Interaction of the Microbiome and the Immune System

Interaction of the Microbiome and the Immune System:

More Than Just a Gut Feeling

2021 Regional Chapter Meeting October 29th, 2021

Thank you to the generous contributors to the Dr. William M. Baird Travel Award Fund in 2021:

We hope all PANWAT members will consider donating to the Dr. William M. Baird Travel Award Fund to

support student and trainee travel awards.

Find out how you can support the fund at the end of this program.

PANWAT Annual Conference, October 29th, 2021 Page 1

2021 PANWAT Officers

Special thanks to our EC Emeritus Member Linda Carlock, MS, DABT

Outreach Coordinator: Paramita Mookherjee, BS, MS, PhD, DABT Global Blood Therapeutics, Inc.

Seagen, Inc.

Seagen, Inc.


2021 PANWAT Regional Meeting Agenda Interaction of the Microbiome and the Immune System:

More Than Just a Gut Feeling

Friday, October 29th 9:00 – 9:10 AM

9:10 – 10:00 AM

10:00 – 10:50 AM

10:50 – 11:00 AM

11:00 – 11:45 AM

Welcome Remarks and Conference Overview Dr. Chris Carosino, PANWAT Vice President, Seagen

Keynote Presentation by Dr. William DePaolo Using the Microbiome to Interrogate the Early Stages of Colon Cancer Moderator: Dr. Julia Cui, PANWAT President, University of Washington

Keynote Presentation by Dr. Diwakar Davar The Inside Scoop on Poop: The Intestinal Microbiome in Cancer Immunotherapy Moderator: Dr. Chris Carosino, PANWAT Vice President, Seagen

Break

Graduate Student Flash Talks: Session 1 Moderator: Dr. Kari Gaither, PANWAT Outreach Coordinator, PNNL

Joe Jongpyo Lim Neonatal Exposure to BPA, BDE-99, and PCB Produces Persistent Changes in Hepatic Transcriptome Associated with Gut Dysbiosis in Adult Mouse Livers

Whitney L. Garcia The Gut Microbiome Influences Host Xenobiotic Metabolism After Exposure to Benzo[a]pyrene and 1-Nitropyre

Sarah Kim Early Life PBDE Exposure Disrupts Gut Microbiome and Microbial Metabolism of Tryptophan

Ryan Lopez Size- and Oxidation-Dependent Toxicity of Graphene Oxide Nanomaterials in Embryonic Zebrafish

11:45AM – 12:10 PM Undergraduate Student Flash Talks Moderator: Joe Lim, PANWAT Senior Graduate Student Representative, University of Washington

John Lam Assessing the Impacts of Cell Culture Medium on Zebrafish Development as a Prelude to Better Assessment of Nanomaterial Safety

Francesca Rossi Association of Ergot Alkaloid Profiles with Insect Tolerance in Grass Cultivars

12:10 – 12:40 PM Lunch break


12:40 – 1:35 PM Graduate Student Flash Talks: Session 2 Moderator: Dr. Lisa Truong, PANWAT Secretary/Treasurer, Oregon State University

Yvonne Rericha Elucidating Mode of Action of Perfluorobutane Sulfonamide Toxicity and Investigating a PFAS Short-Chain Homologous Series in Zebrafish

Connor Leong Development and Validation of a High Throughput Juvenile Zebrafish Depth Preference Behavior Assay

Lindsay Wilson Construction of a Mechanistic Model of Retene Developmental Toxicity Using a Combination of In Vivo, In Vitro, and In Silico Methods

Youjun Suh Regulation of Transporters by Perfluorinated Carboxylic Acids in HepaRG Cells

Abdulaziz Alshehri CYP1B1-mediated Metabolism of Hypoxanthine, A Potential Endogenous Substate Regulates Capillary Morphogenesis.

Break 1:35 – 1:40 PM

1:40 – 2:25 PM Postdoctoral and Fellow Flash Talks Moderator: Dr. Julia Cui, PANWAT President, University of Washington

Kari Gaither Understanding Susceptibility to PAH Exposure Due to Age-related Differences in Metabolism

Subham Dasgupta Phenotypically Anchored mRNA and miRNA Expression Profiling in Zebrafish Reveals Flame Retardant Chemical Toxicity Networks

Lindsey St. Mary Comparative Analysis Between Zebrafish and Automated Live-Cell Assay to Assess 87 Developmental Neurotoxicants

Jacki Coburn Primary Rat Hepatocytes as a Model System for Investigating Xenobiotic-Induced Alternative Splicing

Moumita Dutta Chronic Exposure to Ambient Traffic-related Air Pollution (TRAP) Alters Gut Microbial Abundance and Bile Acid Metabolism in a Transgenic Rat Model of Alzheimer’s Disease

Break 2:25 – 2:30 PM

2:30 – 3:20 PM 2020 PANWAT Achievement Award Lecture – Dr. Bruce Kelman Choose Your Own Adventure: A Career in Toxicology Moderator: Dr. Nadia Moore, PANWAT Past President, Seagen

3:20 – 3:40 PM Award winners and welcome to the next year’s PANWAT Dr. John Clarke, PANWAT Vice President-Elect, Washington State University


Keynote Speaker

Dr. William DePaolo Associate Professor of Medicine at the University of Washington Medical Center | Director of the Center for Microbiome Sciences & Therapeutics (CMiST)

Dr. William DePaolo has recently been appointed an Associate Professor of Medicine at the University of Washington Medical Center and is the recipient of the Lynn M. and Michael D. Garvey endowed chair in Gastroenterology. Dr. DePaolo was also named Director of the Center for Microbiome Sciences & Therapeutics (CMiST), which serve as an intellectual hub for investigators, clinicians and the community interested in the human microbiome. CMiST will offer Innovation Services, host workshops and seminar series, and foster collaboration between clinicians and researchers.

