PANWAT 2019 program final · Programming Workshop – Sunday, October 6th Introduction to Python Programming for Data Analysis and Visualization 8:15 a.m. - 4:00 p.m. Hampton Inn

Computational Toxicology

2019 Regional Chapter Meeting

Boise Centre on the Grove Boise, ID

October 6-7

QUICK GLANCE:

Sunday, October 6th Introduction to Python Programming

8:15 a.m. - 4:00 p.m., Hampton Inn and Suites, 495 S Capitol Blvd.

Welcome Reception

6:30 p.m. - 8:30 p.m., Idaho State Museum, 610 Julia Davis Dr.

Monday, October 7th Scientific Conference

8:30 a.m. – 5:30 p.m., Boise Centre on the Grove, 850 W. Front St.

PANWAT Annual Conference, October 6-7, 2019 Page 1 Boise Centre on the Grove, Boise, ID

PANWAT greatly appreciates the generous support of our 2019 Meeting Sponsors!

Bogus Basin (Platinum) Sponsors: $2500+


2019 PANWAT Sponsors (continued)

Snake River (Gold) Sponsors: $1,500-$2,499


Additional Sponsors: up to $500


Endowment Fund Contributors PANWAT greatly appreciates the

generous contributors:

$2,000 - $2,999

$1,000 – $1,999

Rafael Ponce Katie Sprugel

Robert Tanguay


Endowment Fund Contributors (continued)

$250 - $999 Cecile Krejsa Dennis M. Miller Linda L. Carlock Haley Neff-LaFord Friends of PANWAT: Up to $250

Abigail Brewer Angie Wong Anna Engstrom & Chad Weldy Anne Johnston Annette Myers Anonymous Donor Beth Wong Bukunmi Gesunde Cathie Adams

Christopher M. Carosino Dawn Hayes Jane Heffernan Matt Kadlec Paramita Mookherjee Rosita Rodriguez Siba Ranjan Das Stephen W. Frantz


2019–2020 PANWAT Officers

President: Haley Neff-LaFord, PhD, DABT Seattle Genetics, Inc.

Vice President: Kristen Mitchell, PhD Boise State University

Vice President-Elect: Julia Yue Cui, PhD University of Washington

Secretary/Treasurer: Christopher Carosino, PhD, DABT Seattle Genetics, Inc.

Past President: Nadia Moore, PhD, DABT, ERT JS Held

Postdoctoral Representative: David Scoville, PhD University of Washington

Senior Councilor: Lisa Truong, PhD Oregon State University

Senior Graduate Student Representative: Yvonne Chang, BS Oregon State University

Junior Councilor: Kevin Haines, MSc, DABT NovaTox

Junior Graduate Student Representative: Brianna Rivera, BS Oregon State University

Outreach Coordinator: Paramita Mookherjee, PhD, DABT Omeros

Officer Emeritus: Linda Carlock, MS, DABT Retired


2019 PANWAT Event Schedule - Overview

Hotel*:

Hampton Inn and Suites 495 S Capitol Blvd. Boise, ID 83702

*Free shuttle to the conference center

Programming Workshop – Sunday, October 6th Introduction to Python Programming for Data Analysis and Visualization 8:15 a.m. - 4:00 p.m.

Hampton Inn and Suites 495 S Capitol Blvd. Boise, ID 83702

Welcome Reception – Sunday, October 6th

6:30 p.m. - 8:30 p.m. Idaho State Museum 610 Julia Davis Dr. Boise, ID 83702

Conference and Registration – Monday, October 7th: 8:30 a.m. – 6:00 p.m.

Boise Centre on the Grove 850 W. Front St. Boise, ID 83702


2019 Regional Meeting Agenda

Computational Toxicology Boise Centre on the Grove, Boise, ID, October 6-7, 2019

Sunday, October 6th 8:15 AM – 4:00 PM COMPUTER PROGRAMMING WORKSHOP (REGISTRATION REQUIRED) Introduction to Python Programming for Data Analysis and

Visualization, Hampton Inn and Suites, 495 S Capitol Blvd 6:30 PM – 8:30 PM WELCOME RECEPTION Idaho State Museum, 610 Julia Davis Drive Monday, October 7th Location: Boise Centre on the Grove, 850 W Front St 7:30 - 8:30 AM Registration, Poster Set-Up, and Continental Breakfast 8:30 - 8:40 AM Welcome Remarks and Conference Overview

Dr. Haley Neff-LaFord, PANWAT President, Seattle Genetics, Inc.

MORNING SYMPOSIUM: CURRENT APPLICATIONS OF COMPUTATIONAL SCIENCE

8:40 - 9:40 AM Omic and Bioinformatic Analyses: Examples from Aryl Hydrocarbon Receptor-Mediated Hepatotoxicity Dr. Tim Zacharewski, Michigan State University

9:40 - 10:10 AM Utilizing QST and QSP Modeling to Inform Clinical and Nonclinical

Development of Zymeworks’ Azymetric™ Biparatopic Platforms Dr. Rupert Davies, Zymeworks 10:10 - 10:30 AM Coffee Break and Poster Viewing 10:30 - 11:30 AM Perspectives on the Development, Evaluation and Application of In

Silico Approaches for Predicting Toxicity Dr. Grace Patlewicz, NCCT, US EPA

11:30 - 12:00 PM Data Science in Healthcare: Demystifying Data Science

John Girgis, Health Catalyst


LUNCH AND POSTER SESSION 12:00 - 12:30 PM Lunch (buffet lunch at Boise Centre) 12:30 - 1:45 PM Poster Attendance and Viewing

AFTERNOON SYMPOSIUM: RECENT ADVANCES IN TOXICOLOGY

1:45 - 2:05 PM Building a Predictive Flame Retardant Classification Model Using the

Multi-Dimensional Embryonic Zebrafish Platform Dr. Lisa Truong, Oregon State University

2:05 - 2:20 PM Gut Microbiome Critically Impacts PCB-induced Changes in Hepatic

Transcriptome Metabolic Fingerprints Joe Jongpyo Lim, University of Washington 2:20 - 2:40 PM Development of iPSC-Derived Human Neuroprogenitor cells (hNPCs)

and Neurons to Explore the Environmental Etiology of Guam Parkinson-Dementia Complex (PDC) Dr. Anna Chlebowski, Western University of Health Sciences

2:40 - 2:55 PM Beyond pathway analysis: leveraging a weighted gene co-expression

analysis to link gene sets with PAH-related cancer risk and regulatory values in 3D human bronchial epithelium Yvonne Chang, Oregon State University

2:55 - 3:10 PM Differential hepatic recovery after sub-lethal microcystin-LR

exposure in healthy versus nonalcoholic steatohepatitis rats Tarana Arman, Washington State University

3:10 - 3:25 PM Coffee Break and Poster Viewing 3:25 - 3:40 PM Utilizing Genome-wide Transcriptomics in Zebrafish to Discover and

Characterize a Novel Biomarker for Aryl Hydrocarbon Receptor Activation Prarthana Shankar, Oregon State University

3:40 - 4:00 PM Unraveling mechanisms of tris(1,3-dichloro-2-propyl) phosphate-

induced epiboly arrest in zebrafish Dr. Subham Dasgupta, University of California, Riverside, CA

4:00 - 4:20 PM Early-life PBDE Exposure in Mice Persistently Dysregulates Xenobiotic Biotransformation and Epigenetic Modification Genes in Adult Liver Dr. David Scoville, University of Washington


PANWAT 2019 TOXICOLOGY ACHIEVEMENT AWARD PRESENTATION

4:20 - 5:10 PM Data, Mechanism, Synthesis: The Toxicologist’s Craft Dr. Cecile Krejsa, Kartos Therapeutics

CLOSING EVENTS

5:10 - 5:25 PM Awards and Closing Announcements

Dr. Kristen Mitchell, PANWAT Vice-President, Boise State University Dr. Haley Neff-LaFord, PANWAT President, Seattle Genetics, Inc.

