• Annex 1. State of validation of biomarkers.

      The Nofer Institute of Occupational Medicine, 2007
    • Appendix 1. DNA adducts.

      The Nofer Institute of Occupational Medicine, 2007
    • Appendix 2. Heterocyclic aromatic amines.

      The Nofer Institute of Occupational Medicine, 2007
    • Appendix 3. 1-Hydroxypyrene.

      The Nofer Institute of Occupational Medicine, 2007
    • Appendix 4. Oxidative damage to DNA.

      The Nofer Institute of Occupational Medicine, 2007
    • Bioactive components in foods.

      Manson, Margaret M.; Linseisen, Jakob; Rohrmann, Sabine; Sotiroudis, Theodore G.; Kyrtopoulos, Soterios A.; Hayes, John D.; Kelleher, Michael O.; Eggleston, Ian M.; de Kok, Theo M.; van Breda, Simone G.; et al. (The Nofer Institute of Occupational Medicine, 2007-04)
    • Biomarkers of carcinogen exposure and early effects.

      The Nofer Institute of Occupational Medicine, 2006
      The purpose of this review is to summarise the current situation regarding the types and uses of biomarkers of exposure and effect for the main classes of food-derived genotoxic carcinogens, and to consider some aspects of the intercomparison between these biomarkers. The biomarkers of exposure and early effects of carcinogens that have been most extensively developed are those for genotoxic agents and for compounds that generate hydroxyl radicals and other reactive radical species, and it is on these that this review is mostly concentrated.
    • Challenges from new technologies and new biomarkers.

      Vineis, Paolo; Vermeulen, Roel; Geneletti, Sara; Minelli, Cosetta; Taioli, Emanuela; Thompson, John; Stromberg, Ulf; Kirsch-Volders, Micheline; Raluca, Mateuca; Matullo, Giuseppe (The Nofer Institute of Occupational Medicine, 2007)
    • Comments and suggestions for future work.

      The Nofer Institute of Occupational Medicine, 2007
    • Conclusions.

      Farmer, Peter (The Nofer Institute of Occupational Medicine, 2007)
    • Correlations among biomarkers.

