Cluster of research into endocrine disruption in Europe

Coordinating European Environmental and Human Health Research into Endocrine Disruption

newsletter

The end of CREDO

With many of the participating projects completed, the CREDO cluster is now nearing the end of its lifetime. For this, the last issue of the newsletter, we have asked partner projects for a summary of their findings, and the response has been overwhelming. We have received enough material to fill more than a double issue. Without a doubt, the cluster has greatly contributed to improving our knowledge about endocrine disruption. This could not have been possible without the enthusiasm of all the labs and workgroups involved and their willingness to collaborate across project boundaries. The last years have shown that we have succeeded in creating a research focus for endocrine disruption in Europe and beyond. This is also the time to look ahead to the future. The founder projects of the cluster were set up to provide an integrated approach to wildlife and human health problems, and the cluster has shown how powerful such an approach can be. The question now in many peoples minds is whether we will have the opportunity to build on this expertise in the forthcoming Seventh Framework Programme. I would like to thank the cluster for all the support and goodwill during these four years. Andreas Kortenkamp Cluster coordinator

COMPRENDO: We got the ball rolling can we call it quits?

The potential for endocrine active chemicals (EACs) to cause serious health problems in humans and wildlife and, in some cases at extremely low doses, raises the very real question as to whether their presence in the environment allows normal development, reproduction and aging of individuals. The costs of future medical treatment of endocrine diseases and disorders that may be caused by exposure to EACs and the costs of removal of these compounds from the relevant environmental media are likely to be very high. Three and a half years of COMParative REsearch into the effects of ENDOcrine disrupters has been completed. The following aims to provide a brief overview on the projects achievements, as well as to highlight the gaps in knowledge in endocrine disruption research and where we tried to fill in the blanks. COMPRENDO was set up to improve the understanding of the impacts of endocrine active compounds on aquatic wildlife and humans. The projects focus was on the investigation of potential effects of androgenic and anti-androgenic compounds (AACs) in a range of diverse test systems covering invertebrate, vertebrate and human-relevant test models. A wide range of organisms were exposed to EACs with similar test designs for a comparative assessment and evaluation on the sensitivity of the model systems and endpoints under investigation. Doses of the test substances administered included environmentally relevant concentrations. Sediments originating from field-sampling in European regions with low and high environmental quality standards (Falandysz et al. 2006) were used in mixture studies to assess the responses of amphibians, crustaceans and molluscan test species for comparable patterns in biological effects induced by toxic and hormonally active chemicals. Baseline endocrinology research was a key activity and was conducted to address gaps in knowledge and to advance our
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In this edition 1 5 6 7 8 10 11 12 14 16 18
COMPRENDO: we got the ball rolling EURISKED: multi organic risk assessment UV filters target thyroid biosynthesis News from EDEN Bone development and homeostasis What have we learned from ENDOMET? EASYRING: non-invasive testing methods EDERA: more predictive risk assessment Genetic markers in testis endocrine disruption Biomimetic and biological sensor systems Environment and health research under FP7

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understanding of the possibilities for applying new animal models to predict for possible human health effects. This work also set out to investigate new test species for chemical testing. Residue analyses of EACs in foodstuffs, for all participating EU countries of the COMPRENDO consortium, was reported recently in the July 2006 issue of the CREDO newsletter. Ladykillers in a mans world? Sexual differentiation and development are endpoints reported to be especially susceptible to alterations from exposure to endocrine disrupting chemicals. COMPRENDO investigated how susceptible sexual determination, differentiation and development were to EACs in a range of different test species. Was it possible to produce animals of an indeterminate gender (with sexual organs that were neither explicitly male nor explicitly female) on exposure to AACs? We investigated this with more than 30 chronic laboratory-based exposure experiments. We carried out numerous assays for enzyme activity and steroid determinations and analyses of EAC residues in human tissues and cell lines, rats, amphibians, fish, echinoderms, crustaceans and molluscs. The results of these experiments demonstrated that all test systems were affected for the test compounds TBT, TPT, linuron, diuron, fenarimol, vinclozolin, p,p-DDE. Androgen mimicking substances, such as organotins and some pesticides, resulted in the induction of virilization, reduced reproductive performance, accelerated sexual maturity, reduced larval development, reduced skeletal density, abnormal sex ratios, changes in sex steroid titres and enzyme activities and various effects on the reproductive organs under histopathological investigation. Conversely, feminisation effects, including reduced egg diameters, and a lack of spermatids and spermatophores in males were observed under anti-androgen exposure. In most of the experiments, the observed effect concentrations were in the environmentally relevant range. COMPRENDO's contribution to the OECD testing toolbox Our studies suggested that the reproductive and developmental effects of the test compounds were often comparable between invertebrate groups and with the vertebrates studied. Indeed, the data generated suggested that some of our invertebrate test models might provide powerful surrogates for vertebrate species for the identification of endocrine hazards in Tier 1 testing. Two of the organisms, specifically the calanoid copepods (Acartia tonsa) and the prosobranch snail Potamopyrgus antipodarum, used in COMPRENDO are being considered by the OECD as suitable test organisms for the OECD guideline programme on testing of chemicals (including for the assessment of endocrine disruption). The draft OECD method on calanoid copepods (Acartia tonsa) has been prepared and a ring-test for validation is currently running (OECD 2005; Kusk & Wollenberger 2005). The prosobranch
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snail Potamopyrgus antipodarum has been agreed by the OECD Ad hoc Expert group on Invertebrate Testing as the preferred molluscan species for the development of a test guideline. As a first step toward validation and generation of a draft guideline with P. antipodarum, COMPRENDO prepared a standard operating procedure (SOP) for the culture of this species, and a SOP for a 56 day reproduction test (water and sediment; Schmitt et al. 2006; the document is available on request from the co-ordinator). We await to see which of the European governments will support and drive the implementation of the snail test at the OECD. Of mice and men Animal models are used in virtually every field of biomedical research. Almost all medical knowledge and treatment, especially that in the last century, has involved work with laboratory animals. While the past 50 years has seen, remarkable advances in medicine, new models for endocrine diseases are needed and the COMPRENDO work sought to develop some of these. The significance and ability to extrapolate results obtained from animal models to human health is an area that needs far greater research. A good knowledge of comparative anatomy, physiology and function is essential for the animal model of interest. Within COMPRENDO we gathered extensive data sets of this nature for several test species. Animal models may be found throughout the animal kingdom, and knowledge about human physiology has been achieved in species that seem far removed from the human in terms of evolutionary development. A good example is the importance of the fruit fly for the original studies of basic genetics. The EU chemical strategy (REACH) proposes that industrial chemicals are managed more effectively in order to protect human health and the environment. Our results support the need for EU legislation to drive the precautionary principle for substances with endocrine-disrupting properties. Currently the number of vertebrate animal experiments proposed under REACH is unacceptably high, emphasising further the need for suitable non-vertebrate tests (as developed under COMPRENDO). Comparative Endocrinology COMPRENDO has also made efforts to elucidate the similarities and differences in steroid metabolism in a wide range of test species and animal phyla (Janer et al 2005a-d, Janer et al. 2006, Lavado et al. 2006). It is important to note that as well as the striking similarities between vertebrate and invertebrate responses to AACs, there are also differences in the patterns of responses to the different chemicals. For example the effects of AACs on steroidogenesis differ between fish and molluscs and between molluscs and crustaceans.

COMPRENDO

By studying steroidogenesis in these organisms it became obvious that these differences in responses to some of the AACs were in part due to differences in the steroidogenic pathways. Notwithstanding this, these differences in steroidogenic pathways may also occur between 'closely related' vertebrate phyla (e.g. fish and amphibians) as well as between vertebrates and invertebrates. There are still considerable gaps in our knowledge on the basic endocrinology of most animals, including even for some of the most widely used vertebrate animal models and COMPRENDO provided important contributions to the nature of steroidogenesis in a range of vertebrate phyla. The work under COMPRENDO has identified the existence of some highly conservative configured pathways in the endocrine system. In view of the EU-wide ban of vertebrate animal testing in the cosmetics industry that will come into effect in 2009 and the general ban in 2013, greater knowledge of the hormone systems of invertebrate test species is much needed, if they are to present us with a reasonable alternative. Molecular approaches for unravelling the pathways of effects of AACs It can be expected that almost all pollutants that adversely affect humans and wildlife will do so by interfering with gene expression. The current knowledge about this effect level of environmental chemicals is extremely limited. Although investigations into the responses of individual genes to EACs have started (Filby et al. 2005), there is a need for a more comprehensive and, additionally, a comparative approach. A number of hormone-mimicking chemicals are known to have multiple biological effects at various levels of biological integration, which in turn means that they will have multiple pathways of action (e.g nonylphenol; Harris et al. 2001) and recent studies have shown this (Filby et al. 2006). Molecular approaches potentially offer an effective method for screening and testing for chemical effects across multiple axes. Significant advances have been made in applying gene arrays (measuring the expression of many genes simultaneously) in mammals to study the mechanisms of cancer induction (Perou et al. 2000; Schaner et al. 2003) and in elucidating the mechanisms of action of environmental oestrogens (Moggs et al. 2004). Less attention has been focused on the development of arrays for environmental impact assessment of chemicals in wildlife. Recently, an international and multi-stakeholder initiative in the USA identified gene arrays as a technique that is likely to play a central role in the future development of ecotoxicology research and for the regulatory testing of chemicals. It is now well established that there are a wide range of chemicals that are capable of disrupting the androgen axis