5:25 - 6:00 PM Closing Reception (at Boise Centre on the Grove)

Hosted by Seattle Genetics


2019 Toxicology Achievement Award

Cecile Krejsa, PhD VP, Translational Sciences, Kartos Therapeutics

Dr. Krejsa has led early- and late-phase nonclinical and clinical translation programs for diverse small molecule and protein therapeutics in oncology, autoimmune and allergy indications. Her previous roles include Senior Director, Pharmacology & Toxicology at Acerta Pharma (2014-2018); Senior Program Scientist, Experimental Medicine at Seattle Genetics (2011-13); Principal Scientist, Mechanistic and Applied Pharmacology at ZymoGenetics (2004-09); and Senior Scientist, BioPrint™ at Cerep (2001-04). She served on Leadership Council for the Society of Toxicology PANWAT Chapter (2003-11, including President 2009-10). Dr. Krejsa received her doctorate in Toxicology from the University of Washington (2000) and is an Affiliate Professor at the University of Washington Department of Environmental Health Sciences.


Invited Speaker Biosketches and Abstracts

(in order of presentation)


Timothy Zacharewski, Ph.D. Professor Department of Biochemistry & Molecular Biology, Center for Integrative Toxicology Michigan State University East Lansing, MI Timothy Richard Zacharewski is a Professor in the Department of Biochemistry & Molecular Biology and the Institute for Integrative Toxicology at Michigan State University. He obtained his undergraduate degree in chemistry from the University of Guelph in

Canada, a Ph.D. in Toxicology from Texas A&M University in the laboratory of S. Safe, and received a Medical Research Council of Canada Post Doctoral Fellowship to study with Professor Pierre Chambon in Strasbourg, France. In 1992, Dr. Zacharewski accepted an Assistant Professor position in the Department of Pharmacology & Toxicology at the University of Western Ontario. In 1997, he relocated to Michigan State University where he is now a Professor in the Department of Biochemistry & Molecular Biology and the Center for Integrative Toxicology. His research interests include using complementary omic and bioinformatics approaches to elucidate the mechanisms of toxicity of environmental pollutants, drugs, chemicals and natural products in in vitro and in vivo models. ABSTRACT: Omic and Bioinformatic Analyses: Examples from Aryl Hydrocarbon Receptor-Mediated Hepatotoxicity 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that elicits a broad spectrum of effects mediated by the aryl hydrocarbon receptor (AhR). Despite the conservation of the AhR signaling pathway, the mechanisms associated with the toxicity of TCDD and related compounds remain poorly understood. My lab investigates the species- and sex-specific effects of AhR ligands using omic approaches that are integrated with complementary histopathology and computational/bioinformatic analyses to examine temporal and dose-dependent responses in in vitro and in vivo models. In this presentation, I will provide examples of how we use computational/bioinformatic tools and other resources to analyze, integrate and interpret disparate data sets as we investigate the effects of TCDD and related compounds on the progression of steatosis to steatohepatitis with fibrosis.


Rupert Davies, Ph.D. Director, Translational Sciences Zymeworks Seattle, WA Dr. Rupert Davies currently serves as a Director of Translational Sciences at Zymeworks, where he is responsible for the development of drug candidates from the hit discovery stage through to IND-enabling studies and into FIH clinical trials. Dr. Davies was part of the Zymeworks team that enabled the advancement of ZW25 and ZW49 into the clinic and he and the team are now focused on combining Zymework’s Azymetric™ Bispecific and ZymeLink™ Antibody Drug Conjugate (ADC) platforms to develop drug candidates with expanded therapeutic windows. Throughout his career, he has worked on a broad array of drugs modalities from small molecules and peptides to RNA mimics and antibodies, including five candidates that are currently being evaluated in clinical trials and two approved drugs. Prior to his current role, Dr. Davies worked in various roles at Amgen as well as small start-ups and medium-sized biotechs. He received his PhD in Bioengineering from the University of Utah and completed a post-doctoral fellowship in Bioengineering at the University of Washington. ABSTRACT: Utilizing QST and QSP Modeling to Inform Clinical and Nonclinical Development of Zymeworks’ Azymetric™ Biparatopic Platforms Pharmacokinetic/Pharmacodynamic Modeling and Therapeutic Index Estimation: • Inclusion of biparatopic binding stoichiometry drives more precise fit of PK data in cynos and humans • Utilization of novel pharmacodynamic parameter to evaluate effect of dose concentration

and regimen on efficacy • Estimated delivery of toxin to four compartments: tumor, on-target organs/tissues and off-

target organs/tissues associated with free toxin and FcGammaR2a binding of the antibody • Therapeutic index estimation with different doses and dose frequencies


Grace Patlewicz, Ph.D. Research Chemist National Center for Computational Toxicology (NCCT) US Environmental Protection Agency Research Triangle Park, NC Dr. Grace Patlewicz is currently a research chemist at the National Center for Computational Toxicology within the United States Environmental Protection Agency (EPA). She started her career at Unilever, United Kingdom, before moving to the European Commission Joint Research Centre in Italy and then to DuPont in the United States. A chemist and toxicologist by training, her research interests have been focused on the development and application of (Q)SARs and read-across for regulatory purposes. She has authored ~120 journal publications and book chapters, chaired various Industry groups and has contributed to the development of technical guidance for (Q)SARs, chemical categories and Adverse Outcome Pathways (AOPs) under various Organisation for Economic Co-operation and Development (OECD) work programmes. ABSTRACT: Perspectives on the Development, Evaluation and Application of in silico approaches for predicting toxicity Changes in regulations in the industrial chemicals and cosmetics sectors in recent years have prompted a significant number of advances in the development, application, and assessment of non-testing approaches, such as (Q)SARs and read-across. Many efforts have also been undertaken to establish guiding principles for performing read-across within category and analogue approaches. This presentation offers a perspective, as taken from these sectors, of the current status of non-testing approaches, their evolution in light of the advances in high-throughput approaches and constructs such as adverse outcome pathways. The presentation will illustrate these perspectives drawing from 2 main approaches – 1) Exposure based waiving approaches such as the Threshold of Toxicological Concern (TTC) and their utility in risk-based prioritization decision contexts and 2) Read-across approaches – transitioning towards a hybrid harmonized objective read-across workflow and the application of chemical grouping approaches to identify and evaluate PFAS (per and polyfluorinated substances) toxicities. This abstract does not necessarily represent U.S. EPA policy.


John Girgis Technical Director Health Catalyst Boise, ID John Girgis is a Technical Director at Health Catalyst, a leading software solutions provider for healthcare outcomes improvements. With a background in electrical engineering and computer science, John has over 20 years working in software development and data warehousing. His focus is on helping hospitals build and run the data warehouses needed to support data science and data analytics solutions. More recently, John

has also gone “back-to-school” to apply his software and data skills toward biomolecular science and improving adoption of data science methods for accelerating research in human disease management and treatments. ABSTRACT: Data Science in Healthcare: Demystifying Data Science In today's world of rapidly evolving technology, application of data science to healthcare is becoming more prevalent and is helping hospitals deliver improved patient outcomes. How is this work being done and what can we learn from the application of data science in healthcare? Moreover, the rate of evolution of data science requires periodic examination of our understanding of computational/bioinformatics methods and tools. Industry terms like Big Data, Machine Learning, and even "Data Science" itself, are often misunderstood or become clouded with technical jargon. This presentation will use the healthcare industry as an example to help clarify the application of data science. In addition, I will present a common framework for applied data science that can be used to support scientific research and investigation.


Abstracts for Platform Presentations

(in order of presentation) Building a predictive flame retardant classification model using the multi-dimensional embryonic zebrafish platform Lisa Truonga, Skylar Marvelb, David M. Reifb, Michael T. Simonicha, and Robert L. Tanguaya

aDepartment of Environmental and Molecular Toxicology, the Sinnhuber Aquatic Research Laboratory, and the Environmental Health Sciences Center at Oregon State University, Corvallis, OR; bBioinformatics Research Center, North Carolina State University, Raleigh, NC Flame retardants chemicals (FRCs) commonly added to many consumer products present a human exposure burden associated with adverse health effects. Under pressure from consumers, FR manufacturers have adopted some purportedly safer replacements for first generation brominated diphenyl ethers (BDEs). There is limited toxicity data available to estimate the potential adverse effects of the second and third generation organophosphates and other alternative chemistries. There is a need to establish methods to quickly and efficiently screen chemicals for toxic effects. In this study, we built a 61-FRC library and systemically screen for developmental neurotoxic effects in the embryonic zebrafish model. This data was then compared to publicly available data generated in a battery of cell-based in vitro assays from ToxCast and Tox21 and other alternative models. Of the 61 FRCs, 19 of 45 that were tested in the ToxCast assays were bioactive in more than 2 assays (when grouped by assay type). The embryonic zebrafish data was compared to the 12 previously classified developmental neurotoxic FRCs, which revealed the multi-dimensional zebrafish assay was bioactive in 10/12. As the zebrafish data was high predictive of potential developmental and neurotoxicity, we built a classification model using 12 physico-chemical properties and 3 embryonic zebrafish assays and achieved a balance accuracy of 83.33%. Beyond safer FRCs, this work illustrates the power of a multi-dimensional in vivo platform to expands our ability to predict the hazard potential of new compounds based on structural relatedness, ultimately leading to reliable structure-toxicity prediction without in vivo testing. Gut Microbiome Critically Impacts PCB-induced Changes in Hepatic Transcriptome Metabolic Fingerprints Joe Jongpyo Lim1, Xueshu Li2, Hans-Joachim Lehmler2, Dongfang Wang3, Haiwei Gu3, and Julia Yue Cui1 1Department of Environmental and Occupational Health Sciences, University of Washington; 2Department of Occupational and Environmental Health, University of Iowa; 3Arizona Metabolomics Laboratory, School of Nutrition and Health Promotion, College of Health Solutions, Arizona State University


