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endocrine effects of plastic     Προσθέθηκε στις: 28/05/2007
  Endocrine Effects of Marine Plastics on Kelp Bass Baker, M.E., Moore, C.J. and Andrady, A. (2002)Endocrine Effects of Marine Plastics on Kelp Bass Baker, M.E., Moore, C.J. and Andrady, A. (2002) Research Study Proposal by UC San Diego Department of Medicine and Algalita Marine Research Foundation (Algalita.org) Introduction. Plastic resin pellets in the form of small granules of 0.1 to 0.5 centimeters are widely distributed in the ocean. At this size, plastic pellets are consumed by fish, which can have profound disruptive effects on their endocrine system. Compounds in plastics such as bisphenol have been known to have affect the estrogen response in male and female fish and other vertebrates [1-4]. Recently, Mato et al. [5] showed that plastics accumulate other toxic chemicals such as PCBs and DDE, an anti-androgen [6]. This increases the danger and urgency of controlling plastics in the marine environment. We propose to study the effects of plastics in the marine environment as one step in providing information that can be used in regulating disposal of plastics. For this pilot proposal, we begin with studies on the estrogen response in kelp bass. Later, we will extend our studies to responses to other steroids (e.g. androgens, glucocorticoids, progestins, vitamin D3) and hormones (e.g. thyroid hormone), which have not been studied in as much depth as the estrogen response. Specific Aim 1. To study the effect of marine plastics on the estrogen response in kelp bass. Rationale. Kelp bass can be conveniently grown in tanks and are easy sources of serum for analyses of vitellogenin synthesis and for analysis of plastics and other xenobiotics. We also propose to extract compounds in serum for measurement of inhibition of estradiol binding to its receptor. We propose to use kelp bass as marine equivalents of "canaries in the mine". Methods. Vitellogenin synthesis. A widely used and sensitive assay for estrogenicity is the induction of vitellogenin synthesis in male fish [7]. The background is very low, which makes this a very sensitive assay. Antibodies to bass vitellogenin are available. This permits us to use Western analysis and ELISA assays to monitor vitellogenin synthesis. Binding to estrogen receptor. We will study the binding to the ER of compounds in the serum of kelp bass that have consumed marine plastics and serum of the control fish that do not consume plastics. There are two estrogen receptor (ER) isoforms: ER-alpha and ER-beta [8,9]. Both receptors will be studied, since they have different affinities for some steroids and phytochemicals. We have extensive experience with using the dextran-coated charcoal method for determining binding of estradiol to the ER [10] and other steroids to their receptors [11]. Hydrophobic compounds in serum will be extracted with ethyl acetate. The extract will be dried and then resuspended in either ethanol or DMSO and added to tubes containing ER and radioactive estradiol. Two different incubation conditions will be used in the binding assay: incubation overnight at 4C or for 2 hours at 22C and 1 hour at 4C. Unbound steroid will be removed by incubation for 5 minutes with dextran-coated charcoal. Experiments in our laboratory have shown that there is not appreciable change in specific binding to the ER for incubations of 15 minutes with dextran-coated charcoal. We will use a 200-fold excess of DES to measure non-specific binding, which will be subtracted from total binding to get specifically bound counts. Estrogenic activity of marine xenobiotics in cell culture. We also will determine if the extracts have hormonal activity by studying the effect of extracts on the growth of an estrogen-dependent cell line, such as MCF-7 cells. An advantage of these studies is that they also determine whether xenobiotics are further metabolized to compounds that bind the ER. These studies also determine whether xenobiotics or their metabolites activate the hormone receptor and fully stimulate gene transcription (full agonist activity) or partially stimulate gene transcription (partial agonist) or inhibit