxo-7,8-dihydro-2'-deoxyguanosine
6. 1-Thyminebutanoic acid cyclobutane dimer
It was acknowledged that priorities might need to be reconsidered if partners’ research needs changed or a particular hapten was found not to be the most appropriate for the intended purpose.
Under the ECNIS2 project the synthesis of structurally defined haptens for antibody production was continued. It is based on the concept of preparing the modified deoxynucleoside to which a C4-linker is selectively attached at the 5'-OH group of the deoxyribose moiety to allow covalent coupling of the hapten to a carrier protein. The synthesis of the modified deoxynucleosides frequently entails a multistep sequence, sometimes in conjunction with sophisticated protecting group techniques to allow efficient synthetic access to the target structure. The target structures for antibody production comprise etheno-dA, N2-ethyl-dG, PhIP-C8-dG, dTg, 8-oxo-dG, and dT<>dT. As final hapten the 5'-O-hemisuccinate is prepared from each of the above mentioned adducts, except for dT<>dT, to achieve protein conjugation.
A number of companies were considered for the production of antisera. These companies were evaluated, based on their experience of the tasks to be undertaken as well as cost. Specifically, the company was required to have experience in the conjugation of haptens containing carboxylic acid functions, such as hemisuccinates, using the N-ethyl-N(3-dimethylaminopropyl)-carbodiimide (EDC) method. In addition to conjugation, the company was required to perform the following: immunisation and antisera production; hybridoma fusion and screening; large scale culture; protein G purification (500 ml); and subtype determination. AMS Biotechnologies (Europe) Ltd, an ISO9001 accredited, global supplier of biotechnology reagents and equipment, was found to meet the criteria.
Haptens were conjugated to both chicken gamma globulin and bovine serum albumin. Conjugation to two proteins allows for immunisation with one conjugate and screening against the other, with no risk of cross-reactivity towards the carrier protein used. Progress in production of haptens and antibodies is summarised in the Table 2.

At the ECNIS2 first Annual Meeting in Copenhagen in November 2011, it was decided to identify the antibodies of most interest to partners and to determine the experience of respondents in characterising antibodies by circulating a questionnaire. Five partners expressed specific preferences and two indicated a general interest. The first priority was 1,N6-etheno-2'-deoxyadenosine, with 22 points. 5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 1-thyminebutanoic acid cyclobutane dimer both scored 18 points. At a videoconference held in February 2012, it was decided to focus primarily on the top three haptens. Antibodies have been raised to 1,N6-etheno-2'-deoxyadenosine, N2-Ethyl-2'-deoxyguanosine and 1-thyminebutanoic acid cyclobutane dimer. Given that antibody clones to 1,N6-etheno-2'-deoxyadenosine and 1-thyminebutanoic acid cyclobutane dimer did not react to adducted DNA, it was recommended that they be tested against DNA hydrosylates. Synthesis of thymine glycol was continued by BIU using, in part, the organisation’s own budget and is now finished. Synthesis of the 5'-O-hemisuccinate hapten is nearing completion.
After further consultation between the partners, it was decided to undertake a final prioritisation exercise to encompass partners’ needs for chemical standards as well as haptens/antibodies. The questionnaire was circulated to members of EIEC as well as ECNIS2 partners so that the needs of potential future collaborators could be accommodated. Greatest interest was expressed in 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-O-hemisuccinate and 1-thyminebutanoic acid cyclobutane dimer closely followed byN2-(2'-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(dG-C8-PhIP)-5'-O-hemisuccinate (which is in preparation).
Further discussion concerned funding for antibody production and characterisation. The antibodies being developed are sufficiently specialised to compete in the open market but further characterisation is necessary before their commercial potential can be determined. Advice is being sought on funding and patent issues.
At present storage of chemicals, haptens and antibodies is being undertaken at BIU without cost to ensure their stability. As the ECNIS2 project ended on 31 May 2013, the agreement concerning the ownership and supply of antibodies has been revised, updated approved by NMO and signed.

3. Virtual core facility for analytical techniques
The list of techniques to be included involved compiling those included in the initial surveys and ‘virtual’ core facility. Scanning of recent papers by ECNIS2 colleagues and other publications in the field generated more relevant techniques. The resultant list was circulated among ECNIS2 and EIEC partners for comment. Further refinement resulted in the grouping of related techniques and the assignment of keywords to each (uses, acronyms) for additional power in searching the database. All available techniques are presented in table 3.