Polychlorinated biphenyls (PCBs) are ubiquitously detected in the body and have been linked to numerous diseases. The liver serves as a central hub for the metabolism of xenobiotics and metabolites. Xenobiotics-induced gut dysbiosis is recognized as a critical regulator of disease susceptibility. We hypothesized that PCBs-mediated changes in the metabolic fingerprints and hepatic transcriptome are regulated by enterotype and dose. Ninety-day-old female conventional (CV) and germ-free (GF) C57BL/6 mice were orally exposed to the PCB Fox River Mixture (synthetic PCB mixture, 6 or 30 mg/kg) or corn oil (vehicle, 10 ml/kg), once daily for 3 consecutive days. Organs were collected 24 hours after the final dose. RNA-Seq was conducted on liver, and endogenous metabolites were quantified in liver and serum by LC-MS. Enterotype was the primary factor in clustering the transcriptomic and metabolomic signatures within the same exposure. The numbers of PCB-regulated genes were higher in CV than in GF conditions. The prototypical target genes of the major xenobiotic-sensing transcription factors PXR, CAR, and AhR were more readily induced by PCBs in CV compared to GF conditions, indicating the effect of PCBs on the hepatic transcriptome act partly through the gut microbiome. In addition to the dysregulation of xenobiotic biotransformation and inflammation genes, the folding of incorrect proteins pathway was down-regulated by PCBs, especially in CV conditions. At the high PCB dose, NADP and arginine appear to interact with the drug-metabolizing enzymes in the Cyp1-3 family, Dhcr7, and Nqo1 in CV mice. In GF exposure groups, hepatic glucose was down-regulated, whereas fructose 6-phosphate and glucose 6-phosphate were up-regulated, indicating increased PCBs-mediated glucose utilization is potentiated by lack of gut microbiota. Our findings demonstrate that habitation of the gut microbiota drives PCBs-mediated hepatic responses, possibly due to enterohepatic circulation. Development of iPSC-Derived Human Neuroprogenitor cells (hNPCs) and Neurons to Explore the Environmental Etiology of Guam Parkinson-Dementia Complex (PDC) A.C. Chlebowski1, Y. Yang2, N. Siddique2, T. Siddique2, P.S. Spencer3, J.C. Steele4, G.E. Kisby1 1Basic Medical Sciences, Western University of Health Sciences, Lebanon, OR; 2Northwestern University Feinberg School of Medicine, Chicago, IL; 3Department of Neurology and Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 4Formerly, Department of Medicine, Guam Memorial Hospital, Tamuning, Guam The Parkinsonism-Dementia Complex (PDC) is one phenotype of a disappearing neurodegenerative disease (Guam ALS-PDC) that shows clinical and neuropathological relationships with amyotrophic lateral sclerosis (ALS), atypical parkinsonism and Alzheimer’s disease. ALS-PDC has been linked with both genetic and environmental factors (i.e., certain metal ions, cycad plant neurotoxins), but evidence from human and animal studies is inconclusive. Patient-specific induced pluripotent stem cells (iPSCs) that generate neural progenitor cells (hNPCs) and neurons provide a powerful in vitro system to explore the underlying cause of PDC. We derived iPSC lines from lymphocytes of a PDC-affected Guam female patient and an age-matched healthy Guamanian using non-integrating episomal plasmids. iPSCs were cultured for a minimum of 10 passages and characterized with established pluripotency markers (Oct4, SSEA-4, TRA-1-60, Sox2) prior to the generation of hNPCs and neurons. Protocols for development of the Guam iPSC-derived NPCs and neurons matched those for the NIH-established iPSC line ND50031 (healthy control). An embryoid body protocol (STEMCell Technologies) was used to


derive hNPCs from the iPSC lines and the BrainPhys™ hNPC Neuron Kit (STEMCell Technologies) was used to differentiate hNPCs into mature neurons. hNPCs from all iPSC lines were immunoprobed with established neuroprogenitor markers (nestin, Sox2, Pax6), and neurons immunoprobed for markers of maturing neurons (β-tubulin, Map2, doublecortin). hNPCs and neurons from the ND50031 and Guam lines were comparable based on morphology and immunocytochemical neural markers. Studies are underway to differentiate the iPSC derived hNPCs into neurons and glial cells to examine their response to metal ions and cycad toxins, notably whether neuropathological changes match those that characterize Guam PDC. These studies should clarify the role of environmental factor(s) in ALS-PDC and possibly other related neurodegenerative diseases. Beyond pathway analysis: leveraging a weighted gene co-expression analysis to link gene sets with PAH-related cancer risk and regulatory values in 3D human bronchial epithelium Yvonne Chang1, Julia E. Rager2,3, Susan C. Tilton1,4 1Environmental and Molecular Toxicology Department, Oregon State University, Corvallis, OR; 2Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, The University of North Carolina at Chapel Hill; 3Institute for Environmental Health Solutions, The University of North Carolina at Chapel Hill, Chapel Hill, NC; 4NIEHS Superfund Research Program, Oregon State University, Corvallis, OR, Polycyclic aromatic hydrocarbon (PAH) exposures are linked to numerous adverse outcomes in humans, with cancer as the greatest concern. The cancer risk associated with PAH exposures is commonly evaluated using the relative potency factor (RPF) approach, which estimates PAH mixture carcinogenic potential based on the sum of relative potency of individual PAHs in rodent cancer bioassays, compared to benzo[a]pyrene (BAP), a reference carcinogen. This study set out to combine a novel human in vitro bronchial epithelial cell (HBEC) model with transcriptomic and bioinformatic approaches to evaluate molecular mechanisms of direct relevance to PAH cancer risk. Significantly regulated genes from HBEC treated with PAHs and PAH mixtures were analyzed using a two-tiered WGCNA approach, first to identify gene sets co-modulated to RPF, and then to link genes to a more comprehensive list of regulatory values, including inhalation-specific risk values. Over 3,000 genes from cell cycle regulation, inflammation, DNA damage, and cell adhesion processes were found to be significantly correlated and co-modulated with increasing RPF/BAPeq. Co-modulated genes were linked to additional cancer-relevant risk values, including inhalation unit risks and oral cancer slope factors. These gene sets represent potential biomarkers that could be used to evaluate cancer risk associated with PAH mixtures. Here, we demonstrated a novel manner of integrating gene sets with chemical toxicity equivalence estimates through WGCNA to understand potential mechanisms. Similar studies could further inform the incorporation of organotypic human in vitro models into evaluating cancer risk associated with chemical exposures.