gene transcription (antagonist). Protocols and pitfalls (e.g. removal of phenol red from growth medium) for studying estrogenicity of compounds in MCF-7 cells have been developed [12]. Moreover they can be extended later to other hormone dependent cell lines such as androgen-dependent LNCaP cells. Analysis of serum for phthalate esters and other plastic-derived compounds. Dr. Anthony Andrady has developed techniques for analysis of phthalate esters and other plastic-derived compounds. He will advise us on performing these assays. Analysis of steroids and other hormones. We will determine the effect of marine plastics on androgen, estrogen, glucocorticoid levels. Fish use 11-keto-testosterone as the main androgen instead of testosterone and dihydrotestosterone [13]. Fish also use different progestins than land animals [13]. Dr. Andrady will assist us in assays using HPLC and GC. Antibodies are available for cortisol, estradiol, estrone, testosterone, thyroid hormone and insulin. We have a laboratory at UC San Diego that routinely measures these hormones in serum. The above constitutes the main research for this pilot project. We have expertise for measuring binding of all classes of steroids to their receptors. Thus, in later studies we determine the effects on the binding of androgens, glucocorticoids, progestins and vitamin D3 to their receptors. We also can study the effects of compounds associated with marine plastics on the growth of androgen dependent cell lines, such as LNCaP cells. Lastly, we have expertise in steroid dehydrogenase assays. Hydroxysteroid dehydrogenases such as 11beta-hydroxysteroid dehydrogenase (11beta-HSD) regulate the concentration of cortisol and other glucocorticoids. 17beta-hydroxysteroid dehydrogenase (17beta-HSD) regulates the concentration of androgens and estrogens. Phthalates and other compounds that bind to either 11beta-HSD or 17beta-HSD would have profound affects on steroid hormone concentrations. References 1. White, R; Jobling, S; Hoare, SA; Sumpter, JP; Parker, MG. Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology, 135, 175-182 (1994). 2. Tyler, CR; Jobling, S; Sumpter, JP. Endocrine disruption in wildlife: a critical review of the evidence. Critical Reviews in Toxicology, 28, 319-361 (1998). 3. Soto, A.M., Justica, H. Wray, J.W., Sonnenschein, C. p-Nonyl-phenol: an estrogenic xenobiotic released from "modified" polystyrene. Environ. Health Perspect. 92, 167-173 (1991). 4. Sonnenschein, C; Soto, AM. An updated review of environmental estrogen and androgen mimics and antagonists. J. Steroid Biochem. & Molec. Biol. 65, 143-150 (1998). 5. Mato, Y., Isoibe, T., Takada, H., Kanehiro, H., Ohtake, C, Kaminuma, T. Plastic resin pellets as a transport medium for toxic chemicals in the marine environment. Environ. Sci. Technol. 35, 318-324 (2001). 6. Kelce, WR; Stone, CR; Laws, SC; Gray, LE; Kemppainen, JA; Wilson, EM. Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature 375, 581-585 (1995). 7. Sumpter, JP; Jobling, S. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environmental Health Perspectives 103, Suppl 7, 173-184 (1995). 8. Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson J-A. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors ? and ?. Endocrinology 138, 863-870 (1997). 9. Kuiper, GGJM., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag, P.T.,van der Burg, B., Gustafsson, J-A. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ?. Endocrinology 139, 4252-4263 (1998). 10. Baker ME, Sklar DH, Terry LS, Hedges MR. Diethyl Pyrocarbonate, a histidine selective reagent, inhibits estrogen binding to receptor protein in rat uterus cytosol. Biochem. Int. 11, 233-238 (1985). 11. Baker ME, Terry LS. Diethylpyrocarbonate, a histidine selective reagent, inhibits progestin binding to chick oviduct cytosol. Steroids 42, 593-60 (1983). 12. Nagel, SC; vom Saal, FS; Welshons, WV. Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum. J. Steroid Biochem. & Molec. Biol. 69, 343-357 (1999). 13. Baker, M.E. Adrenal and sex steroid receptor evolution: environmental implications. J. Molec. Endocrinol. 26, 119-225 (2001). Source: www.mindfully.org

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