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Currently used progestin measurement systems either use human cell derived reporter gene bioassays (van der Linden et al., 2008, Viswanath et al., 2008) thus do not necessarily result in ecologically significant data, or require rather elaborate additional instruments (Vulliet et al., 2008, Vanderford et al., 2003). In conclusion, only skilled laboratory workers are able to conduct these expensive and time-consuming measurements.

Therefore, our main aim was the creation of a cheap and simple progestin measurement system that can be used in any biosafety level 1 laboratory. When our biological system is ready even untrained personnel will be able to measure progestin concentrations in water samples without any elaborate equipment. A low-tech photometer will suffice for quantitative measurements – qualitative measurements will be possible without any instruments at all. Also, costs will be low due to the easy cultivation of yeast in petri dishes and the possibility to regrow the measurement system virtually infinitely.

Measurement will be conducted by first resuspending yeast cells in low iron medium and adding a defined amount of D-luciferin solution to the tube. Then a defined volume of a water sample will be transferred to the tube. After incubation (some hours) luminescence can be measured. In the end, the progestin contamination is analyzed by comparing measured data to a standard curve.

With the help of the Tübingen Yeast Progestin-measurement System (TYPS) it will be possible to map the progestin contamination of entire rivers from source to estuary. With this data it will be possible to easily determine the polluters of surface waters. Once polluters are identified countermeasures can be devised for every specific case in order to reduce progestin contamination.

When we were presenting our project on SynBio-Day in Tübingen many people have asked us why we did not create a biological system that actively eliminates progestin from (for example) treated sewage effluent. Of course we would really like to efficiently eliminate progestin from waterbodies but there are both technical and ethical concerns with this.

In order to eliminate progestins we could either construct an organism (GMO) that takes up progestins via endocytosis and produces cytoplasmic enzymes that destroy the contaminants or we could create an organism that produces progestin-digesting exoenzymes.

Any organism that ingests progestins in order to destroy these substances must necessarily have contact with the contaminated water i.e. has to be added to the sewage water in sewage treatment plants (STP). In this case one would have to extract the GMOs from the treated sewage effluent before it enters regular waterbodies due to biosafety concerns. Even sessile / basifixed GMOs (e.g. plants) would pose a certain risk since parts of that organism (like leaves) could become loose, leave the STP, and regrow outside of the STP. In any case, horizontal gene transfer cannot be ruled out. A reliable and cost-efficient kill-switch could solve some of these biosafety problems though.

Exoenzymes are problematic in their own way since sewage water contains various microorganisms and innumerable proteases. Any protein that is introduced to sewage water will surely be digested in a matter of minutes thus be rendered useless. Therefore, STPs would have to constantly add huge amounts of exoenzymes to the sewage water. We believe that such systems could never become cost-efficient.

References

VAN DER LINDEN, S. C., HERINGA, M. B., MAN, H.-Y., SONNEVELD, E., PUIJKER, L. M., BROUWER, A. & VAN DER BURG, B. 2008. Detection of Multiple Hormonal Activities in Wastewater Effluents and Surface Water, Using a Panel of Steroid Receptor CALUX Bioassays. Environmental Science & Technology, 42, 5814-5820.

VANDERFORD, B. J., PEARSON, R. A., REXING, D. J. & SNYDER, S. A. 2003. Analysis of Endocrine Disruptors, Pharmaceuticals, and Personal Care Products in Water Using Liquid Chromatography/Tandem Mass Spectrometry. Analytical Chemistry, 75, 6265-6274.

VISWANATH, G., HALDER, S., DIVYA, G., MAJUMDER, C. B. & ROY, P. 2008. Detection of potential (anti)progestagenic endocrine disruptors using a recombinant human progesterone receptor binding and transactivation assay. Molecular and Cellular Endocrinology, 295, 1-9.

VULLIET, E., WIEST, L., BAUDOT, R. & GRENIER-LOUSTALOT, M.-F. 2008. Multi-residue analysis of steroids at sub-ng/L levels in surface and ground-waters using liquid chromatography coupled to tandem mass spectrometry. Journal of Chromatography A, 1210, 84-91.