Endocrine disrupting chemicals are substances that affect its homeostasis. In the last 50 years, there have been recordings about growth of incidence and prevalence of health issues due to disruption of endocrine system, including different kinds of hormone-dependent cancers, diabetes, obesity, metabolic syndrome, reduced development of the organism and reduced fertility. Because the main source of endocrine disrupting chemicals for a human represent different substances in food, among which we also count natural compounds, isoflavonoids, and synthetic bisphenols, we have focused in the master's work mainly on them.
The purpose of the master's work was to evaluate agonistic estrogenic activity of selected isoflavonoids and bisphenols on epithelial human cervical cancer cell line, with the help of stably transfected transactivation assay in vitro. The used HeLa-9903 human cell line was stably transfected with gene for human estrogen receptor alpha and enzyme luciferase gene construct. After the agonist binds on mentioned receptor, the two produce the luminous signal and its potency enables us to measure the activity of testing substances. We also verified the reporter cells' viability with the citotoxic assay. For substances that caused supra-maximal luciferase activity, we also performed competition assay. With its help we verified, if the luminous signal was really due to gene's induction after binding of selected substance on human estrogen receptor alpha, or it may have induced the detecting system through some other mechanisms.
The results among positive-working substances have shown that their estrogenic activity was concentration-dependent. The values of the concentrations, corresponding to 10% (PC10) and 50% (PC50) of the positive control, were in nM or low µM range in testing both isoflavonoids and bisphenols. The majority of tested isoflavonoids caused supra-maximal luciferase activity at concentrations, >1 μM. With competition assays for some of the isoflavonoids we have proven, that they induced responses by their activation of the human estrogen receptor alpha and through some other, yet not known mechanism. Genistein, formononetin, ononin and prunetine proved to be the strongest or the most effective agonists among tested isoflavonoids. They were followed by biochanin-A and daidzein, as well as R,S-equol and S-equol. Even more weaker agonists were calycosin and glycitin, while the weakest one, cladrin, was little above the border of agonistic activity. Puerarin didn't have agonistic activity. All tested bisphenols were proven to be strong agonists. Comparing their agonistic activity, bisphenol F was the most effective agonist, followed by bisphenol A and bisphenol AF, with similar effects, and bisphenol S with the lowest effectiveness.
Because of the supra-maximal luciferase activity, which was caused by the majority of isoflavonoids, further researches about their specific estrogenic activity will be necessary. In this case, the development of new methods in vitro and/or in vivo, that would give us more reliable results, will be inevitable.
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