Interlaboratory validation trials
The 32P-postlabelling interlaboratory trials led to the production of a set of DNA standards and the development of a standardized protocol, use of which reduced interlaboratory variability in quantitation of DNA adducts. Modifications to the protocol reduced the amount of radioactivity used and shortened the assay time. Reducing the volume of the incubation mixture, by evaporating the DNA digest to dryness prior to the labelling reaction was also found to result in increased incorporation of radioactivity. Using the alternative chromatographic approach of HPLC, considerable progress was made in reducing run times from ~60 minutes to ~15 minutes, representing a significant increase in potential throughput. Autosampling strategies were considered involving freezing of samples or addition of ethanol to minimise autoradiolysis. Improved columns and solvent gradients were also developed or employed to improve the resolution and quantitation of adducted material in these shorter elution runs.
Identification of the important parameters in an assay and the use of standards was considered to be the most practical approach to reduce interlaboratory variabiliy in results. The further development of high through-put methods for DNA adduct analysis that are cost- and time-effective is necessary to enable complex, life-course exposures to be studied.
DNA adducts are now well-validated biomarkers of exposure to environmental carcinogens, in part due to activities carried out within ECNIS and ECNIS2. A number of detection methods can be used, including postlabelling, immunochemical, fluorescence, electrochemical and mass spectrometric. What has become clear is that tobacco smoking is frequently a confounding factor in studies of, for example, human exposure to air pollution, occupational exposure to carcinogens and effect of diet on DNA adduct formation in human tissues. It is thus important to understand the nature and origins of smoking-induced DNA damage, and to take account of its effects, whenever conducting human biomonitoring studies. Smoking-related DNA adducts identified include those formed by BaP, PAHs, 4-aminobiphenyl, reactive oxygen species, products of lipid peroxidation, formaldehyde, acetaldehyde, acrolein, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotine (NNN) and an as-yet-unidentified ethylating agent; smoking-related protein adducts have been found to derive from BaP, 4-aminobipenyl, NNK, NNN, ethylene (oxide), acrylamide (glycidamide) and acrylonitrile.
There have been thus far about 8 medium-to-large studies (n>200; up to ~1000 subjects) involving analyses of DNA adducts in human populations. In most cases these have examined the relationship between adduct levels and xenobiotic metabolising gene or DNA repair gene polymorphisms (e.g. GSTM1, GSTP1, GSTT1, CYP1A1, CYP1B1, XPA, XPD), with a range of significant and non-significant associations reported. Other large studies have been conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC) prospective study. These include investigation of the association between aromatic/bulky DNA adducts and exposure to environmental carcinogens, risk of breast cancer and risk of gastric and colorectal cancer .
During the course of the ECNIS and ECNIS2 projects, there have been some advances in the throughput of several of the common methods for adduct detection in molecular epidemiology studies, including for postlabelling a reduction in the amounts of radioactivity needed for analysis, and some initial attempts at simplified chromatography. For immunoassays, a higher sensitivity and throughput chemiluminescence immunoassay for PAH-DNA adducts has been developed . Mass spectrometry of DNA adducts has also achieved higher sensitivity such that it can be applied to smaller samples of human material.
With the emergence recently of the ‘exposome’ concept, which equates to the totality of environmental exposures from conception onwards , characterising the exposome represents a considerable challenge, requiring untargeted analysis for adducts with high throughput. Adductomics, namely the identification of exogenous and endogenous electrophiles through their trapping as modifications to DNA and protein, is thus a component of the exposome approach (Kanaly et al. 2006; reviewed by Rappaport et al. 2012). There is a need to develop current methods further to meet this challenge.
The validation of DNA and protein adducts as biomarkers of exposure to genotoxic agents means that such analyses can assist in identifying causative disease-related events in human populations. To move to the bigger scale of population study demanded by the exposome concept, further developments of existing methods are required to achieve cost- and time-effective high throughput analyses.
The search for alternatives to the use of peripheral blood mononuclear cells (PBMCs) in large-scale studies of DNA repair capacity (DRC) ruled out lymphoblastoid cell lines (LCLs) as a suitable surrogate. Cryopreserved cells appeared to be a satisfactory alternative to fresh cells for the assays tested. The development of high through-put fluorometric assays showed promise for the assessment of DRC in samples from population studies and important practical information about the stability of the activity in stored samples was obtained. Application of the panel of DRC assays that are now available to genotype-phenotype correlation studies has already allowed identification of new functional gene variants and haplotypes. Ultimately, these methods may improve prediction of individual DRC and susceptibility to environmental genotoxins.