(Fisher 2004; Schulte-Oehlmann et al. 2000), as well as the oestrogen pathway/function (Harris et al. 2001) and the thyroid axis (Crofton et al. 2005, Opitz et al. 2005). Adding further weight to the likely importance of AACs in the environment, recently in the UK, a nationwide study of effluents emanating from sewage treatment works, found that anti-androgenic activity was equally as widespread as oestrogenic activity (Tyler et al. unpublished data) further supporting the timeliness of the COMPRENDO research focus on AACs. The prerequisite for any biological action regarding AAC effects is the existence of androgens and their corresponding receptors in test species. In vertebrates, the existence of androgens and corresponding AR is well established. COMPRENDO investigated whether androgens and androgen-like receptors (AR) and oestrogen and oestrogen-like receptors (ER) were present in several groups of invertebrates (echinoderms, crustaceans and molluscs) and how hormonally active substances might interfere with these targets in invertebrates in comparison to vertebrates. Receptor binding studies for ER/AR revealed the existence of oestrogen and androgen specific binding sides in Paracentrotus lividus, Antedon mediterranea, and Marisa cornuarietis whereas in Hyalella azteca only an androgenic binding side (not interfering with ecdysteroids) was detected. The following nuclear receptors have now been detected: in Marisa ER-like, ERR-like, E75-like, RAR-like and RXR-like, in Paracentrotus; ERR-like, RAR-like and RXR-like, in Hyalella, ERR-like. The full sequence for Marisa ER-like was obtained under COMPRENDO. Molecular cloning and sequencing approaches, however, found no androgen receptors in any of these three invertebrate species, despite the existence of binding sites in tissue preparations for all. The function of the ER receptors in invertebrates have not yet been clarified. COMPRENDO further cloned and sequenced almost 100 sequences for genes known to play key roles in sexual development and function in fish and established a multi-species cDNA macro-array prototype for fish. The array was fully validated for use in a range of fish species including the zebrafish and fathead minnow. The first application of the macro-array to an exposure of fathead minnow to the aromatase inhibitor fenarimol demonstrated an excellent match between the molecular responses and the induced phenotypic effects. Closing the COMPRENDO chapter opening new doors Comparative studies constituted one of the cores of the COMPRENDO research and some of the findings have highlighted the potential of invertebrates for use in chemical testing for signalling potential effects in vertebrates. More extensive research on user-friendly tests and techniques are required to facilitate the testing for the legal obligations of end users and corporate clients (e.g. authorities or industry).
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COMPRENDO has brought two new invertebrate species to the OECD for consideration and has developed a multi-fish species gene array for the enhancement of chemical testing regimes and systems. These tools are also likely to improve the protection of human health, for example as applied to the monitoring of water quality and effectiveness of wastewater treatment plants, or in the assessing for compliance and threshold of biological effects of chemicals and their mixtures. The work under COMPRENDO has further served to highlight that we perhaps need to be less restrictive in our approach to chemical testing. Understanding the basic biology and endocrinology of a greater number of animals (both invertebrate and vertebrates) is likely to help us to develop more effective and sensitive testing systems for chemicals of concern, such as EACs, and thus develop more effective warning systems for the adverse health effects of chemicals. It should also be emphasised that an understanding of chemical effects in invertebrates is not important only for their potential use and development as surrogates for some aspects of vertebrate testing, but also for the protection of invertebrate fauna in the environment per se, as they play a pivotal role in all ecosystems. Little is known about potential effects of endocrine disrupters in wild invertebrate populations and there is the very real need for field- & bio-monitoring studies to investigate for possible impacts of EACs on invertebrates, as has been shown for some vertebrate populations.

COMPRENDO was a journey of discovery. The ambitious and diverse nature of the work under COMPRENDO meant that re-iterative evaluation of the work, needs, and achievements was essential throughout the life of the project. Delivery on many aspects of the science was made greater through linkages of the work with other research projects and indeed necessitated some resourcefulness on behalf of the partners. In the rapidly moving science of molecular biology and gene arrays, team members also had to be highly adaptive and flexible to deliver on some of the challenges set. COMPRENDO was European research at its best. In addition to a valuable contribution to our understanding of endocrine disruption, COMPRENDO has also served to forge strong international research linkages between member states of the EU and to develop great friendships. To those who funded and supported COMPRENDO, we should like to express our sincere gratitude. We hope the European Union, as our benefactor, will have the staying power that will inevitably be required to utilise the findings from COMPRENDO and from other projects in the CREDO cluster to more forward-thinking political actions to regulate hormone mimicking substances.

Ulrike Schulte-Oehlmann, Susan Jobling, Charles Tyler and Jrg Oehlmann on behalf of the COMPRENDO consortium www.comprendo-project.org

References
Crofton, K.M., Craft, E.S., Hedge, J.S., Gennings, C., Simmons, J.E., Carchman, R.A., Carter Jr., W.A., DeVito, M.J. (2005). Thyroid-hormone-disrupting chemicals: Evidence for dose-dependent additivity or synergism. Environ. Health Perspect. 113: 1549-54. Falandysz J., Albanis T., Bachmann J., Bettinetti R., Bochentin I., Boti V., Bristeau S., Daehne B., Dagnac T., Galassi S., Jeannot R., Oehlmann J., Orlikowska A., Sakkas V., Valsamaki V. & Schulte-Oehlmann U. (2006). Some chemical contaminant of surface sediments at the Baltic Sea coastal region of the Gulf of Gda sk, the Brda River outlet in inland Poland, and at refn erence sites in the Wadden Sea, Germany, with special emphasis on androgenic and anti-androgenic compounds. J. Environ. Sci. Health: submitted. Filby, A.L., and Tyler, C.R. (2005). Molecular characterization of estrogen receptors 1, 2a and 2b and their tissue and ontogenic expression profiles in fathead minnow (Pimephales promelas). Biol. of Reprod. 73:648-662. Filby, A.L., Thorpe, K.L. and Tyler, C.R. (2006). Multiple molecular effect pathways for an environmental oestrogen in fish. J. Mol. Endocrinol. Accepted, in press. Fisher J.S. (2004). Environmental anti-androgens and male reproductive health: Focus on phthalates and testicular dysgenesis syndrome. Reproduction 127: 305-15. Harris, C.A., Santos, E.M., Janbakhsh, A., Pottinger, T.G., Tyler, C.R., Sumpter, J.P. (2001). Nonylphenol affects gonadotropin levels in the pituitary gland and plasma of female rainbow trout: Environ. Sci. Technol. 35: 29092916. Janer, G., LeBlanc, G.A. & Porte, C. (2005a). Androgen metabolism in invertebrates and its modulation by xenoandrogens. A comparative study. Ann. N.Y. Acad. Sci. 1040: 354-356. Janer, G., LeBlanc, G.A. & Porte, C. (2005b). A comparative study on androgen metabolism in three invertebrate species. Gen. Comp. Endocrinol. 143: 211-221. Janer, G., Lavado, R., Thibaut, R. & Porte C. (2005c). Effects of 17b-estradiol exposure in the mussel Mytilus galloprovincialis: A possible regulating role for steroid acyltransferases. Aquat. Toxicol. 75: 32-42. Janer, G., Sternberg, R.M., LeBlanc, G.A. & Porte, C. (2005d). Testosterone conjugating activities in invertebrates: Are they targets for endocrine disruptors? Aquat. Toxicol. 71: 273-282. Janer, G., Bachmann, J., Oehlmann, J., Schulte-Oehlmann, U. & Porte, C. (2006). The effect of organotin compounds on gender specific androstenedione metabolism in the freshwater ramshorn snail Marisa cornuarietis. J. Steroid. Biochem. Mol. Biol. 99: 147-156.

Janer, G., Lyssimachou, A., Bachmann, J., Oehlmann, J., SchulteOehlmann, U. & Porte, C. (2006). Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: The effect of xenoandrogenic compounds. Steroids, 71: 435-444. Kusk, K.O., Wollenberger, L. (2005). Validation of a full life-cycle test with the copepod Acartia tonsa. Institute of Environment & Resources, Technical University of Denmark, 56 pp. Lavado, R. Barbaglio, A., Candia Carnevali, M.D. & Porte, C. (2006). Steroid levels in crinoid echinoderms are altered by exposure to model endocrine disruptors. Steroids, in press. Moggs, J.G., Tinwell, H., Spurway, T., Chang, H-S., Pate, I., Lim, F.L., Moore, D.J., Soames, A., Stuckey, R., Currie, R., Zhu, T., Kimber, I., Ashby, J., Orphanides, G. (2001). Phenotypic anchoring of gene expression changes during estrogen-induced uterine growth.Environ. Health Perspect. 112: 1589-1606. Perou, C.M., Serlie, T., Eisen, M.B. (2000). Molecular portraits of human breast tumours. Nature 406 (6797): 747-752. Schaner, M.E., Douglas, T.R., Ciaravino , G., Srlie, T., Troyanskaya,O., Diehn, M., Wang Y.C., Duran, G.E., Sikic , T.L., Caldeira, S., Skomedal, H., Tu, I-P., Hernandez-Boussard , T., Johnson, S.W., O'Dwyer, P.J., Fero, M.J., Kristensen, G.B., Brresen-Dale, A.-L., Hastie, T., Tibshirani, R., van de Rijn, R., Teng, N.N., Longacre, T.A., Botstein, D., O. Brown, P., Sikic, B.I. (2003). Gene expression patterns in ovarian carcinomas. Environ. Health Perspect. 112:1589-606. OECD (2005). OECD Draft Guidelines for Testing of Chemicals. Proposal for a new Guideline. Calanoid Copepod Development and Reproduction test with Acartia tonsa. 2005. Paris, France, Organisation for Economic Cooperation and Development. Edited by Kusk, K.O. and L. Wollenberger. Opitz, R., Braunbeck, T., Bgi, C., Pickford, D.B., Nentwig, G., Oehlmann, J., Tooi, O., Lutz, I. & Kloas, W. (2005). Description and initial evaluation of a Xenopus metamorphosis assay for detection of thyroid system-disrupting activities of environmental compounds. Environ. Toxicol. Chem. 24: 653-664. Schmitt, C., Duft, M., Brandelik, C., Schulte-Oehlmann, U., Oehlmann, J. (2006). SOP for testing of chemicals: Reproduction test with the prosobranch snail Potamopyrgus antipodarum for testing endocrine active chemicals. Part I: Culturing of Potamopyrgus antipodarum. Part II: Reproduction test using water exposure. Part III: Reproduction test using spiked sediment. J.W. Goethe University of Frankfurt, Department of Ecology and Evolution Ecotoxicology, Germany. Schulte-Oehlmann, U, Watermann, B., Tillmann, M., Scherf, S., Markert, B., Oehlmann, J. (2000). Effects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part II: Triphenyltin as a xeno-androgen. Ecotoxicology 9: 399-412.

