Detection Of Estrogenic Activity Research Articles

Replacing animal-derived components in in vitro test guidelines OECD 455 and 487

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns.In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis.All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium.Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations.The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites.A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos.Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.

Detection of estrogenic activity in Slovenian wastewaters by bioassay

Abstract Estrogenic activity has been detected in aquatic ecosystems across the world. However, there is a lack of such data for Slovenian wastewaters and surface waters. The Slovenian monitoring program of effluents discharged into surface waters does not require that emissions of natural and synthetic estrogens into aquatic environments be assessed and controlled. In our study, we assessed the potential estrogenicity of wastewater samples from three wastewater treatment plants using a yeast estrogen screen assay (YES assay). Due to the high inhibition of yeast growth in samples obtained during our first sampling period, it was impossible to detect any estrogenic activity. An additional silica gel clean-up step reduced the toxicity of samples collected during our second sampling period; as a result, we were able to record up to 95% relative estrogenic activity inhibition. Deconjugation of the estrogens did not significantly influence our results. We detected estrogenic activity using a YES assay in almost all influent and effluent samples tested, which suggests that the wastewater treatment plants (WWTPs) examined do not effectively remove (xeno)estrogens from wastewaters. Our results suggest that a YES assay is an appropriate screening method for monitoring estrogenic activity in effluents. However, prediction of the potential impacts of wastewater (xeno)estrogens on aquatic organisms require additional in vitro and in vivo assays.

Detection of Estrogenic Activity from Kraft Mill Effluents by the Yeast Estrogen Screen

Estrogenic activity of kraft pulp mill effluents (P. radiata, E. globulus and mixed -50% E. globulus and 50% P. radiata) was evaluated by the yeast estrogen screen assay. The estrogenic activity values were relatively low, ranking between 1.475 and 0.383 ng/L of EE2 eq. (Estrogenic equivalent of 17 alpha-ethynylestradiol), where the highest value corresponds to the E. globulus effluent and the lowest value to the P. radiata effluent. Analysis by solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) of chemical compounds present in all three effluents detected at least five major groups of organic compounds, corresponding to fatty acids, hydrocarbons, phenols, sterols and triterpenes. Comparison of analytical and biological data suggests that sterols could be the cause of the estrogenic activity in the evaluated effluent.

Detection of estrogenic activity in herbal teas by in vitro reporter assays

Herbal teas have become popular as alternatives to caffeinated beverages during past two decades. However, toxicological studies of herbal teas have been limited and the safety of herbal teas thus remains unknown. We focused on the estrogenic activities of herbal teas since some of their ingredients are similar to those used in herbal remedies for menopause relief and therefore contain phytoestrogens. To investigate the potential estrogenic activity of extracts prepared from herbal tea mixtures commercially available and to provide useful information for the safety assessment of those products, we initially screened the estrogenic activity in extracts of 15 different herbal teas by an assay using recombinant yeast cells expressing the human estrogen receptor (YES). A distinct estrogenic activity was thus detected in the ethanolic extracts from four herbal tea mixtures. Licorice root was specified as a ingredient responsible for the estrogenic activity in those extracts. In contrast, the aqueous extracts of all herbal tea mixtures we tested exhibited distinct estrogenic activity in YES, thus suggesting the existence of various ingredients that contain estrogenic constituents extractable with water. Among them, the extract of peppermint tea exhibited the highest estrogenic activity. The estrogenic activity in extracts of herbal tea mixtures and specified ingredients were thereafter confirmed by a reporter assay system using transiently transfected HEK293 cells.

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17β-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182 780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100 000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.

Detection of estrogenic activity in Flemish surface waters using an in vitro recombinant assay with yeast cells

Numerous environmental chemicals possess estrogen-like properties. At elevated doses, natural estrogens and environmental estrogen-like chemicals are known to produce adverse effects on humans and wildlife. Sources of potential exposure to endocrine disrupting compounds have to be identified for risk and hazard assessment. Extracts prepared from 16 selected water samples taken in Flemish rivers, effluents of municipal wastewater treatment plants and reservoirs for drinking water production were analysed for estrogenic activity with a cellular bioassay. Yeast cells, which are stably transfected with the DNA sequence of hER and which contain expression plasmids with the reporter gene lac-Z, encoding the enzyme beta-galactosidase, were used to measure receptor binding. Flemish rivers showed the highest estrogenic potency, compared to effluents of waste water treatment plants and reservoirs which showed low induction factors (beta-galactosidase production) relative to solvent control conditions. By comparison with a standard curve for 17 beta-estradiol (E2), estrogenic potency in water samples was calculated as E2-equivalents and ranged from below detection limit (approximately 2.75 ng E2/l) up to 81.4 ng/l E2-equivalents. About 7 water samples had more than 10 ng/l E2-equivalents. These elevated levels of E2-equivalents are likely to exert significant adverse effects on reproduction success of wildlife, which should be verified with in vivo studies.

Detection of estrogenic activity in berseem (Trifolium Alexandrinum)

Detection of estrogenic activity in berseem (Trifolium Alexandrinum)