European Standards Committee on Urinary DNA Lesion Analysis (ESCULA).
Exposure of organisms to environmental insults such as xenobiotics or radiation can lead to excess production of reactive oxygen species (ROS) culminating in oxidative stress. Modification of cellular components, especially DNA, lipids and proteins, can result from interaction with ROS. These interactions can give rise to DNA base modifications such as 8-oxo-7,8-dihydroguanine (8-oxoGua) or, indirectly, to adducts such as 1,N6-etheno-2′-deoxyadenosine (dA). Damage to DNA may result in mutations or nonmutational effects such as telomere shortening.
The principal methods used for analysis of urinary products of oxidatively damaged DNA are chromatographic, e.g. high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS, HPLC-gas chromatography (GC)/MS, HPLC-electrochemical detection (ECD), or immunoassay, especially competitive enzyme-linked immunosorbent assay (ELISA). Because of the complexity of urine, chromatographic methods require a ‘clean-up’ step such as column switching to separate the fraction of interest before analysis by MS or ECD. ELISA has been widely used because it is cheap, easy to use, requires no pretreatment of urine, and is amenable to high through-put
To investigate the discrepancies between the chromatographic and ELISA techniques and to improve interlaboratory agreement, a consortium of academic and industrial participants, the European Standards Committee on Urinary (DNA) Lesion Analysis (ESCULA) , was established in 2006. The establishment of ESCULA enabled large-scale international validation trials of methods for the determination of urinary 8-oxodGuo to be carried out. The first of these studies evaluated agreement within and between techniques and between laboratories.
In order to improve inter-assay agreement and also to investigate intra-individual variability, a further large-scale study was undertaken under the auspices of ESCULA . Twenty-six laboratories were sent samples and 25 data sets were returned from 18 of these. Samples included: 8-oxodGuo-spiked PBS, 8-oxodGuo-spiked urine, and urine samples from healthy and head or neck cancer patients; the latter were included to test the effects of disease-related alterations in urine composition on 8-oxodGuo analysis. Each laboratory used its own sample preparation and analytical method (MS, ECD or ELISA) but with calibrants supplied by the study organisers.
For 8-oxodGuo spiked into buffer, the values determined by the MS and ECD methods showed close agreement, whereas the results determined by ELISA differed from these and among laboratories. All methods showed rank order agreement. For the 8-oxodGuo-spiked urine samples, the MS and ECD determined values differed little but the ELISA values were on average 4.2-fold higher. Analysis of 8-oxodGuo levels in urine samples from healthy subjects and cancer patients showed that the major difference in values originated from ELISA versus chromatographic techniques. For chromatographic techniques, difference between subjects was the most important determinant of 8-oxodGuo level; for ELISA, interlaboratory variation and residual variation were the most important contributors.
To determine intra-individual variability in excretion of 8-oxodGuo, timed urine samples were collected over 24 hours from 26 healthy Swedish subjects on two days, a week apart. The samples were analysed for 8-oxodGuo by UHPLC-MS/MS and ELISA. Good correlation was found for 8-oxodGuo excretion between spot samples and the 24 hour values although adjustment for creatinine or specific gravity (SG) lessened this. Within-individual variation in excretion of 8-oxodGuo was moderate (20% for first void, creatinine-corrected samples; 17% for 24 hour excretion).
In conclusion, the use of calibrants improved intra-technique agreement for chromatographic techniques to the extent that these methods can be used for pooling of results in large-scale studies. ELISA variability was greater than for the chromatographic methods and this method cannot be used to determine absolute levels of 8-oxodGuo. For urine sampling, creatinine or SG-adjusted first void samples are recommended if 24 hour sampling is not possible.
Some evidence in the literature supports the idea that enzymatic degradation of 8-oxodGTP by the 8-oxodGTPase activity of NUDT1 (MTH1) is an important, if not the primary, source of urinary 8-oxodGuo, also implying that dGTP in nucleotide pools is an important target for oxidants. Knock-down of NUDT1 decreases 8-oxodGuo excretion in response to ionising radiation and knock-in of NUDT1 increases baseline excretion of 8-oxodGuo.
Funding from the ECNIS second call was provided for a project entitled ‘Contribution of diet, cell turnover and DNA repair to production of urinary DNA damage products: validation of biomarkers of DNA damage and repair’ led by Peter Farmer and Mark Evans, University of Leicester in collaboration with Ryszard Oliński, Bydgoszcz, and David Phillips, Institute of Cancer Research, Sutton, UK. Yusaku Nakabeppu, Kyushu University, Fukuoka, Japan and Leon Mullenders and Alex Pines of Leiden University Medical Center, The Netherlands, were non-ECNIS collaborators.
The contributions of NUDT1 and nucleotide excision repair (NER)/transcription-coupled repair (TCR) activity to the production of urinary 8-oxodGuo were investigated using wild-type animals, genetically modified nudt1-/- and nudt1+/- mice, and mouse models of Cockayne syndrome B (lacking functional TCR), xeroderma pigmentosum complementation group A (lacking functional TCR and global genome repair) and xeroderma pigmentosum complementation group C (lacking functional global genome repair). Urine samples were collected monthly for up to 12 months from all mice. Analysis of urinary 8-oxodGuo was performed at all time points, and 8-oxo-7,8-dihydroguanine (8-oxoGua), the oxidised nucleobase, at selected time points; additionally the level of 8-oxodGuo was determined in brain, lung, liver and kidney harvested from mice at the end of the study. Data from this investigation proved inconclusive regarding the role of NUDT1 in urinary 8-oxodGuo production (manuscript in preparation), prompting extension of investigations to in vitro work, and provided data implicating the role of other pathways. This on-going in vitro work involves use of NUDT1 knock-out cell lines, and examination of 8-oxodGuo excretion and gene expression.
The growing interest in the field of oxidative stress, and the increasing numbers of publications have led to inconsistencies in nomenclature of oxidatively damaged nucleobases. Because ESCULA recognises good communication to be important to scientific understanding, a sub-group of ESCULA participants together with a number of external experts in oxidative stress combined to promote discussion of terminology and provide recommendations for good practice . The aim was to remove doubt about the lesions being discussed and to simplify literature searches.
Biomarker stability during sample collection, handling, transport and storage is important for both large-scale population and clinical studies. In work additional to that funded by ECNIS2, Marcus Cooke, University of Leicester, and colleagues have devised novel strategies to maintain sample stability and also facilitate transport and storage, which has led to a patent application.
Future work will include the continuing evaluation of methods for the collection, handling, transport and storage of urinary biomarkers of oxidatively damage nucleic acids to ensure their stability. The contribution of the activity of the Nudix hydrolase family of enzymes and (2′-deoxy)ribonucleotide pools as sources of modified (2′-deoxy)ribonucleotides in urine will be further investigated. The repertoire of lesions routinely measured in urine will be extended, beginning with the oxidatively modified ribonucleoside, 8-oxo-7,8-dihydroguanosine. Finally, the potential merger of the European Standards Committee on Oxidative DNA Damage (ESCODD), the European Comet assay Validation Group (ECVAG) and ESCULA to create a European Standards Committee on Urinary and DNA damage Analysis is under discussion.
From the participants of a relatively simple, early interlaboratory trial, ESCULA has grown with the addition of further research groups and small/medium enterprises to become a leading authority on nucleic acid-derived biomarkers of oxidative stress in urine.

