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

Automated screening by Opera

Samples: Luke Iwanowicz & Vicki Blazer, USGS-BRD

Plate 1

Samples: Luke Iwanowicz & Vicki Blazer, USGS-BRD

Automated screening by Opera

Plate 2

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!