Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay.
The present study was aimed to develop a sensitive, fast and user friendly progesterone receptor transactivation assay using recombinant yeast cells, Saccharomyces cerevisiae, modified to express human progesterone receptor (PR) and progesterone response element (PRE) driving the expression of green fluorescent protein. Stimulation of cells with increasing concentrations of progesterone resulted in significant elevation in fluorescence activity, with the minimum effective dose of progesterone being 0.1 nM. RU486, significantly inhibited progesterone induced transactivation and non-progesterogenic steroids failed to transactivate PR till 10 microM concentrations. About 7 different chemicals (mostly pesticides or their metabolites) like DDT and its metabolites, nonylphenol, endosulfan were screened in this assay system for their role in transactivation and they were all found to be anti-progestative and IC50 values within the range of 3-20 microM. Further, the assay was used to analyze the endocrine disrupting activity of extracted water samples from leather industries known for their high content of various chemicals and it was found to be rich in anti-progestative compounds. It resulted in about 30% reduction in transactivation. In conclusion, we demonstrated that this yeast based bioassay provides a rapid and robust assay for high throughput screening of (anti)progestative compounds from various sources.
Chatterjee
Shamba
S
Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, Uttarakhand, India.
Kumar
Vikas
V
Majumder
Chandrajeet B
CB
Roy
Partha
P
D001681:Biological Assay; D015153:Blotting, Western; D004353:Drug Evaluation, Preclinical; D052244:Endocrine Disruptors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006727:Hormone Antagonists / Q000494:pharmacology; D007220:Industrial Waste / Q000032:analysis; D008024:Ligands; D015735:Mifepristone / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011374:Progesterone / Q000819:agonists / Q000037:antagonists & inhibitors; D011372:Progestins / Q000037:antagonists & inhibitors*; D012313:RNA / Q000302:isolation & purification; D011980:Receptors, Progesterone / Q000187:drug effects / Q000235:genetics; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics; D015533:Transcriptional Activation / Q000187:drug effects
D052244:Endocrine Disruptors; D006727:Hormone Antagonists; D007220:Industrial Waste; D008024:Ligands; D011372:Progestins; D011980:Receptors, Progesterone; D049452:Green Fluorescent Proteins; D015735:Mifepristone; D011374:Progesterone; D012313:RNA