ESCULA recommendations for best practice
1. Chromatography, coupled with EC or MS detection are both acceptable.
2. Presently, ELISA approaches cannot be recommended for the accurate quantification of 8-oxodG in human urine.
3. Use of common calibrants improves inter-laboratory agreement, particularly for mass spectrometric methods, allowing data from different laboratories to be pooled
4. For mass spectrometric assays, use of stable isotopically labelled internal standards is essential - both single point calibration or multiple point, external calibration curves are acceptable.
5. 24 h excretion is the gold standard, but spot samples are also acceptable:
- timed samples for excretion rates (nmol/h)
- correction for creatinine (nmol 8-oxodG/mmol creatinine)
- or SG in first morning voids (e.g. nM-SG 1.018)
6. Little influence of disease status, at least with respect to cancer, is on urine composition and measurement of urinary 8-oxodG.
7. Broadly, many of these recommendations can apply to other urinary damaged nucleic acid products, until their individual assessment can be achieved.


European Comet Assay Validation Group (ECVAG)
The use of the comet assay as a test for DNA damage has increased dramatically during the last 20 years and publications providing comet assay results number about 500 annually. DNA damage can be measured as strand breaks or oxidized purines by enzymatic digestion of DNA with the Fpg enzyme. Recent novel versions of the comet assay include measurements of repair activity of bulky DNA adducts and oxidatively damaged DNA. However, despite the widespread use of the comet assay, there is no consensus on the normal level of DNA damage in lymphocytes or tissues, and assessment of DNA repair activity by a comet assay-based method is still in its infancy. Furthermore, many features of the assay (slide preparation, electrophoresis) affect both intra-assay variability and inter-assay reproducibility. However, an important experience gathered from the use of the comet assay for genotoxicity is the necessity for reproducibility and for agreement on true levels of values, e.g. DNA damage or DNA repair incisions, in the various applications of the method. For the purpose of validation of the comet assay in DNA damage and repair, the European Comet Assay Validation Group (ECVAG) was established. It is the objective of ECVAG to reach consensus on the likely level of DNA damage in lymphocytes and procedures, and values, of the DNA repair incision assay. ECVAG also intends to develop a bona fide internal standard to minimise both intra- and inter-experimental variability in the measures of comet assay endpoints.

ECVAG Partners are:
Andrew Collins, University of Oslo, Norway
Marcus Cooke, University of Leicester, UK
Roger Godschalk, Maastricht University, The Netherlands
George D.D. Jones, University of Leicester, UK
Blanca Laffon, University of A Coruña, Spain
Adela López de Cerain, University of Navarra, Spain
Giuseppe Matullo, University of Turin, Italy
Peter Møller, University of Copenhagen, Denmark
Lennart Möller, Karolinska Institutet, Stockholm, Sweden
David Phillips, Kings College, London, UK
Patrizia Riso, University of Milan, Italy
Michael N. Routledge, University of Leeds, UK
Maciej Stępnik, Nofer Institute of Occupational Health, Łódź, Poland
João Paulo Teixeira, National Institute of Health, Porto, Portugal
Ulla Vogel, National Research Center for the Working Environment, Copenhagen, Denmark

In the first round of validation trials, funded by ECNIS project, 4 trials have been performed:
- Assessment of variation in the level of DNA strand breaks coordinated by University of Copenhagen (11 laboratories)
- measurement of Fpg-sensitive sites coordinated by Karolinska Institute (10 laboratories)
- measurement of DNA repair incision activity coordinated by University of Maastricht (10 laboratories)
- developing a robust internal standard material coordinated by University of Leicester (10 laboratories)