Differential hepatic recovery after sub-lethal microcystin-LR exposure in healthy versus nonalcoholic steatohepatitis rats Tarana Arman, Katherine D. Lynch, Johnny T. Aldan, Baron J. Bechtold and John D. Clarke Department of Pharmaceutical Science, Washington State University, Spokane, WA 99202, USA

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease that can advance to cirrhosis and hepatocellular carcinoma due to continual exposure to exogenous stressors such as poor diet or toxicants. Microcystin-LR (MCLR) is a hepatotoxic cyanotoxin known to cause a NASH-like phenotype and exacerbate preexisting diet-induced NASH. Previous data indicate the liver can recover after MCLR-induced hepatotoxicity in healthy rodents. We hypothesize that MCLR-induced hepatotoxicity in diet-induced NASH will not recover due to continued exogenous stress from a poor diet. Male Sprague-Dawley rats were divided into two groups and fed either a control or a high fat/high cholesterol (HFHC) diet for eight weeks. The animals were then subdivided into two treatment groups receiving intraperitoneal injection of either vehicle or 30 µg/kg MCLR every other day for two weeks. Animals from each diet/treatment group were euthanized at one of three time points: at the completion of the MCLR exposure period, after 2 weeks of recovery, and after 4 weeks of recovery. Diet regimens were maintained for the entirety of the study. MCLR toxicity altered fasting plasma biochemistries in both healthy and NASH groups (triglycerides increased 54-97%, insulin increased ~150%, glucose decreased ~60%). After MCLR exposure, plasma ALT increased 122% in the control group and plasma cholesterol levels increased 210% in the NASH group. All plasma biochemistries recovered after four weeks. In contrast, MCLR-induced liver fibrosis recovered after four weeks in the healthy group but not in the NASH group. After four weeks of recovery the MCLR treated NASH group has less liver steatosis and more liver inflammation compared to the vehicle treated NASH group. These data suggest that despite recovery in plasma biochemistries, continued stress of a poor diet after MCLR exposure impairs hepatic recovery mechanisms and resembles a burnt-out NASH phenotype. (Funding: 4R00ES024455) Utilizing Genome-wide Transcriptomics in Zebrafish to Discover and Characterize a Novel Biomarker for Aryl Hydrocarbon Receptor Activation Prarthana Shankar*, Mitra C. Geier*, Ryan S. McClure**, Gloria R. Garcia*, Jane K. La Du*, Lisa Truong*, Katrina M. Waters**, Christopher M. Sullivan*, Robert L. Tanguay* *Department of Environmental and Molecular Toxicology, Oregon State University **Biological Sciences Division, Pacific Northwest National Laboratory The aryl hydrocarbon receptor (AHR) is activated by several chemicals including polycyclic aromatic hydrocarbons (PAHs) and TCDD. AHR activation is well understood, but the roles of downstream genes are largely unknown. We conducted transcriptomics in 48-hours post fertilization (hpf) zebrafish developmentally exposed to 16 PAHs. Using the Context Likelihood of Relatedness program, we inferred a network that links the PAHs based on coordinated responses of genes that are altered as a function of PAH exposure. The PAHs formed two major clusters: Cluster 1 consisted of the less biologically active PAHs, while Cluster 2 induced AHR2 (zebrafish ortholog of human AHR)-dependent Cyp1a expression in the skin and/or vasculature. In addition to cyp1a and cyp1c1, one of the most elevated transcripts by all Cluster 2 PAHs was wfikkn1


(WAP, kazal, immunoglobulin, kunitz and NTR domain-containing protein), a potential novel biomarker for AHR2 activation. When we exposed AHR2-null and Wildtype zebrafish to DMSO or TCDD, and quantified the expression of wfikkn1, the significant induction of wfikkn1 was AHR2-dependent. To functionally characterize wfikkn1, we generated a wfikkn1 CRISPR-Cas9 mutant zebrafish line which appears morphologically normal but is hyperactive in a photomotor response assay at 120 hpf. To identify genes/pathways associated with wfikkn1, Wildtype and mutant zebrafish were exposed to DMSO or TCDD, and mRNA from 48-hpf zebrafish was sequenced. We identified over 200 differentially expressed genes between each pair of treatment combinations. The significant GO term processes associated with wfikkn1 mutants include ATP-binding, immunoglobulin, and iron binding processes. ATP-binding, transmembrane, and protein kinase processes were uniquely altered in TCDD-exposed wfikkn1 mutants compared to TCDD-exposed Wildtype zebrafish. We are currently investigating in situ RNA expression and in vitro Wfikkn1 protein activity to understand downstream transcriptional events. Unraveling mechanisms of tris(1,3-dichloro-2-propyl) phosphate-induced epiboly arrest in zebrafish Subham Dasgupta, Sara M.F.Vliet, Vanessa Cheng, Constance A. Mitchell, David C. Volz Department of Environmental Sciences, University of California, Riverside, CA Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organohalogen flame retardant that migrates from end-use products into indoor dust, raising concerns about potential impacts on human embryonic development. Using zebrafish as a model, we showed that initiation of TDCIPP exposure during cleavage/early-blastula (0.75-3 h post-fertilization, or hpf) results in epiboly defects by 6 hpf. We also studied the downstream effects of epiboly defects on the developmental trajectory and found that TDCIPP-exposed embryos display an irreversible disruption of medoserm formation, dorsoventral patterning, and development of RBC and heart later in development. Therefore, using a combination of pharmacologic and epigenetic strategies, sought to identify potential mechanisms involved in TDCIPP-induced epiboly arrest. As data available from the U.S. EPA’s ToxCast program suggested that TDCIPP may target certain nuclear receptors, we first exposed embryos to TDCIPP in the presence or absence of 70 different nuclear receptor ligands from 0.75-6 hpf and then quantified epiboly defects at 6 hpf. Based on this initial screen as well as follow-up pre-treatment studies, we found that ciglitazone (a peroxisome proliferator-activated receptor γ, or PPARγ, agonist) and 17β-estradiol (E2, an estrogen receptor α, or ERα, agonist) significantly mitigated TDCIPP-induced epiboly arrest in a concentration-dependent manner. Moreover, bisulfite amplicon sequencing revealed that co-exposure to E2 (but not ciglitazone) from 0.75-2 hpf alleviated TDCIPP-induced effects on CpG methylation at 2 hpf, suggesting that, contrary to ciglitazone, the protective effects of E2 on epiboly may be linked to differences in epigenetic reprogramming. Overall, our results point to a complex interplay among nuclear receptors and cytosine methylation in both the modulation and alleviation of epiboly defects induced by TDCIPP.


Early-life PBDE Exposure in Mice Persistently Dysregulates Xenobiotic Biotransformation and Epigenetic Modification Genes in Adult Liver David K. Scoville, Joseph L. Dempsey, and Julia Yue Cui Department of Environmental and Occupational Health Sciences, University of Washington The former flame retardants polybrominated diphenyl ethers (PBDEs) are bioaccumlative and readily detectable in humans. Exposures to environmental toxicants during development can alter adult disease risks. We have previously shown that acute exposure to PBDEs in adult mice alters expression of xenobiotic biotransformation genes in liver. Here, we tested our hypothesis that early-life exposure to PBDEs would result in hepatic xenobiotic biotransformation gene expression changes that persist into adulthood. Male and female C57BL/6J mice were exposed to PBDE congeners BDE-47 (5, 50, 150 μmol/kg), BDE-99 (1, 5, 50 μmol/kg), or corn oil (vehicle, 10 ul/g) once per day via intraperitoneal injection on postnatal days (PND) 2-3. Tissues were collected at PND5 (acute response), PND15 and 30 (adolescent ages), and PND60 (adulthood). Liver gene expression was quantified with RNA-Seq. Reads were aligned to mm10 using HISAT2. Genes were counted using featureCounts. DESEQ2 was used for differential expression analysis. Neonatal exposure to BDE-47 and -99 significantly altered the liver transcriptomes of adult male and female mice. Overall, BDE-99 altered the expression of more genes than BDE-47. However, the magnitude and direction varied by dose, sex and age. Males exposed to high-dose BDE-99 had the largest number of genes dysregulated at PND5. In contrast, low-dose BDE-99 males exhibited the largest number of dysregulated genes at PND60. Both congeners down-regulated genes in females at PND60. Similar trends were observed in xenobiotic biotransformation genes. At PND60, several Cyp2, Cyp4, Gst, and Ugt genes were regulated by low-dose BDE-99. Genes that epigenetically regulate transcription, including histone lysine demethylases (Kdm), were also regulated by BDE-99 through Day60. Inverse correlations were observed between Kdm5d and certain P450 isoforms (Cyp2b and Cyp4a) both at PND30 and PND60. Kdm5d is known to demethylate H3K4me3, an important regulatory epigenetic mark.


