dianaa.stavreva% lrbge,%nci,nih% - … innovations alliance foundation conference...
TRANSCRIPT
A high-throughput, fluorescence-based cell assay to detect endocrine-disrupting chemicals (EDCs) in water sources
Diana A. Stavreva LRBGE, NCI,NIH
Water 2.0 Water Innovations Alliance Foundation Conference
February 26th, 2013, NYC
Endocrine Disruptor is an exogenous substance or mixture that alters funcMon(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populaMons (WHO/I 2002)”
.Characteris*cs of EDCs • Wide range of effects – Endocrine signaling governs all Mssues/organs – Nuclear and membrane receptors, neurotransmiWers, etc.
• Low dose effects
• Persistent and latent effects – Developmental exposure -‐ most sensiMve window – TransgeneraMonal effects (dioxin, BPA, phthalates)
• Ubiquitous sources – PharmaceuMcals – Industrial products – Consumer products –Personal care products
Presence of EDCs in the environment, especially in water, is a major health concern, but the actual level of this contamination is largely unknown.
We need better detection methods!
!
Our solution: fluorescent protein - tagged receptors for detection of various classes of EDCs.
Translocation of the receptor from the cytoplasm to the nucleus will indicate the presence of biologically active EDCs interacting with this particular receptor.
Glucocorticoid receptor biology
15-30 min
Visualization of receptor translocation
GFP-GR
Hormone
DAPI Overlay
- Hor
mon
e +
Hor
mon
e
Geographic location of the collection sites Polar Organic Chemical Integrative Sampler (POCIS)
Glucocorticoid activity in water samples
Samples: Luke Iwanowicz, USGS-BRD, Leetown Science Centre, WV
Testing 11 HPLC fractions from sample SS97 for glucocorticoid activity (GR translocation)
HPLC: Paul Klausmeyer, SAIC-Frederick
Gas Chromatography – Mass Spectrometry (GC-MS) analyses of fraction A (sample SS97)
• Sample SS97 is posiMve for both, glucocorMcoid and androgen acMvity. • Chemical methods did not recognize any known glucocorMcoid.
Mass spectra of peaks 1-3 compared with AES 2010 database suggested similarities to known androstane-class compounds.
GC-MS: Paul Klausmeyer, SAIC-Frederick
This screening method could be automated
Concentration-dependent translocation of GFP-GR and GFP-AR in response to known hormones
Opera automated imaging analysis system
Geographic location of collection sites and their contamination with glucocorticoid and androgen activity
27% and 35% of the samples are positive for glucocorticoid and androgen activity, respectively.
Chimeric nuclear receptors as a tool to screen for EDCs
Elizabeth Martinez 2005. JSMB 2006.Methods in Enzymology
ER ligands translocate the GR-‐ER chimera to
the nucleus.
GR-‐ER chimera-‐ stable cell line
Using this approach we can create chimeric receptors capable of detecting various types of hormonal activities.
• We developed a high-throughput cell-based assay to detect biologically active endocrine disrupting chemicals in water sources.
• We detected previously unrecognized glucocorticoid activity in US water.
• We are looking for commercial partners to further develop and implement this method.
Summary
A provisional patent covering this technology has been filed: U.S. Patent Application No. 61/656,473.
NIH, NCI LRBGE
Gordon Hager
Mia Sung Songjoon Baek Tom Johnson Lou Schiltz Tina Miranda Lars Grontved Mary Hawkins John Pooley Bethrice Thompson Lyuba Var0covski Lenka S@xova Ido Goldman
Daniel Sack Anuja George
Collaborators: • Paul Klausmeyer, Natural Product Support Group SAIC-Frederick, MD
• Luke Iwanowicz, Vicki Blazer, USGS-BRD, Leetown Science Center, WV
• Ty Voss, HiTF, NCI
Acknowledgments
Thank you!