DePaolo Lab Research Mission Our bodies are home to an enormous microbial ecosystem with more than 100 trillion microbes. Astonishingly, for every one of our genes there is an estimated 145 microbial genes. Collectively, these microorganisms and their gene products are referred to as the microbiome and this community of microbes performs essential functions that help maintain human health. Shifts in the composition, distribution and/or function of the microbiome have not only been implicated in diseases of the GI tract, but also play important roles in systemic diseases such as autism, hematological cancers and neurological disease. Due to the inherent plasticity of the microbiome, the manipulation of its communities has the potential for significant therapeutic applications to treat numbers of diseases.

The goals of the DePaolo Lab are to explore how intestinal perturbations caused by genetics and/or environmental factors (e.g., diet, infection, pollution) alter the microbial landscape of the intestine and to use this newly acquired knowledge to develop strategies and therapies to maintain or restore ecological harmony. To accomplish these goals the DePaolo Lab utilizes an integrated and multidisciplinary approach that incorporates both clinical human samples with relevant animal models of disease and pairs –omics data with novel assays to define actual biological function. Through careful analysis and holistic approaches the DePaolo Lab hopes to define new pathways, identify new targets and gain mechanistic insight into the relationship between the environment, human genes and the microbiome. pandemic.

Keynote Presentation: Using the Microbiome to Interrogate the Early Stages of Colon Cancer


Keynote Speaker

Dr. Diwakar Davar Assistant Professor of Melanoma and Phase I Therapeutics in the Division of Hematology-Oncology in the Department of Medicine at University of Pittsburgh

Dr. Davar’s initial work evaluated outcomes of melanoma patients treated with HD IL-2; and their data suggested that HD IL-2 administration in a non-ICU setting did not compromise outcomes. They subsequently evaluated whether the addition of immunomodulatory doses of pegylated IFN (peg-IFN) improved upon PD-1 blockade in advanced PD-1 naïve melanoma; and they showed that the combination resulted in 61% ORR with 46% 2-year PFS and that response was associated with IFN-γ gene expression and CD8 T cell infiltrate.

By studying the gut microbiota of patients treated with anti-PD-1 in melanoma, we identified unique gut microbial signatures of response and immune-related adverse events to anti-PD-1 therapy. In this work,

we developed the concept of “gut microbial communities (microbiotypes)” and their variable association with clinical outcomes to anti-PD-1, and how their non-uniform geographic distribution contributed to observed discrepancies between previously published cohorts. Concurrently, we developed a protocol evaluating responder-derived fecal microbiota transplant (FMT) in PD-1 refractory melanoma. In this first-in-human effort, we demonstrated that microbiome modulation reversed resistance to immunotherapy in a subset of melanoma patients by altering levels of immunosuppressive myeloid cells and were published in Science (Davar et al. 2021).

We have additionally developed novel studies including a neoadjuvant study of TLR9 agonist CMP-001 in high-risk resectable melanoma. We demonstrated that the neoadjuvant nivolumab/CMP-001 combination produced high pathologic response rates with evidence of immune activation peripherally and intra-tumorally. These results have prompted several follow-on studies including a pilot neoadjuvant study evaluating 18F- AraG as an integrated imaging biomarker; a randomized phase II neoadjuvant trial in ECOG-ACRIN (EA6194; co-PI Davar); and a national phase II/III study of nivolumab/CMP-001 compared to nivolumab. Separately, I have contributed to the development and implementation of the first-in-human studies of TIGIT mAb (BMS-986207), Tim-3 mAb (TSR-022), GITR mAb (TRX-518), CTLA-4 NF (BMS-986218), anti-IL-8 (BMS-986253), and prostaglandin E2 (PGE2) receptor 4 (EP4) antagonist (BMS-986310).

Dr. Davar’s research interests lie in translational cancer immunotherapy; and specifically focuses on exploring the role of the gut microbiome, DC agonists such as TLR9 agonists and key immune checkpoints in cancer immunotherapy.

Keynote Presentation: The Inside Scoop on Poop: The Intestinal Microbiome in Cancer

Immunotherapy The intestinal microbiome has been implicated in various aspects of cancer from carcinogenesis to the response of anti-cancer therapies including chemotherapy and immunotherapy. Using large datasets and multi-omic approaches to high-dimensional data analyses, Dr. Davar and his team have uncovered unique microbiota signatures of response and toxicity to PD-1 immune checkpoint inhibitors. We have leveraged this understanding to develop approaches to drug the microbiome in PD-1 refractory melanoma and other cancers; and to develop approaches to treat immune-related adverse events (irAEs).


