Watershed Ecosystem and Human Health Risk Assessment Process

1. Gene Expression Signatures as Bioindicators of Exposure and Health Effects Related to Environmental Contaminants

2. Ecosystem & Human Health Risk Assessment • Risk Assessor / Risk Manager Dialogue • Identify Goals and Assessment Endpoints • Preparing Conceptual Model • Developing Analysis Analysis • Data Sources: • DNR • Public Health • County • MI CGI • Research Reports Stressor Exposure Watershed Modeling Receptor Response Community Response • Resource Use & Exposure Communicating Results to Risk Managers and Stakeholders Watershed Ecosystem and Human Health Risk Assessment Process

3. Limitations to Environmental Risk Assessment • Ecological risk assessment • Difficulties in assessing exposure and related health effects in the field • Traditionally relies on population distribution and biodiversity analysis • Time consuming, expensive, lacks precision • Human health risk assessment • Extremely difficult to isolate environmental causes of clinical health effects • Shortcomings of epidemiology studies due to diverse lifestyles

4. Biotechnology: Molecular Based Eco Risk Assessment (WMU) • In the laboratory, define gene expression patterns in healthy versus ill animals exposed to environmental contaminants. • Using biotech tools, measure gene expression patterns in wildlife (or humans) exposed to contaminants. • Gene expression predicts exposure and cryptic health effects. • Use data to determine cleanup priorities.

5. Gene Expression as Diagnostics for Health and Disease • 30,000 genes are expressed in humans. • Many are expressed during good health (normal physiology). • During illness, some are over expressed (immune system genes, beta amyloid gene in Alzheimer’s disease), while some are under expressed (dopamine synthesis genes in Parkinson’s disease).

6. Gene Expression • Genes sequences code for • Complementary mRNA sequences code for • Amino acid sequences that are • Proteins that underlie living processes.

7. Initial Proof of Concept Studies • Load tadpole and fish tissues with PCB concentrations found in Kalamazoo River wildlife • Short approach: use DMSO to carry PCBs into animals to desired concentration (1 day to 1 week exposure) • Long approach: feed PCB laced food over weeks to appropriate tissue levels • Observe behavior, morphology, mortality, gene expression signatures

8. Induction of Apoptosis CPP32ß (caspase-3) ICE (interleukin-1 converting enzyme) Endocrine Control Retinoic acid receptor Thyroxine receptor POMC (pro-opiomelanocortin) Neurological Function D2 dopamine receptor Nerve growth factor (NGF) Control of cell cycle/Cell structure/Metabolic control p53 ß-actin GAPDH Response to xenobiotics p450 Multi-functional p53 ICE Bioindicator Genes

9. Gene Expression in 18 Day Old Tadpoles: Bioindicators of Exposure • Exposure to low levels of Aroclor 1254 (5 and 50 ppb) increased gene expression • NGF • ß-actin • CPP32ß • ICE • POMC • p53 % Gene expression/Control % Survival/DMSO % Gene expression/Control % Survival/DMSO % Gene expression/Control % Survival/DMSO

10. Decreases in Gene Expression Are Predictive Bioindicators • Decreased gene expression at high doses (700 ppb) correlated with decreased survival and the onset of adverse health effects • NGF • ß-actin • Decreases in gene expression occurred in tadpoles exposed to 300 ppb and greater % Gene expression/Control % Survival/DMSO

11. Gene Expression Signatures as Bioindicators of Exposure to PCBs and Related Health Effects in Developing Xenopus Frogs and carp, Cyprinus carpioProject Coordinator: Charles F. Ide, Ph.D. Professor of Biological Sciences and Director, Environmental InstituteJay C. Means, Ph.D. Professor of Environmental Chemistry and Toxicology, Associate Director, Environmental InstituteCo-Investigator: Anna M. Jelaso, Ph.D. Assistant Professor, Environmental Institute Co-Investigator: Marla A. Fisher, Ph.D. Postdoctoral Researcher, Department of Environmental and Molecular Toxicology, NCSUDr. Bharti Katbamna, Professor of Speech Pathology and AudiologyLibby Lehigh-Shirey, Ron Celestine - Graduate Students

12. New Frog Gene Expression Work • DMSO assisted Aroclor 1254 exposure studies (2 day exposures) showed that tissue levels <50 ppm showed increases in gene expression, no external health effects; levels >100 ppm showed decreases in gene expression and overt health effects and mortality • Need to determine if tissue levels < 100 ppm produce cryptic health effects in real word exposure (dietary) setting (can gene expression analysis reveal cryptic health effects that will ultimately alter fitness)

13. New Gene Expression Work • Pilot work shows frogs exposed to low levels of PCBs (50ppb, DMSO) or through diet (12, 24 ppm) showed slow metamorphic rates • Important for frog population fitness due seasonal limitations on frog lifestyle • Determine gene expression changes underlying PCB induced changes in metamorphic rate

