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Abstract
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.
Original language | English |
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Place of Publication | Søborg |
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Publisher | Technical University of Denmark |
Number of pages | 77 |
Publication status | Published - May 2011 |
Fingerprint
Dive into the research topics of 'Mechanistic evaluation of endocrine disrupting chemicals'. Together they form a unique fingerprint.Projects
- 1 Finished
-
Design, fabrication and testing of support structures for biomimetic water filters
Taxvig, C., Nellemann, C., Granby, K., Kortenkamp, A. & Andersen, H. R.
Technical University of Denmark
01/03/2008 → 18/05/2011
Project: PhD
Prizes
-
DTU´s Young Researcher Award 2011
Taxvig, C. (Recipient), 2011
Prize: Prizes, scholarships, distinctions
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