2021 Toxicology Achievement Award Bruce Kelman, PhD, DABT, ATS, ERT Senior Vice President, J.S. Held, LLC

Bruce Kelman has more than 40 years of experience in toxicology and has served as a consultant and expert in numerous investigations across North America, South Korea, and the United Kingdom. He has evaluated numerous claims of personal injury and health impacts from chemicals and drugs and has presented a variety of health risk concepts to policy makers, government regulators, citizen groups, and individuals. Dr. Kelman also regularly interprets standard and non-standard tests for drugs of abuse including ethanol, opioids, methamphetamine, cocaine, and cannabis.

Dr. Kelman holds a PhD from the University of Illinois and has been certified in toxicology by the American Board of Toxicology since 1980. He is also certified in toxicology by the Academy of Toxicological Sciences. Bruce is a Registered Toxicologist with both the United Kingdom and

EUROTOX registries. His research includes more than 130 scientific publications.

Dr. Kelman’s support of PANWAT goes back to its origin. He joined in 1984 as a charter member and served on one of the first executive councils (councilor, 1985-87). Although he briefly left (1990-93), he quickly returned, rejoined and has remained an involved member: he served on PANWAT’s presidential

chain from 2005-2009; he helped lay the foundation for establishing PANWAT’s Endowment Fund (which was established in 2009); and he ensured annual PANWAT sponsorship by the consulting

company (Veritox) he founded in 1998.

Achievement Award Lecture: Choose Your Own Adventure: A Career in Toxicology


Graduate Student Flash Talk Abstracts

Session 1 1. Neonatal Exposure to BPA, BDE-99, and PCB Produces Persistent Changes in Hepatic Transcriptome Associated with Gut Dysbiosis in Adult Mouse Livers Joe Jongpyo Lim, Moumita Dutta, Joseph L. Dempsey, Hans-Joachim Lehmler, James MacDonald, Theo Bammler, Cheryl Walker, Terrance J. Kavanagh, Haiwei Gu, Sridhar Mani, and Julia Yue Cui University of Washington, Seattle WA Recent evidence suggests that complex diseases can result from early life exposure to environmental toxicants. Polybrominated diphenyl ethers (PBDEs), and polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) and remain a continuing risk to human health despite being banned from production. Developmental BPA exposure mediated-adult onset of liver cancer via epigenetic reprogramming mechanisms has been identified. Here, we investigated whether the gut microbiome and liver can be persistently reprogrammed following neonatal exposure to POPs, and the associations between microbial biomarkers and disease-prone changes in the hepatic transcriptome in adulthood, compared to BPA. C57BL/6 male and female mouse pups were orally administered vehicle, BPA, BDE-99 (a breast milk-enriched PBDE congener), or the Fox River PCB mixture (PCBs), once daily for three consecutive days (postnatal days [PND] 2 to 4). Tissues were collected at PND5 and PND60. Among the three chemicals investigated, early life exposure to BDE-99 produced the most prominent developmental reprogramming of the gut-liver axis, including hepatic inflammatory and cancer-prone signatures. In adulthood, neonatal BDE-99 exposure resulted in a persistent increase in Akkermansia muciniphila throughout the intestine, accompanied by increased hepatic levels of acetate and succinate, the known products of A. muciniphila. In males, this was positively associated with permissive epigenetic marks H3K4me1 and H3K27, which were enriched in loci near liver cancer-related genes that were dysregulated following neonatal exposure to BDE-99. Our findings provide novel insights that early life exposure to POPs can have a life-long impact on disease risk, which may partly be regulated by the gut microbiome.



Session 1 2. The Gut Microbiome Influences Host Xenobiotic Metabolism After Exposure to Benzo[a]pyrene and 1-Nitropyre Whitney L. Garcia, Carson J. Miller, Gerard X. Lomas, Kari A. Gaither, Kimberly J. Tyrrell, Jordan N. Smith, Kristoffer R. Brandvold, Aaron T. Wright Pacific Northwest National Laboratory Polyaromatic hydrocarbons are an abundant class of environmental contaminants that have varying levels of carcinogenicity depending on their individual structures. The gut microbiome is a key contributor to xenobiotic metabolism; however, the exact mechanism is unknown. This study sought to determine the role the gut microbiome has in determining the rates of metabolism, specifically through profiling active cytochrome P450 enzymes before and after exposure to two structurally different polycyclic aromatic hydrocarbons, benzo[a]pyrene and 1-nitropyrene. Ten-week-old C57BL/6J conventional and gnotobiotic mice were orally exposed to polyaromatic hydrocarbons or to vehicle alone. Additionally, baseline metabolic rates were evaluated in unexposed conventional and gnotobiotic mice. Following treatment, activity-based protein profiling was conducted, and metabolic rates of metabolism were measured. Gnotobiotic mice exhibited higher rates of metabolism for benzo[a]pyrene and 1-nitropyrene compared to conventional mice in unexposed conditions. Upon exposure to benzo[a]pyrene and 1-nitropyrene, conventional mice exhibited higher rates of metabolism with gnotobiotic mice lacking the ability to metabolize 1-nitropyrene. In unexposed conditions, cytochrome P450 enzymes 2d10 and 2d26 were upregulated in gnotobiotic mice with no cytochrome P450s with elevated activity in conventional mice relative to gnotobiotic mice. The lack of a gut microbiome downregulated cytochrome 1a1 activity in benzo[a]pyrene exposure conditions, with no detection of cytochrome 1a1 activity in gnotobiotic and conventional mice upon exposure to 1-nitropyrene. This study revealed a novel interaction between the gut microbiome, the rate of oxidative liver metabolism, and active P450 profiles, in the host, in unexposed conditions, and upon exposure to two structurally different polycyclic aromatic hydrocarbons. Collectively, these data indicate the importance of the gut microbiome in xenobiotic metabolism



