Development and application of in vitro and in vivo reporter gene assays for the assessment of (xeno-)estrogenic compounds in the aquatic environment

In recent years, both scientific and public concern about the possible threat of estrogenic compounds in the environment that may impact the reproduction of humans and wildlife has increased. Many substances have been demonstrated to possess estrogenic potency using in vitro test systems, and these compounds have been identified in the environment using chemical analysis. However, up until now, it has been difficult to rapidly estimate the total biologically active estrogenic potency in environmental mixtures, as well as predict the exposure of and possible effects on organisms. The research presented in this thesis describes the development and application of two new bioassays to determine the biologically active exposure of natural and xenobiotic estrogens (xeno-estrogens) in the environment and predict the estrogenic effects of such exposure on fish. These new bioassays may help to determine if organisms are exposed to levels of (xeno-)estrogens which may impact their reproduction or endocrine functioning.An in vitro estrogen receptor (ER)-mediated chemical activated luciferase reporter gene expression (ER-CALUX) assay was developed by stable transfection of an ER regulated luciferase reporter gene in the T47D human breast cancer cell line. The ER-CALUX is very sensitive and highly responsive to natural estrogens, such as estradiol (E2), with a detection limit of 0.5 pM E2, an EC50 of 6 pM E2 and a maximum induction of 100-fold at 30 pM relative to solvent controls. The presence of anti-estrogenic activity can be determined by co-incubation with E2, and was demonstrated for ICI 182,780, TCDD and tamoxifen. A variety of xeno-estrogenic compounds are estrogenic in the ER-CALUX assay, including pesticides, alkylphenols, phthalates and phytoestrogens, though with a potency 10,000 to 100,000 times less than E2. In comparison to a widely used recombinant yeast screen, the ER-CALUX assay was more sensitive to both (xeno-)estrogens and anti-estrogens. Given the exquisite sensitivity of the ER-CALUX assay, it can be an ideal screening tool for determining estrogenic activity in complex mixtures from various environmental matrices, such as water, sediment and particulate matter. A number of sediment extracts obtained from industrialized areas such as the Port of Rotterdam showed high estrogenic potency. In addition, a simple method was developed to determine if estrogen metabolites present in human urine and fish bile could be reactivated into active estrogens. Estrogen glucuronides in urine obtained from human males and females were deconjugated by enzymatic hydrolysis following incubation with ß-glucuronidase or live E. coli cells, indicating that bacterial hydrolysis as present in wastewater treatment plants may form a secondary source of estrogenic substances in the environment. Also, glucuronides in bile samples from male fish showed estrogenic activity following deconjugation. These conjugated metabolites could also form a secondary source of estrogen exposure in fish due to enterohepatic recycling. High estrogenic activity of deconjugated bile metabolites from male bream correlated with elevated estrogenic potency in water extracts as well as vitellogenin induction in blood of the same fish.An in vivo reporter gene assay using transgenic zebrafish was developed by stably introducing an estrogen binding sequence linked to a TATA box and luciferase reporter gene into the genome. This is the first report of an in vivo reporter gene assay to assess exposure to estrogen modulating substances. Exposure of the transgenic zebrafish to E2 during juvenile stages revealed the period of gonad differentiation to be the most responsive early life stage. In this juvenile period, E2 induced luciferase activity in a dose-dependent manner. Also the natural estrogens estrone (E1) and 17α-estradiol, as well as the synthetic estrogens diethystilbestrol and ethinylestradiol (EE2), and the organochlorine pesticide o,p'DDT demonstrated estrogenic activity in vivo in this test system. In adult male transgenic zebrafish, short-term exposure to E2 revealed a number of tissues such as liver, bone, muscle and scales to be a potential target organ for E2. However, the testis was the most sensitive and responsive target tissue to estrogens. Exposure to E2 resulted in dose-response related luciferase induction in the testis. Immunohistochemistry revealed the Sertoli and germ cells to be the main target cells of E2 in the zebrafish testis. ERαandβmRNA was detected already in young embryos (24 hours post fertilization) and both subtypes were highly expressed during the period of gonad differentiation. In adult male transgenics, tissues with the highest luciferase activity (testis and liver) expressed both ERαandβmRNA.Exposure of ER-CALUX cells is very direct without interaction with environmental factors influencing bioavailability and accumulation and without the toxicokinetics of an intact organism. In the transgenic fish assay, however, these factors do play a role, resulting in concentrations in the cell which are sometimes difficult to predict using an in vitro system. Both assays exhibited high sensitivity to natural and synthetic estrogens such as E2, E1 and EE2, though differences in their relative potencies to E2 were found. EE2 was 100 times more potent than E2 in the transgenic fish, whereas it was only slightly (1.2 times) more potent than E2 in the ER-CALUX assay. The highly accumulating xeno-estrogen o,p'DDT was estrogenic in both assays, with similar potencies relative to E2. On the other hand, xeno-estrogenic chemicals with which are readily metabolized in vivo such as nonylphenol and bisphenol A were estrogenic in vitro but did not induce reporter gene expression in vivo in short- term exposures. Estrogenic activity was also found in mixtures reflecting concentrations of (xeno-)estrogens found in effluents of a domestic wastewater treatment plant, using both the ER-CALUX and transgenic zebrafish assays. In the transgenic zebrafish, levels of estrogenic activity found in this effluent exceed levels reported in the literature necessary to induce vitellogenin induction and inhibit testicular growth and reproduction in other fish species. This indicates that fish at this particular location may be exposed to (xeno-)estrogens at levels that may impact their reproductive success.This study resulted in the development of two important new biological tools to identify estrogenic compounds. These tools are very valuable in determining if substances in the environment are present at levels that may interfere with reproduction or endocrine functioning in organisms. The in vitro ER-CALUX assay with T47D cells is the most sensitive and responsive stably transfected reporter gene assay reported to date and has already proven indispensable in rapidly and cost-effectively determining total biological potency of estrogenic chemicals in complex environmental mixtures. The in vivo transgenic zebrafish assay is the ideal complement to the ER-CALUX assay because environmental chemistry and toxicokinetics of compounds are taken into account. This assay is important for predicting the estrogenic effects of chemicals on fish. The surprising result that the male testis, and not the liver, is the most sensitive and responsive target tissue to estrogens underlines the vulnerability of the male reproductive organs and provides a scientific basis for the world-wide observance of male gonad abnormalities in wild populations of fish. These newly developed techniques have demonstrated that though probably not widespread, there are some localized areas of concern of elevated (xeno-)estrogenic exposure in the Dutch aquatic environment.