Introduction. Accumulation of xenoestrogenic compounds in waterways is a world-wide public health problem.

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1 Use of a human breast cancer cell proliferation assay as an exposure assessment tool for total bioaccumulated xenoestrogens in channel catfish caught in Pittsburgh s three rivers. Frank Houghton PhD, Conrad Volz DrPH, MPH, Yan Liu BS Env Eng, Christopher Price, Mary Elm BS, Devra Lee Davis Phd,, MPH, Maryann Donovan MPH, PhD, and Patricia Eagon PhD. Department of Gastroenterology, Hepatology and Nutrition, School of Medicine, University of Pittsburgh, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Center for Environmental Oncology, University of Pittsburgh Cancer Institute.

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

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 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 CHCl 3 /CH 3 OH (9:1 v/v) Reduced to residue using N 2 gas Stored at -20 O C under N 2 Solubilized in ethanol/glycerol (3:1 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) PI= Absorbance of treated well Mean of negative control wells (n=10)

10 Estrogen response profile (idealized) Control Estradiol 1/4000 1/3000 1/2000 1/1500 1/1000 1/500 1/200 1/ Strong Response Moderate Response No Response Proliferation Index Treatment

11 Estrogen response profile (channel catfish 138) Control E2 Control 1/4000 1/3000 1/2000 1/1500 1/1000 1/500 1/200 1/ no response Proliferation Index Treatment

12 Estrogen response profile (channel catfish 100) Control E2 Control 1/4000 1/3000 1/2000 1/1500 1/1000 1/500 1/200 1/ moderate response Treatment Proliferation Index

13 Estrogen response profile (channel catfish 47) Control E 2 Control 1/4000 1/3000 1/2000 1/1500 1/1000 1/500 1/200 1/ strong response Treatm ent Proliferation Index

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

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

16 Results The strongest proliferative responses were from catfish caught near the steel works on the Monongahela River and the Point. The weakest proliferative responses were from extracts from catfish caught upstream on the Allegheny River

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

18 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.

19 Acknowledgements Community participants: Support: Venture Outdoors Clean Water Action Bassmasters Individual local anglers 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

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