1. Estrogenicity of tissue extracts from white bass and channel catfish
caught along the Three Rivers of Pittsburgh, Pennsylvania
Frank D. Houghton, Jr.1, Yan Liu2, Christopher J. Price1, Mary S. Elm2, Maryann Donovan2,3,
Devra Davis2,3, Conrad D. Volz2,3, and Patricia K. Eagon1,2,3
1VA Medical Center; 2University of Pittsburgh; 3CEO, University of Pittsburgh Cancer Institute, Pittsburgh, PA
# 3458
Accumulation of endocrine-disrupting
substances from manufacturing, agricultural and
residential sites in waterways is an enormous and
universal environmental problem.
Such waterways are important sources of
drinking water, recreation, and subsistence fishing
by the local community.
Examples of endocrine disruption in fish
include the feminization of male fish exposed to
effluents from sewage treatment plants, as well as
masculinization of female fish exposed to treated
waste water from paper mills.
Introduction
Our concerns:
Are xenoestrogens present in fish captured in
the Allegheny, Monongahela, and Ohio Rivers in
Pittsburgh?
What are the implications for the health of the
rivers, the communities that use water from these
rivers, and the fishers who subsist on fish caught
from these rivers?
labeled E2. Fish tissue extracts were tested at dilutions of 1/20 to
full-strength. The mixtures were incubated at 4°C overnight, and
bound ligand was separated from free by P6 resin spin columns.
• Cell proliferation assay (CPA): Human breast cancer cell
lines used were ER-positive MCF-7 and ER-negative BT-20.
Cells were incubated for 72h with estradiol (1nM) or fish extracts
diluted in medium to final concentrations of 1/4000-1/100 of the
original extracts. Cell growth was detected by use of CellTiter 96
Aqueous One Solution Cell Proliferation Assay (Promega).
Proliferation in test wells is expressed compared to proliferation
in untreated and estradiol-treated cells.
Methods
• Fish Capture: Fish were caught by local anglers, placed on ice,
and transported to the laboratory for immediate dissection.
• Extraction of fish: a one-gram sample including skin, muscle,
and fat, was taken perpendicular to the lateral line from each
white bass (n=7) and channel catfish (n=21). Each sample was
homogenized and extracted with chloroform:methanol (9:1). The
organic phase was evaporated under nitrogen, and the residue was
stored under nitrogen at -20°C. For use in assays, residues were
dissolved in EtOH:glycerol (70:30).
• In vitro competitive estrogen receptor (ER) binding assay:
Cytosol was prepared from mature rabbit uteri, and aliquots were
incubated with 5nM [3H]-estradiol (E2) in the absence and
presence of test substances. Diethylstilbestrol (DES) was used as a
positive control in concentrations of 1-10,000 times that of the
White BassWhite Bass
(Morone chrysops)
White bass are silvery in color with a milky
white belly. In Pennsylvania the white bass is native to the western
counties, especially Lake Erie and the Ohio River watershed. They prefer
large open clear water with a firm bottom and water depths of less than
30 feet. Adult white bass feed primarily on small fishes and larger insects.
Life expectancy is usually 3 to 5 years.
Competition of white bass extracts
in an in vitro ER binding assay
PercentofControl
0 1xDES; 1/20 100xDES; 1/5 10000xDES; 1/1
Competitor concentration (fold excess DES; extract dilution)
0
25
50
75
100
125
Fish # 77
Fish # 13
Fish # 44
Fish # 120
Fish # 5
Fish # 40
DESControl (No competitor)
Effect of white bass #120 extract
on MCF-7 cell growth
0.0
0.5
1.0
1.5
2.0
2.5
ProliferationIndex
Untreated
E 2
Control
1/4000
1/3000
1/2000
1/1500
1/1000
1/500
1/200
1/100
Treatment
Moderate response
Effect of white bass #40 extract
on MCF-7 cell growth
0.0
0.5
1.0
1.5
2.0
2.5
ProliferationIndex
Untreated
E 2
Control
1/4000
1/3000
1/2000
1/1500
1/1000
1/500
1/200
1/100
Treatment
Strong response
Effect of channel catfish #47 extract
on MCF-7 cell growth
0.0
0.5
1.0
1.5
2.0
2.5
ProliferationIndex
Untreated
E 2
Control
1/4000
1/3000
1/2000
1/1500
1/1000
1/500
1/200
1/100
Treatment
Strong response
Channel catfishChannel catfish
(Ictalurus punctatus)
The channel “cat” has a slender body with a deeply forked tail. The upper
jaw is longer than the lower jaw, with long, black barbels around the
mouth. They are found statewide. They prefer areas with deep water, i.e.