Abstracts for Poster Presentations

(in alphabetical order, first author)

Effects of Common E-Cigarette Compounds on In-Vitro Viability and Glutathione as Potential Modulators of Susceptibility Michael L. Anderson1, Dianne Botta1, Collin C. White1, Lung-chi Chen2, Terry Gordon2 and Terrance J. Kavanagh1

1Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA; 2Department of Environmental Medicine, New York University, New York, NY In 2017, 2.9% of adults reported having used an e-cigarette at some point. More concerning is that 20.8% and 4.9% of high school and middle school students, respectively, also reported using e-cigarettes at least once. However, comparatively little research has been done on the potential hazards and effects of e-cigarettes. Four specific chemicals have been commonly associated with vaping: the pyrolysis products formaldehyde and acetaldehyde; and the flavoring agents cinnamaldehyde and diacetyl. In addition to assessing cytotoxicity, we are investigating whether the antioxidant glutathione (GSH) modulates susceptibility. In vitro experiments were carried using two immortalized mouse liver cell lines (wild-type cells, and GSH deficient Gclm null/β-galactosidase knock-in cells). Cell viability at multiple doses of each compound was measured using the alamarBlue assay. DDAOG, a β-galactosidase substrate, was used as a proxy for Gclm promotor activity. Data were analyzed using Microsoft Excel, R, or Prism 8. Both cell lines dosed with formaldehyde showed decreased viability (though not statistically significant, pending re-run) starting at 400 µM. Cinnamaldehyde caused decreased viability starting around 500 µM in wild type cells; Gclm null cells showed decreased viability at 250 µM with a moderate resurgence up until 750 µM, after which viability dropped off again. With both compounds, Gclm null cells exhibited greater sensitivity than wild type cells, suggesting that GSH plays a key role in detoxification. Acetaldehyde and diacetyl showed little effect on cell viability in either cell line, even up to 1 mM. These results do not preclude the potential for hazard, since liver cells tend to have high metabolic and detoxification activity and may not indicate effects in other cell types (e.g. lung cells). Future studies will assess effects on GSH, oxidative stress and DNA damage, and whether these in vitro results predict in vivo effects. Supported by NIH R01CA239253. Metabolic Response to Benzo[a]pyrene Toxicity in Normal Human Bronchial Epithelial Cells Kelley Bastin, Brianna Rivera, Lisbeth K. Siddens, Monica Maier, David E. Williams, Susan C. Tilton Oregon State University Polycyclic aromatic hydrocarbons (PAH) are contaminants released into our environment from incomplete combustion processes associated with wood burning, fossil fuels and cigarettes. Some PAHs are carcinogenic and excessive exposure can result in several forms of cancer, including


lung. When PAHs are inhaled, cells metabolize them to reactive metabolites that can cause mutations and alteration of gene signaling leading to tumor growth. The role of metabolism in susceptibility of chemically-mediated toxicity and respiratory disease in humans is not well understood and investigations are hindered by lack of adequate research models. Most standard in vitro culture systems lack the capacity to metabolize chemicals and may not adequately represent toxicity in humans. Therefore, the objective of our study is to evaluate the metabolic capacity of primary human bronchial epithelial cells (HBEC) cultured in 3D as a model to study toxicity of PAHS. Cells were treated for 48 hr with the PAH benzo[a]pyrene (BaP; 10-500ug/ml) and collected for evaluation of transcriptional biomarkers and formation of BaP metabolites. Global gene expression data was analyzed by benchmark modeling to identify genes and pathways sensitive to dose-response treatment. DNA damage, xenobiotic response and oxidative stress were among the most significant pathways regulated by BAP treatment and dose-dependent changes in transcripts for several Phase I and II enzymes have been confirmed by qPCR. In addition, preliminary data shows formation of BAP metabolites in cells and media from 3D HBEC as measured by UPLC. Future studies will measure metabolic enzyme activity by activity-based protein proteomics to correlate with gene expression data and metabolism studies. Overall, these studies will help to determine the relevance of in vitro 3D culture models for studying chemical toxicity in the lung. Differences in Inflammation Induced by Silver Nanoparticles and Quantum Dots in Glutathione Deficient and Normal Mice is Influenced by Lung Clearance Rates D Botta, CC White, LM McConnachie, SC Schmuck, DK Scoville, CM Schaupp, X Gao and TJ Kavanagh Department of Environmental and Occupational Health Sciences, University of Washington Engineered nanomaterials (ENMs) have utility in a number of commercial, industrial and medical applications. Many ENMs have been shown to elicit inflammatory responses in the lungs of rodents exposed via inhalation or aspiration/instillation. We previously published that a relatively low dose (6 ug/kg BW) of CdSe quantum dots (QDs) caused significant lung inflammation at 8 hr when delivered by nasal installation to male Gclm wild type (WT) and heterozygous (HT) mice but not Gclm knock-out (KO; glutathione (GSH) deficient) mice. Using IC-PMS analysis, we also observed in that study that QDs were cleared from the lungs of Gclm KO mice but not Gclm WT or HT mice. Furthermore, in unpublished studies, all female Gclm genotypes responded similarly to the males after QD instillation at both 8 and 24 hr. In this study, we report on the lung inflammatory response (%neutrophils in bronchoalveolar lavage determined by FACS or cytospin counts at 24 hr) of male and female Gclm WT, HT and KO mice to silver nanoparticles (AgNPs; 250 ug/kg BW) delivered by oropharyngeal aspiration. The delivered dose of AgNPs was estimated by examining light side-scatter in lavaged cells using FACS. Side-scatter was generally correlated with the %neutrophils. Based on side-scatter the combined three Gclm genotypes and both sexes, AgNPs were retained, suggesting that Ag was not cleared from the lungs of these mice. In contrast to QD instillation, aspiration of AgNPs caused a significant lung inflammatory response in both male and female mice, irrespective of Gclm genotype. These results indicate that Gclm genotype differentially influences the clearance of QDs but not AgNPs from the lungs of mice, which has implications for ENM induced lung inflammation in humans carrying genetic


polymorphisms in GCLM and other GSH-related genes. Supported by NIH/NIEHS P30ES007033, U19ES019545, T32ES007032 and T32AG000057. Transcriptome RNA-seq Reveals Non-Alcoholic Fatty Liver Disease Mediated by AhR Activation Giovan N. Cholico1, Kenneth A. Cornell1,3,4, Kristen A. Mitchell1,2

1Biomolecular Sciences Graduate Programs, Boise State University, Boise, ID 2Department of Biological Sciences, Boise State University, Boise, ID

3Department of Chemistry and Biochemistry, Boise State University, Boise, ID 4Biomolecular Research Center, Boise State University, Boise, ID Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disorders that ranging from simple steatosis to fibrosis and cirrhosis. Recent evidence indicates that activation of the aryl hydrocarbon receptor (AhR) by the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) elicits NAFLD and liver fibrosis. We have previously shown that TCDD increases liver damage, inflammation, and myofibroblast activation during experimental liver fibrosis elicited by chronic administration of carbon tetrachloride (CCl4). In this project, we used mice with AhR-deficient hepatocytes (AhRDHep) to investigate mechanisms of TCDD-induced fibrosis and NAFLD. AhRDHep and control (AhRfl/fl) mice were treated with 1.0 ml/kg CCl4 every four days for 5 weeks, and TCDD (100 μg/kg) was administered during the final week of the experiment. Mice were euthanized, and liver tissue was collected for histological and gene expression analyses. TCDD treatment markedly increased liver damage, inflammation, myofibroblast activation, and steatosis in CCl4-treated AhRfl/fl mice but not in CCl4-treated AhRDHep mice. In general, these endpoints were not markedly increased by CCl4 or TCDD treatment alone. RNA-seq analysis of gene expression corroborated the histopathological findings and further revealed that TCDD administration produced a NAFLD-like gene expression profile in CCl4-treated AhRfl/fl mice. Notably, TCDD reduced the expression of genes related to glycolysis, gluconeogenesis, and glycogen synthesis in CCl4-treated AhRfl/fl mice. Based on these findings, we conclude that a single dose of TCDD is sufficient to produce NAFLD in mice with preexisting liver injury, and this occurs through a mechanism that requires AhR signaling in hepatocytes. Evaluating child blood lead levels in a mining community after 30 years of remediation activities AM Feldpausch, RA Schoof, C Van Landingham Ramboll US Corporation Substantial remediation of mine wastes has occurred over the past 30 years and a residential metals abatement program has been active for nearly 15 years in the mining and mineral processing community of Butte, MT. To assess the effectiveness of these programs in reducing lead exposures, a 2014 study evaluated blood lead in venous samples collected from children ages 1-5 during 2003 through 2010. Individual blood lead records were assigned to two neighborhood districts, Uptown and Flats, to also evaluate differences in blood lead levels (BLLs) between neighborhoods. Uptown