      Gyorffy, Erika; Anna, Livia; Rudnai, Peter; Kovacs, Katalin; Schocket, Bernadette (The Nofer Institute of Occupational Medicine, 2006)
      An extensive literature survey on correlation of biomarkers resulted in the following preliminary conclusions: • No significant correlation between individual pairs of DNA adducts was found in a number of the 32P-postlabelling and immunoassay studies, indicating limited overlapping of the substrate specificity of the different DNA adduct methods. • Attempts to show correlations between a group of related DNA adduct structures and a chemically specific single DNA adduct structure by using the same type of methodology gave controversial results. These suggest the co-existence of both closely linked and independent metabolic activation pathways for complex mixtures of xenobiotics, and may also reflect differences in the kinetics of DNA adduct formation and elimination. • A larger number of studies revealed a positive correlation between DNA adduct levels in target and surrogate tissues than did not find a correlation. Correlation may depend on exposure dose and the metabolic capacity of the corresponding tissues. • In the majority of the studies there was a positive correlation between DNA adduct levels in tumour and normal tissues, suggesting similarities in the xenobiotic activation/elimination processes of the tumour and normal tissues. However, the levels of DNA adducts found suggested organ specificity. • There was a positive correlation for different urinary metabolites and urinary mutagenicity in most of the studies. • No correlation between DNA adducts and urinary polycyclic aromatic hydrocarbon metabolites was generally found, but stratification by genotype suggested that correlation might be present. • Correlation was more probable between structurally specified protein adducts and urinary polycyclic aromatic hydrocarbon metabolite 1-hydroxypyrene than with less specific xenobiotic-protein structures. • Stratification of the study population for confounding factors, such as smoking status, may reveal hidden correlations. • Cytogenetic biomarker studies also gave complex results. Examples of both positive correlation and lack of correlation with exposure markers were found. Molecular epidemiological studies of cancer that include exposure biomarkers have greatly increased in number in recent years, the rationale being to measure the biologically relevant aspect(s) of exposure. However, the use of biomarkers to measure exposure is not a panacea. Most biomarker-based studies, of both prospective and retrospective design, rely on a single biological sample. Most exposures in cancer epidemiology are time-related variables, and both carcinogenesis models and empirical evidence strongly point towards the importance of induction and latency periods in cancer onset, the need to separate the role of duration and intensity of exposure, and the decrease in effect after cessation of exposure. While most biomarkers measure recent exposure, it is possible to apply them in the measurement of temporal changes, e.g. by collecting repeated samples from subjects enrolled in prospective studies or from a sample of the original cohort. Another important parameter is the in vivo lifetime of the biomarker after it has been generated by a carcinogen exposure. Biomarker-based epidemiological results are subject to the methodological problems affecting all types of observational studies, namely bias and confounding. A further problem in the measurement of exposure biomarkers in retrospective studies is their possible dependence on the disease process. With the increasing use of biomarkers of dose and effect of carcinogens and the possible input of these data into the regulatory area, it is essential that the methodology be standardised and wherever possible internationally accepted protocols be established for this. The need to validate exposure biomarkers before their application in population-based studies arises from the variability in biomarker-based measurements of exposure, which can be due to interindividual sources (e.g. differences between exposed and unexposed individuals, usually the component of variability of primary interest), intraindividual sources (e.g. variability in hormonal levels) and observer sources, including measurement error. This is the domain of so-called transitional studies, which aim to characterise the biomarker itself rather than the underlying biological phenomenon. This is the area in which most work is needed in the near future: efforts such as the ECNIS Network of Excellence will be instrumental in providing a logical framework for the development and validation of biomarkers of exposure with the ultimate goal of their application in molecular epidemiological studies. • There was a positive correlation for different urinary metabolites and urinary mutagenicity in most of the studies. • No correlation between DNA adducts and urinary polycyclic aromatic hydrocarbon metabolites was generally found, but stratification by genotype suggested that correlation might be present. • Correlation was more probable between structurally specified protein adducts and urinary polycyclic aromatic hydrocarbon metabolite 1-hydroxypyrene than with less specific xenobiotic-protein structures. • Stratification of the study population for confounding factors, such as smoking status, may reveal hidden correlations. • Cytogenetic biomarker studies also gave complex results. Examples of both positive correlation and lack of correlation with exposure markers were found. Molecular epidemiological studies of cancer that include exposure biomarkers have greatly increased in number in recent years, the rationale being to measure the biologically relevant aspect(s) of exposure. However, the use of biomarkers to measure exposure is not a panacea. Most biomarker-based studies, of both prospective and retrospective design, rely on a single biological sample. Most exposures in cancer epidemiology are time-related variables, and both carcinogenesis models and empirical evidence strongly point towards the importance of induction and latency periods in cancer onset, the need to separate the role of duration and intensity of exposure, and the decrease in effect after cessation of exposure. While most biomarkers measure recent exposure, it is possible to apply them in the measurement of temporal changes, e.g. by collecting repeated samples from subjects enrolled in prospective studies or from a sample of the original cohort. Another important parameter is the in vivo lifetime of the biomarker after it has been generated by a carcinogen exposure. Biomarker-based epidemiological results are subject to the methodological problems affecting all types of observational studies, namely bias and confounding. A further problem in the measurement of exposure biomarkers in retrospective studies is their possible dependence on the disease process. With the increasing use of biomarkers of dose and effect of carcinogens and the possible input of these data into the regulatory area, it is essential that the methodology be standardised and wherever possible internationally accepted protocols be established for this. The need to validate exposure biomarkers before their application in population-based studies arises from the variability in biomarker-based measurements of exposure, which can be due to interindividual sources (e.g. differences between exposed and unexposed individuals, usually the component of variability of primary interest), intraindividual sources (e.g. variability in hormonal levels) and observer sources, including measurement error. This is the domain of so-called transitional studies, which aim to characterise the biomarker itself rather than the underlying biological phenomenon. This is the area in which most work is needed in the near future: efforts such as the ECNIS Network of Excellence will be instrumental in providing a logical framework for the development and validation of biomarkers of exposure with the ultimate goal of their application in molecular epidemiological studies.
    • Dietary vitamins, polyphenols, selenium and probiotics: biomarkers of exposure and mechanism of anticarcinogenic action.

      The Nofer Institute of Occupational Medicine, 2007-04
      The major aim of this review was to summarize the state-of-the-art in use of biomarkers for some anticarcinogenic food components and to identify knowledge gaps, especially those of relevance for the partners of the NoE ECNIS and its contacts. Since this is a vast area, only certain selected topics, as outlined below, are considered in detail. The important links found between use of a substance as a biomarker and its mechanisms of action led to a further aim, that of reviewing the mechanisms of action of some of the most promising anticarcinogenic compounds.
    • Epidemiological concepts of validation of biomarkers for the identification/quantification of environmental carcinogenic exposures.