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EURISKED

Multi-organic risk assessment of selected endocrine disrupters

Objectives The ultimate goal of the EURISKED project was to determine multi-organic effects of a variety of endocrine disrupters (EDs). These are: nonylphenol, bisphenol A, dibutylphtalate, octyl-methoxycinnamate (OMC), 4-methylbenzylidene camphor (4-MBC), benzophenone-2, procymidon, linuron, resveratrol, 8-prenylnaringenin, genistein, oestradiol-benzoate, and androstandiol. Utilizing a variety of reporter cell systems, the first goal of the project was to determine steroidal (oestrogenic, androgenic, thyromimetic gluco- or mineralocorticoid or progestational) effects. On the basis of these results, reporter gene transfected mice were to be investigated with those substances which proved to have effects in the cell biological experiments. In parallel to these reporter cell assays, experiments in immature and adult female and male rats as well as in steroid receptor deleted (K.O.) animals were performed. Effects of the endocrine disrupters were to be measured by physiological, molecular and morphological means. Steroids and also thyroid hormones have profound effects in many other organs outside the reproductive tract. It was therefore the major goal of this project to study these effects outside the reproductive tract. As a control, targets within the reproductive tract needed to be incorporated in the study design as well. Scientific achievements There is now clear indication that a number of the test substances, which are widely used as food additives, cosmetics or plasticizers have profound effects on many organs. This could be verified in the various reporter assays utilizing either gene transfected cells from different organ-specific primary cells with specific responses to steroids, or reporter gene transfected mice. The experiments with the reporter cells were completed within the first two years, those with the reporter animals were finished by the end of the project. Utilizing these assays, it became clear that the endocrine disrupters had effects in a variety of cells stemming from different organs. Some are oestrogenic, some anti-androgenic, others are unexplainable at present and may involve other nuclear or non-nuclear receptor mediated processes. As expected, many of the proposed EDs have effects outside of the reproductive tract. So far, the brain, the pituitary gland, the liver, the bone, fat tissue and of major importance the thyroid gland were shown to be target of some of the studied EDs. Plant derived so-called phytoestrogens stimulate bone formation but also uterine and mammary gland tissue. By means of histology, including immunocytochemistry, it was shown that all uterine parts (endometrium, myometrium) were stimulated by the phytoestrogens, whereas 4-MBC and OMC had very mild uterotropic effects. Linuron and procymidone were ineffective. 8Prenylnaringenin, a hop-derived flavonoid, caused formation of polypoid structures in the endometrium, an effect which has never been published before. In the mammary gland all phytoestrogens clearly stimulated the formation of the the nuclear proliferation marker Proliferating Cell Nuclear Antigen (PCNA) and of the progesterone receptors, both are very typical oestrogenic effects. The higher doses of the phytoestrogens stimulated also development of mammary gland ducts and milk production. If occurring also in the human this would endanger the uterus and the mammary gland to develop malignant tumours. In the thyroid, the phytoestrogens and the UV screens inhibited either thyroidperoxidase or the iodide symporter, and in other organs the thyroid hormone deiodinases were affected, which may endanger the organism to become hypothyroid. Some of the tested UV filters proved to be oestrogenic, an undesired effect if the substances are resorbed transcutaneously. Upon approval through the ethical committee, five female and six male human probands were subjected to treatment with two commercially available sunscreens with a high UV protection factor. One company responded to the request about information on the amounts of OMC in their product. Surprisingly 10% of the whole sun milk was OMC. The other sunscreen contained OMC and 4MBC in unknown amounts. Extraction of these substances and HPLC separation and UV detection indicated that up to 10g of 4MBC and OMC were present in 100ml of the second commercially available sunscreen. The back and front part of the trunk of the test subjects were exposed to known amounts of the UV screens and blood samples were collected through indwelling antecubital vein catheters prior to and up to 8 hours after initial exposure. Exposure was then continued for another 3 days and on the fourth day the same treatment and blood withdrawal regimen was applied. Extraction, HPLC separation and UV detection did not detect any of the two suncreens. In animal experiments it was also shown that both sunscreens are rapidly metabolized and also the metabolite could not be detected in the serum of the subject. Extracts of selected blood samples were subjected to the highly sensitive method of mass spectrometry and again no indication of presence of either of the two UV screens or their metabolites could be demonstrated. Hence, the measurement performed so far did not result in detectable serum levels. The two pesticides tested inhibited testosterone-induced growth of prostates and seminal vesicles. This anti-androgenic effect may have adverse effects in pubertal boys, not only in the development of prostate or seminal vesicles but also in bone maturation. Prior to the establisment of the EURISKED project it was shown that the prostate expresses high amounts of the oestrogen receptor of the beta subtype (ER). It was therefore another focus of interest to investigate the effects of
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ER in the prostate. By means of histological and immuocytochemical analysis and utilizing the ER knock-out mouse model, it could be demonstrated that the ER appears to have anti-proliferative effects in the prostate, thereby having possibly protecting effects against the development of prostate cancer. From the two UV screens, 4MBC proved to be a pure ER agonist, which may therefore have a potential in the treatment of prostate cancer. In a detailed study about antenatal effects of the sunscreens, neuronal and maturational parameters were investigated and 4MBC at a relatively high dose exerted clear effects in the animals which were perinatally treated and investigated at the age of 12 weeks. Significant effects were observed in the prostate, thyroid gland, uterus, ovary and also molecular endpoints like IGF1, progesterone gene expression were altered. In another experiment, the effects of 4MBC and benzopheneone-2 on pubertal development was investigated and a delay of puberty and a variety of adverse effects in a number of organs including the uterus and the bone were observed. Two consortium members investigated the effects of the arylhydrocarbon receptor (AhR) in some more detail. It could be shown that genistein and resveratrol were AhR agonists, while 17-oestradiol, 4MBC and OMC were AhR antagonists. The biological meaning of these findings are currently unclear. Stimulation of the AhR results in formation of CYP1A1 enzymes which are involved in detoxifiaction of environmental toxicants. Socio-economic relevance and policy implications The consortium is confident that the results furthered our knowledge about the safety of some food additives, pesticides and of some UV screens, which will lead to political activities to direct the use of these compounds. Information made available to consumers will help them make balanced decisions about their risks of exposure to these chemicals. Conclusions It appears that the choice of EDs and of animal models was appropriate and could lead to recommendations resulting in risk assessment guidelines for humans. The considerable amount of samples generated during the funding period of the project as well as scientific publications documenting the results of the project are deliverables of the project.

Thyroid hormone biosynthesis

A target for endocrine disruption by UV filters In recent years it has become clear that, in addition to the reproductive system, the thyroid hormone axis is a major target of endocrine disruption. In our investigations performed as part of the EURISKED project we examined the effects of selected compounds, including certain UV filters, on thyroid hormone biosynthesis. UV filters are produced in amounts of several hundreds of tons per year, and their main use is in skin protection products against sunburn, photo aging and skin cancer. For example, 4-methylbenzylidene-camphor (4MBC) is contained at concentrations of up to 4% (w/w) in commercial preparations. Another UV filter, benzophenone 2 (BP2), is no longer permitted in sun lotions within the EU, however it is still contained in other cosmetics to prevent their UV-induced damage. The concentrations of BP2 in these cosmetics are much lower than the concentrations allowed for other UV filters in sun protection products, but since BP2 is found in a wide variety of products, it is likely that it is applied to human skin virtually every day. In vitro and in vivo effects of 4-MBC and BP2 on the thyroid hormone axis A prerequisite for the synthesis of the iodide-containing thyroid hormones is the uptake of iodide into the hormone-producing thyroid cells. This process is catalyzed by the socalled sodium iodide symporter (NIS) and can be examined in a thyroid cell culture model, the rat-derived cell line FRTL-5. We found that 4-MBC decreased iodide accumulation in this cell line in a dose-dependent manner, starting at a concentration as low as 100mol/L. This effect was due to a reduced expression of the NIS protein, as determined by Western blotting. Thyroid hormone biosynthesis is catalyzed by thyroid peroxidase (TPO). We developed an in vitro enzyme assay based on human recombinant TPO (hrTPO) which was stably transfected into the human thyroid carcinoma cell line FTC-238. Using this assay we demonstrated that BP2 inhibited hrTPO with IC50 values of 450 and 370mol/L, respectively, in the guaiacol and the iodide oxidation reaction, two classical methods to measure peroxidase activities. These IC50 values are one to two orders of magnitude lower than those determined for methimazole (MMI) and propylthiouracil (PTU), two TPO inhibitors used as anti-thyroid drugs in the therapy of hyperthyroidism (figure 1). Preincubation of hrTPO with a combination of BP2 and hydrogen peroxide (a necessary cofactor for TPO), but without substrate, inactivated the enzyme. This effect was prevented by the presence of micro-molar amounts of iodide in the preincubaton mixture, indicating that effects of BP2 on TPO may be especially detrimental under conditions of iodide deficiency. To examine the effects of the two UV screens in vivo, rats were treated with increasing doses of 4-MBC and BP2. Already after 5 days application, both compounds dosedependently decreased T4 and increased TSH serum levels. This is a constellation typical for hypothyroidism, a condition

Wolfgang Wuttke Division of Clinical and Experimental Endocrinology, University of Gttingen, Germany, on behalf of the EURISKED consortium. www.eurisked.org

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Figure 1: Dose response curves and IC50 values for the inhibition of hrTPO by a) MMI, b) PTU and c) the UV filter BP2 in the guaiacol oxidation assay.

Consequences A ban of 4-MBC is already under consideration by EU authorities and our findings have deepened the concern over this substance. As for BP2, data on human exposure and skin absorption are needed in order to facilitate a new evaluation of the risks of its use as a UV stabilisator in cosmetics. Cornelia Schmutzler, Inka Hamann and Josef Khrle Institute for Experimental Endocrinology, Charit Berlin, Germany www.eurisked.org

of pathologically low thyroid performance. 4-MBC also caused goitre in rats, both after short-term (5 days) and longterm (12 months) administration in rats (Jarry et al. 2004; Schlumpf et al. 2004; Inka Hamann, unpublished). Risks for human health? Several recent publications, among them Janjua et al. (2004), show that UV filters can penetrate the human skin, and in the case of 4-MBC, reach serum levels of ~80mol/L. There are no data for BP2, but benzophenone 3, which is chemically closely related to BP2, was detected in human serum at a concentration of ~1.3mol/L. These are concentrations at which effects were seen in our in vitro assays. Thus, it cannot be excluded that UV filters may also have adverse effects on thyroid hormone biosynthesis in humans. According to our data, the effects of UV filters on TPO and on the NIS may combine with and aggravate effects of low iodine supply. This finding has to be considered in the context that iodine deficiency is still a problem in many parts of the world (including industrialised countries such as Germany).

References
Janjua NR, Mogensen B, Andersson AM, Petersen JH, Henriksen M, Skakkebaek NE, Wulf HC (2004). Systemic absorption of the sunscreens benzophenone-3, octyl-methoxycinnamate, and 3-(4-methyl-benzylidene) camphor after whole-body topical application and reproductive hormone levels in humans. J. Invest. Dermatol. 123, 57-61. Jarry H, Christoffel J, Rimoldi G, Koch L, Wuttke W (2004). Multi-organic endocrine disrupting activity of the UV screen benzophenone 2 (BP2) in ovariectomized adult rats after 5 days treatment. Toxicology 205, 87-93. Schlumpf M, Schmid P, Durrer S, Conscience M, Maerkel K, Henseler M, Gruetter M, Herzog I, Reolon S, Ceccatelli R, Faass O, Stutz E, Jarry H, Wuttke W, Lichtensteiger W (2004). Endocrine activity and developmental toxicity of cosmetic UV filters-an update. Toxicology 205, 113-122.

