1. Use of a human breast cancerUse of a human breast cancer
cell proliferation assay as ancell proliferation assay as an
exposure assessment tool forexposure assessment tool for
total bioaccumulatedtotal bioaccumulated
xenoestrogens in channel catfishxenoestrogens in channel catfish
caught in Pittsburgh’s threecaught in Pittsburgh’s three
rivers.rivers.
Frank Houghton PhD, Conrad Volz DrPH, MPH, Yan LiuFrank Houghton PhD, Conrad Volz DrPH, MPH, Yan Liu
MPH, Christopher Price, Mary Elm BS, Devra Lee DavisMPH, Christopher Price, Mary Elm BS, Devra Lee Davis
PhD, MPH, Maryann Donovan MPH, PhD, and Patricia KPhD, MPH, Maryann Donovan MPH, PhD, and Patricia K
Eagon PhD.Eagon PhD.
Department of Gastroenterology, Hepatology and Nutrition, School ofDepartment of Gastroenterology, Hepatology and Nutrition, School of
Medicine, University of Pittsburgh, Department of Environmental andMedicine, University of Pittsburgh, Department of Environmental and
Occupational Health, Graduate School of Public Health, University ofOccupational Health, Graduate School of Public Health, University of
Pittsburgh, Center for Environmental Oncology, University of PittsburghPittsburgh, Center for Environmental Oncology, University of Pittsburgh
Cancer Institute.Cancer Institute.

2. Accumulation of xenoestrogenic
compounds in waterways is a
world-wide public health problem.
Fish are sensitive sentinels of
waterway xenoestrogen
contamination.
Introduction

3. Effects of xenoestrogens on
male fish
• Disruption of testicular development
• Elevation of serum vitellogenin levels
• Conversion to intersex phenotype

4. Potential sources of
xenoestrogens in Pittsburgh’s
three rivers
• Legacy waste from steel mill sites
• Untreated sewage
• Residential waste
• Agricultural runoff

5. Routes of exposure to
xenoestrogens in water
• Recreational activities
• Subsistence fishing
• Municipal drinking water

6. Aim
To assess the efficacy of a
cell proliferation assay (CPA)
to determine the presence of
estrogenic substances in the
flesh of channel catfish
caught in Pittsburgh’s three
rivers

7. Methods
• Fish caught
• Location of fish catch recorded
• Biometric parameters recorded
• Fish fillets extracted
• Extracts tested for estrogenicity
via cell proliferation & estrogen
receptor binding assays

8. Extraction Method
• 1g (±5%) flesh, skin, and fat
• Extracted with CHCl3/CH3OH (9:1 v/v)
• Reduced to residue using N2 gas
• Stored at -20O
C under N2
• Solubilized in ethanol/glycerol (70:30
v/v)

9. Cell proliferation assay (CPA)
Cell lines used:
MCF-7: Estrogen receptor-positive
BT20: Estrogen receptor-negative
Cell treatments:
Physiological estrogen (estradiol, 10-9
M)
Fish extracts (varying doses)
Medium only (negative control)
Cell number assessed: CellTiter96
Proliferation index (PI)
Absorbance of treated well
Mean of negative control wells (n=10)
PI=

10. Estrogen response profile
(idealized)
C
ontrol
Estradiol1/40001/30001/20001/15001/10001/5001/2001/100
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
No Response
Strong Response
Moderate Response
Treatment
ProliferationIndex

11. Estrogen response profile
(channel catfish 138)
C
ontrolC
ontrol
2
E
1/40001/30001/20001/15001/1000
1/500
1/200
1/100
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
Treatment
ProliferationIndex no response

12. Estrogen response profile
(channel catfish 100)
C
ontrolC
ontrol
2
E
1/40001/30001/20001/15001/1000
1/500
1/200
1/100
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
Treatment
ProliferationIndex
moderate response

13. Estrogen response profile
(channel catfish 47)C
o
n
tro
lC
o
n
tro
l
2
E
1
/4
0
0
01
/3
0
0
01
/2
0
0
01
/1
5
0
01
/1
0
0
0
1
/5
0
0
1
/2
0
0
1
/1
0
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
Treatment
ProliferationIndex
strong response

14. CPA Results
• N=21
• 1 extract exhibited a strong response
• 13 extracts displayed a moderate
response
• 7 extracts produced no response

15. Highland Park
Point State Park
Kittanning,
Armstrong County
Monongahela River

16. Results
In general, extracts that are stimulators
of cell proliferation also compete for ER
binding (data not presented)

17. Conclusions
These results support the
hypothesis that bioaccumulated
xenoestrogens are highest in
channel catfish caught nearest to
legacy-contaminated locations
and areas of untreated sewage
overflows.

18. Community participants:
Venture Outdoors
Clean Water Action
Bassmasters
Individual local anglers
Support:
The DSF Charitable Trust & The Heinz Foundation
through the Center for Environmental Oncology,
University of Pittsburgh Cancer Institute, and
The Veterans Research Foundation of Pittsburgh
Acknowledgements