A second round of trials was performed under the ECNIS2 project. The University of Copenhagen undertook the development of a set of ‘reference conditions’. Five parameters were identified that were considered crucial for the variation in the comet assay. These included: 1) the percentage of agarose in which the cells are embedded, 2) the strength of the electrophoretic field, 3) the duration of the alkaline treatment, 4) the duration of the electrophoresis, and 5) settings in different image analysis systems. Samples were distributed to the participating laboratories who analysed these by their own comet assay procedure and the ‘standard protocol’.
The trial included 14 laboratories. It showed a reduction in the interlaboratory variation in oxidatively damaged DNA in MNBCs from healthy humans, although only half of the laboratories successfully adopted the standardized comet assay protocol. The trial showed that 52.8%, 35.0% and 12.3% of the variation in Fpg-sensitive sites was explained by interlaboratory variation, variation between samples and residual variation for the laboratory-specific protocols, respectively. The analysis of Fpg-sensitive sites by the standard protocol showed that the interlaboratory variation, variation between samples and residual variation explained 23.1%, 59.6% and 17.3% of the total variation, respectively. The results from this trial showed that it was possible to reduce the interlaboratory variation in Fpg-sensitive sites in MNBC by use of a standard assay protocol in which a few critical steps are made consistent or standardized between laboratories. In contrast, there was little reduction in the interlaboratory variation in strand breaks in MNBCs in this trial, which was probably because there was a low level of strand breaks in the cell from the healthy human subjects.
An investigation of the intra- and interlaboratory variation in DNA damage from MNBCs was coordinated by Karolinska Institutet. The analysis encompassed measurements of strand breaks (basic comet assay) and Fpg-sensitive sites. Coded samples of MNBCs were distributed to the participating laboratories. Each laboratory received three set of samples (to be analysed on three different days of analysis). The investigation used a Latin square design with samples from three donors. Each sample set consisted of nine samples (three donors and three identical samples per donor per day). This was a balanced design where the interlaboratory variation, intra-day variation, day-to-day variation and sample variation could be distinguished.
The 14 participating laboratories used their own comet assay protocols to measure the level of DNA damage in MNBCs and coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the calibration curve samples, the dose of ionizing radiation, interlaboratory variation, intralaboratory variation and residual variation contributed to 60.9%, 19.4%, 0.1% and 19.5%, respectively, of the total variation. In the MNBC samples, the interlaboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intralaboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of Fpg-sensitive sites in the MNBC samples indicated that the interlaboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intralaboratory (0.2%) and inter-subject (0.3%) variation again contributed less to the overall variation. The most important finding in this trial was that the variation within each laboratory was relatively low. This is reasuring, although one should also note that the intralaboratory variation is similar to the inter-subject variation (Ersson et al. 2013).
A survey of variation in DNA damage in different European countries was led by Maastricht University. The focus was on the detection of DNA strand breaks (basic comet assay) and Fpg-sensitive sites in MNBCs from samples collected in The Netherlands, Sweden, UK, Poland, and Portugal. This created a wide range of samples originating from populations in the north, south, east and west of Europe. Identical samples of these cells were distributed to 14 participating laboratories for analysis of DNA damage. The blood samples were obtained from young, healthy women.
In keeping with all the previous ECVAG trials, there was substantial variation in the levels of DNA damage when they were expressed in the primary comet assay values, whereas the transformation of the values to lesions/106 bp by the calibration curve sampels reduced interlaboratory variation. The CoV between laboratories decreased for both DNA strand breaks (68% to 26%) and Fpg-sensitive sites (57% to 12%) by calibration of the primary comet assay values with reference standard samples. The levels of DNA strand breaks in the samples from The Netherlands and Poland (0.56-0.61 lesions/106 bp) were significantly higher compared with the three other countries (0.41-0.45 lesions/106 bp). In contrast, there was no difference between the levels of Fpg-sensitive sites in MNBCs from healthy donors in the five different countries (0.41-0.52 lesion/106 bp). The results thus indicate that the level of Fpg-sensitive sites in MNBCs does not differ substantially between healthy humans from different countries in Europe, whereas the level of DNA strand breaks might differ or that this particular endpoint is more sensitive to variations in isolation of MNBCs between laboratories. Continuation of work on the development of robust internal standard materials for the comet assay, to the point that they can be considered for use in future interlaboratory studies, was led by the University of Leicester. To date, the work has demonstrated that using BrdU-substituted reference cells as true internal standards leads to substantial reductions in the CoV for intra- and inter-experimental measures of damage formation and repair. However, at present, the internal standard material is still inappropriate for wide-scale use and further development is needed. Conditions need to be established for the consistent and dependable preparation of the BrdU-substituted internal standard materials, and to permit the long-term storage of internal standard materials.
At present it is not possible to promote a standard comet assay protocol that will be adopted by all laboratories. In addition, it is very difficult to convince researchers to report their comet assay results in other than the primary comet assay values that they have always used. Consequently the literature contain results expressed as tail moment, tail length, %DNA in tail and visual score. The most important recommendation from ECVAG is to use the same comet assay unit. The transformation of primary comet assay values to lesions/106 by calibration curves is the preferred way forward because it bypasses the discussion of which primary comet assay endpoint is correct and the values are expressed in a unit (lesions per unaltered basepair) that is easy to understand for researchers who are not familiar with the comet assay.