NEWS FROM EDEN

The EDEN project has recently been granted an extension of six months, to the end of May 2007. This was triggered by force majeur delays in catching fish and obtaining tissue specimens. The extension will enable EDEN to complete urgently needed exposure assessments for multiple chemicals in fish and human tissues. Such assessments were rarely carried out in the past. Instead, single chemicals were measured, leaving the issue of combined exposures unanswered. The development of new biomarkers useful for examining endocrine disruption in humans is making progress, as are studies of the role of the KISS peptin in puberty. New data about sperm quality in Germany await final analysis, due to appear in 2007. Considerable progress has also been made with our understanding of how endocrine disrupters work together as mixtures, and current work focuses on the interplay of substances with dissimilar modes of action. An expert workshop was held to explore options of incorporating this new knowledge into better chemicals regulation. The EDEN team is currently busy with finalising a document about mixtures regulation which will be published in 2007. Andreas Kortenkamp University of London School of Pharmacy, UK www.edenresearch.info
www.credocluster.info November 2006 Page 7

BONETOX

Bone development and homeostasis new critical targets in toxicology

Bone quality and function have not traditionally been the focus of toxicological research. However, in recent years a number of chemicals and food contaminants have been identified that interfere with bone development and remodelling. Among those it has been demonstrated that aryl hydrocarbon receptor (AhR) ligands, such as 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), have the ability to alter bone structure and function at doses that are close to human background exposure levels. The BoneTox project aims to clarify whether AhR-mediated endocrine mechanisms can explain the observed bone toxicity. Alterations in heart and tooth development are additional physiological outcomes studied in the project. The molecular focus of the project is on the dioxin, oestrogen and retinoid (vitamin A) signalling pathways. This article covers bone and retinoid results that have been or are in the process of being published. Methods and experimental models Method development is essential to the BoneTox project since few molecular biology and chemical methods have been adapted to the analysis of hard tissues. Similarly, bone measurements of small animals are only standardised to a limited extent to accommodate for toxicological studies. The animal models cover both traditional experimental lines of rats and mice, and several genetically modified mouse strains. In addition, human epidemiological studies are ongoing, but to a limited extent. Bone studies in rats Combined gestational and lactational TCDD exposure has shown bone defects mainly in the offspring of the most sensitive rat line studied (Miettinen et al., 2005). The defects included decreases in bone mineral density (BMD), bone length and cross-sectional area of cortex. Mechanical testing revealed significantly reduced breaking force and stiffness of the bones. The effects were time- and dose-dependent, with earlier exposure resulting in more severe defects. Most of the defects were almost recovered in one-year old offspring. These results indicate that dioxins affect the developing bones by interfering with bone growth and mechanical strength, and that the effects are mainly reversible. The effect on bone of early life exposure to a chemical mixture that mimics pollutants found in the blood of the Canadian Inuit population has been studied in the rat (unpublished data). Preliminary results indicate that several bone parameters revealed from pQCT measurements were affected both in dams and offspring at doses of about 500 times human intake levels. Changes observed in the offspring were evident in young and young adult pups, whereas older offspring showed clear, although not complete, recovery. Effects on bone by the brominated flame retardants hexabromocyclododecane (Olausson et al., 2006), brominated

Tibia

Femur

Maternal dose of TCDD (g/kg)

Figure 1. Peripheral quantitative computed tomography (pQCT) scans on tibial and femoral diaphysis in rats on postnatal day (PND) 35 after different maternal doses (0, 0.1, 0,3, 1 g/kg) of TCDD displaying the changes in cross-sectional geometry. (Image by Hanna Miettinen)

diphenyl ethers (pentaBDE (Litens et al., 2006) and decaBDE) and tetrabromobisphenol A are studied in a series of 28-days repeat dose and/or 1-generation toxicity studies in the rat. This work is done in collaboration with the EC project FIRE (QLRT-2001-00596). Bone studies in mice Sex steroids are important in maintaining the skeleton of both males and females. BoneTox partners have been characterising the bone phenotypes of oestrogen receptor and (ER and ER) inactivated mice (Moverare et al., 2003). These studies demonstrated that ER is the most important oestrogen receptor for the regulation of several different bone parameters including trabecular thickness, trabecular number and cortical thickness. To obtain a global perspective of the effect of oestrogen on bone proteins, whole bones from control and ovariectomised mice treated with oestradiol were used for proteome analysis (Pastorelli et al., 2005). Several novel oestrogen-regulated proteins were found, and the changes in the bone protein profile suggest that oestradiol affects a broad spectrum of metabolic pathways in bone. Bone studies in other mouse lines, modified with regard to RAR, AhR and RALDH1are ongoing. Bone studies in vitro Analyses of the localization and action of AhR in bone cells have revealed a strong expression of AhR both in osteoblasts and osteoclasts (Ilvesaro et al., 2005). The activity of osteoclasts was not affected by TCDD exposure, indicating that TCDD does not have direct effect on mature osteoclasts. However, continued studies have shown that TCDD could effectively disturb the osteoclastogenesis (unpublished data). Retinoids The retinoid (vitamin A) system plays an essential role in embryonic development, reproduction, growth and overall survival (Mark et al., 2006). Scientists in BoneTox have clearly demonstrated that the retinoid system is a sensitive target for TCDD and other AhR ligands (Fletcher et al., 2005a; Nilsson

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BONETOX

intracellular binding proteins for retinoids displayed significantly lower hepatic retinoid levels compared to wild-type mice, and following TCDD exposure these mice were almost entirely depleted for hepatic retinoid stores (Hoegberg et al., 2005). Dissemination All the results gained in the BoneTox project are disseminated during scientific meetings and are published promptly in scientific journals. Several thesis projects have been concluded while others are still ongoing. A special session on bone and tooth toxicology, with many BoneTox contributors, has been held at the Dioxin 2006 conference in Oslo, Norway. The BoneTox project will be finalized at the end of 2006.

Louise Lyrens, Maria Herlin and Helen Hkansson Institute of Environmental Medicine, Karolinska Institutet, Sweden, on behalf of all BoneTox participants. www.imm.ki.se/bonetox/
Figure 2. Rat primary bone cells grown on bovine bone slice and visualized by confocal microscopy. AhR is stained red by rhodamine conjugated secondary antibody, and F-actin is demonstrated by fluorescein isothiocyanate (FITC)-conjugated phalloidin. The actin ring containing multinuclear cell is an osteoclast while the surrounding cells are osteoblasts. Scale bar is 50m. (Image by Joanna Ilvesaro)

References
Fletcher N, Giese N, Schmidt C, Stern N, Lind PM, Viluksela M, Tuomisto JT, Tuomisto J, Nau H, Hakansson H. (2005a). Toxicol Sci. 86(2), 264-72.

and Hakansson, 2002). Altered tissue levels of multiple retinoid forms, as well as altered retinoid specific enzyme activities and expression profiles have been observed at low AhR ligand exposure levels. Through the collaboration with FIRE new knowledge will be gained about the structural requirements for disturbances in the retinoid system by brominated flame retardants, some of which are suspected to interfere with the AhR. A new retinoic acid metabolite, 9-cis-4-oxo-13,14-dihydroretinoic acid has been identified as a novel and sensitive indicator of TCDD exposure (Schmidt et al., 2002, Fletcher et al., 2005a). The liver level of this metabolite was markedly decreased in TCDD-treated rats at all timepoints analysed in a 112 day time-course single dose study (unpublished data). The metabolite level was also decreased in the liver of dams and offspring exposed to a chemical mixture based on the blood profile seen in the Canadian Inuit population (unpublished data). The biological function and activity of the metabolite is currently being further investigated. To identify novel genes and pathways that may be associated with dioxin-induced retinoid disruption, global analyses of gene expression after exposure to TCDD has been carried out (Fletcher et al., 2005b), and studies have been performed in wildtype and mutant mice lacking one or more isotypes of RAR, RXR or intracellular binding proteins for retinoids (CRABP1, CRABP2, CRBP1). Treatment with TCDD resulted in loss of approximately the same amount of retinoids in the different receptor knockout lines, with the exception of RXR-null mice, which did not lose any retinoids upon TCDD exposure (Hoegberg et al., 2005). Mice lacking all three

Fletcher, N., Wahlstrom, D., Lundberg, R., Nilsson, C.B., Nilsson, K.C., Stockling, K., Hellmold, H. and Hakansson, H. (2005b). Toxicol Appl Pharmacol 207, 1-24. Hoegberg, P., Schmidt, C.K., Fletcher, N., Nilsson C.B., Trossvik, C., Schuur, A.G., Brouwer, A., Nau, H., Ghysenlinck, N.B., Chambon, P., Hkansson, H. (2005). Chem Biol Interact 156, 25-39. Ilvesaro, J., Pohjanvirta, R., Tuomisto, J., Viluksela, M. and Tuukkanen, J. (2005). Life Sci 77, 1351-66. Litens, S., Lyrens, L., van der Ven, L. and Hkansson, H. (2006). in: Dioxin 2006, Oslo, Norway. Mark M, Ghyselinck NB, Chambon P. (2006). Annu. Rev. Pharmacol. Toxicol. 46, 451-480. Miettinen, H.M., Pulkkinen, P., Jamsa, T., Koistinen, J., Simanainen, U., Tuomisto, J., Tuukkanen, J. and Viluksela, M. (2005). Toxicol Sci 85, 1003-12. Moverare, S., Venken, K., Eriksson, A-L., Andersson, N., Skrtic, S., Wergedal, J., Mohan, S., Salmon, P., Bouillion, R., Gustafsson, J-A., Vanderschueren, D., Ohlsson, C. (2003). Proc Natl Acad Sci USA 100, 13573-8. Nilsson CB and Hakansson H. (2002). Crit Rev Toxicol. 32(3), 211-32. Olausson, H., Herlin M., van der Ven, L., van der Kuil, A., Verhoef, A., Leonards, PEG., Piersma, AH., Vos, JG., Hkansson, H. (2006). In: Dioxin 2006, Oslo, Norway. Pastorelli, R., Carpi, D., Airoldi, L., Chiabrando, C., Bagnati, R., Fanelli, R., Moverare, S. and Ohlsson, C. (2005). Proteomics 5, 4936-45. Schmidt, C.K., Volland, J., Hamscher, G. and Nau, H. (2002). Biochim Biophys Acta 1583, 237-51.

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ENDOMET

What have we learned from ENDOMET?

The ENDOMET project Dysregulation of ENDOgenous steroid METabolism potentially alters neuronal and reproductive system development: effects of environmental plasticisers was set up to determine whether environmental plasticisers (EPs) could interact with human neuronal and reproductive systems. It was also envisaged that the programme would provide in vitro tests, largely using human cell lines, which could be used to identify potential endocrine disrupters (EDs). Now that ENDOMET has finished (June 2006), the results from all the groups are being summarised and collated to give a series of key achievements and questions. Non-genomic pathways EDs are classically described as acting as agonists/antagonists at steroid receptors, the so-called genomic pathway. Although this certainly occurs, the mode of action of many EPs, especially the phthalates and alkyl phenols, can best be explained by their effects on steroid sulphation, steroid synthesis, cell signalling and cell transport. In particular, phthalates, which in vivo have major endocrine-disrupting potential, have been shown to act on this non-genomic basis. Tissue specificity Even when genomic aspects are considered, there appears to be tissue specificity: although human neuronal cell lines contain oestrogen and androgen receptors, they are relatively insensitive to these genomic effects of EPs (apart from those of bisphenol-A), unlike the human reproductive cell lines. Neuronal enzymes Some EPs affect the neuronal enzyme dehydroepiandrosterone (DHEA) sulphotransferase (SULT 2A1) which converts DHEA and pregnenolone into their corresponding sulphates. This process is known to be essential for memory formation and retention. Many EPs are fat-soluble. If they were not metabolised, could they cross the blood brain barrier, accumulate and then have subtle effects over a lifetime on memory and cognitive function? At present we do not have the answers but this seems a possibility, particularly as EPs can also affect neuronal cell signalling. Foetal development Many EPs affect the thyroid system and also alter iodide uptake into the cell, so indirectly modulating thyroid function. As the thyroid is critically important in neurodevelopment, could EPs accumulate in the foetus and alter brain structure and/or function?

In vitro profiling We finally studied 16 plasticisers and orphan phenols (phenolic compounds believed to have ED activity in vivo, such as o-phenylphenol) and used 23 in vitro tests. Cluster analysis of the results, linked with those from our genomic/proteomic arrays, has shown that ED activity can be predicted using a relatively small number of tests. We are currently aiming to patent this to provide in vitro profiles which could be used in identification of compounds as EDs. This development of in vitro tests would reduce the use of animals in reproductive toxicology and would potentially be compatible with the requirements of the REACH programme.

