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The objectives of the project were the follwoing:

Ultimate goal of the EURISKED project was to determine multi-organic effects of a variety of endocrine disrupters (EDs) and these are: Nonylphenol, Bisphenol A, Dibutylphtalate, Octyl-methoxycinnamate (OMC), 4-methylbenzylidene camphor (4-MBC), Benzophenone-2, Procymidon, Linuron, Resveratrol, 8-Prenylnaringenin, Genistein, Estradiol-benzoat, Androstandiol. Utilizing a variety of reporter cell systmes the first goal of the project was to determine steroidal (estrogenic, 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 endocrined disrupters were to be measured by physiological, molecular and morphological means. Steroids, but 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.

The scientific achievements of the project can be summarized as :

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 finished within the first 2 years, those with the reporter animals have been 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 estrogenic, 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. 8-Prenylnaringenin, 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 estrogenic 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, the iodide symporter, 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 estrogenic, an undesired effect if the substances are resorbed transcutaneously. Upon approval through the ethical committee, 5 female and 6 male human probands were subjected to treatment with 2 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 suscreen 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 100 ml 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 continuted for another 3 days and on the 4^th day the same treatment and blood withdrawal regimen was applied. Extraction, HPLC separation and UV detection did not detect any of the 2 sucreens. 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. With a much higher sensitivity extracts of selected blood samples were subjected the highly senisitive method of mass spectrometry and again no indication of presence of either of the 2 UV screens or their metabolites could be demonstrated. Hence, the measurement performed so far did not result in detectable serum levels.

The 2 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 exrpesses high amounts of the estrogen receptor of the beta subtype (ERß). It was therefore another focus of interest to investigate the effects of ERß in the prostate. By means of histological and immuncytochemical analysis and utilizing the ERß knock-out mouse model it could be demonstrated that the ERß appears to have anti-proliferative effects in the prostate, thereby having possibly protecting effects against the development of prostate cancer. From the 2 UV screens, 4MBC proved to be a pure ERß 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 dosis 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 Benhopheneone-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 arylhydrocarbone receptor (AhR) in some more detail. In a bioassay to determine the effects of stimulation of inhibition of the AhR it could be shown that genistein and resveratrol were AhR agonists, while E2, 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.

The results of the 3 years funding period enables the consortium to estimate the putative danger for the human.

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 good and will allow recommendations resulting in risk assessment for humans. These might result in guidelines. 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.