1 Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark2 National Food Institute, Technical University of Denmark
BACKGROUND: This PhD project is part of the research area concerning effects of endocrine disrupters at the National Food Institute at DTU in Denmark. Endocrine disrupting chemicals (EDCs) have proved to be important for improper development of the male reproductive organs and subsequent for the potential inability to reproduce. Most recently, it was found that combinations of chemicals - each at a concentration where the single compound gave no effect - led to significant effects in experimental animals. There is a need for more knowledge about the mechanisms behind the observed effects, to be able to detect effects and predict mixture effects. In addition, a new hypothesis have emerge concerning a potential role of exposure to endocrine disrupting chemicals, and the development of obesity and obesity related diseases. AIM: This PhD project aimed to gain more information regarding the mechanisms behind the effects of EDCs. The focus of the current project has been on the use of in vitro assays to investigate: • The endocrine disrupting potential of phytoestrogens (PEs) and mixtures of PEs • How PEs and mixtures of PEs affect pathways involved in the development of obesity • The use of in vitro metabolising systems in conection with in vitro testing of EDCs METHODS: Twelve dietary relevant phytoestrogens (PEs) either alone or in mixtures were analysed in a battery of in vitro bioassays designed to look for effects on: a) steroid hormone production in human adrenal corticocarcinoma cells (H295R steroid synthesis assay), b) aromatase activity in human JEG-3 choriocarcinoma cells, c) estrogenic activity using the human MCF-7 cell proliferation assay, d) interaction with the androgen receptor (AR) in a reporter gene assay, e) effects on adipogenesis in the 3T3-L1 preadipocyte cell line, and f) effect on PPAR α and γ using a transactivation assay. For the in vitro metabolism studies, ten selected EDCs: five azole fungicides, three parabens, and two phthalates, were tested in vitro in the T-screen assay to determine possible changes in the ability of the EDCs to bind to and activate the thyroid receptor (TR) after biotransformation. The two in vitro metabolising systems applied were human liver S9 mix and PCB-induced rat microsomes. The ability of the two selected in vitro metabolising systems to metabolise the ten test compounds, as well as the evaluation of the endogenous metabolic capacity of the GH3 cells, applied in the T-Screen assay, were examined using LC-MS analysis RESULTS: The results showed that all the tested PEs and PE-mixtures increased estradiol production in the H295R cells. The mixture containing all tested PEs, as well as the isoflavonoids also decreased testosterone production in H295R cells, indicating an induced aromatase activity. Furthermore, many of the tested PE-mixtures significantly stimulated MCF-7 human breast adenocarcinoma cell growth, and induced aromatase activity in JEG-3 choriocarcinoma cells. In the PPAR transactivation assay, the different PE-mixtures had stronger effect on PPARγ than on PPARα, with some mixtures showing PPARγ agonistic effects while others had more PPARγ antagonistic effects. Furthermore, the tested single PEs, as well as mixtures of PEs had an inhibitory effect on lipid accumulation in vitro, although at higher concentrations than nutritionally relevant. In the in vitro metabolism studies no marked difference in the effects in the T-screen assay was observed between the parent compounds and the tested metabolic extracts. The GH3 cells themselves significantly metabolised the two tested phthalates, dimethyl phthalate (DMP) and diethyl phthalate (DEP). The two in vitro metabolizing systems tested gave an almost complete metabolic transformation of the tested parabens and phthalates, with a recovery rate of the parent compounds of less than 1%. However, a difference was found between the human S9 and rat microsome assay systems when looking at the metabolism of the azole fungicides. The PCBinduced rat microsomes gave a statistically significant difference between the amount of parent compound before and after treatment with the microsomes for four out of the five azole fungicides tested. When using the human liver S9, no significant metabolic transformation of the azole fungicides was detected. CONCLUSIONS AND PERSPECTIVES: Overall, the results from the studies presented in this thesis support the evidence suggesting that nutrition relevant concentrations of PEs, both alone and in mixtures, induce various endocrine disrupting effects. The main effect seems to be an estrogenic effect mediated both at the receptor level as seen in the MCF-7 cell proliferation assay, but also at the level of steroid synthesis, as seen in the H295R cell assay. Additionally, many PEs, as well as mixtures of PEs have an inhibitory effect on lipid accumulation in vitro, an effect that could involve the estrogen receptor, and also a result that could suggest a beneficial effect of PEs with regard to obesity. However, the role of the different players involved in adipogenesis and lipolysis is still not understood. Therefore, based on the current results, the influence of PEs on adipogenesis and their effects on the different pathways involved in the development of obesity and obesity related diseases remains unclear, and needs further investigation. Finally, the results and qualitative data from the in vitro metabolising studies show that an in vitro metabolising system using liver S9 mixtures or hepatic rat microsomes could be a convenient method for the incorporation of metabolic aspects into in vitro testing for endocrine disrupting effects.