Members of the ENDOMET consortium, Rosemary Waring, David Ramsden University of Birmingham, UK Christian Behl Johannes Gutenberg-Universitat, Germany Eva Bonefeld-Jrgensen University of Aarhus, Denmark Ulrich Loos University of Ulm, Germany Maria Fickova and Sona Scsukova Institute of Experimental Endocrinology, Slovakia

http://endomet.bham.ac.uk/

Selected publications Many of the results from ENDOMET have been published in a special issue of Molecular and Cellular Endocrinology (MCE), December 2005 volume 244 (1-2), see below.
Ghisari M and Bonefeld-Jorgensen EC, Impact of environmental chemicals on the thyroid hormone function in pituitary rat GH3 cells, MCE 244, 31-41, 2005. Waring RH and Harris RM, Endocrine disrupters: a human risk?, MCE 244, 29, 2005. Harris RM, Kirk CJ and Waring RH, Non-genomic effects of endocrine disrupters: inhibition of estrogen sulfotransferases by phenols and chlorinated phenols, MCE 244, 72-4, 2005. Turan N, Waring RH and Ramsden DB, The effects of plasticisers on 'sulphate supply' enzymes, MCE 244, 15-9, 2005. Wenzel A, Franz C, Breous E and Loos U, Modulation of iodide uptake by dialkyl phthalate plasticisers in FRTL-5 rat thyroid follicular cells, MCE 244, 6371, 2005. Breous E, Wenzel A and Loos U, The promoter of the human sodium/iodide symporter responds to certain phthalate plasticisers, MCE 244, 75-8, 2005. Mlynarcikova A, Kolena J, Fickova M and Scsukova S, Alterations in steroid hormone production by porcine ovarian granulosa cells caused by bisphenol A and bisphenol A dimethacrylate, MCE E 244, 57-62, 2005. Mlynarcikova A, Fickova M and Scsukova S, Ovarian intrafollicular processes as a target for cigarette smoke components and selected environmental reproductive disruptors, Endocrine Regulations 39 (1), 21-32, 2005.

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EASYRING

Rapid non-invasive testing methods

EASYRING final report Endocrine disrupting chemicals (EDCs) can cause adverse effects on the reproductive biology of vertebrates. These compounds may have a marked impact on reproductive biology by mimicking or antagonising the effects of naturally occurring sex steroids or other natural hormones, such as via (anti)oestrogenic and (anti)androgenic action. Starting from this knowledge, the project Environmental Agents SusceptibilitY assessment utilizing existing and novel biomarkers as Rapid non-invasive testING methods (EASYRING) undertook its research programme as a true ring. The project moved full circle, starting from the environment and the River Lambro (the most polluted tributary of the River Po, Northern Italy), by means of in vitro and in vivo studies and mathematic models, to improved information on the environmental levels of some known EDCs and their biological effects on reproduction. Several specific tasks were undertaken to meet these broad aims, both in aquatic species (the common carp, Cyprinus carpio, and the African clawed frog, Xenopus laevis, as models) and in small mammals (mice). This has generated large amounts of multidisciplinary data, much of which has already been or is due to be disseminated in scientific papers and at international meetings. These tasks were successfully completed at the conclusion of the project, in December 2005. River contamination Focussing on the aquatic environment, moderately low level contamination of the River Po and particularly of the middle section were documented by chemical analyses of water samples collected upstream and downstream at the confluence of the River Lambro. By contrast, the results of sediment and macro invertebrate analyses suggested a clear distinction between the upstream and downstream stretches of the middle River Po, showing the consequences of EDC loads from its polluted tributary. The discrepancy between sediment and overlying water is not surprising, and can be explained by the moderate lipophilicity of EDCs and the higher recording capacity of bed sediments compared to grab water samples. Furthermore, fish sampled downstream at the confluence of the River Lambro showed a trend towards increasing plasma levels of vitellogenin (VTG), the confirmed biomarker for oestrogenicity, altered steroid plasma levels and morphological alteration of gonads and liver. Consequently, the first step of EASYRING was to use different in vitro assays (two recombinant stable human cell lines, MVLN and MDA-kb2, in parallel with the yeast oestrogen screen (YES) and the yeast androgen screen (YAS) as a preliminary screening of the water and sediment fractions in order to gain more knowledge about the presence of EDCs. The screening revealed the presence of a mix of compounds with different mode of actions, namely oestrogenic, androgenic and anti-androgenic compounds. Information from tests consistently identified a small number of fractions as being mainly responsible for oestrogenicity. Chemical analyses undertaken on these fractions identified a small number of known oestrogenic chemicals, i.e. oestrone (E1), 17-oestradiol (E2), oestriol (E3), nonylphenols mix (NPs),

bisphenol A (BPA), and t-octylphenol (tOP). The three natural oestrogens and the three industrial chemicals identified in the highly oestrogenic fractions were chosen to be used in the environmental mixture (1x Lambro) for in vivo exposures of common carp and X. laevis. With this premise and with the aim of interpreting the alterations found in feral fish of the River Po, the effects of graded concentrations of the most interesting of the above-mentioned oestrogenic chemicals as well as of their mixture, were examined during the second year of the project in such a way as to mimic environmentally relevant levels and compositions. Early development of gonads and sexual differentiation are very sensitive to hormonal imbalance and therefore to the action of those chemicals which can affect the complex functions of the reproductive system. To investigate these processes, and specifically how the River Lambro may affect them, carp fingerling which had undifferentiated gonads and still under development, were exposed to the 1x Lambro mixture for five months. The study was completed by two other treatments in which carp fingerlings were exposed to River Po bed sediments, collected upstream and downstream of the confluence of the River Lambro. In this way, carp were also exposed to the natural mixtures of EDCs present in the main Italian river particularly after the net enrichment transported by the River Lambro. Alteration of gonad differentiation, gonad morphology, sex ratio and steroid ratio and plasma VTG induction were indeed observed in carp juveniles exposed to environmental mixtures and downstream sediment. Dipstick assay A specific goal of EASYRING was to develop a new non-invasive system for the easy detection of VTG directly in the field in order to assess oestrogenic responses in the carp, a common fish found in European waterways and regularly used in environmental monitoring programmes across Europe. Our data showed that VTG can be detected in fish mucus using a lateral-flow immunoassay (LFIA; dipstick assay) performed within a few minutes of sampling, at VTG concentrations low enough to detect oestrogenic effects in the environment. To verify the applicability of the dipstick tool and its related protocol, the participants operated in the presence of personnel from ARPAL, the Regional Environmental Protection Agency. Comparison of VTG levels in plasma and mucus of the same fish using a quantitative sandwich ELISA previously established indicates a positive correlation in almost all groups of carp investigated. However, mucus VTG in some fish groups did not reflect plasma VTG as linearly as observed in more controlled laboratory studies, and care should therefore be taken when using the non-invasive strategy in the field. Apparently, exposure to anti-oestrogens may disturb the VTG turnover such that mucus and plasma VTG are no longer in equilibrium. Possibly, a blood sample should be taken, which could be analysed by the LFIA in a few minutes in the field. From larger specimens, blood sampling could also be a nondestructive strategy. Further studies should be performed to elucidate this. Alberta Mandich University of Genoa, Italy www.easyring.org
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EDERA

Twenty years of basic research to be translated into more predictive risk assessment
Endocrine disrupters (EDs) are environment and food contaminants known to alter metabolic functions of higher animals by interfering with the activity of a specific class of receptors: the intracellular receptors (IR). Because a significant number of man-made compounds show endocrine disrupting properties, regulatory agencies are recognizing the importance of defining acceptable limits of ED concentrations in the environment and are starting to discuss the extent to which currently available methods are applicable to risk assessment of ED. Thanks to in-depth investigation carried out on the IR mechanism of action over the last twenty years, we now know that IRs are finely regulated transcription factors controlling the activity of selected promoters in concert with general or cell specific co-regulators (co-activators and co-repressors). The interactions with co-regulators explain why synthetic IR ligands were found to exert cell- and promoter-specific action. Application of molecular biology tools revealed that IRs targeted by EDs are present in most, if not all, cells in mammals, and are profoundly involved in the control of most metabolic functions, pointing to the widespread undesired effects that ED might exert. Thus, in view of the newly acquired awareness on IR mechanism of action and functions there is a growing concern on the validity of the methodologies so far applied to identify EDs and to predict their harmful effects. Indeed, current in vivo and in vitro methodologies restrict the analysis to very specific organs or cell types and, therefore, are not sufficient to predict the potential consequences of EDs after short or long-term exposure. The recent emphasis on generating in vitro model systems for toxicological analysis has certainly discouraged the development of models that are suitable to envision the whole spectrum of effects on the body, which is required when dealing with endocrine disruption that is, by definition, a living organism. Novel opportunities Molecular imaging techniques applied to animal engineering now provide novel opportunities to create model systems that portray the systemic activities of a specific molecule in real time by means of non invasive technologies. For the first time, this enables measurement of the biological activity of a given compound without pain or sacrifice of the laboratory animal. These models are particularly suited to the study of transcription factors, such as IR, because of the facility to assess the expression of genes encoding bioluminescent or fluorescent proteins, the so-called reporter genes. Typically, a reporter system for the study of IR-dependent transcription is generated by integrating into the mouse genome a hormoneresponsive promoter driving the exogenous reporter gene (figure 1). With the aid of European funds (BRIDGE-BIOTCT92-0308), our laboratory planned and produced a first paradigmatic reporter mouse, with the ERE-Luc that provides major insights on ER physiology.
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Figure 1: Reporter mice for in vivo studies of receptor activity. The ERELuc reporter mouse was generated by the integration of a transgene carrying the firefly luciferase gene under the control of a duplicated oestrogen responsive element (ERE) located upstream of a minimal thymidine kinase gene. Insulator sequences prevent any influence of the surrounding chromatine on the synthetic transgene. When the substrate luciferine is made available, the luciferase enzymatic activity results in the production of photons which can be detected by the use of a CCD camera and with the aid of specific algorithm that derives the intensity of the photon emission in each area of the mouse an image can be generated. An appropriate scale of colours can be generated in order to visualize the intensity of photon emission.