Session 1 3. Early Life PBDE Exposure Disrupts Gut Microbiome and Microbial Metabolism of Tryptophan Sarah Kim, Hao Li, Haiwei Gu, Sridhar Mani, and Julia Y. Cui University of Washington, Seattle, Washington Polybrominated diphenyl ethers (PBDEs) are previously used as flame retardants that bio-accumulate in human compartments including breast milk and blood. They are highly prevalent in fatty food and human breast milk because of the lipophilic nature. In a previous study, it showed that developmental exposure to PBDEs is associated with increased diabetes prevalence in humans and animal studies. Gut microbiome is also an important modifier of host xenobiotic biotransformation. It is known that gut microbiome can critically modulate metabolic syndrome including obesity and diabetes. Pregnane X receptor (PXR) is a xenobiotic sensing nuclear receptor responsible for xenobiotic detoxification and regulation of energy metabolism. PXR gene variants are shown to be associated with inducing metabolic syndrome. It is also known that PXR is activated by indole 3-propionic acid (IPA), which is a microbial metabolite from tryptophan. A recent epidemiology study showed that serum IPA is associated with lowered risk of type 2 diabetes and lower grade inflammation on high-risk individuals (Tuomainen et al., 2018). However, very little is known to what extent early life PBDE exposure procures adult onset of diabetes and the underlying mechanisms. We hypothesized that early-life PBDE exposure causes persistent dysbiosis in pups including microbial metabolism which leads to dysregulated PXR signaling. We used humanized PXR mice to generate humanized specific conditions with 4 groups: standard diet, DE-71 diet, standard diet + IPA, and DE-71 diet + IPA. IPA was administered in the drinking water. Tissues were collected at Day 21 and 3 months. Intestinal contents were used for metagenomic shotgun sequencing and serum for metabolite analyses. One-way ANOVA statistical analysis with the p-value of less than 0.05 was used for all data. We have identified that maternal exposure to DE-71 produced persistent gut dysbiosis and alterations in microbial tryptophan metabolism. Overall there were more DE-71 mediated obesity-related microbial changes at the 3 months time point as compared to the 21-days time point. IPA co-exposure normalized certain DE-71 regulated bacteria. In addition, sex- and IPA-specific modulation of the gut microbiome as well as microbial metabolites of tryptophan were noted. In conclusion, early-life exposure caused persistent dysbiosis in pups including altered microbial metabolism and may contribute to dysregulated PXR signaling.



Session 1 4. Size- and Oxidation-Dependent Toxicity of Graphene Oxide Nanomaterials in Embryonic Zebrafish Ryan M. Lopez, Joshua R. White, Lisa Truong, Robyn L. Tanguay Oregon State University, Corvallis, Oregon Graphene and its derivatives are two-dimensional hexagonal lattices that possess desirable physicochemical properties leading to an ever-expanding list of industrial and biomedical uses. Graphene oxides (GOs) are becoming a popular graphene alternative and while there have been human safety studies, investigations of developmental toxicity are lacking. The use of distinct GOs are rapidly increasing, outpacing our efforts to understand their GO-specific impacts on biological systems. The overall goal of this study is to further understand the developmental toxicity of structurally distinct GOs on early life stage zebrafish. Our previous work advanced the use of the early life stage zebrafish as an ideal model to interrogate the interactions between engineered nanomaterials (ENMs) and biological systems. We investigated GO 250 x 250 nm (sGO), 400 x 400 nm (mGO), 1 x 1 μm (lGO); partially reduced GO (prGO) 400 x 400 nm; reduced GO (rGO) 400 x 400 nm, and 2 x 2 μm which first underwent extensive materials characterization under support from the NHIR Consortium. GOs were stabilized in water (GOs in water (GOW)), while prGO and rGOs were stably dispersed in sodium cholate. Zebrafish were exposed to 0, 2.32, 5, 10.7, 23.2, and 50 μg/mL of each GO at 6 hours post fertilization (hpf) until 120 hpf. Developmental toxicity was induced in a GO-specific manner, with mGOW as the most toxic material. We determined that mGOW-specific developmental toxicity manifested in the yolk syncytial layer (YSL). Additionally, sodium cholate stabilization significantly increased GOW toxicity. The observed GO-specific developmental effects are likely indicative of size-and oxidation-dependent toxicity and reveals the importance of identifying structure-specific toxicity which is particularly relevant in biomedical applications of GOs.