14. Gene Origin Function Change in gene expression CRF (corticotropin releasing factor) Hypothalamus Stimulates ACTH release; Stimulates TSH release in amphibians  POMC (pro-opiomelanocortin) Pituitary Precursor to ACTH and Melanin  TSH (thyroid stimulating hormone) Pituitary Stimulates TH production in thyroid gland  TR-beta (thyroid hormone receptor) Target tissue Forms heterodimer with RXR; Binding of TH  D2 (Type II Deiodinase) Target tissue; mainly the brain Converts T4 into T3  D3 (Type III Deiodinase) Target tissue Converts T4 and T3 into rT3 and T2, biologically inactive forms 

15. New Gene Expression Work • Perform chronic 60 and 90 day dietary exposures (0, 12, 50, 100, 200 ppm Aroclor 1254) • Analyze previous gene battery including thyroid system genes • Analyze new endocrine disruption gene battery (wnt4a, sox3, ER, AR, CYP17, CYP19)

16. New Gene Expression Work • PCB exposure in utero reduces low frequency hearing in humans • Established in mammalian models, but not relevant ecosystem models • Expose developing frogs to PCBs (dietary) and assay development of auditory function using Auditory Brainstem Response Methods • Correlate with gene expression analysis for genes involved in development of vestibulo-auditory brain circuits (NGF, CX 43, CX 31) • Will establish a model system for assaying cryptic contaminant induced health effects in wildlife, and will provide basic data regarding genes controlling auditory system development

17. New Gene Expression Work • High atrazine levels present in the St. Joseph River watershed and in Lake Michigan • Controversial morphology and histology based studies claim that atrazine feminizes male frogs at <environmental levels • Settle controversy by exposing frogs to atrazine (0.1, 25 ppb) and measuring gene expression for genes that determine sexual phenotype (wnt4a for males, sox3 for females; also CYP19 and CYP17 aromatase, and for ER and AR) in treated and untreated animals • Also, pilot data shows that low level atrazine treatment speeds up metamorphosis, so measure expression of thyroid system genes • Should establish a molecular basis for endocrine disruption effects of atrazine at environmental levels

18. New Gene Expression Work - Carp • Carp are the most contaminated fish in the Kalamazoo River (up to 164 ppm PCBs) • Exposed carp in the laboratory through diet (12 ppm Aroclor 1242) for 1, 2, 3, 4 months • Gene expression analysis showed upregulation P4501A (bioindicator of exposure) • Gene expression changes also present in carp caught from contaminated river sites versus cleaner sites • Increased liver (hepatopancreas) macrophage aggregates in carp caught from contaminated river sites versus cleaner sites

19. Kalamazoo River: Superfund Site Due to PCB Contamination

20. PCB Levels in Carp Tissues Laboratory Exposed Carp Kalamazoo River Carp Muscle PCBs (ug/g) Days Fed PCBs (A1242)

21. PCB Induced p4501A1 Gene Expression in Carp Laboratory Exposed Carp Kalamazoo River Carp P450 1A1 mRNA (ng/ul) Days Fed PCBs (A1242)

22. Histopathology: Kalamazoo River Carp Macrophage Aggregates Example macrophage aggregate showing lipofuscin, hemosiderin, melanin Gamori’s Prussian Blue, no counterstain • Hepatocyte and exocrine pancreas macrophage aggregates • Stain: H&E

23. Histopathology Quantification: PCB Sites Increased MA Area and MA # • % Macrophage Aggregate Area • significantly increased in carp from PCB vs reference sites (ANOVA p=0.026; n=17 reference, n=10 PCB) • Despite carp from PCB sites are younger/smaller • Increase not from age/size • Macrophage Aggregate #/Area • significantly increased in carp from PCB vs reference sites (ANOVA, p=0.0453; n=17 reference, n=10 PCB) • Significantly different among sites (p=.0079) • Lake Allegan higher than Trowbridge, Ceresco, Morrow

24. Meaning of Increased Macrophage Aggregates in PCB exposed carp • Carp hepatopancreas macrophage aggregates consist primarily of lipofuscin • Increase in MA densities in carp hp from PCB sites  Increase in lipofuscin  Increased need for lipid clearance and storage in MAs in PCB exposed carp

25. Correlation of hepatopancreas MA Densities with hepatopancreas PCBs • PCBs are major contaminant in Kalamazoo River • In general, carp from reference sites: low r, high p • Carp from PCB sites: high r, low p • Lake Allegan carp: MA# significantly correlated with PCB levels • Suggests distinct carp population at this site (Kalamazoo RiverDams) • Suggests population responding to PCBs (vs stunted growth?)

26. Carp Model • Carp contaminates with high levels of PCBs are found in the Kalamazoo River Superfund Site • CYP1A mRNA, an indicator of exposure to PCBs, was elevated in carp liver from PCB contaminated sites compared to upstream reference sites • Macrophage Aggregate densities were elevated in carp liver from PCB contaminated sites compared to reference sites • Molecular data plus histopathology data suggest carp in PCB exposed sites are responding to exposure • Data will be viewed in light of claims by PRP scientists that contaminated organisms in the river are in good health