The EDERA programme was initiated with the aim of testing the suitability of the ERE-Luc mouse as a novel model system to: i.) identify presence of oestrogenic compounds in food and environment; ii.) provide a complete pattern of the body districts perturbed by xenoestrogens; iii.) assess the potential risk of acute or chronic exposure to xenoestrogens. Bioluminescence imaging EDERAs main task was to generate protocols based on in vivo bioluminescence imaging and demonstrate their applicability to toxicological and risk assessment analysis. This main goal was achieved by means of a series of more specific tasks. With comparative studies involving the quantitative analysis of photon emission in vivo and luciferase enzymatic activity ex vivo we could demonstrate that imaging is a method applicable to obtain a map of the effects of different concentrations of a given xenoestrogen in space and time (figure 2). The faithfulness of luciferase as reporter of ER transcriptional activity was shown by comparing the effect of administration of oestrogens of different origin (the natural hormone 17-oestradiol; the phytoestrogen genistein and the synthetic oestrogens p,p-DDT and BHC) on the accumulation of luciferase or transcripts from endogenous ER target genes (such as progesterone receptor, CYP17 and the oestrogen receptors themselves). In a second subtask, the imaging-based methodology was applied to the study of the effects of long-term exposure to food oestrogens like the phytoestrogen genistein or to complex mixture of oestrogens such as soy milk. Our

EDERA

experiments showed, for the first time, that phytoestrogens accumulate in the body and in long-term exposures interfere with ER signalling in a tissue selective manner. Most interestingly, treatment with soy milk generates a state of activation of ERs which differs significantly from that which was shown with genistein: in long-term treatment we observed a slight activation of ERs in liver, but a very significant activation in testis, indicating that the pure substance, genistein, may have effects significantly different than mixtures of phytoestrogens even if enriched in genistein itself. The model system proved particularly sensitive to exposure to different concentrations of xenoestrogens because it could show different responses to administration of pure or dilute soy milk and the state of ER activity was directly proportional to the amount of soy milk ingested. These data point to the necessity to better investigate the potential ED activity of soy milk, particularly when administered to infants of both sexes. Another EDERA subtask tested the possibility to generate a reporter system discriminating ER subtype specific oestrogens. To this aim, we bred the ERE-Luc mouse into the BERKO mouse.

Preliminary results, to be further confirmed, demonstrated the validity of the system in which luciferase is activated only by ER specific compounds. The last subtask of the EDERA project addressed the construction of novel reporter vectors for the generation of reporter animals to be used to identify and study ED acting via androgen and thyroid hormone receptors. Replacing, reducing and refining Overall we feel that the outcome of the EDERA project is very positive in terms of environmental and food protection because it has clearly shown the major advantages associated to the use of reporter animal technology with respects to the currently available model systems. In our view, the reporter mouse technology is an excellent candidate to REPLACE the existing tests that, for their nature, are unable to provide an overall view of oestrogenic activity in the whole organism. By means of non-invasive in vivo imaging technology, the methods provide the opportunity to REDUCE the number of animals to be used in the in vivo tests first of all because animal sacrifice is not needed, and secondly the possibility to follow the endocrine effects in time in the same animal reduces the need to use large numbers of animals in each experimental group to reach significant data and also reduces the need for control groups (the effects of a treatment is evaluated versus the baseline state of activity of the receptor on the same animal). Last, but not least, the technology will REFINE current methods by providing for the first time the possibility to study the effect of EDs systemically and, after long-term exposure even to low doses, our methodology will eliminate the pain created by animal testing and abolish the necessity of animal sacrifice. Thus, we feel that the work carried out by the EDERA consortium has shown the advantages of novel transgenic animal models for the study of EDs effects and exposure. The studies carried out with the ERE-Luc mouse need to be extended to generate other reporter systems, perhaps enabling study of the activity of more than one IR in the same animal. Studies along these lines are currently being carried out in new consortia under the Sixth Framework Programme for Research and Technological Development. Within the NoE CASCADE, EMIL and DIMI we are collaborating in the generation of novel, bimodal imaging vectors and new reporter animals; within the STREP EWA and the IP CRESCENDO we are pursuing in the investigation of the physiological changes of ER activity during development and aging; and within the STREP EXERA we will collaborate with several SMEs to test the feasibility of the use of reporter mice for the generation of 3d cultures of specific target tissues for high-throughput analysis of EDs.

Figure 2: Time course analysis of p,p-DDT activity in ERE-Luc mouse. Male ERE-Luc mice fed with oestrogen-deprived diet were injected with 50 g/kg of 17-oestradiol or with p,p-DDT and photon emission analyzed at different times. The upper panel shows a typical portray of the image generated by the CCD camera is shown. In the lower panel is the semi-quantitative analysis of the photon detected in selected body districts.

Adriana Maggi University of Milan, Italy www.edera.unimi.it

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GENDISRUPT

Genetic markers in testis endocrine disruption

Achieved and achievable
The mechanisms of action of endocrine disrupters (EDs) are not well known either at the molecular, cellular, tissue or organism levels, or during the critical periods of development. EDs may act as endocrine deregulators and generate gene deregulation depending on the genetic background of the organisms. Consequently, analysis of the genetic effects of these compounds on developing gonads could facilitate assessment of the mechanisms of action of EDs upon the individuals and the progeny of future generations by genetic transmission. The mammalian testis and the biological process of cell differentiation to spermatozoa (spermatogenesis) have been identified as targets of special interest, due to several reports and epidemiological studies showing increases in male infertility and testicular cancer incidence. Our research aimed to evaluate EDs and their effects on animals and humans during development, including fetal and neonatal periods. A genetic approach The GENDISRUPT project took a genetic approach and focused analysis on the effects on, and the genetic susceptibility of testicular cells to selected endocrine disrupters. Two main models were used for this: mouse and human. The mouse model was based on both in vitro and in vivo approaches. The in vitro approaches were carried out on primordial germ cells (PGCs) and Sertoli cells. The in vivo approaches, based on mice, were designed such that exposure of mothers in pre-mating period could show epigenetic effects, exposure during embryonic development to indicate prenatal effects, and exposure during the pubertal period to define effects during puberty. Several compounds with reported oestrogenic activity were tested in the mouse model: Bisphenol A (BPA), which is a component of polycarbonate plastics and is a contaminant possessing a weak oestrogenic activity. Lindane, the gamma isomer of hexachlorocyclohexane (HCH), is one of the oldest synthetic pesticides still in use and affects reproductive function in animals: decreased spermatid numbers and Sertoli cell fragmentation have been reported. A phthalate, mono(2-ethylhexyl)phthalate (MEHP), the toxic metabolite of the widespread plasticiser component di(2-ethylhexyl)phthalate (DEHP). Phthalate esters were found to cause malformations in the reproductive tract of intra-uterine exposed males. Zearelanone (ZEA), a nonsteroidal phytoestrogen from the group of resorcylic acid-lactones has been reported to cause malformations in the reproductive tract and germ cell apoptosis in the testis. 17-oestradiol (E2) was also studied, as a natural oestrogenic compound.

Key objectives The main objectives of this work were to undertake: 1. selection and characterisation of genes showing deregulation of expression, by the action of EDs, during the development and differentiation of mouse testicular cells; 2. a comparative analysis of gene deregulation in relation to treatments, dosage and cell types affected; and 3. phenotypic evaluation of the effect of EDs in mouse testis development. The analysis of gene expression was mainly carried out on commercial microarray platforms which contain most of the mouse transcriptome. This approach allowed us not only to define molecular markers for gene expression deregulation as an effect of EDs in testis development, but also to define quantitatively the effects of exposure period and dose. We could also address specific effects of EDs, such as "low dose effect" or the emerging question of epigenetic effects. In parallel, a specific prototype of microarray (GENDISRUPT-1) containing 300 specific oligonucleotides (from genes selected in a previous EC project, BIO4-CT96-0183) was also designed and evaluated. Subsequent studies were undertaken using functional genomics and proteomics approaches. The combination of both approaches can facilitate the understanding of gene regulation (and deregulation) and the potential effect of gene deregulation in the cells and biological systems under study. Detailed comparative anatomical analysis, testis histopathology and hormonal status were carried out on the same samples from ED-exposed mice that were evaluated for gene expression deregulation. The developing gonad Although the reproductive system is considered a main target for EDs, scant information is available on the effects of oestrogens and EDs on the development of embryonic gonad, and in particular, of PGCs. In the mouse, several conditions are known to influence PGC development and to result in sterility or development of germ cell tumours. To date, there have been few studies which have included a thorough investigation of the expression of oestrogen receptors (ER and ER) in gonadal somatic cells and germ cells during the crucial period of PGC development. Human studies With regard to the human studies, an aim of this project was to identify molecular markers that might explain genetic susceptibility to endocrine disruption in humans, focusing on male infertility and testicular cancer. These two traits are thought to be complex diseases in which the interactions of environmental (i.e. EDs) and genetic susceptibility factors could condition their development. The human studies were focused on clinical cases of testicular carcinoma and infertility. Analysis of single nucleotide polymorphisms (SNPs) of target genes and linkage analysis of quantitative trait loci (QTLs) can address the genetic susceptibility to EDs. Data mining processing and high-throughput genotyping methods have enabled some gene associations with male infertility to be established.

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The image in the top bar shows a partial view of microarray analysis.

Genetic associations with male infertility Nineteen candidate genes were selected for genetic association studies of human male infertility and testicular cancer and 46 SNPs were selected to perform these studies. However, only 37 of these SNPs were validated in our population and high-throughput genotyping protocols were designed for these 37 polymorphisms. To perform the genetic association studies, we recruited a total of 167 DNA samples from idiopathic infertile men, 1000 DNA samples of people from the general Spanish population, 101 DNA testicular tissue samples from testicular cancer patients, and 7 testicular tissue samples from healthy donors. To perform these analyses, a total of 28 SNPs were evaluated within eleven candidate genes and a total of 13,543 genotypes were obtained. The findings support a genetic basis of human male infertility and we observed associations of the ESR1, KIT, KITLG and PTEN genes with male infertility. These observations are in accordance with the possible existence of male infertility susceptibility factors within these genes in humans. An important finding is the implication of the ESR1 gene in male infertility. It is interesting to note that these results have been also observed in three independent series from Greece, Japan and Italy. Multilocus analyses of FSHR, ESR1, ESR2, CYP19A1 and NRIP1 revealed the existence of genetic interaction between these genes and their possible implication in the human male infertility. Preliminary evidence of loss of heterozygosity in the PTEN gene has been detected in human testicular cancer. Further studies of this gene in a larger group of testicular cancer patients could reveal if this observation can be considered as a common event in testicular cancer pathology. Microarray studies The results of mouse studies using microarrays were obtained by analysis of more than 3 million individual gene data. This represents an enormous effort of analysis never done before to evaluate comparative gene expression deregulation induced by EDs in developing testis. As a consequence of this analysis, which is still in progress, we have the following observations to report: MEHP is the ED which has demonstrated the strongest effect, based on the highest level of gene deregulation; MEHP and ZEA exposure define specific gene expression signatures by microarray analysis; The highest deregulation effects, for all the EDs analysed, was on mice that were exposed to the different EDs throughout all life stages up to analysis at 4 weeks postnatal; However, MEHP also induced remarkable deregulation when it was given to mothers during the embryonic period, in spite of the fact that animals were not exposed during the pubertal period. In general, E2 showed lower effects on gene deregulation in testis than the other EDs tested. A low dosage effect was evident in the longest period of exposure to MEHP and lindane. A proteomic profile of the soluble proteins expressed at different stages of mouse testis development was carried

out and 42 proteins were identified. Conspicuous variations in their accumulation (representing up or down-regulation) were detected. In general it may be concluded that the effects of different EDs analysed during testis development do not follow the same pathways at the level of gene deregulation. Furthermore, most of the EDs analysed in this study have a pattern of gene deregulation which is distinctive from that of E2. Morphologically visible effects Studies on the possible effects of ED treatments on mouse testis development also revealed some morphologically visible effects on the seminiferous epithelium, including especially higher numbers of apoptotic cells and of diploid spermatids. Results obtained from the study of PGCs provide the first indication that exposure to oestrogens during embryonic life may have profound effects on germ cell growth and differentiation through the action of gonadal somatic cells and the molecular pathways identified. Embryonic exposure to high levels of oestrogens or EDs constitutes a risk for PGC transformation in pluripotent tumorigenic cells. This process could be at the origin of germ cell tumour formation in the testis. An in vitro assay has been set up to identify and quantify oestrogenic effects on fetal testis cells. After validation, this assay might represent the basis of a novel test for rapid oestrogenic and anti-oestrogenic activity screening and investigation into the molecular pathways underlying the effect of these compounds on fetal gonads. Conclusions From these studies, it is apparent that the complex mechanisms of endocrine disruption could be involved in testis development. The type of compound, the dose, and the developmental stage of exposure could operate on different gene expression patterns and consequently affect different molecular pathways. Establishing a defined pattern of gene expression associated with specific compounds and with mixtures of potential EDs will be one way to design and validate analytical tools based on microarrays and new technologies in development. GENDISRUPT has contributed to the application of genetic analysis to identify molecular markers and in the knowledge gained on the mechanism of action of environmental chemicals on animal and human reproduction. Further studies will support and enhance the results obtained in GENDISRUPT towards controlling the adverse effects of endocrine disrupters and other reprotoxicants.