Undergraduate Student Flash Talk Abstracts

1. Assessing the Impacts of Cell Culture Medium on Zebrafish Development as a Prelude to Better Assessment of Nanomaterial Safety John Lam, Ryan M. Lopez, Lisa Truong, Michael T. Simonich, and Robyn L. Tanguay Oregon State University, Corvallis, Oregon Nanoparticles (NPs) are widely used in modern society and can be found in consumer products from novel medicines to construction materials. Currently, NP impacts on the environment and human health remains understudied. Studies have investigated NP effects, but the materials, experimental conditions, and platforms (in vivo or in vitro) used are highly diverse. In the absence of systematic toxicological assessments, it remains challenging to identify NP specific features associated with toxicity. Most in vitro cell-based NP screening approaches use media rich in ionic species (e.g., RPMI supplemented with fetal bovine serum (FBS)). These media constituents are critical for cellular viability and function, though studies have shown that toxicity of certain NPs vary when comparing conclusions in cell culture and zebrafish models. Zebrafish can be used to systematically compare the toxicity of NPs, as this model has a number of well understood advantages. We previously determined that zebrafish can develop normally in low ionic solutions (ultra-pure water, UP), but cell culture systems cannot tolerate UP alone. The goal of this study was to determine if zebrafish could adapt to common cell culture conditions to allow more direct NP toxicity result comparisons. Embryonic zebrafish were dechorionated and exposed to varying percent concentrations of FBS and RPMI with a mix of antibiotics (Amphotericin B, Penicillin, and Streptomycin; AB) in UP from 6 to 120 hours post fertilization (hpf); resulting mortality and morphological effects were recorded at 24 and 120 hpf. We determined that using 1% of the AB mix was essential to prevent bacterial growth. In this assay, developing zebrafish had a maximum tolerable percentage of 4% FBS and 50% RPMI. The results of this study lay the foundation for the reassessment of NP zebrafish toxicity under these conditions to permit better comparisons with existing cell culture NP toxicity data.


Undergraduate Student Flash Talk Abstracts 2. Association of Ergot Alkaloid Profiles with Insect Tolerance in Grass Cultivars Francesca Rossi1, Leanna Van Slambrook2, Navneet Kaur2, and Jennifer Duringer1 1 Department of Environmental & Molecular Toxicology, Oregon State University, Corvallis, OR, USA, 2 Department of Crop and Soil Sciences, Oregon State University, Corvallis, OR, USA The sod webworm (Chrysoteuchia topiaria Zeller) is one of the most destructive insect species found within grass seed operations in Oregon. During early life stages, larvae feed on the root systems leading to weakened crop stands and poor seed yields. Endophytes are mutualistic fungi species that co-exist within plants, with some producing secondary compounds known as mycotoxins which can serve as natural insect deterrents. Ergot and peramine alkaloid mycotoxins expressed in some grass species have been shown to be effective insecticides, depending on their concentration and chemical profile. The present work quantified ergot alkaloids, ergoline alkaloids, and peramine across tall (n=10, TF) and fine fescue (n=8, FF), orchard grass (n=3, OG) and perennial ryegrass (n=11, PRG) via tandem liquid chromatography-mass spectrometry (LC-MS/MS).Total mycotoxin concentrations were compared to insect resistance measured through no choice bioassays. It was found that, as a whole, ergot alkaloids averaged 91±87, 24±28, 23±13, and 39±26 ng/g; ergoline alkaloids 275±479,2,479±7740, 173±228 and 1,665±5,170 ng/g; and peramine 1,280± 26, 24,506 ±22, 45± 0 and 17,241± 29 ng/g in TF, FF, OG and PRG samples that contained these compounds, respectively. The average percent mortality of the sod webworm on TF, FF, OG and PRG cultivars was 51%, 35%, 52% and 49%, respectively. When correlating percent mortality to mycotoxins detected, it was found that plants with high mortality had elevated levels of peramine and chanoclavine. This project advances the goal of protecting Oregon’s grass seed industry from sod webworm predation by providing alternative, sustainable pest prevention strategies through the use of naturally occurring endophytes. Further work will be conducted measuring the insect’s preference between cultivars of grass species and endophyte infection/mycotoxin expression.