ECNIS Repository
ECNIS Repository (http://ecnis.openrepository.com) is an electronic platform for storing and providing access to digital documents dealing with the matters of environmental hazards for cancer. It is an Open Access service. It enables reading, copying and printing full texts of the publications. Ensures efficient propagation of the scientific research results. The authors may deposite their works in the Repository through the editor (in the Scientific Library of the Nofer Institute of Occupational Medicine - NIOM) or directly on-line. The protocol OAI-PMH (Open Archives Initiative Protocol for Metadata Harvesting) enables searching Repository contents by Google or Yahoo! search engines. The author submits his/her work in the form of a pre- or postprint. Before the article is placed in the Repository, the publishers’ policy on free accessibility of the publication is verified in the SHERPA data base (http://www.sherpa.ac.uk/romeo/). DSpace software has been used to develop the Repository. The system is hosted by BioMedCentral. The ECNIS Repository is registered in the Directory of Open Access Repositories (OpenDOAR), Registry of Open Access Repositories (ROAR) and in the DRIVER Project - a shared platform for document repositories of the European Research Area.

Stages of placing articles in the ECNIS Repository:
1. Registration in the Repository (MyDSpace)
2. Depositing of publication
• Input of publication metadata
• Attach full text (pdf)
• Accept licence
• Provide access
3. Retrieval of publications
• Search – simple and advanced search after typing specific data (e.g. author or keywords),
• Browse – browsing the content of the data base by indices (collection name, publication title, author(s), keywords, publication dates).
4. Internet accessibility
CNRI Handle System (Corporation for National Research Initiatives Handle System). Each work is assigned an unique ID. This enables retrieval of the publication on the Internet.

Statistical data (24 July 2013)
Number of records (ECNIS - Environmental Cancer Risk, Nutrition and Individual Susceptibility and Article with annotation) - 524.
Articles 295
Articles with annotation 184
Chapters 32
Reports 7
Thesis 6

Additional functions:
- Connotea, Del.ico.us, Digg, LinkedIn, Citeulike, Facebook, Stumble It! social bookmarking
- exporting of bibliogaphic notes to RefWorks, EndNote, RefMan, BibTex
- direct linking to PubMed
- searching by MeSH and Keywords.

ECNIS Repository has so far 2530 visitors with 16415 items seen and 2687 downloads.

Training instruments
ECNIS network has established an education and training program, with three main components (i) training courses, (ii) a web-based training course, and (iii) a fellowship program.

Training course
Two training courses have been held: “Oxidative Stress-Disease, Methods and Concepts”, Karolinska Institutet, Stockholm, Sweden, August 27 –31, 2012, and « Nutrition and Environmental Carcinogens », Nofer Institute of Occupational Medicine, Lodz, 8-9 May 2013.

A course entitled “Oxidative Stress-Disease, Methods and Concepts”, was organized at Karolinska Institutet, Stockholm, Sweden, in August 27 –31, 2012. The course was based on oxidative stress in health, disease, exposure, food, vitamins, chronic diseases, nano structures and much more. It was a lot of "cross-over" between the participants subjects that ranged from chronic disease in general to how, and when, or not to, take vitamin supplements, after heavy physical activity. We showed how to do analyses of oxidative lesions by chemical (8-oxo-dG) and biological (comet assay) methods. There were 25 participants in the course.

A second course, entitled “NUTRITION AND ENVIRONMENTAL CARCINOGENS”, was organized at the Nofer Institute of Occupational Medicine in May, 2013. Lectures, with faculty from several ECNIS participating institutions, covered topics related to epidemiology and toxicology of environmental carcinogenesis with particular focus on nutritional factors, oxidative stress- oxidatively modified DNA lesions, and omics as well as gender differences in health risk. Practical training for course participants was also held with novel techniques for determination of the heavy metals (ICP-MS technique) or biomonitoring of dioxins in human milk presented. The course was 46 participants (30 ECNIS2 members) - students from ECNIS institutions and collaborating institutions.

Web-based training course with new modules
New modules have been prepared for a comprehensive course in environmental epidemiology and biostatistics, based on the Summer School held by the International Prevention Research Institute. The modules are being finalized and uploaded on the already existing web-based training course that was developed during ECNIS (2006-2010) and the first reporting period of ECNIS2. The current version of the web-based course is on (http://www.episat.org/episat/courses/)

Web-based course
A web-based course has been established within the framework of the ECNIS project entitled ‘Molecular epidemiology: principles and applications’. This course is targeted to researchers who take part in human studies, but lack basic education in epidemiology or molecular epidemiology.

Fellowship program
ECNIS2 fellowships were awarded to post-doctoral students and junior scientists from laboratory within or without the Consortium to visit a laboratory in the Consortium for an indicative duration of 1-2 months, to work on a collaborative project related to the objectives of ECNIS2 and to obtain advanced training. In particular two fellows from NIOM, Poland spent their fellowships at: National Research Centre for the Working Environment , Copenhagen, Denmark and at Nanosafety Research Center, Finnish Institute of Occupational Health Helsinki, Finland. The first one investigated the genotoxic potential of carbon-based nanomaterials, by analysing genotoxicity of BAL cells and assessed by comet assay and the other studied genotoxic potential of ZnO.Two other fellows coming from non ECNIS institution i.e. Department of Epidemiology, Instituto di Ricerche Farmacologiche "Mario Negri", Italy were provided training at the Institute for Translational Epidemiology - Mount Sinai, NY under the supervision of ECNIS senior scientist. Both of them had received training in epidemiological methods and first one performed a comprehensive meta-analysis of the relationship between alcohol consumption and primary liver cancer, and the other investigated association between allium vegetables intake and risk of head and neck cancer performing pooled analysis of data from the International Head and Neck Cancer Epidemiology Consortium.

