A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection.
A public concern is continuously arising about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions. These substances, defined as endocrine disrupting compounds (EDC) represent an heterogeneous class of molecules either steroidal or not, sharing the ability of interfering with the endocrine system via nuclear receptor signaling pathways. Therefore there is an urgent need for high throughput screening systems able to detect EDCs and evaluate their biological activity. However, little attention has been dedicated to the development of assays for androgen-like compounds. The present work describes the development and optimization of a new rapid and sensitive bioluminescent yeast-based bioassay for androgen-like compounds in a 96-well microplate format. The bioassay is based on recombinant Saccharomyces cerevisiae cells modified to express human androgen receptor (hAR) and containing the sequence androgen response element (ARE) which drives the expression of Photinus pyralis luciferase, used as reporter gene. A recombinant yeast strain constitutively expressing luciferase was used as external control to correct the light signal accordingly to cell viability and sample matrix aspecific effects. The bioassay responds to testosterone as reference androgen in a concentration-dependent manner from 0.05 to 1000 nM allowing an accurate and precise quantitative evaluation in aqueous environmental samples down to 10(-11)mol/L. Other known androgen-like compounds exhibit similar dose-response behavior, thus permitting the use of the bioassay for an overall detection of androgen-like effect in environmental samples.
Michelini
Elisa
E
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. elisamichelini@yahoo.it
Leskinen
Piia
P
Virta
Marko
M
Karp
Matti
M
Roda
Aldo
A
D000728:Androgens / Q000008:administration & dosage / Q000032:analysis; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004784:Environmental Monitoring / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013739:Testosterone / Q000008:administration & dosage* / Q000032:analysis
D000728:Androgens; D008164:Luminescent Proteins; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D013739:Testosterone; D008156:Luciferases