Biomarkers of Exposure to Hazardous Substances (2017-2022) The UC - PowerPoint PPT Presentation

Biomarkers of Exposure to Hazardous Substances (2017-2022) The UC Davis Superfund Research Center conducts research to: a) Improve understanding of the mechanisms by which hazardous chemicals produce adverse health affects, b) Develop, validate and integrate novel methods to evaluate chemical exposures, levels of contamination, and health risks, and c) Develops innovative remediation strategies to reduce hazardous substance exposure and toxicity. These activities improve the ability of the National Superfund Program to address legacy and emerging contaminants and associated transformation products to more comprehensively protect the U.S. population from health risks posed by hazardous substances.

Biomarkers of Exposure to Hazardous Substances Project PI 1. Optimizing Bioremediation Tom Young, Frank Loge 2. Nanosensing Platforms Tingrui Pan 3. Immunochemical BioMarkers Natalia Vasylieva 4. Cardiac Toxicity Nipavan Chiamvimonvat 5. Endoplasmic Reticulum Stress Fawaz Haj, Christophe Morisseau

Core C: Community Engagement - Dr. Beth Rose Middleton, PI In response to intensive forestry management and illegal marijuana groves, collaborative research with the Yurok Tribe Environmental Program (YTEP) will: • Conduct environmental sampling to identify contaminants and their concentrations • Implement field deployable assays for use by YTEP partners • Collaboratively identify culturally and ecologically appropriate remediation strategies

Community Engagement Core - Dr. Beth Rose Middleton, PI The Community Engagement Core works to develop meaningful bi- directional communication strategies between university and tribal researchers and community partners to apply UCD Center research to address community concerns. Broadly, the chemical detection technologies, remediation strategies and training opportunities aim to provide communities with autonomous methods for addressing environmental health problems within their community while training scientists on developing equitable, respectful, and responsible projects with community partners.

Core A: Analytical Chemistry Core - Dr. Jun Yang, PI Develop analytical methods to detect hazardous chemicals for the variety of UCD-SRP projects. Validate alternative analytical methods such as: • Immunoassays • Cell-based assays

sEH inhibition and EpFA block Endoplasmic Reticulum Stress (ER Stress) ROS Glucose Phospholipase A2 IRE1 Arachidonic Acid Nucleus α P CYP450 P O XBP1s O H ATF6 ERAD O 14,15 EET P P R K P E sEH sEH inhibitor ATF6(N) P O elF2 α O H CHOP H O ATF4 14,15 DHET Wagner et al. 2017 O H

Project 5: Monitoring Endoplasmic Reticulum Stress Caused by Chronic Exposure to Chemicals, Dr. Fawaz Haj and Dr. Christophe Morisseau Investigate new mechanistic insights into the effects of chronic exposure of Superfund (SF) chemicals on endoplasmic reticulum (ER) stress. Effects of SF chemicals on ER stress by • Altering gene expression • Inhibition • Competition for catalysis • Increasing reactive oxygen species • BLOOD AND URINARY BIOMARKERS OF DISRUPTION OF THE ER STRESS PATHWAY TO MONITOR XENOBIOTIC EXPOSURE AND POSSIBLY DRIVE THERAPEUTIC INTERVENTION.

Project 4: Critical Role of Mitochondrial Oxidative Stress (MOS) in Chemical Induced Cardiac Toxicity, Dr. Aldrin Gomes (mitochondria) and Dr. Nipavan Chiamvimonvat (heart) Investigate molecular mechanisms of chronic exposure to Superfund chemicals on mitochondrial oxidative stress (MOS) and proteasome dysfunction Target Analytes: • Pesticides • Antimicrobials • HaHs/PaHs • Commercial Chemicals • Pharmaceuticals • CELL, BLOOD AND URINARY BIOMARKERS OF DISRUPTION OF MITOCHONDRIA TO MONITOR XENOBIOTIC EXPOSURE AND POSSIBLY DRIVE THERAPEUTIC INTERVENTION.

DEVELOP BIOMARKERS TO DETECT FUNDAMENTAL PROCESSES OF TOXICITY DEPRESSION CANCER TOOTH EpFA: EETS EEQS EDPS DECAY THE MITOCHONDRIAL ROS ER STRESS AXIS MPTP NEUROPATHIC TRICLOSAN PAIN PARKINSON ’ S PARAQUAT DIABETES INDOMETHACIN HEART CARBONTET FAILURE NITROPHENOLS INFLAMMATION DICLOFENAC IBD FIBROSIS

Pr Project oject 4 - Monitoring Mitochondrial Oxidative Stress and Cardiac Toxicity Caused by Chronic Exposure to Chemicals Dr. Nipavan Chiamvimonvat, Project Leader Dr. Aldrin Gomes, Co-Leader

Over Ov erall all aims aims Hypothesis : chronic exposure to xenobiotics and/or non- steroidal anti-inflammatory drugs (NSAIDs) leads to mitochondrial oxidative stress (MOS) that results in proteasome dysfunction, apoptosis, tissue fibrosis and cardiac toxicity. Focus : Heart health related diseases. Approach : used cell based assay and in vivo models to test effect of exposure to SF chemicals and/or NSAIDs on mitochondrial stress, proteasome dysfunction, apoptosis, fibrosis and associated alterations of cell, plasma and urine profile as a biomarker. Deliverable : Easier methods to monitor mitochondrial oxidative stress as a marker of xenobiotic exposure.

