Detecting estrogenic activity in water samples withestrogen-sensitive yeast cells using spectrophotometry and fluorescencemicroscopy
Environmental estrogens are environmental contaminants that can mimic the biological activities of the female hormone estrogen in the endocrine system, i.e. they act as endocrine disrupters. Several substances are reported to have estrogen-like activity or estrogenic activity. These include steroid hormones, synthetic estrogens (xenoestrogens), environmental pollutants and phytoestrogens (plant estrogens). Using the chromogenic substrate ortho-nitrophenyl-{beta}-D-galactopyranoside (ONPG) we show that an estrogen-sensitive yeast strain RMY/ER-ERE, with human estrogen receptor (hER{alpha}) gene and the lacZ gene which encodes the enzyme {beta}-galactosidase, is able to detect estrogenic activity in water samples over a wide range of spiked concentrations of the hormonal estrogen 17{beta}-estradiol (E2). Ortho-nitrophenol (ONP), the yellow product of this assay can be detected using spectrophotometry but requires cell lysis to release the enzyme and allow product formation. We improved this aspect in a fluorogenic assay by using fluorescein di-{beta}-D-galactopyranoside (FDG) as a substrate. The product was visualized using fluorescence microscopy without the need to kill, fix or lyse the cells. We show that in live yeast cells, the uptake of E2 and the subsequent production of {beta}-galactosidase enzyme occur quite rapidly, with maximum enzyme-catalyzed fluorescent product formation evident after about 30 minutes of exposure to E2. The fluorogenic assay was applied to a selection of estrogenic compounds and the Synchrotron-based Fourier transform infrared (SR-FTIR) spectra of the cells obtained to better understand the yeast whole cell response to the compounds. The fluorogenic assay is most sensitive to E2, but the SR-FTIR spectra suggest that the cells respond to all the estrogenic compounds tested even when no fluorescent response was detected. These findings are promising and may shorten the duration of environmental water screening and monitoring regimes using yeast-based estrogen assays, and the development of biosensors for environmental estrogens designed to complement quantification methods.
- Research Organization:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE Director. Office of Science. Office of AdvancedScientific Computing Research. Office of High Energy Physics, LaboratoryDirected Research and Development; UC Berkeley Water Resources InstituteProject No. W-944
- DOE Contract Number:
- DE-AC02-05CH11231
- OSTI ID:
- 889626
- Report Number(s):
- LBNL-59880; TRN: US200704%%97
- Resource Relation:
- Conference: 2006 Symposium - Safe Drinking Water: WhereScience Meets Policy, The University of North Carolina at Chapel Hill,Chapel Hill, North Carolina, U.S.A., March 16-17,2006
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
DRINKING WATER
ENZYMES
ESTROGENS
FEMALES
FLUORESCEIN
FLUORESCENCE
GENES
HORMONES
LYSIS
MICROSCOPY
MONITORING
POLLUTANTS
SPECTRA
SPECTROPHOTOMETRY
STEROID HORMONES
STRAINS
SUBSTRATES
YEASTS
estrogenic activity estrogen yeast assay fluorogenicchromogenic EDC FTIR