Rapid and Sensitive Reporter Gene Assays for Detection of Antiandrogenic and Estrogenic Effects of Environmental Chemicals

Rapid and Sensitive Reporter Gene Assays for Detection of Antiandrogenic and Estrogenic Effects... Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were cotransfected with the human androgen receptor expression vector and the mouse mammary tumour virus (MMTV) 2 -luciferase vector using the new nonliposomal transfection reagent FuGene. Stimulation of the cells for 24 h with the synthetic androgen receptor agonist, R1881 (10 nM), resulted in a 30- to 60-fold induction of luciferase activity. The classical antiandrogenic compounds hydroxy-flutamide, bicalutamide, spironolactone, and cyproterone acetate together with the pesticide(metabolite)s, vinclozolin, p,p′-DDE, and procymidone all potently inhibited the response to 0.1 nM R1881. Compared to the traditional calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription. To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast cancer cells with an estrogen response element–luciferase vector. Thus, FuGene may prove to be valuable in diverse reporter gene assays involving transient transfections for screening of potential endocrine disruptors for (anti)androgenic and (anti)estrogenic properties. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicology and Applied Pharmacology Elsevier

Rapid and Sensitive Reporter Gene Assays for Detection of Antiandrogenic and Estrogenic Effects of Environmental Chemicals

Loading next page...
 
/lp/elsevier/rapid-and-sensitive-reporter-gene-assays-for-detection-of-ew8RA36iPR
Publisher
Elsevier
Copyright
Copyright © 1999 Academic Press
ISSN
0041-008x
DOI
10.1006/taap.1998.8598
Publisher site
See Article on Publisher Site

Abstract

Reports on increasing incidences in developmental abnormalities of the human male reproductive tract and the recent identifications of environmental chemicals with antiandrogenic activity necessitate the screening of a larger number of compounds in order to get an overview of potential antiandrogenic chemicals present in our environment. Thus, there is a great need for an effective in vitro screening method for (anti)androgenic chemicals. We have developed a rapid, sensitive, and reproducible reporter gene assay for detection of antiandrogenic chemicals. Chinese Hamster Ovary cells were cotransfected with the human androgen receptor expression vector and the mouse mammary tumour virus (MMTV) 2 -luciferase vector using the new nonliposomal transfection reagent FuGene. Stimulation of the cells for 24 h with the synthetic androgen receptor agonist, R1881 (10 nM), resulted in a 30- to 60-fold induction of luciferase activity. The classical antiandrogenic compounds hydroxy-flutamide, bicalutamide, spironolactone, and cyproterone acetate together with the pesticide(metabolite)s, vinclozolin, p,p′-DDE, and procymidone all potently inhibited the response to 0.1 nM R1881. Compared to the traditional calcium phosphate transfection method, this method has the advantage of being more feasible, as the assay can be scaled down to the microtiter plate format. Furthermore, the transfection reagent is noncytotoxic, allowing its addition together with the test compounds thereby reducing the hands-on laboratory time. This assay is a powerful tool for the efficient and accurate determination and quantification of the effects of antiandrogens on reporter gene transcription. To extend the application of FuGene, the reagent was shown to be superior compared to Lipofectin for transfecting MCF7 human breast cancer cells with an estrogen response element–luciferase vector. Thus, FuGene may prove to be valuable in diverse reporter gene assays involving transient transfections for screening of potential endocrine disruptors for (anti)androgenic and (anti)estrogenic properties.

Journal

Toxicology and Applied PharmacologyElsevier

Published: Mar 1, 1999

References

  • Effect of toxaphene on estrogen receptor functions in human breast cancer cells
    Joergensen, E.C.B.; Autrup, H.; Hansen, J.C.
  • Assessment of the Alamar Blue assay for cellular growth and viability in vitro.
    Nakayama, G.R.; Caton, M.C.; Nova, M.P.; Parandoosh, Z.
  • Synergism between androgens and protein kinase-C on androgen-regulated gene expression
    Ruiter, de P.E.; Teuwen, R.; Trapman, J.; Dijkema, R.; Brinkmann, A.O.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create folders to
organize your research

Export folders, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off