Research “Estrogenic compounds effect on the physiology and reproduction of frogs”   Project background Endocrine-disruptors are a group of compounds that are able to alter human and other animals natural hormone functions. Some of these compounds are found naturally in plants, but the majority is artificially manufactured and is found for example in pesticides, industrial chemical used in the production of certain products such as plastics, and in sewage from household products (detergents, soaps) and excreted contraception. The environment, including animals and humans, are increasingly exposed to endocrine-disruptors. It is known, that these compounds can influence the hormone system by either mimic, block or alter the natural function of the body’s hormones. This can cause both physiological and behavioural changes. It is still not fully elucidated, however, precisely what changes these compounds cause and which compounds are dangerous (1).   Many of the endocrine-disruptors are termed estrogenic or anti-androgenic, because they influence the sexhormone profile, the development of male- and female sexorgans and secondary sexcharacteristics and hereby create fertility problems. As mentioned, endocrine-disruptors can alter the normal hormonal functions by several means. Some of these compounds mimic or partly mimic the sex steroid hormones (estrogens and androgens (testosterone)) by binding to hormone receptors or influence the cell signalling pathways that the hormone-receptor complex initiates. Other endocrine-disruptors block or alter the hormones binding to the receptors. Finally, some block or alter the production or breakdown of either the hormone itself or its receptor (2). In contrast to natural steroid hormones, many of the synthetic endocrine disruptors are slow to break down in the environment and can accumulate in nature and in fat and muscle tissue in animals. In Denmark an study of brown trout (Salmo trutta) and roach (Rutilus rutilus) in sewage effluent receiving streams, has caught the attention of the public because grave changes in the reproductive system of the fish was found. In this study they found immature oocytes in the testicular tissue (intersex) of male fish, as well as females with apparently normal ovaries but with small areas of sperm cells in various stages of development. Moreover, in some males the testicles contained vacuoles resulting in an apparent negative effect on the development of the sperm cells (3). In frogs (Xenopus laevis), where larvae had been exposed to the estrogenic pesticide Atazin in ecologically relevant doses, several individuals had multiple gonads or were hermaphrodites with both ovaries and testicles. Also retarded gonadal development and testicular oocytes were found (4). In Denmark we have eight different species of frogs and three species of toads, all of which have declined sharply in numbers over the past 50 years. The reason for this decline is probably mainly due to loss of habitat, but also due to deterioration in the quality of remaining habitats. Frogs are closely connected to the aqueous environment and are therefore extremely sensitive to pollution. As mentioned, laboratory experiments has shown that eggs, larvae or juvenile frogs that have been exposed to endocrine-disruptors, has both morphological and hormonal changes that might disrupt or altogether abolish reproduction. However, it is not known if wild frogs have similar changes. In Denmark this has not been investigated. To conserve the remaining frog population in nature it is therefore important to find out more about if endocrine-disruptors are a treat to wild frogs. Documenting information (cause and effect) about endocrine-disruptors is still sparse, and there is still a lot of work that needs to be done. The aim of my project is to look at immediate and long-term reproductive consequences in frogs, both physiological, anatomical and behavioural, of exposure to ecologically relevant doses of endocrine disrupting compounds. The general hypothesis that will be tested is if certain endocrine-disruptors, administered during early development, even in small doses, will change the reproductive ability in adult frogs. A list of questions will sought answered: -         What potentially endocrine disrupting compounds causes alteration in the reproduction of frogs, and in what doses? -         What physical, morphological and behavioural abnormalities in frogs are caused by the endocrine disrupting compounds? -         Is there in Denmark found any wild frogs with reproductive abnormalities that may indicate that they have been exposed to endocrine-disruptors? I have chosen to test the compounds ethinylestradiol, nonylphenol and maybe one or more of the estrogenic pesticides that are used by farmers in Denmark. Ethinylestradiol is a synthetic estrogen found as one of the main ingredients in the contraceptive pill. A large percentage (approx. 26%) of the ingested ethinylestradiol is excreted and is found in the sewage effluents in doses that in the laboratory can cause reproductive abnormalities (4). Nonylphenol is used in cleaning agents, surface active ingredients, cosmetics, foaming agents, paint and a long list of other products. Doses of nonylphenol found in the Danish aquatic environment also here exceed concentrations that in the laboratory have caused physical abnormalities in fish (4). Finally, a list of pesticides that are under suspicion of being endocrine disrupting today used or has previously been used in the agricultural field in Denmark. Studies show that some of these pesticides and their breakdown products can be widely found in streams and lakes (Danish Environmental Protection Agency (4)).   The project is divided up into three phases. First of all, the immediate consequences of exposure to endocrine-disruptors will be studied. Here I want to do a morphological and histological assessment of the development of the reproductive tissue of juvenile Xenopus tropicalis exposed to the described estrogenic compounds during development (egg and larvae). In the second phase I will look closer at the long-term consequences of this exposure using adult frogs. I will look at the sexhormone profile (by measuring circulating concentrations of androgens and estrogens in both sexes), analyse for vitellogenin (induction of vitellogenin synthesis in males of this female egg precursor is a biomarker for abnormal steroidhormone function (5)), egg/sperm development (egg mass and sperm quality), size of larynx (size is influenced by testosterone), and sexual behaviour observations (calling frequency and duration – the males calling is testosterone dependent and important in courtship). The final phase will be fieldwork with a study of the frequency of reproductive deformities in Danish frogs collected at different locations (lakes and ponds) with a known concentration of endocrine-disrupting chemicals. The studies will commence in April 2003.  Contact Publications CV

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