      The Nofer Institute of Occupational Medicine, 2007
      The present report has been prepared by a group of European scientists in the context of the EU-funded Network of Exellence ECNIS whose goal is to provide effective biomarkers for the study of the relationships between environmental toxicants, dietary habits and cancer. The use of biomarkers in cancer epidemiology has a rather long history (the wording "molecular epidemiology" was originally proposed by Perera and Weinstein in 1982) and great successes have been achieved, like the investigation of the predictive ability of chromosome aberrations, the relationship between aromatic amines in tobacco, the NAT2 genotype and bladder cancer, or the mechanisms by which benzene induces leukaemia. However, many biomarkers are introduced into research without proper validation, and this hampers successful research, introducing bias or simply blurring existing associations. In addition, new and complex issues are emerging through the introduction of high-throughput technologies, like the expected large number of false positives, or the complex interplay between environmental exposures, intermediate markers, confounders and disease. Causality assessment has really become a challenge. For these reasons, we think it can be useful to summarize the main criteria for biomarker validation (Chapter 1); to offer some insights into new technical and statistical developments for the management and interpretation of biomarker data (Chapter 2); and to offer some examples of existing information (usually sparse) on biomarker validation (Chapter 3).
    • The epidemiological theory: principles of biomarker validation.

      Vineis, Paolo; Gallo, Valentina (The Nofer Institute of Occupational Medicine, 2007)
    • Ethics and data protection in human biomarker studies.

      Casteleyn, Ludwine; Dumez, Birgit; Jamers, An; Van Damme, Karel (The Nofer Institute of Occupational Medicine, 2010)
      Human biomarker studies in environmental health are essential tools to study the relationship between health and environment. The development of relevant research potential and the setup of bio-monitoring surveys should ultimately lead to a better understanding and prevention of environmentally induced adverse health effects. In this volume we review ethics and data protection in environmental health studies using human biomarkers. The question is raised whether study participants are adequately and equally protected throughout Europe and whether at the same time progress in environmental health related studies is safeguarded. The collection of human samples and data is subject to regulations and rules of different kinds, from deontological codes to data protection laws, from the local to the international level. Within an EU context, the probably most important international references in this respect are the Data Protection Directive (95/46/EC), the Oviedo Convention, more in particular its Additional Protocol concerning biomedical research and Rec(2006)4 on research on biological material of human origin. A critical analysis of research experiences in various EU countries shows that difficulties, ambiguities or even inconsistencies exist in the way ethical and juridical challenges are framed and being dealt with within and across countries. The diverse implementations of EU regulations or international guidelines in domestic law may in particular hinder transnational research and bring about inequalities in the level of protection. Overall, in research on biological material of human origin emphasis is primarily on decisional autonomy and protection of the individual's rights whilst the collective need to protect health as a public asset is relatively less valued. This is particularly striking when personal data or samples collected for a specific research purpose could be of value in a new study. This secondary use often remains very complex or almost impossible, although there are strong and recognized arguments for facilitation of such further use in the context of environmental health research where public interest comes to the forefront and risks are minimal, on the condition that adequate collective protection and control against improper use of samples or data can be installed, and breach of confidentiality or any use of data which would be not in line with the subject's moral stakes can be excluded. An increasing demand exists for adapted communication strategies at all stages of a study, not only at the individual level, but also at the collective level, and including the time of translation of results in preventive actions and policy making. The vital role of communication is obvious. Each communicative act may affect trust in the study at hand and in science in general. Expertise in the field of social sciences is therefore demanded. Even though many improvements regarding the legal and ethical challenges in human studies have already been implemented in the EU in the last years, these issues are not well known among the actors involved and efforts should be devoted to better education and dissemination of information and enhanced transparency targeting researchers but also the general public, the media and the policymakers. Research ethics committees play a pivotal role in assessing to what extent decision making processes fit with both individual and societal interest. Respect for human dignity and equality of moral status of all individuals, social justice, solidarity and democratic participation may thereby be appealing reflections of European values and useful complements to the four conventional bioethical principles (autonomy, beneficence, non-maleficence, and justice) that are widely applied for evaluating policies, programs or activities that may entail risk to human health. To achieve more consistency in the research conditions nationally and internationally an ethics committee at the level of the ‘European research area' could be envisaged, mainly focusing on transnational research. Moreover, the analysis and evaluation of different cases in different situations would build-up an extensive knowledge and experience that may serve as an inspiring starting point for a well informed societal debate. This way, gradually, more consistency in the handling of study proposals and increased transparency in decision making might be reached, lifting the daily practices and the protection of both individual and community interest to a higher level of meeting up with ethical concerns in transnational research. Researchers have the duty to support the translation of research results in preventive actions at individual and at collective level whenever relevant. Participatory processes will facilitate the inclusion of arguments from the societal perspective and increase trust and mutual understanding between all parties involved, add to the legitimacy of the final outcome and the public support for the policy decision-making process, and implement democracy.
    • Introduction.