ECNIS2 website
In view of the ECNIS2 project's intimate links with the original ECNIS project, and having in mind the great success, in terms of visibility, of the latter's website, (1 050 000 visitors and 3 511 000 hits as on May 31, 2013) it was decided that the website of ECNIS2 will form part of the ECNIS website (www.ecnis.org) in the form of a link appearing on the site's main page. The website content for the ECNIS project (i.e. Management, ECNIS Policy, Education & Training, Partner) were grouped under The ECNIS Project. The new link was created with an ECNIS2 sub-page containing information on the project's aims and activities. Other aspects of ECNIS2 dissemination, such as news and events, the ECNIS repository, partner-only area etc. make use of the already existing corresponding areas of the ECNIS website. Other activities, including the announcement on the website of news and events of relevance to the project and the updating of the ECNIS Repository, are in operation.

In total, 1 050 000 visitors have been counted on 31 May 2013 by the web server. . 636 000 visitors have been counted at the end of ECNSI NoE on 31 October 2010; it means that in the course of ECNIS2 there were 414 000 visits.

The ECNIS webpage counted 3 511 000 hits (a hit is counted each time someone views a file while a visit happens when someone visits website). There were 2 710 000 hits at the end of ECNIS NoE project in 2010. It means that in the course of ECNIS2 there were 801 000 hits.

The ECNIS help desk on ethics intends to operate for researchers facing questions related to transnational research only in the specific research domain of food, environment and cancer and implying the use of human biomarkers. The rationale of the helpdesk on ethics was developed and explained in the first year and during the second ECNIS2 year adapted and restructured. The helpdesk on ethics and data protection now gives an overview of ethical and privacy aspects that have to be handled within the context of human biomarker studies in environmental health. It lists the tasks necessary to comply with legal obligations and provides practical advice with a check list on what to do when starting up a study. It contains many frequently asked questions (FAQs) issuing different relevant topics, in a legal as well as practical way.

During the two years of ECNIS2, the ethics and privacy helpdesk was consulted 754 times. ECNIS team has elaborated on several issues that seemed to emerge many questions and difficulties, as experienced during informal contacts and during ‘questions and answers’ moments at lectures at seminars and congresses. Two major topics were handled: the focus on communiation and facilitation of secondary use of personal data/biological samples.
Good communication at all stages of a research project from the start till the dissemination of results at the individual and the collective level is of overriding importance. Experiences show that communication aspects of research are still very much overlooked or underestimated, due to many reasons, when it does not relate to communication towards science. Three practical missives are presented: i) train the recruiter; ii) involve communication experts; iii) introduce participatory approaches.
Secondly, information is spread on how to proceed in case of further use of data and/or samples. For current regulations on secondary use, the EU Data Protection Directive (Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the protection of individuals with regard to the processing of personal data on the free movement of such data), the Additional Protocol to the Convention on Human Rights and Biomedicine, concerning Biomedical Research and the recommendation Rec (2006)4 regarding secondary use of data/samples are the main references.

Several exemptions exists and circumstances in which re-using data or samples is allowable are defined. It has to be kept in mind that different situations may call for different praxis. The appropriate balance between protection of the study participant and research progress may also depend on the nature of the data and samples. In addition, human biomarker research in environmental health may entail less or no risk for the study participant as compared to for example clinical trials. To balance the interests at stake, each information and consent form should be carefully assessed by both researchers and ethics committees. Wishes of donors that their samples should not be used for some specific study should always be respected.

Gender Help Desk
Gender Help Desk have been incorporated onto the ECNIS webpage in month 6th of ECNIS2 project. Besides Guideline for Mentor Programme and Guideline for Leadership Programme, which were elaborated within Gender Issues Panel of ECNIS it also contains a link to Nature journal special issue “WOMEN IN SCIENCE”
The website statistics show that guidelines provided have drawn substantial attention of ECNIS website visitors:
Guideline for Mentor – 580 visitors
Guideline for Leadership – 709 visitors

Following development of draft of the Organization formula for the transforming existing ECNIS structure into ECRECEF further consultations with the ECNIS members, members of European Institute of Environmental Cancer and stakeholders took place during the second ECNIS2 year. The European Institute of Environmental Cancer has a form of centrally coordinated ten Polish research institutions, which has been included on a Polish Road Map for Research Infrastructure and to be granted by Polish Ministry of Science. Altogether as many as 120 researchers contribute to the EIEC’s work, including 30 senior staff, and 34 experienced scientists (postdocs). The network includes specialist in toxicology, chemistry, molecular biology, biochemistry, epidemiology (molecular cancer epidemiology), biostatisticians, bioinformatics and oncologists.
In particular the subject of further cooperation was thoroughly discussed during the Final ECNIS Meeting (May, 2013) (joint ECNIS-EIEC meeting), where integration after ECNIS cessation, and plans for future activities were considered. In view of lack of funding for support of ECRECEF from EC after ECNIS2 completion the only potential way for durable integration and support for some key elements of ECNIS2 is through Polish national funding. Both ECNIS and EIEC members agreed to continue ECNIS ideas under EIEC umbrella. All the planned activities will be performed with close collaboration with ECNIS Network of Excellence. ECNIS will retain name as a well know “trademark” with its key elements within EIEC structure.