is located at higher elevation and includes the city center located atop historical mine workings whereas the Flats are an area at lower elevation with newer housing and commercial areas. These districts were compared using a linear mixed regression model. Significant declines in BLLs were observed over time in both neighborhoods. Statistically significant differences in BLLs were seen between Uptown and Flats in the most recent test years, when the geometric mean BLLs were at their lowest values. Uptown geometric means declined from 3.55 µg/dL in 2003-2004 to 1.71 µg/dL in 2009-2010, while geometric means in the Flats declined from 3.42 µg/dL to 1.44 µg/dL during the same time span. An update to this study is underway, in which 2,317 BLL records from 2012 through 2017 are being evaluated. The blood lead limit of detection (LoD) is 3.33 µg/dL for the current dataset where BLLs were measured using a point-of-care device, compared to the LoD of 1 µg/dL obtained previously. Given that almost 80% of BLLs are now below the current LoD, calculation of mean BLLs is not meaningful. Assessment of remediation effectiveness is focused on evaluating the percent of children with BLLs greater than the reference level of 5 µg/dL. Comparison with NHANES data is confounded by lack of confirmation of elevated BLLs in venous samples. Leveraging the zebrafish model to evaluate the hazard potential of metal oxide nanomaterials Kimberly Hayward, Lisa Truong, Michael Simonich, and Robert L. Tanguay Department of Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory, Oregon State University Metal oxide Nanoparticles (MONPs) measuring between 1 and 100nm in diameter are a rapidly expanding technology due to their potential application in medicine, sensing, energy storage, and consumer products. Their potential undesirable effects on human health remain largely unknown. The National Institutes of Environmental Health Sciences (NIEHS) developed the Nanomaterials Health Implications Research Consortium, to systematically evaluate the potential for adverse health effects from exposure to nanomaterials across human relevant systems. Metal oxides are a Consortium priority for structure-bioactivity assessments. A suite of eight MONPs with physicochemical properties characterized by the Harvard School of Public Health-NIEHS Nanosafety Center, were supplied by the Consortium. We exposed zebrafish to individual MONPs (0, 2.32, 5, 10.7, 23.2, or 50 µg/ml; n=32/conc.) from 6 hours post-fertilization (hpf) to 120 hpf. Over two dozen morphology and behavioral endpoints were assed at 24 and 120 hpf. None of the metal oxides were associated with aberrant morphology at 120hpf, but WO3 (15 nm), ZnO (50 nM), TiO2 (100 nm), SiO2 (15 nm) Al2O3 (30 nm), and both CeO2 (10 and 30 nm) produced abnormal photomotor behavior. Our next step is to determine if developmental life stage only exposure to MONPs from 6 – 96 hpf produce behavioral or respiratory fitness deficits in adult zebrafish as we are testing the hypothesis that early life stage exposures will lead to adult deficits or disease. Our ultimate goal is to establish a systems level approach that anchors these complex phenotypes to underlying transcriptome changes. The work is supported by NIEHS grant U01 ES027294.


Evaluating Biomarkers in Response to Polycyclic Aromatic Hydrocarbon Exposure in Diseased and Normal Human Bronchial Epithelium C. HUYNH*, Y. Chang, K. Bastin, B. Rivera, L.K. Siddens and S.C. Tilton Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331 Polycyclic aromatic hydrocarbons (PAHs) are known to cause adverse health effects in the human lung, including cancer and elevated inflammation. We evaluated biomarkers relevant for PAH toxicity in the 3D human primary bronchial epithelial cell (HBEC) model collected from normal and asthmatic donors. We previously found PAHs to decrease cellular barrier function integrity, which can lead to increased permeability, inflammation, and oxidative stress of the airway epithelium and play a role in the pathogenesis of lung disease. Therefore, we hypothesized that inflammation pathways may be dose-responsive for PAH exposure when evaluating diseased and normal cells susceptibility to chemical insult. Primary HBEC from normal and asthmatic donors were exposed to benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC) or coal tar extract (CTE) at concentrations ranging 0.02-2 ug/ml in PBS with 1% DMSO. Trans-epithelial electrical resistance (TEER) was used to evaluate barrier function. Lactate dehydrogenase leakage assay was performed to determine cytotoxicity. TEER showed a dose dependent decrease in asthmatic cells. Asthmatic cells seem to be more sensitive to DBC treatment than the normal cells. RNAseq from normal cells treated with concentrations of BaP 10, 100 and 500 µg/ml was used to identify dose-responsive pathways and biomarkers. Using K-Means and hierarchical clustering, significant clusters of dose-responsive genes were identified. Significant gene clusters were uploaded on to a commercial Metacore software for functional enrichment analysis of gene expression data. Inflammation and apoptosis pathways were identified to exhibit a dose response across BaP treatments in normal cells. We will also analyze RNAseq results from DBC and CT-treated normal cells to identify dose-responsive pathways and genes. Next steps include identifying sensitive biomarkers that exhibit dose response in these pathways in normal HBEC and comparing gene expression using qPCR in diseased HBEC.

Hepatocyte aryl hydrocarbon receptor is required for extracellular matrix degradation after combined carbon tetrachloride and 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in mice Natalie L. Johnson1, Giovan N. Cholico2, Kristen A. Mitchell2,3, Michele T. Pritchard1,3 1University of Kansas Medical Center, Department of Pharmacology, Toxicology and Therapeutics and the 3Liver Center, Kansas City, KS; 2 Biomolecular Sciences Graduate Programs, Boise State University; 3Department of Biological Sciences, Boise State University The liver is the most regenerative organ and can heal without scarring. However, after relentless injury, the liver scars and fibrosis occur due to an imbalance between extracellular matrix (ECM) synthesis and degradation, favoring ECM accumulation. Hyaluronan (HA) is a major ECM molecule dysregulated in chronically wounded tissue and contributes to scarring. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant and an aryl hydrocarbon receptor (AhR) ligand. Previous work from our group showed that TCDD-induced AhR activation


exacerbates liver injury, inflammation, and hepatic stellate cell activation during experimental liver fibrosis induced by chronic carbon tetrachloride (CCl4) administration. This effect of TCDD was abrogated in mice with AhR-deficient hepatocytes (AhRΔHep), but unexpectedly, the extent of liver fibrosis was unchanged between TCDD-treated AhRΔHep mice and control (AhRΔfl/fl) mice. Here, our objective was to determine if increased hyaluronan content or reduced ECM degradation was responsible for equivalent fibrosis in these two groups of mice. We performed a hyaluronan binding protein (HABP) assay to localize HA and in situ zymography to evaluate gelatinase activity in livers from AhRfl/fl and AhRΔHep mice after treatment with CCl4, TCDD, both chemicals (cotreatment), or no treatment. HABP staining was observed around the bile ducts in all mice regardless of genotype and treatment. Cotreatment with TCDD and CCl4 significantly increased HA content in the livers of AhRfl/fl mice (p<0.001), but not in AhRΔHep mice. In situ zymography showed that cotreatment increased gelatinase activity in AhRfl/fl mice compared to control mice (p<0.01) and mice exposed to TCDD alone (p<0.01). In contrast, there was no increase in gelatinase active among any AhRΔHep treatment groups. Together, these data suggest that hepatocyte AhR expression is required for ECM degradation after CCl4/TCDD-induced fibrosis. Exogenous AhR Activation Exacerbates the Biliary Response to Bile Duct Ligation in Mice Sarah E. Kobernat1, Alyssa L. Celedon2, Bradley Hiedemann2, Kenneth A. Cornell1,3, Kristen A. Mitchell1,2 1Biomolecular Sciences Graduate Programs, 2Department of Biological Sciences, 3Department of Chemistry and Biochemistry, Boise State University Liver fibrosis, which is a pathological condition characterized by the excessive accumulation of extracellular matrix, occurs in response to hepatic injury and inflammation. Damage and inflammation cause the conversion of quiescent hepatic stellate cells (HSCs) to activated myofibroblasts characterized by the synthesis of collagen type I. We previously reported that AhR activation by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) increased liver damage, inflammation, and HSC activation in the liver of mice subjected to bile duct ligation (BDL), which is a model of experimental liver fibrosis. In response to BDL, bile acids accumulate in the liver and elicit hepatocyte damage and inflammation, leading to myofibroblast activation and periportal fibrosis. Recent reports show that AhR activation by TCDD dysregulates bile acid homeostasis, which led us to speculate that dysregulated bile acid homeostasis may contribute to the increased liver damage and inflammation observed in TCDD-treated BDL mice. To test this, male C57Bl/6 mice were treated with TCDD (20 μg/kg) or peanut oil (vehicle) one day prior to BDL or sham surgery. Mice were euthanized 3, 7, or 14 days after surgery. Histological and biochemical analysis revealed that TCDD treatment exacerbated liver injury, inflammation, and myofibroblast activation 14 days post-BDL, and it also produced a 3-fold increase in hepatic bile acid levels. In addition, exposure to TCDD was found to decrease the expression of genes involved in bile acid synthesis and export but increase expression of genes involved in alternative trafficking and synthesis pathways. TCDD was also found to increase the biliary epithelial cell proliferation that is observed in response to BDL. This raises the possibility that TCDD treatment may exacerbate liver damage, inflammation, and HSC activation by a mechanism involving modulating the biliary response to BDL.