Jess del Mazo, Pedro P. Lpez-Casas and Mara Paz Department of Cell Biology and Development, Centro de Investigaciones Biolgicas (CSIC), Spain http://gendisrupt.cib.csic.es/

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MENDOS

Developing biomimetic and biological sensor systems for EDCs

Many international organisations, such as the European Commission, the Organisation for Economic Co-operation and Development and the World Health Organisation, as well as national governmental departments, such as the US Environment Protection Agency, have launched cooperative international activities to test and assess thousands of synthetic chemicals found in many common products for their potential effect on the hormonal systems of humans and animals in order to address reproduction related disorders. The main conclusion of most reports resulting from these activities up to now was the urgent need to establish reliable test methods. Both in vivo assays and in vitro screening systems are required to assess the endocrine disrupting potential of chemicals and to test for the interference of compounds with the oestrogenic and androgenic pathways or thyroid system. The general goal of the MENDOS project on biomimetic optical sensors for environmental endocrine disrupter screening has been to provide novel analytical technology for screening endocrine disrupting chemicals (EDCs), both in terms of costeffective tools for rapid pre-screening of environmental samples as well as clarification of potential EDCs modes of action and risk assessment. The project has focused on developing novel biomimetic recognition materials based on molecularly imprinted polymers (MIPs) together with highly sensitive miniaturised optical transducers. In addition, alternative approaches based on whole cell-based biosensors and bioassays as well as DNA microarray technology have also been followed. Due to the large number of chemicals with potential endocrine effects, screening methods should be capable of detecting many classes of compounds in a reasonably short time (high throughput screening) and offer the possibility of on-site analysis to identify sources of contamination in surface waters and sources of drinking water. Current technologies barely accommodate these requirements since they rely almost exclusively on biological/biochemical assays, which suffer from the general disadvantages of biosensors/bioassays, including sophisticated preparation and handling, lack of stability, use of (radio)labeled components or biomarkers, limited storage capability and subsequent high costs. Almost all of these drawbacks might be avoided by the introduction of novel biomimetic screening systems based on integrated optical sensor technology, as a non-animal artificial receptor-based fast screening method for environmental EDCs. The combination of optical sensor technology and artificial biorecognition layers mimicking the biofunctionality of a receptor/antibody with the advantageous properties of polymers should be designed to be applicable in environmental monitoring schemes. However, as no single test can assess all potential EDCs in the environment, the MIP sensors were developed in comparison to new biosensor systems based on cellular systems. While the MIP sensor should identify EDCs by their structure, the bioassays should respond to a broader range of EDCs by measuring cellular effects and allow detection of EDCs in complex samples with relevance
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for hazard and risk assessment to the environment and of human health. Biomimetic recognition materials Several main technological developments in the field of biomimetic and biological sensor systems for EDCs were achieved during the 3-year MENDOS project. The studies on biomimetic recognition materials resulted in the development of MIPs which selectively could recognize priority pollutants such as 17-oestradiol, benzo[a]pyrene (BAP), diethylhexylphthalate and atrazine. These MIPs were obtained both in bulk format as well as in the form of micro- or nanospheres useful for application in binding assays or as stationary phases in chromatography or solid phase extraction (SPE). Next to the selectivity, MIP development studies have considered particle size for the respective applications. With the proper synthetic strategy, the size and morphology of the imprinted spheres can be rationally controlled by varying the polymerisation conditions, the nature of the cross-linker, the monomer concentration and the polymerisation temperature (figure 1). For chromatographic evaluation (e.g. HPLC) or SPE, MIP particles with dimensions ranging from 10 to 25m are usually applied, compared to binding assays with MIPs immobilised at a chemical sensing interface where smaller particles (less than 1m) with a narrow size distribution are required. The main advantage of using MIPs as synthetic sorbents with antibody-like binding properties is their biomimetic recognition ability, which enables them to selectively extract one or several structurally related analytes at trace concentrations from complex environmental mixtures the separation of oestrogens from natural organic matter was obtained in a simple two-step extraction procedure. In addition, MIPs are stable under harsh chemical conditions, so that a wide variety of binding and eluting reagents can be applied without loss of recognition performance. It has furthermore been demonstrated that the developed MIP sorbents favourably compare to commercially available C18-based SPE materials in terms of selectivity and recovery, providing an advanced sample preparation tool for selective extraction of oestrogens from complex aqueous samples (figure 2).

Figure 1 (above left): Scanning electron micrographs (SEM) of imprinted micro and nanospheres (images provided by B. Mizaikoff; published in Wei et al. Biosens. & Bioelectr. 21, 1943-1951 , 2006). Figure 2 (above right): Total ion chromatogram (TIC) and selected ion chromatogram of spiked river water at 17-oestradiol 50 ng/L after MIP based SPE (images provided by B. Mizaikoff; published in Wei et al. Biosens. & Bioelectr. 21, 1943-1951 , 2006).

MENDOS

Similarly, molecularly imprinted polymers in the form of bulk polymers or microspheres for the target analyte BAP were successfully prepared using 4-VP and DVB as functional and cross-linking monomers. The resultant MIPs showed good recognition towards BAP over the nonimprinted polymers while other tested polyaromatic hydrocarbons showed significantly reduced binding. MIP films were also obtained by assembly of MIP-nanospheres via linear-polymer interlayers. The advantage of this coating technique is that it is reversible, that it can be done in situ even in a flow-cell, and that it can be done with substrates having a high aspect ratio, that are difficult to coat by spincoating. Using atrazine imprinted particles in surface plasmon resonance (SPR) mode, it has been shown that the layers did respond to the presence of the analyte. Optical sensor technology In terms of optical sensor technology, both novel interferometric transducers as well as SPR based optical sensing platforms have been developed. In particular, (i.) a mobile SPR sensor based on prism coupling, spectral modulation and wavelength division multiplexing and (ii.) a high-throughput SPR system based on angular spectroscopy of surface plasmons on an array of diffraction gratings (figure 3). The SPR sensor method was combined with antibodies and MIPs for detection of selected EDCs. Antibody-based SPR sensors detected selected EDCs (e.g. atrazine, 2,4-dichlorophenoxyacetic acid, BAP, and 4-nonylphenol) in sub-ng/ml concentrations. Biosensor systems In parallel, biosensor systems were developed in order to get valid screening methods with biological relevance. A wholecell bioassay, consisting of genetically modified eukaryotic cells using a mammalian vector carrying a Luc-reporter gene, under the control of the Drosophila melanogaster hsp22 stress inducible promoter was developed. This assay was shown to give a sensitive signal, dependent on both toxicity and bioavailability of the tested compounds such as heavy metals, genotoxic agents and endocrine disrupters. By optimisating culture of the human H295R cell line and a nonradioactive detection method (ELISA), an aromatase assay was developed as a test system for the interference of xenobiotic chemicals with steroid metabolism. This specific endpoint, in addition to the interference with hormone receptors, is of high relevance to be included in the endocrine disruption test strategy. Furthermore, a system for assessing the oestrogenic activity of a sample in situ has been established based on immobilised chemo-sensitive luminescent yeast cells. It was demonstrated that luminescent yeast based hydrogel bioassays are applicable to the analysis of environmental water samples, potentially as a primary screening tool and as the biological and chemical immobilisation parts in the construction of fibre-optic biosensors. The possibility of long-term storage, easy and cost-effective preparation, no need for continuous cell cultivation, and only 2.5 hours assay time, make the immobilised oestrogen inducible yeast-based hydrogel a promising alternative to the recombinant yeast reporter gene assays that are currently used.

Figure 3: A laboratory prototype of a high-throughput SPR sensor based on spectroscopy of surface plasmons on an array of diffraction gratings (left) and detail of the sensor chip (right) (development at IREE, images kindly provided by J. Homola).

DNA chips for detection of EDCs Finally, DNA chips holding oligo DNA probes for hormone responsive genes were successfully developed for the detection of EDCs. Several dedicated chip surface chemistries were applied and evaluated for quality and performance characteristics. The epoxy surface showed to be most appropriate for the effective binding of NH2 modified 50bp long oligonucleotides. The up and down regulation of the oestrogen regulated genes by EDCs could be detected in ZR75 breast cancer cells using this oligo DNA chip. Detection of anti-androgenic action of EDCs has been feasible utilizing 22RV prostate cancer cells. While the identification of individual EDCs is not possible in environmental samples, it could be shown that DNA chip-based assays are able to reveal the presence of oestrogenic compounds based on their hormonal activity even in low concentrations. The main objective envisaged for the MENDOS project was the development of field-deployable monitoring systems for EDCs in the aqueous environment. Although it has not yet been possible to establish a fully functional MIP-based optical sensor platform for EDC monitoring, significant advances have been made in MIP technology and optical sensor equipment, thus setting the basis for successful future implementation of highly sensitive biomimetic optical sensors. The latter, as well as biosensors and DNA chips could, after further refinement and validation, be used as tools to screen for oestrogenic activity of environmental samples or chemical test compounds, which is of relevance for monitoring water quality (Water Framework Directive) and chemical safety (REACH).

