Evaluation of in vitro assays for determination of estrogenic activity in the environment

Evaluation of in vitro assays for determination of estrogenic activity in the environment

7 Conclusions

Background

Recent studies in a number of countries have shown that the aquatic environment can possess estrogenic activity capable of influencing the fauna.
(Xeno)estrogens are believed to reach the aquatic environment mainly by means of municipal and industrial sewage outfalls. However, agricultural drainage may also be a route for (xeno)estrogens to enter the aquatic system.
In the aquatic environment, estrogenic activity has primarily been ascribed to the natural steroids, 17b-estradiol (E2), estrone (E1) and estriol (E3), and the synthetic estrogen, ethinylestradiol (EE2). To a lesser extent, xenoestrogenic chemicals, such as alkylphenols and bisphenol A, may also contribute to the estrogenic activity in the aquatic environment.
In vitro assays measure the total estrogenic activity of an environmental water sample. The total estrogenic activity in the sample is then compared to the activity of E2 and expressed as estradiol equivalents (EEQ).
Total estrogenic activity (expressed as EEQ values) of sewage treatment plant influents have been reported to be 0.6-153 nanograms per litre. In the effluents, EEQ values are usually below 25 nanograms per litre, although values of up to about 150 nanograms per litre have been reported in the USA.
In surface water, the EEQ values found are generally from below 1 nanogram up to 15 nanograms per litre, although values of up to about 80 nanograms per litre have been reported in one study.
The EEQ levels found in some aquatic systems are sufficient to cause estrogenic effects in fish in laboratory experiments.

Sample preparation for in vitro analysis

The assessment of estrogenic activity in environmental water samples begins with sample collection and storage until analysis. However, detailed descriptions of the modes of water sample collection and storage for in vitro investigations are lacking in most papers.
Some studies suggest that there may be loss of compounds during the extraction procedure, particularly when employing solid-phase extraction.
The filtration and extraction procedures for environmental samples should be properly validated and optimised before the onset of extensive monitoring studies.

Evaluation of in vitro assays

Several in vitro assays have been developed to screen for estrogenic activity, including estrogen receptor binding assays, MCF-7 cell proliferation assay s (E-screen) and reporter gene assays
The detection limits for the various assays differ:
- ER binding: 272 ng E2/l
- E-screen: 0.27 ng E2/l
- Mammalian-based reporter gene assay (ER-CALUX): 0.1 ng E2/l
- YES assay (yeast-based reporter gene assay): 1-3 ng E2/l
By including the concentration factors employed when assaying environmental samples, the detection/quantification limit for the whole method can be determined:
- E-screen: 0.07-0.14 ng EEQ/l
- ER-CALUX: 0.001 ng EEQ/l
- YES assay: 0.03 ng EEQ/l.
Estrogen receptor binding assays have poor sensitivity and are not easily amenable to automation, thereby limiting their utility for monitoring studies of environmental samples.
The E-screen has the limitation, that a positive response cannot be attributed strictly to estrogen receptor agonists, since a range of non-estrogenic substances has been found to influence the proliferation of MCF-7 cells, at least in some cell lines. In addition, considerable inter-laboratory variability has been observed in test results from the E-screen. Finally, the long assay time is considered impractical for extensive monitoring studies.
Reporter gene assays seem to be a suitable choice for monitoring environmental matrices for estrogenic activity. Reporter gene assays are divided in the mammalian-based and the yeast-based assay s.
Some mammalian-based reporter gene assays are highly sensitive. However, mammalian cells are more difficult and expensive to cultivate. The yeast-based reporter gene assay (YES) is less sensitive and does not always detect antagonistic activity. However, it is more simple and inexpensive to perform.
The final choice of which reporter gene assay to employ (mammalian-based or yeast-based) depends on the importance of a lower detection limit versus the importance of ease of use and lower costs.
Reporter gene assays could be proposed as a first step for identifying environmental samples with estrogenic activity. However, they should be complemented by in vivo assays, taking into account the complexity of processes occurring in whole organisms, for assessment of potential adverse effects on the fauna