I am running an experiment on EDCs at different pHs. To see the effect of the endocrine disruptor, I am using the YES assay. Does pH and ethanol affect yeast response on YES assay ? I found that the estrogenicity after degradation is higher than the parent compound. Why does this occur?
There are a lot of 'depends' in my answers, because the question needs some more detail. Usually the YES assay is used with sample extracts in Ethanol, which you allow to evaporate before addition of the yeast because ethanol is toxic to yeast. So yes, ethanol will affect the response if you have enough in the sample to affect mortality. The OD620 blank normalisation in the calculation is quite robust at dealing with some amount of mortality, but it does not take into account any physiological changes done by the ethanol at sub-lethal levels.
pH will also have an effect at some point on its range. Extremely high or extremely low pH will be toxic, in between there will be an acceptable range where yeast growth is normal, and two ranges where it is below normal, but still alive.
I seem to recall that we accidentally made up some yeast media at a pH of about 5 and that killed the culture.
Sometimes chemicals degrade into more active forms via various processes. Often this is through biological processes (bioactivation) in bacteria, or in the liver of larger organisms. Several pharmaceuticals use this process to create active drugs at the site of need from harmless precursors.
I hope this is of some help.
Check out my YES paper in Science of the Total Environment in 2010. With such a short assay time (samples sit on the yeast for only two hours) we have found that very little affects the assay as the yeast grow very little in this time frame. We can even measure estrogenic activity directly in human urine with this method. In addition, for particularly dirty wastewater samples we spin the yeast out of the sample media before adding fresh media and substrate/lysis buffer and using this method very little affects the enzyme reaction needed to convert the substrate to product also. Regarding the first comment, ethanol is toxic to the yeast typically above a certain percentage (around 1% depending on the strain) but that can easily be avoided by making a more concentrated extract if working with extracts. Any extracts and/or hormone stocks in ethanol or DMSO are never added at anything higher than 1% in our assays.
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