      Akesson, Bjorn (The Nofer Institute of Occupational Medicine, 2007-04)
    • Introduction.

      Kyrtopoulos, Soterios (The Nofer Institute of Occupational Medicine, 2007)
    • Oxidative damage.

      Olinski, Ryszard; Loft, Steffen; Nair, Urmila; Nair, Jagadeesan (The Nofer Institute of Occupational Medicine, 2006)
      Free radical attack upon DNA generates, among other changes, modified bases. The amount of 8-oxo-7,8-dihydroguanine, one of the most critical lesions in human lymphocytes, was found to be 1.2/106. Gua by a mass spectrometric approach. Spurious oxidation occurring during sample preparation is the major problem in measurement of oxidised bases in DNA. Exposure to ambient air particles and benzene is associated with higher levels of 8-oxo-7,8-dihydroguanine and other oxidatively modified bases. Studies of the effects of antioxidant supplements and antioxidant-rich food have given contradictory results. New techniques allowing the simultaneous determination of 8-oxo-7,8-dihydro-2’-deoxyguanosine, 8-oxo-7,8-dihydroguanine and 5-methylhydroxy-uracil in urine samples have revealed that the combined amount of these compounds excreted into urine of healthy human subjects corresponds to about 2800 oxidative modifications of guanine per cell per day. Cohort studies are required to assess whether an increased level of biomarkers of oxidative DNA damage is associated with an increased risk of developing cancer. Lipid peroxidation resulting from chronic inflammatory processes can result in production of excess reactive oxygen and reactive nitrogen species and DNA-reactive aldehydes, including 4-hydroxy-2-nonenal, malonaldehyde, acrolein, and crotonaldehyde. Alkylation of DNA bases by these reactive electrophiles is thought to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced lipid peroxidation. The specific and sensitive methods developed for the detection of these potentially mutagenic adducts in human tissues and in urine have been applied to the analysis of DNA from a variety of normal and disease-related samples.
    • Phosphotriesters: Long lifetime biomarkers.

      Le Pla, Rachel C.; Jones, G. Don D.; University of Leicester, UK (The Nofer Institute of Occupational Medicine, 2006)
      In addition to reacting with DNA bases, many genotoxic agents react with the oxygen of the internucleotide phosphodiester linkages to form phosphotriester adducts. Several studies have shown that phosphotriester lesions have a half-life that exceeds that of any other DNA alkylation product. The relative abundance of phosphotriesters depends upon the alkylating character of the agent. The role of phosphotriester adducts in carcinogenesis is unknown. Mutation resulting from phosphotriester formation has not been fully studied, but such adducts may influence cellular function by altering the binding of proteins to DNA.
    • Sensitivity and specificity of techniques for the identification of biomarkers.

      Farmer, Peter B.; Phillips, David; Moller, Lennart; Singh, Raj; van Schooten, Frederik-Jan; Godschalk, Roger; Mateuca, Raluca; Kirsch-Volders, Micheline (The Nofer Institute of Occupational Medicine, 2006)
      The approaches described for the determination of DNA adducts mostly reach sensitivity limits in the range of 1 adduct/107–109 nucleotides and are thus applicable for studies of environmental exposure to genotoxins. Availability of DNA may be a limiting feature and the kind of sample required will determine how a method is used in human biomonitoring studies. Protein adducts are generally stable and are therefore very suitable for use as biomarkers of exposure. The sensitivity of the mass spectral approaches for these assays has been shown to be sufficient for detection of adducts at low pmol/g protein levels. However, there is a lack of a screening method for characterisation of exposures to complex mixtures and no really high throughput analytical methods,preferable for large-scale human molecular epidemiological studies, exist. Structural chromosomal aberrations in peripheral blood lymphocytes have been widely used in occupational and environmental settings as a biomarker of early effects of geno-toxic carcinogens. The predictivity of chromosomal aberrations as a biomarker for increased cancer risk may depend on the composition of the cohorts included in the study. The use of micronuclei as a measure of chromosomal damage has become a standard assay in both genetic toxicology testing and human biomonitoring studies. Analysis of re-sults from European cohorts indicated that subjects with cancer had a significant increase in frequency of micronuclei.