The MIT-ROS-ER stress axis

Effect of xenobiotics on cell viability, Reactive Oxygen Species (ROS) production, and mitochondrial membrane permeability (MMP) Cell viability in H9c2 cardiac cells incubated with 50 µ M CCl 4 , 100 µ M paraquat, 20 µ M naphthalene, 10 µ M diclofenac (DIC) for 24 h. Pre-treatment with 20 µ M mito-Tempol (MT) prevented reduced cell vitality caused by CCl 4 . H 2 0 2 , 200 µ M.

Effect of xenobiotics on Cardiac Cell Viability Relative Cell Viability (%) 100 120 20 40 60 80 0 Control Celecoxib (30uM) ** CCL4 (50uM) ** Celecoxib (30uM) + CCL4 (50uM) ** H2O2 (200uM) **

Xenobiotic exposure affects mitochondrial electron chain transport activity and proteasome activity β 1 β 2 β 5 Mitochondrial complex l activity is decreased by naphthaline (20 µ M) and paraquat (100 µ M) but not CCl4 (20 µ M) or DIC (20 µ M). Lower figures show proteasome dysfunction occurs in hearts of ibuprofen treated mice

Reducing mitochondrial electron transport chain activity increases ROS and reduces cell viability Ibuprofen treated mice Complex I Activity (Abs 340nM) 0.35 Female Heart 8D ROS 0.34 0.33 ** 0.32 0.31 0.30 0.29 0.28 0.27 0 20 40 60 80 100 120 140 160 180 200 Control Rotenone Complex I Activity (Abs 340nM) IB 0.36 Male Heart 8D 0.35 0.34 0.33 0.32 0.31 0.30 ROT – Rotenone (a Complex I activity inhibitor) 0.29 0.28 0 20 40 60 80 100 120 140 160 180 Controkl Rotenone IB

Current Model SF Chemicals Cardiomyocytes Mitochondrial dysfunction; Complex I and III inhibited NSAIDs ROS Δψ de creased UPS dysfunction ER stress CARDIOTOXICITY Oxidized proteins Proteasome Antioxidants Cell Death Transfection

Con Concl clusion ons s • CCl 4 naphthalene, paraquat induces cardiac toxicity, mitochondrial stress and proteasome dysfunction. • Mitochondrial-stress is induced by other xenobiotics: diclofenac, ibuprofen, naproxen. Fu Futu ture Dir Direction ections s • Expand target analysis (Pesticides, HaHs/PaHs, Commercial Chemicals and Pharmaceuticals). • Determine cell, blood and urinary biomarkers of mitochondrial dysfunction to monitor Xenobiotic exposure and possibly drive therapeutic intervention.

Acknowledgements • Bruce Hammock • Natalia Vasylieva (project 3) • Fawaz G. Haj (Project 5) • Christophe Morisseau (Project 5) • Jun Yang (core A) • Daniel Tancredi (core B) Funding from : NIEHS/Superfund Research Program P42 ES004699

Project 4 - Monitoring Mitochondrial Oxidative Stress and Cardiac Toxicity Caused by Chronic Exposure to Chemicals Nipavan Chiamvimonvat, MD Division of Cardiovascular Medicine

Mortality rate of cardiovascular disease surpasses that of cancer Cardiovascular Disease Cancer Circ 131(4): e29-322, 2015

Cardiac fibroblasts • Cardiac fibroblasts account for ~75% of all cardiac cells, but contribute only ∼ 10-15% of total cardiac cell volume. • The principal sources of extracellular matrix (ECM) proteins. • A heterogeneous population. • Derived from various distinct tissue niches including resident fibroblasts, endothelial cells, and bone marrow sources.

Roles of cardiac fibroblasts Yue et al , Cardiovascular Res 2011

Molecular mechanisms leading to cardiac fibrosis Wakili et al , 2011

Flow cytometric analysis of the isolated cells from mouse hearts

Recreating disease in a dish hiPSCs and hiPSC-CMs SSEA4 Merged DAPI Background Background Fluorescence Oct3/4 Specific Fluorescence SSEA-4 Specific Oct3/4 + hiPSCs SSEA + hiPSCs 10 3 99% 10 3 <1% 10 3 10 3 <1% 99% 10 2 10 2 10 2 10 2 10 1 10 1 10 1 10 1 0 0 0 0 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Side Scatter Side Scatter hiPSC-CMs DAPI Phalloidin Troponin T Merged

Activation of MAPK in hiPSC-CMs and hiPSC-fibroblasts by TNF- α

Novel Cell-in-Gel Platform Novel 3D Cell-in-Gel Spontaneous APs Single APs 20 mV 20 mV 250 ms 1s

Effects of mechanical stress on Ca 2+ handling

Conclusions • Generation of reliable platform for testing the effects of Superfund chemicals on cardiac myocytes and fibroblasts. • Development of bioassays to test the effects of exposure.

Acknowledgements • Bruce Hammock • Aldrin Gomes (Project 4) • Fawaz G. Haj (Project 5) • Christophe Morisseau (Project 5) • Jun Yang (core A) • Ye Chen-Izu • Padmini Sirish Funding from : NIEHS/Superfund Research Program P42 ES004699