Hilda Witters VITO (Flemish Institute for Technological Research), Environmental Toxicology Unit, Belgium Michael Jakusch ARC Seibersdorf research GmbH, Austria

www.mendos.org

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Research Framework Programme Seventh News: MENDOS

Perspectives for environment and health research activity in the Seventh Framework Programme (FP7)
Introduction The European Commissions Environment and Health (E&H) Action Plan, adopted in 2004, has as its main aim to improve the understanding of the link between environmental factors and health. The implementation of the goals of this action plan through research has started in FP6 via funding of several large- and small-scale research projects on topics identified as priorities in the Action Plan. In FP6 a new approach was introduced for integrated environment and health risk assessment methodologies. FP6 projects such as NOMIRACLE or INTARESE will examine the concepts towards integrated risk assessment and link the environment and health data in a more effective way to also better support environment and health-related policy making. Along this line, the concept full-chain approach was introduced, putting more emphasis on the identification of pollution sources, exposures (including human biomonitoring), links to health effects, and economic valuation. The third new concept in FP6 dealt with the economic valuation models and tools to be developed for the area of environment and health in order to support impact assessment. The efforts will continue and intensify during FP7. Although new knowledge has been or is being generated both in FP5 and FP6 projects, there is still a lack of essential information in many areas on how environmental factors interfere with human health and well-being. We do not know to any great extent how multiple environmental exposures occurring during the whole human lifecycle from the moment of conception to aging and senescence can result in adverse health effects. Due to the complexity of the issues involved, only an integrated approach focusing on multiple stressors, pathways and effects, using scientific advances and tools such as ecotoxicological tools and modelling, can at medium or long term give us more definitive ideas as to the extent of environmental factors on the burden of disease at individual or population level. Justification and rationale The draft work programme 2007 will continue implementing the E&H Action Plan through research. The impact pathway approach adopted in FP6 will be further strengthened. The efforts to be supported in the first two years of the programme will build on work undertaken in previous programmes. In FP7, special attention will be given to combined effects of stressors and the role of confounding factors. The focus will be on vulnerable groups and emerging issues will not be neglected. The aim will be to generate new evidence and at the same time develop/enhance models that can help us in better understanding and foresee health and environment interactions. To continue supporting informed policy making, research will focus on new and/or improved tools for prevention strategies with emphasis on air pollution and epidemiological research, with applications in biomonitoring. The ultimate aim will be to
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aid policy makers in identifying prevention strategies through a better understanding of the full chain of cause effect relationships: sources emissions concentrations exposures and health effects. Research actions such as high-resolution GIS or validation of biomarkers for epidemiological research or biomonitoring, developed through new technologies based on omics methodologies, will aid the understanding of the precise relationships between environment and disease. Ultimately, this information may allow us to identify ways to reduce or prevent disease by pinpointing biochemical and molecular functions that have been perturbed by environmental stressors. Emerging issues to be tackled include health impacts of global change and certain man-made chemicals, keeping in mind realistic exposure scenarios. On the effect side, focus will be on research dealing with health impacts that continue to be prevalent and may be increasing, such as respiratory and other effects of air pollution, to be tackled by establishing a large European cohort study, declining reproductive health, the reasons for which remain uncertain, and neurodevelopmental effects. Short-term goals The work programme for the first call for proposals in FP7 will continue contributing to the goals of the E&H Action Plan by tackling existing knowledge gaps via a series of small and large-scale multidisciplinary research actions. The efforts to be supported will partly build on work undertaken in previous framework programmes. Priority will be given to issues which were largely neglected in FP6. These issues include the long-term impacts of air pollution (whether outdoor or indoor) and chemicals on human health. Research support for biomonitoring pilot activity an important activity of the E&H Action Plan will be provided. Support for new and emerging issues and approaches have not been forgotten, such as the use of GIS approaches to support environmental epidemiology and to investigate the health impacts of drought and desertification. Specific efforts have been made to introduce international co-operation more strongly in the environment and health area and to link to international initiatives such as GEO in which environment and health is also a well-identified priority. The impact pathway approach adopted in FP6 will therefore be further strengthened. Medium-term goals Later calls for proposals will continue supporting environment and health research by focusing on environmental stressors of greatest importance for European populations. Particular focus will be on vulnerable population groups such as children and ageing population. The approach taken is practical, combining projects on specific targeted issues such as health impacts of electromagnetic fields with those which will take a more integrated approach in support of policy making such as integrated health risk assessment of exposure to multiple urban risk factors such as noise, air pollution, and chemicals,

taking into account socio-economic factors. The general aim will be to generate new evidence and at the same time develop/enhance risk and impact assessment models and tools that can help us in better understanding and deciphering health and environment interactions. The ultimate aim will be to aid policy makers in identifying prevention strategies through a better understanding of the full chain of causeeffect relationships: sources emissions concentrations exposures and health effects economic valuation. Short and medium term priorities for research are presented in Annex 2. Cross-thematic approaches and links In FP6, environment and health research was implemented through three thematic areas: Health, Food Quality & Safety, and Environment research. In FP7, environment and health research has been identified as an own activity under Theme 6 Environment (including Climate Change). The aim is to focus on multidisciplinary research activities on interactions of environmental risk factors and human health, taking into account exposures via different exposure routes. In Theme 1 Health, focused research on major diseases, such as cancer and cardiovascular diseases, including chronic diseases and public health research on disease prevention, will complement the E&H research activity. In addition, research on food, nutrition and diet-related diseases and disorders will be the subject of Theme 2 Food, Agriculture and Biotechnology. These areas are seen more complementary to the approach taken in the environment and health area. In some specific cases, possibilities for coordinated calls between the E&H activity and the other two themes could be envisaged. International collaboration will be enhanced via specific topics especially relevant to third countries. The research efforts will also support the goals of specific international initiatives such as GEO or other international activities, such as sustainable development or water and health initiatives. Efforts will be made to enhance synergies between the EC Environment and Health activity and similar efforts elsewhere, e.g., in the USA, possibly via co-ordinated calls.

Work programme 2007 To build up the work programme for 2007, the environment and health research activity in FP7 is divided in three areas: 1. Health effects of exposure to environmental stressors Indicative topics for first call: Indoor air pollution in Europe: an emerging environmental health issue Environmental factors and their impact on reproduction and development 2. Integrated approaches for environment and health risk assessment Indicative topics for first call: European network on human biomonitoring European cohort on air pollution Drought and desertification-related public health and socio-economic impacts 3. Delivery of methods and decisions to support tools for risk analysis and policy development Indicative topics for first call: Geographical information systems in support for environment and health research ERA-NET for environment and health Conclusion Taken together, the multidisciplinary research efforts to be supported in FP7, linking environmental issues to health, should contribute in a significant manner to the main aim of the E&H Action Plan, namely understanding the links between environmental stressors and health impacts, taking into account individual susceptibilities and focusing on certain priority health outcomes. Other key driving forces behind the adopted research strategy are the Earth Observation initiative, and the Sustainable Development Strategy with its focus on health impacts of environmental chemicals. Thematic Strategies on the Urban Environment and Air Pollution as well as the Programme of Community Action in the Field of Public Health are also important initiatives identifying future research needs. The first calls are likely to be launched at the end of 2006 or the beginning of 2007.

Contact: Dr Tuomo Karjalainen/Dr Kirsi Haavisto European Commission, DG Research Directorate I (Environment) B-1049 Brussels, Belgium tuomo.karjalainen@ec.europa.eu kirsi.haavisto@ec.europa.eu Note: The indicative topics and priorities given in this paper are subject to change.

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Weybridge +10 workshop

European workshop on the impact of endocrine disrupters The European Union (EU) has embarked on massive research efforts on endocrine disrupters and to date has launched scientific projects worth more than 140 million Euros. A lot of this research has now been completed, and new findings concerning the effects of chemicals have emerged. Progress has been made in pinpointing human life stages particularly vulnerable to endocrine disruption, and new data about male reproductive health and wildlife effects have come to light. To review the implications of these new findings for EU policy making, an international workshop was held in Helsinki (November 8-10, 2006), ten years on from the 1996 European Workshop on the Impact of Endocrine Disrupters on Human Health and Wildlife (so-called Weybridge meeting). International experts and policy makers from EU member states assessed the new research findings, identified knowledge gaps, defined future research priorities and considered the implications for policy making and chemicals regulation. The Helsinki workshop was held under Finlands presidency of the EU and organised by the Academy of Finland, with cooperation from the European Commission's Research Directorate-General and the European Environment Agency. Results of the 2006 Helsinki workshop will be published as a consensus document. For more information see www.aka.fi/euseminars

Participating projects
COMPRENDO Coordinator: Dr Ulrike Schulte-Oehlmann www.comprendo-project.org EDEN Coordinator: Dr Andreas Kortenkamp www.edenresearch.info EURISKED Coordinator: Prof. Wolfgang Wuttke www.eurisked.org FIRE Coordinator: Prof. Antoon Opperhuizen www.rivm.nl/fire

Associated projects
ACE BONETOX EASYRING EDERA ENDOMET GENDISRUPT MENDOS SENSPESTI

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CREDO coordination
For further information and to be kept informed of developments within CREDO please contact: Dr Andreas Kortenkamp andreas.kortenkamp@pharmacy.ac.uk Tel/fax: +44 20 7753 5908 Dr Ragnor Pedersen ragnor.pedersen@pharmacy.ac.uk Tel/fax: +44 20 7753 5811 Centre for Toxicology The School of Pharmacy,University of London 29-39 Brunswick Square, London WC1N 1AX United Kingdom

Copenhagen workshop on endocrine disrupters

Consumer products and endocrine disrupters: possible effects on human populations May 28 May 31, 2007, Copenhagen, Denmark The fourth Copenhagen workshop on endocrine disrupters to be held at Rigshospitalet is intended to facilitate an exchange of information and views both within the scientific community and with experts engaged in regulation and policymaking. The workshop will focus on possible effects of exposures to endocrine disrupters present in our everyday life e.g. in our food, cosmetic and our homes. The role of mixed exposures and links between effects in laboratory animals and observations in wildlife and humans will also be addressed. Effects both on reproduction and non-reproductive organs will be discussed. The meeting is supported by the Danish Ministry of the Environment. More information is available at www.reproduction.dk

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Contact at the European Commission: Dr Tuomo Karjalainen Tuomo.Karjalainen@cec.eu.int Dr Kirsi Haavisto Kirsi.Haavisto@cec.eu.int Dr Andreas Kortenkamp, 2006 Disclaimer While every effort has been made to provide correct information, we assume no responsibility for the accuracy of the information. Although this describes EU-funded projects, neither the European Union nor the European Commission shall be held responsible for the content, nor the views expressed here.

Endocrine disrupting contaminants diet interaction database (EDID)

Diet is a major source of exposure to contaminants such as endocrine disrupters. It is also a major modifier of the organisms susceptibility to xenobiotics, through the intake (adequate, unbalanced or deficient) of nutrients and natural bioactive substances. Limited knowledge is available on the interactions among xenobiotics and substances naturally present in food commodities, even though more information would be highly relevant to the more general issues of food safety and prevention. The Italian Istituto Superiore di Sanit (ISS) website on endocrine disrupters has been updated with a section dedicated to a brand new database named EDID: Endocrine Disrupting Contaminants Diet Interaction Database. EDID is a database of studies present in the international literature, either on experimental systems, on animal populations or humans, which aims to be easy to consult, periodically updated and to stimulate further research in this field. EDID has been created as part of the ISS special project on endocrine disrupters. The database may be accessed at www.iss.it/inte/edid/cont.php?id=110&lang=2&tipo=17

The CREDO cluster is funded by the European Commissions Fifth Framework Programme for research, technological development and demonstration activities in the European Community. A joint project funded by two thematic programmes: Quality of Life and Management of Living Resources Programme, and Energy, Environment and Sustainable Development Programme.

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ISSN 1744 - 1978