Modeling the Effects of Glomerular Dysfunction on the Proximal Tubule Using a Microphysiological System Kevin A. Lidberg1, Ranita Patel2, Catherine K. Yeung3,4, Jonathan Himmelfarb4, and Edward J. Kelly1,4 1Department of Pharmaceutics, University of Washington, 2Department of Nephrology, Seattle Children’s Hospital, Seattle, WA, 3Department of Pharmacy, University of Washington, 4Kidney Research Institute, Seattle, WA Proximal tubule epithelial cells (PTECs) treated with albumin in vitro generate reactive oxygen species, secrete cytokines (e.g CCL2, IL-8), and produce fibrogenic molecules (e.g. TGFβ, fibronectin). This suggests that the proximal tubule may play a role as pathological mediator in the progression of proteinuric renal disease. Here, we treated a cell-based microfluidic model of the human renal proximal tubule with human serum or albumin from human serum to explore how altered glomerular filtrate composition can drive PTECs to acquire a maladaptive phenotype. Exposure of PTECs to 2% human serum increases the expression of marker of proliferation Ki-67 (KI-67) and secretion of kidney injury molecule 1 (KIM-1) and interleukin-6 (IL-6). Albumin treatment did not promote IL-6 secretion, but increased KIM-1 secretion in a donor-dependent manner. Co-treatment with cetuximab (anti-EGFR), tocilizumab (anti-IL-6R), or ruxolitinib (JAK1/2 inhibitor) did not inhibit serum-induced secretion of KIM-1 or IL-6, indicating that these processes may be independent of STAT3. Transcriptomic profiling by RNA-seq revealed that the expression of 820 genes were significantly affected by treatment with 2% human serum and Ingenuity Pathway Analysis™ identified the transforming growth factor beta and tumor necrosis factor alpha pathways as the two most likely upstream regulators mediating the transcriptional changes. Together, these data show PTECs become pro-inflammatory with serum treatment and indicate that this microphysiological system can be used to identify the signaling pathways that trigger a switch in PTEC phenotype. Investigating the Reproductive Risks of Prolonged Smoke Exposure among Wildland Firefighters: A pilot concept Luke Montrose and Ann Brown Boise State University and University of Idaho The frequency and intensity of wildfires in the Northwest has increased in recent years. This demands that wildland firefighters engage in the suppression of fires for extended durations throughout the fire season, which now lasts up to 5-months of the year. Such changes in fire behavior have increased the occupational health risk for chronic, high concentration smoke exposure for wildland firefighters. Thus, there is an urgent need to bolster the relatively sparse data on long term occupational health impacts for wildland firefighters. Using the pilot study concept discussed here, we will examine the effects of extended occupational exposure to wood smoke components on the reproductive health of wildland firefighters.


Systems Toxicological Investigation of Per- and Polyfluoroalkyl Substances (PFAS) Yvonne Rericha, Lisa Truong, and Robert Tanguay Department of Environmental and Molecular Toxicology, Sinnhuber Aquatic Research Laboratory, Oregon State University Per- and polyfluoroalkyl substances (PFAS) are persistent compounds that are frequently detected in the environment, biota, and humans. In recent years, toxicological studies have addressed a wider range of PFAS, however, there are still thousands yet to be assessed. To investigate PFAS toxicity, we will use in vivo testing to understand structure-activity relationships, inform physiologically-based pharmacokinetic (PBPK) models, and explore mechanisms of action. We developed a high-throughput screening (HTS) platform to evaluate individual PFAS using early life stage zebrafish. Dechorionated embryos will be statically exposed at 6 hours post fertilization (hpf) in individual wells of 96-well plates. Measured developmental/larval endpoints will include embryonic photomotor responses at 24 hpf, larval locomotor responses and 18 morphological endpoints at 120 hpf. For each compound, we will run a preliminary concentration response curve, using a range of concentrations between 0 and 100 µM (n= 12 at each concentration). Definitive testing using 36 fish at each concentration will follow to better define a concentration response curve, enabling calculation of EC10, EC50, EC80, etc. To further elucidate mode of action, we will analyze differentially affected biological pathways via full-genome RNA sequencing of 48 hpf embryos exposed to select PFAS at the defined EC80. Since humans are exposed to PFAS primarily by ingestion, we will also conduct juvenile dietary exposure studies, with select compounds, spanning 17 to 45 days post fertilization. At the conclusion of these studies, we will measure fecundity and adult behaviors that include social interaction, anxiety, predator avoidance, and cognition. Through in vivo screening, structure-activity analysis, and transcriptomics, we aim to gain enormous insight into the mechanisms of PFAS toxicity. Toxicity testing of anti-parasitic MTN inhibitors against mammalian cells Swainston K, Smith T, Cornell KA Department of Chemistry and Biochemistry, Boise State University, Boise, ID Giardia intestinalis (GI) is the most common protozoan parasite in the U.S. It is contracted by consumption of cysts that are passed in the feces of humans, domestic animals, and wildlife and frequently contaminate watersheds in Western states. The cysts pass through the stomach and excyst to the form trophozoites that infect the proximal small intestine, where they cause severe flatulence, diarrhea, abdominal cramps, nausea, vomiting, dehydration, and weight loss. Drugs like metronidazole (MTZ), tinidazole, and nitazoxanide are usually prescribed, but there are increasing cases of treatment failure. Our prior work has shown that the parasite enzyme 5’ Methylthioadenosine nucleosidase (MTN) is an excellent target for drug development as it is unique to the parasite and absent from humans. In silico screening of allosteric drugs against crystallographic models of the parasite MTN has identified 36 potential MTN inhibitors, a number of which show in vitro anti-parasitic activity. In an effort to demonstrate that the compounds do not show undesired off target effects in humans, we have performed preliminary in vitro cytotoxicity screening of these compounds against cultured normal human fibroblasts and human


fibrosarcoma cells. Our initial studies show that most of the compounds show little effect against human cells, even at high (100 µM) concentrations. The results of these initial studies will be presented. In Vitro Comparison of Biotoxicity of Reduced Graphene and Graphene Oxide Nanoparticles Lucas Uecker, Ray Hamilton, Andrij Holian Center of Environmental Health Sciences, University of Montana Sub groups of graphene nanoparticles have been increasing in popularity for commercial applications due to the particle ' s unique chemical properties and low-cost. This study evaluated the biotoxicity of reduced graphene oxide (rGO) and graphene oxide (GO) particles (6.25, 12.5, 25, 50, and 100 µg/ml) on C10 lung epithelial cells and C57BU6 alveolar macrophages isolated from mice. Evaluations included measurements of cell viability and cell death, along with the release of proinflammatory cytokines from both cells. There was a significant decrease and greater decrease in cell viability of C10 cells when exposed to rGO particles compared to GO. However, there was no measurable release of inflammatory cytokines from the C10 cells, which included TNF-, IL-33, IL-13, and IL-1. Similar to the epithelial cells, alveolar macrophages also showed a significant decrease in cell viability and an increase in cell death after being exposed to rGO particles that was greater than the response to GO. The macrophage cells were tested for IL-1P release (a product of NLRP3 lnflammasome activation). Similar to the toxicity outcomes, there was a significant dose response showing higher levels of IL-1P response in rGO. In summary, the results demonstrated that the rGO particle is more bioactive in both models when compared to GO. Identification of Predictive Biomarkers Associated with Antibiotic Associated Nephrotoxicity in Cystic Fibrosis Patients Using a Kidney Microphysiological Device Kirk P. Van Ness PhD DABT, Edward J. Kelly, PhD and Pavan Bhatraju, MD MSc University of Washington The longevity of individuals living with cystic fibrosis (CF) through successful treatments and lung transplantation has brought with it unforeseen problems and complications: one being the emergence of acute kidney injury from antibiotic treatment stemming from colonized pulmonary microbes in these patients. We have employed a kidney microphysiological device system (MPS)with cultured human proximal tubule epithelial cells (PTECs) to identify potential sensitive renal biomarkers arising from exposure to aminoglycosides and polymyxins commonly used to treat CF patients. Specifically, we exposed PTECs to various concentrations of Polymyxin B (PMB), Polymyxin E (colistin) and tobramycin for 72 hours and collected daily effluents as a source for nephrotoxicity biomarker discovery. Biomarkers associated with epithelial cell injury KIM-1 (Kidney Injury Molecule 1) and NGAL (Neutrophil Gelatinase-Associated Lipocalin), inflammation (soluble Fas Receptor, Fas Ligand and TNF RI) and apoptosis (caspase-cleaved cytokeratin 18 and intact cytokeratin 18) were chosen to evaluate kidney chip effluents by ELISA.