clear, warm lakes and moderately large to large rivers, with clean sand,
gravel or rock-rubble bottoms. Adult channel catfish are bottom feeders
that use smell and taste to locate food. They are omnivorous and will eat
insect larvae, crayfish, mollusks, small fish, and dead fish.
Competition of channel catfish extracts
in an in vitro ER binding assay
PercentofControl
0
25
50
75
100
125
Fish # 35
Fish # 100
Fish # 61
Fish # 94
Fish # 132
Fish # 87
0 1xDES; 1/20 100xDES; 1/5 10000xDES; 1/1
Competitor concentration (fold excess DES; extract dilution)
DESControl (No competitor)
Effect of channel catfish #100 extract
on MCF-7 cell growth
0.0
0.5
1.0
1.5
2.0
2.5
ProliferationIndex
Untreated
E 2
Control
1/4000
1/3000
1/2000
1/1500
1/1000
1/500
1/200
1/100
Treatment
Moderate response
Results: ER binding assays
Bass extracts: Of 6 bass extracts tested, 4 exhibited
moderate or strong competition for ER binding in a
dose-dependent manner; 2 enhanced binding.
Catfish extracts: Of 21 catfish extracts tested, 2
were strong and 7 were moderate competitors,10 were
weak competitors, and 1 was non-competitive.
ER binding assay:
- no competition
-/+ weak competition
+ moderate competition
++ strong competition
↑ enhancement of binding
Cell proliferation assay:
- no response
-/+ weak response
+ moderate response
++ strong response
location
code
Catch Location Species
Fish
ID#
Sex
ER Binding
Assay
MCF-7
Response
BT20
Response
1 Kittanning Channel Catfish 36 Female + - -
1 Kittanning Channel Catfish 35 Female -/+ - -
1 Kittanning Channel Catfish 94 Male + - -
1 Kittanning Channel Catfish 85 Male + -/+ -
1 Kittanning Channel Catfish 37 Male -/+ -/+ -
1 Kittanning Channel Catfish 38 Unknown -/+ -/+ -
2 Monongahela White Bass 40 Female ++ ++ -
2 Monongahela White Bass 44 Male -/+ - -
2 Monongahela White Bass 77 Unknown - -
2 Monongahela Channel Catfish 75 Female - - -
2 Monongahela Channel Catfish 46 Female -/+ -/+ -
2 Monongahela Channel Catfish 87 Male ++ -/+ -
2 Monongahela Channel Catfish 41B Male -/+ -/+ -
2 Monongahela Channel Catfish 47 Male -/+ ++ -
2 Monongahela Channel Catfish 76 Unknown + + -
3 Highland Park White Bass 9 Unknown ND - -
3 Highland Park White Bass 5 Unknown + - -
3 Highland Park White Bass 13 Unknown - -
3 Highland Park Channel Catfish 61 Female + -/+ -
3 Highland Park Channel Catfish 62 Male -/+ - -
3 Highland Park Channel Catfish 64 Male -/+ -/+ -
4 Point Channel Catfish 68 Female -/+ + -
4 Point Channel Catfish 100 Male + + -
4 Point Channel Catfish 69 Male -/+ -/+ -
4 Point Channel Catfish 78 Unknown ++ + -
5 Store White Bass 120 Male + + -
5 Store Channel Catfish 138 Unknown + - -
5 Store Channel Catfish 132 Unknown ++ -/+ -
Summary of ER binding and cell proliferation assays
Results: CELL PROLIFERATION ASSAYS
• Some extracts resulted in strong cellular responses in ER-
positive MCF-7 cells, comparable to their response to
physiological levels of estradiol.