Session 2 1. Elucidating Mode of Action of Perfluorobutane Sulfonamide Toxicity and Investigating a PFAS Short-Chain Homologous Series in Zebrafish Yvonne Rericha, Lisa Truong, and Robyn L. Tanguay Sinnhuber Aquatic Research Laboratory, Deparment of Environmental & Molecular Toxicology, Oregon State University Per- and polyfluoroalkyl substances (PFAS) have been used in a variety of products and are now ubiquitously detected in the environment and humans. Some PFAS cause a variety of adverse health effects with various proposed modes of action. Additional investigation is needed, particularly for lesser-studied short-chain PFAS. We investigated a homologous series of PFAS with 4 fluorinated carbons and different functional head groups: perfluorobutane sulfonamide (FBSA), perfluorobutane sulfonic acid (PFBS), perfluoropentanoic acid (PFPeA), and 4:2 fluorotelomer sulfonic acid (4:2 FTS). We evaluated developmental toxicity in zebrafish across 8 concentrations from 0-100 µM and conducted unbiased RNA sequencing (RNA-seq) to begin to identify underlying toxicity mechanisms. Zebrafish were statically exposed in 96-well plates beginning at 8 hours post fertilization (hpf), with mortality, morphology, and behavior assessments at 24 and 120 hpf. Exposure to all PFAS elicited abnormal larval behavior at 120 hpf. Only FBSA induced significant incidence of morphological effects; 47 µM was selected for RNA-seq exposures for all PFAS, phenotypically anchoring transcriptional changes from FBSA to 70-100% morphological effect. Zebrafish were sampled at 48 hpf, before observable morphological effects. FBSA exposure resulted in the differential expression of 1,909 transcripts, while the other PFAS only impacted the expression of a few transcripts. Gene ontology identified biological processes related to lipid metabolism and biosynthesis and to cellular homeostasis as highly enriched after FBSA exposure. Our findings illustrate that functional head group predicts PFAS toxicity and that transcriptomic effects precede larval morphological effects. We will use this approach to evaluate additional PFAS in the future.



Session 2 2. Development and Validation of a High Throughput Juvenile Zebrafish Depth Preference Behavior Assay Connor Leong1,2, Yvonne Rericha1,2, Dunping Cao1, Lisa Troung1,2, Jennifer Field1 and Robyn L. Tanguay1,2 1Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR Using zebrafish (Danio rerio) to screen and identify potential toxicants at low concentrations includes assays to measure sub-lethal endpoints, such as behavior. We developed a depth preference assay to measure anxiogenic behaviors in juvenile zebrafish. The apparatus was based off the area of a 96 well plate, with 12 rectangular wells (25 x 10 x 15 mm). Each well had a shallow and deep zone, with a ramp between the two zones, holding 1 fish per well (12 fish per array). Readouts from the assay were percent time, distance travelled, and divided into time bins: novel (0-2:00 min), dark (2:01-5:00 min), and light (5:01-10:00 min). To validate the assay, 10 day post fertilization (dpf) wild type zebrafish were run though the assay. The percent time and distance swam were highest in the deep zone, suggesting that the deep zone is associated with the least amount of stress. This assay was used to evaluate larval zebrafish whose parents were fed a PFAS-contaminated diet. The diet consisted of 1 ng/g or 100 ng/g of perfluorohexane sulfonamide (FHxSA) or 6:2 fluorotelomer sulfonate (6:2 FTS). In the light time bin, offspring from the 100 ng/g FHxSA and 100 ng/g 6:2 FTS groups swam significantly further in the deep zone, compared to the controls, an average increase of 17.3 cm and 20.5 cm, respectively. High activity in the deep zone suggests that the treatment groups had higher levels of anxiety because they spent more time in the zone associated with the lowest amount of stress. The assay’s ability to detect behavioral deficits in juvenile zebrafish demonstrates its utility for high throughput behavioral screening.



Session 2 3. Construction of a Mechanistic Model of Retene Developmental Toxicity Using a Combination of In Vivo, In Vitro, and In Silico Methods Lindsay B. Wilson, Katherine J. Schultz, Jordan N. Smith, Katrina M. Waters, Robyn L. Tanguay Oregon State University, Pacific Northwest National Laboratory Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants produced via both anthropogenic and natural sources. Forest fires are a major source of PAHs in the environment, many of which have been associated with human disease. Retene is a frequently detected PAH in environmental sampling and is abundantly produced by forest fires. Biologically, retene has been shown to induce developmental toxicity at low concentrations across animal models, though its mechanism of toxicity has not been elucidated. Some PAHs canonically induce toxicity via the aryl hydrocarbon receptor (AHR) pathway, though a growing body of evidence supports divergent mechanisms of toxicity across PAHs. The zebrafish is a well-establish model in molecular toxicology and has been utilized to interrogate PAH toxicity and the AHR signaling pathway for decades. Retene induces developmental toxicity in zebrafish in an AHR2-dependent manner, though, interestingly, in a metabolically-incompetent, luciferase reporter-based assay, retene did not activate AHR2. We hypothesize that retene is biotransformed to a reactive metabolite which activates the AHR2 pathway, leading to developmental toxicity. Using CRISPR-Cas9-generated knockout zebrafish lines, we have assessed the role of multiple AHR-dependent genes in retene toxicity and biotransformation. Additionally, we have utilized in silico homology modeling of the AHR ligand binding domain and metabolite prediction to identify likely AHR-activating retene metabolites. Finally, we will utilize activity-based probes and protein profiling for identification of metabolic enzymes which catalyze retene bioactivation in isolated larval microsomes. With the combination of these findings, we have begun to construct a proposed complete retene toxicity model. We believe this study will inform our greater knowledge of divergent mechanisms of PAH bioactivity and shed light on the role of xenobiotic metabolism in AHR-dependent toxicity.