We observed an antibiotic-specific biomarker response with elevations of KIM-1 in the effluents at 24 hours with PMB, increased NGAL and FAS concentrations at 24 hours with colistin exposure and increased NGAL concentrations with tobramycin. These data suggest that the mechanisms of nephrotoxicity resulting from antibiotic exposure to human PTECs triggers an antibiotic-dependent biomarker response and follow up analyses with RNA transcript (RNAseq) and microRNA analyses will provide systems biology data to help identify additional biomarker candidates. AhR signaling in hepatocytes is required for maximal hepatic stellate cell activation during TCDD-induced liver fibrosis Shivakumar R. Veereabhadraiah1, Kenneth A. Cornell2,3, Kristen A. Mitchell1,4 1Biomolecular Sciences Graduate Programs, Boise State University; 2Department of Chemistry and Biochemistry, Boise State University; 3Biomolecular Research Center, Boise State University; 4Department of Biological Sciences, Boise State University, Boise, ID The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzon-p-dioxin (TCDD) and related environmental contaminants. Recent evidence indicates that chronic exposure of rodents to TCDD elicits liver fibrosis (scarring), which is a pathological condition characterized by the excessive deposition of extracellular matrix by hepatic stellate cells (HCSs). Upon chronic liver injury and inflammation, HSCs transition from a quiescent state to an activated, myofibroblast-like state, characterized by proliferation, chemokine production, and collagen production. We previously reported that TCDD treatment directly increases HSC activation in vitro. In this study, we sought to determine if in vivo exposure to TCDD elicits liver fibrosis by activating HSCs directly or by increasing liver injury and/or inflammation, which could indirectly increase HSC activation. To test this, Cre-Lox recombination was used to create mice with AhR-deficient hepatocytes (AhRΔHep) or AhR-deficient HSCs (AhRΔHSC); AhRfl/fl mice served as controls. To induce liver fibrosis, female, adult mice were gavaged with TCDD (100 µg/kg) or peanut oil every four days for 92 days. At the end of the experiment, liver damage, inflammation, and HSC activation were measured. TCDD increased expression of the HSC activation markers Col1a1, TIMP-1, and Col3a1 in AhRfl/fl and AhRΔHSC mice but not in AhRΔHep mice. This indicates that AhR signaling in HSCs is dispensable for TCDD-induced liver damage, whereas hepatocyte AhR signaling is required. Increased HSC activation in TCDD-treated mice did not appear to be driven by hepatocyte necrosis, as serum levels of alanine aminotransferase were minimal and consistent across all TCDD-treated mice regardless of AhR expression. Likewise, TCDD treatment increased inflammatory cell infiltration in the liver of all mice, regardless of AhR knockdown. In contrast, TCDD induced hepatic steatosis in both AhRfl/fl and AhRΔHSC mice, but this effect was completely abolished in AhRΔHep mice. We conclude that HSCs are not directly targeted by TCDD during the development of liver fibrosis. Instead, TCDD-induced HSC activation occurs through a mechanism that requires AhR signaling in hepatocytes. At this time, the role of steatosis in TCDD-induced liver fibrosis is unclear.


A Systematic Variation of Experimental Conditions to Help Harmonize Zebrafish Early Life Stage Bioactivity Screening Lindsay Wilson, Lisa Truong, Robert Tanguay Oregon State University Zebrafish are used extensively to discover novel pharmaceuticals, investigate mechanisms of chemical action and for human disease model research. Although the zebrafish model is gaining acceptance for 21st century toxicology research, the lack of harmonized experimental approaches for developmental toxicity screening limits full model adoption. A few identified sources of variability include: the presence/absence of the chorion (an acellular membrane that could pose a barrier to chemicals), the photoperiod, and the precise dosing regimen (daily renewal v. static exposure). To address these variables, we used a systematic approach to vary each experimental condition for nine chemicals and a single nanomaterial. We intentionally selected these materials to represent varied physiochemical properties including photo lability and a range of log KOWs. Utilizing the Tanguay lab early life stage exposure paradigm, we evaluated the ten compounds for bioactivity across three exposure conditions: dechorionated v. chorion on, dark exposure v. light/dark cycle exposure, and static exposure v. daily solution renewals. Embryos were exposed at 6-8 hours post-fertilization (hpf) and screened for 22 morphological and two behavioral endpoints at 120 hpf. We compared the bioactivity profiles across all conditions by calculating concentrations which produced a 50% response (EC50). Preliminary data indicates chorion status affects the bioactivity of two of the chemicals. For estradiol, the presence of the chorion during exposure reduced bioactivity, while the bioactivity of abamectin increased under this condition. The results of this study will provide information necessary to help investigators tailor zebrafish assay conditions to consider the physiochemical properties of test agents. This, we hope, will increase data reproducibility, confidence, and acceptance; moving the field one step closer to using the powerful zebrafish screening platform to broadly predict chemical bioactivity. Transcriptomic profiling of PBDE-exposed HepaRG cells unveils critical lncRNA-PCG pairs involved in intermediary metabolism Angela Zhang, Cindy Yanfei Li, Edward Kelly, Lianne Sheppard, Julia Yue Cui University of Washington Polybrominated diphenyl ethers (PBDEs) are flame-retardants that are known to bioaccumulate in humans through inhalation or ingestion. Although its toxicities are well characterized, little is known about the changes in transcriptional regulation caused by PBDE exposure. Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of transcriptional and translational processes. It is hypothesized that lncRNAs can regulate nearby protein-coding genes (PCGs) and changes in the transcription of lncRNAs may act in cis to perturb gene expression of its neighboring PCGs. The goals of this study were to 1) characterize PCGs and lncRNAs that are differentially regulated from exposure to PBDEs; 2) identify PCG-lncRNA pairs through genome annotation and predictive binding tools; and 3) determine enriched canonical pathways caused by differentially expressed lncRNA-PCGs pairs. HepaRG cells, which are human-derived hepatic cells that accurately represent gene expression profiles of human liver tissue, were exposed to BDE-47 and BDE-99 at a dose of 25 μM for 24 hours. Differentially expressed lncRNA-PCG pairs


were identified through DESeq2 and HOMER; significant canonical pathways were determined through Ingenuity Pathway Analysis (IPA). LncTar was used to predict the binding of 19 lncRNA-PCG pairs with known roles in drug-processing pathways. Genome annotation revealed that the majority of the differentially expressed lncRNAs map to PCG introns. PBDEs regulated overlapping pathways with PXR and CAR such as protein ubiqutination pathway and PPARα-RXRα activation but also regulate distinctive pathways involved in intermediary metabolism. BDE-47 uniquely regulated signaling by Rho Family GTPases and PBDE-99 uniquely regulates JAK/Stat signaling, bile acid biosynthesis, sirtuin signaling pathway, and autophagy. In conclusion, lncRNAs play essential roles in modifying important pathways involved in intermediary metabolism such as carbohydrate and lipid metabolism.

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PANWAT 2019 program final · Programming Workshop – Sunday, October 6th Introduction to Python Programming for Data Analysis and Visualization 8:15 a.m. - 4:00 p.m. Hampton Inn