This is planned to continue links between ECNIS partners, with cooperation with Polish researchers under EIEC consortium. Works related to validation and standardization will be continued within EIEC. The intention after the tenure of the NoE is to maintain a stock of core antibodies and chemicals for distribution to interested parties, to synthesize further antigens and chemicals, and to maintain the virtual facility of analytical laboratories. Within ECNIS a special effort has been made to facilitate the optimal utilization of already existing knowledge and data, through the construction of the "Molecular Epidemiology and Cancer" (MEC) database, which brings together the data of a number of population studies, thus providing increased statistical power to evaluate the influence of various environmental or other factors on cancer risk. The incorporation of omics data emerging from population-based environmental health studies, which are currently relatively limited but are anticipated to increase substantially in the coming few years, along with the development of analysis tools targeted at omics data, would give MEC a unique character and help establish it as valuable resource in environmental carcinogenesis research. During joint meeting of ECNIS2 General Assembly and EIEC a number of new contacts has been established for incorporation of new data to MEC. Within EIEC, further development of ECNIS repository will take place in order to create a major world electronic platform where scientists, stakeholders and layman can find all relevant information concerning environmental cancer risks. This is envisaged to develop cooperation in the coming years within EIEC to continue integrative projects. Three areas were identified as of key importance in future research strategy, namely a) the exploitation in population studies of the power of new technologies of high-density biological analysis (omics) for the development of new biomarkers of exposure and biological effects, b) the utilization, in relation to the above, of existing cohorts/biobanks and, importantly, the construction of new cohorts/biobanks which would be specifically designed to address questions of relevance to the identification of the environmental determinants of cancer at a continent-wide scale, c) the promotion, at a theoretical as well as a practical level, of improved exposure assessment.

Overall conclusions - Impact of the ECNIS2 project
ECNIS2 was established following the completion of the highly successful ECNIS Network of Excellence project with the specific objective of laying the foundations for the establishment of a structure, in the form of a virtual European institute (ECRECEF) to support the long-term coordination of European research in the area of molecular epidemiology of environmental cancer. During its course, ECNIS2 identified a course towards this end which is viable and realistic, in the form of the «European Institute for Research on Environmental Cancer» as part of the Polish Road Map for Research Infrastructure. This is an important achievement given the objective difficulties related to the funding possibilities available for multinational research efforts in Europe. In parallel to the fulfilment of this objective, work conducted under ECNIS2 built upon the achievements of the ECNIS NoE, further increasing the «ECNIS added value» in a number of areas which are of major importance in furthering the coordination of cancer research in Europe.

Potential Impact:
Dissemination measures and plans
A dissemination taskforce has been created, consisting of 9 partners (NHRF - coordinator, NIOM, KI, ULEIC, NIEH, CollegiumMed, KULeuven, Leocordia AB and IPRI). A communication office was also created which, while based at NHRF, has a virtual structure to include, in addition to NHRF, two other partners, NIOM and Leocordia AB in the day-to-day dissemination activities of the network. A communication officer was appointed (M. Botsivali, Athens) who presented the plans on the project's dissemination activities at the project's first general meeting.
In view of the ECNIS2 project's intimate links with the original ECNIS project, and having in mind the great success, in terms of visibility, of the latter's website, it was decided that the website of ECNIS2 should form part of the ECNIS website (www.ecnis.org) in the form of a link appearing on the site's main page. The link opens an ECNIS2 sub-page which contains information on the project's aims and activities. Other aspects of ECNIS2 dissemination, such as news and events, the ECNIS repository, partner-only area etc. make use of the already existing corresponding areas of the ECNIS website.
An electronic brochure have been prepared and circulated to a large number of organisations, networks and individuals.

The project’s activities have been publicised and disseminated using the following means:
- In view of its successful previous operation and high visibility, the website of the original ECNIS project (http://www.ecnis.org) was extended to disseminate information about ECNIS2. During the 2 years of operation of the ECNIS2 project the site received 1 050 000 visitors.
- Production of an electronic brochure and two newsletters, which were circulated to hundreds of recipients and are also available for downloading from the project’s website.
- A large number of research papers related to the objectives of ECNIS and acknowledging the project have been published in international scientific journals during the 2 years of the project’s operation (see Table A1).
- Presentation of the project and its activities at a number of international and national conferences targeting different audiences, including the research community, experts and authorities active in human biomonitoring, exposure assessment, chemicals risk assessment etc (see Table A2). In addition the project consortium maintained contact with other European projects in the area of environmental health and a presentation was given in the context of a conference organised by one of these projects (COPHES, final meeting conference). A database of “Research teams, networks and projects on Molecular epidemiology of carcinogenesis” was constructed and used as a tool for promoting contacts and disseminating ECNIS activities.
- Future dissemination activities will depend on the success or otherwise of the establishment of EIEC/ECRECEF as described in other parts of the Report.

List of Websites:

www.ecnis.org

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Contacto

Kamila Szczesniak, (Head of Department)
Tel.: +48 42 6314 688
Fax: +48 42 6314 839
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