Session 2 4. Regulation of Transporters by Perfluorinated Carboxylic Acids in HepaRG Cells Joe Jongpyo Lim, Youjun Suh, Elaine M Faustman, Julia Yue Cui Department of Environmental & Occupational Health Sciences, University of Washington Perfluorinated carboxylic acids (PFCAs) are widespread environmental pollutants for which human exposure has been documented. PFCAs at high doses were known to regulate xenobiotic transporters partly through PPARα and CAR in rodent models. Less is known regarding how various PFCAs at a lower concentration modulate transporters for endogenous substrates such as amino acids in human hepatocytes. Such studies are of particular importance because amino acids are involved in chemical detoxification and their transport system may serve as promising therapeutic targets for structurally similar xenobiotics. The focus of this paper is to further elucidate how PFCAs modulate transporters involved in intermediary metabolism and xenobiotic biotransformation. We tested the hepatic transcriptomic response of HepaRG cells exposed to 45µM PFOA, PFNA, or PFDA in triplicates for 24h (vehicle: 0.1% DMSO), as well as the prototypical ligands for PPARα (WY-14643, 45µM) and CAR (CITCO, 2µM). PFCAs with increasing carbon chain lengths (C8-C10) regulated more liver genes, with amino acid metabolism and transport ranked among the top enriched pathways and PFDA ranked as the most potent PFCA tested. Genes encoding amino acid transporters, which are essential for protein synthesis, were novel inducible targets by all 3 PFCAs, suggesting a potentially protective mechanism to reduce further toxic results. None of the transporter regulations appeared to be through PPARα or CAR but potential involvement of Nrf2 is noted for all 3 PFCAs. In conclusion, PFCAs with increasing carbon chain lengths up-regulate amino acid transporters and modulate compensatory response of xenobiotic transporters in HepaRG cells.



Session 2 5. CYP1B1-mediated Metabolism of Hypoxanthine, A Potential Endogenous Substate Regulates Capillary Morphogenesis. Abdulaziz Alshehri, Andrew J. Annalora, Nader Sheibani, Andrew D. Patterson, Patrick L. Iversen and Craig B. Marcus. Oregon State University, Corvallis, Oregon Cytochrome P450 1B1 (CYP1B1) is an enzyme that plays a central role in the metabolism of xenobiotics. CYP1B1 also regulates the metabolism and cellular activity of several endogenous substrates, including melatonin, retinoic acid, arachidonate, and estradiol. Mutations in CYP1B1 interfere with the enzyme’s structure/function, and are linked to multiple eye diseases, including primary congenital glaucoma (PCG). Because CYP1B1’s role in the eye is not fully understood, improved knowledge of its tissue-specific metabolic function is needed to guide new therapeutic strategies. Our group uses untargeted metabolomics and a retinal endothelial cell (REC) model derived from both wild-type (WT) and CYP1B1 KO mice, to study CYP1B1’s role in the eye. We have now identified more than 50 novel candidate substrates of CYP1B1 that may regulate eye function. To date, 10 putative substrates have been screened for their effect on capillary growth in a 96-well morphogenesis assay, and two (hypoxanthine and arachidonate) were found to strongly promote capillary morphogenesis in a CYP1B1-dependent manner. Here we measured the potency of hypoxanthine’s stimulatory effect on CYP1B1-mediated capillary growth, in comparison to 4 known substrates of CYP1B1. We performed a 7-point dose-response experiment with hypoxanthine and demonstrated a strong (~20%) stimulation of capillary branching at low nanomolar concentrations (EC50 = ~30 nM). Comparisons of bioactivity among the 4 known CYP1B1 substrates (listed above) indicate that hypoxanthine metabolites stimulate REC cell morphogenesis on a par with all other known substrates, except for estradiol, which inhibits capillary growth. Hypoxanthine metabolites appear to play an underappreciated but potentially important role in regulating eye development and repair processes. Insights gained from this work will inform new therapeutic approaches for treating glaucoma and related eye diseases linked to abnormal CYP1B1 expression and function.



Session 2 6. Characterization of human fecal microbiome and metabolome reveals critical host-microbiome interactions in key developmental pathways Angela Zhang, Moumita Dutta, Sheela Sathyanarayana, Xiaojian Shi, Haiwei Gu, and Julia Yue Cui University of Washington, Seattle Children’s Research Institute, Arizona State University Gut microbiome has been implicated in a variety of different diseases and conditions in humans. The relationship between the host and the gut microbiome is mainly governed by the interactions of microbial-produced metabolites and host receptors. Little is known regarding changes in the microbiome and host metabolites during normal development in humans. It is hypothesized that variations in pharmacokinetics in infants and children may be partially due to variations in their gut microbiota; therefore, understanding the basal composition of the gut microbiome will be vital in the advancement of personalized medicine. Fecal matter from child donors of 4 age groups (1 week-6 months, 7-24 months, 3-5 years, and 5-12 years) were collected (n=12-16 per age group). From whole genome sequencing, GC/MS and LC/MS measurement, we identified significant taxa (ANOVA, p < 0.05) that contribute to the maturation of distinct microbial metabolic pathways. For example, degradation of cholic acid, which is the major host-derived primary BA from cholesterol, was most enriched in the 7-24 months and the 3-5 years age groups,and was mainly contributed by Clostridium scindens and Lachnospiraceae bacterium 5157FAA. Lachnospiraceae bacterium 5157FAA is also a major player in hexitol fermentation to form SCFAs. In addition, within the first year of age, Lactobacillus rhamnosus and Megamonas hypermegale were shown to be the major taxa for hexitol fermentation. Marginal correlation analysis revealed strong host-microbiome interactions within these pathways, suggesting that the gut microbiome plays a key role in modulating the maturation of nutrient homeostasis. This study will be the first to characterize how the microbiome changes over time in a healthy individual during critical developmental period using multi-omics approaches.