• Effects of fish extracts differed with location of fish capture:
Bass extracts (n=7):
• 2 produced cell growth: one from the Monongahela, one from store.
• Extracts of bass from other sites did not exhibit proliferative responses.
Catfish extracts (n=21):
• 1 was strongly stimulatory, 4 moderate, 10 weak, and 6 had no
response.
• Of interest, catfish extracts with strong and moderate proliferative
responses were from fish caught at polluted sites.
• The ER-negative BT-20 cells exhibited no growth
stimulation when treated with the fish extracts or with E2.
Acknowledgements
This study is part of the Community Based Participatory Research Project. Partners include
Venture Outdoors, Clean Water Action, Bassmasters, and individual local anglers. All fish
were caught by angler participants. Information derived from this study will be shared with all
participants and the general public.
The DSF Charitable Trust and The Heinz Foundation provided funding for this project through
the Center for Environmental Oncology (CEO), University of Pittsburgh Cancer Institute.
Results
In general, extracts that compete for
ER binding are also stimulators of
cell proliferation.
The sex of the fish was not a factor in
predicting response in either assay.
Estrogenicity of fish extracts differed
with location of fish capture.
Conclusions
These results suggest that some fish
caught in local waters contain substances that
have estrogenic activity.
The level and activity of these substances
are sufficient to recommend that persons at
risk for endocrine-responsive cancers should
avoid eating locally caught fish.
References
EPA TRI DATA-2005, http://www.epa.gov/tri/, accessed 3/25/07.
Volz, CD, Water quality problems in Southwestern Pennsylvania in Miller,
T. Editor, Regional water management in Southwestern Pennsylvania:
Moving toward a solution. July 2006, University of Pittsburgh, Institute
of Politics.
Volz, CD and C Christen, 2007. Why are water recreationalists most at risk
from waterborne infectious disease? Journal of Occupational and
Environmental Medicine. 49(1):104-105.
Eagon, PK, N Chandar, MJ Epley, MS Elm, EP Brady and KN Rao: Di(2-
ethylhexyl)phthalate-induced changes in liver estrogen metabolism and
hyperplasia. Int. J. Cancer 58: 736-743, 1994.
Rogerson, BJ and PK Eagon: A male-specific hepatic estrogen binding
protein: Characteristics and binding properties. Arch. Biochem.
Biophys. 250: 70-85, 1986.
Duda, RB et al.: pS2 Expression Induced by American Ginseng in MCF-7
Breast Cancer Cells. Annals of Surgical Oncology 3: 515-520, 1996.
Local anglers
Aim:Aim: to determine if estrogenic
substances are present in fish caught
in various parts of Pittsburgh’s
three rivers.
Monongahela River
near the U. S. Steel
Edger Thompson
Works
Highland Park
Locations of Fish Capture in Southwestern Pennsylvania
Point State Park
Kittanning,
Armstrong County
4) Point: Catch site is approximately 2 miles downstream from a
US Steel Plant. It is near over 100 CSOs and aging municipal
sewage infrastructures that overflow into the rivers (Volz, 2006).
5) Store: Channel catfish from a Georgia fish farm and white bass
from Lake Erie were purchased from a local fish market for
comparison purposes.
1) Kittanning: 36 miles upstream from Pittsburgh on the
Allegheny River in Armstrong county. This site is relatively
unimpaired by local industry. Angler focus groups consider fish
from this site “safe” for eating (Volz and Christen, 2007).
2) Monongahela: Catch site at the Braddock Dam, 11 miles
upstream from Pittsburgh, bordered by the Edger Thompson
Works of US Steel. This plant was part of the largest integrated
iron and steel making operations in the world. It remains a
significant EPA Toxic Release Inventory (TRI) site (EPA,
2007). Upstream of this catch site are numerous other TRI sites,
including coking operations.
3) Highland Park: Catch site at the Highland Park Bridge/Dam,
on the Allegheny River. It is significantly downstream of the
relatively few TRI sites on the Allegheny. Additionally, it is
downstream of far fewer combined sewer overflows (CSO).
Pollution at Fish Capture Locations