COMPARISON OF THREE RECOMBINANT GENE YEAST BIOASSAY FOR SCREENING XENOESTROGENIC COMPOUNDS

There has been increased concern regarding the study of endocrine disrupters. The study about the screening method of endocrine disrupters has become an important issue for governments and institutes in many countries. This study focused on improving method of recombinant gene yeast bioassay. The estrogen bioassay employed in this study involves the use of a genetically modified yeast strain to determine the ability of a test agent to trans-activate the estrogen receptor. This yeast strain contains a human estrogen receptor gene (ER alpha) and a reporter gene coding for the β-galactosidase. Test agents with estrogenic activity activate the receptor gene in yeast strain and subsequently result in the production β-galactosidase. The estrogenic activity of chemicals is quantified by determining the activity of the β-galactosidase. In this study, three recombinant gene yeast bioassay including shaking bottle method, 96-well microtiter plate method and single plate method for screening estrogenic activities were compared each other by detecting estrogenic activity of 17β-estradiol. In the beginning of applying recombinant yeast bioassay in our laboratory, the operation procedure was shaking bottle method referred to the designs of Dr. Rehmann. From the beginning of screening a batch of samples (about 30) to the end of the experiment, it generally lasted 2-3 days. Because of the disadvantages of the method, it was improved in our laboratory to use 96-well microtiter plate method. It was shortened to 1 d to screen a batch of samples and made it possible to realize fast monitoring. But this method still needs more than 10h to finish one measurement. Now the method was further improved to be single plate method. In this method, oxylaticase was used as cellular wall cracker substitute for chloroform. Then one measurement could be finished in 4-6h. Comparison of the improved methods was made with different yeast construct in other laboratories by detecting estrogenic activities of six chemicals. Results (evaluated by EC50) are consistent with reference reports. Since the single plate method changed great in experimental operation, its experimental parameters were optimized. 0.50 was selected as optical density of detection solution at 600nm and 1h was selected as reaction time of enzyme. By the improved method of optimized detecting optical dense and β-galactosidase reacting time, the detection time was shortened from several daysto only a half day in screening a batch of samples. Meanwhile, the culture solution and consumed materials were also saved, whereas the sensitivity and accuracy of the method still remain the same as those before improvement. This makes it possible to realize the purpose of fast environmental monitoring for xenoestrogenic compounds.