Postdoctoral & Fellow Flash Talk Abstracts 1. Understanding Susceptibility to PAH Exposure Due to Age-related Differences in Metabolism Kari A. Gaither (1), Whitney Garcia (1,2), Kimberly Tyrrell (1), Aaron T. Wright (1,2), Justin Teeguarden (1,3), Jordan N. Smith (1,3) (1) Pacific Northwest National Laboratory, Richland, WA 99352, (2) Washington State University, Pullman, WA 99163, (3) Oregon State University, Corvallis, OR 97331 Retene and phenanthrene are two prevalent polycyclic aromatic hydrocarbons (PAHs) found at high concentrations in wildfire smoke, for which toxicity data is limited. While phase I cytochrome P450s catalyze reactions to enhance solubility of PAHs, bioactivation can also occur. Further, metabolism varies with developmental stage due to changes in abundance and activity of enzymes. We hypothesize that age alters the rates of detoxification and/or bioactivation in an individual exposed to PAHs which can affect susceptibility. To test our hypothesis, we measured rates of metabolism of retene and phenanthrene in individuals ≤1 yr using human liver microsomes (HLM), n=6. We incubated microsomes with target PAH and measured disappearance of the parent compound using reverse phase HPLC. We then assessed for any differences compared to that of an average adult using pooled HLM (200 individuals). Intrinsic clearance for both chemicals varied greatly among individuals and in pooled HLM. Intrinsic clearance of retene was 0.035 ml/min/mg microsome in the pooled sample, while in individuals ≤1 yr, it ranged from ~2-fold lower to 10-fold higher than the pool (0.015-0.35 ml/min/mg microsome). Of note, a 20-fold variation in intrinsic clearance was seen between individuals. For phenanthrene, intrinsic clearance was 0.56 ml/min/mg microsome in pooled HLM and ranged from 7-fold lower at 0.08 ml/min/mg to 0.6 ml/min/mg microsome in individuals ≤1 yr. Age-related metabolism is a poorly understood but critically important factor in assessing health risk of exposure. These findings reveal high variability in the metabolism of two PAHs in individuals ≤1 yr as compared to an average adult. We are currently working to identify the enzymes responsible for metabolism of these chemicals and metabolites formed from these metabolic reactions. These approaches will aid in efforts to determine human health risk and susceptibility to PAH exposures in individuals, particularly during early maturation.


Postdoctoral Fellow Flash Talk Abstracts 2. Phenotypically Anchored mRNA and miRNA Expression Profiling in Zebrafish Reveals Flame Retardant Chemical Toxicity Networks Subham Dasgupta, Cheryl L Dunham, Lisa Truong, Michael T Simonich, Christopher M Sullivan, Robyn L Tanguay Oregon State University, Corvallis, Oregon The ubiquitous use of flame retardant chemicals (FRCs) in the manufacture of many consumer products leads to inevitable environmental releases and human exposures. Studying toxic effects of FRCs as a group is challenging since they widely differ in physicochemical properties. We previously used zebrafish as a model to screen 61 representative FRCs and showed that many induced behavioral and teratogenic effects. In this study, we selected 10 FRCs belonging to diverse physicochemical classes and executed paired mRNA-micro-RNA (miR) sequencing, enabling us to investigate the role of miRs as posttranscriptional regulators in FRC toxicity. We found widespread disruption of mRNA and miR expression across several FRCs. Neurodevelopment was a key disrupted biological process across multiple FRCs and was corroborated by behavioral deficits. Several mRNAs (e.g., osbpl2a) and miRs (e.g., mir-125b-5p), showed differential expression common to multiple FRCs . These common miRs were also predicted to regulate a network of differentially expressed genes with diverse functions, including apoptosis, neurodevelopment, lipid regulation and inflammation. Likewise, commonly disrupted transcription factors (TFs) such as retinoic acid receptor, retinoid X receptor, and vitamin D regulator were predicted to regulate a wide network of differentially expressed mRNAs across FRCs. Many of the differential mRNA-TF and mRNA-miR pairs were predicted to play important roles in development as well as cancer signaling. Specific comparisons between FRCs TBBPA and its derivative TBBPA-DBPE showed contrasting gene expression patterns that corroborated with their phenotypic profiles. The newer generation FRCs such as IPP and TCEP produced distinct gene expression changes compared to the legacy FRC BDE-47. Our study is the first to establish a mRNA-miR-TF regulatory network structurally diverse FRCs to discover common and unique FRC targets that will help better understand their mechanisms of toxicity.


Postdoctoral Fellow Flash Talk Abstracts 3. Comparative Analysis Between Zebrafish and Automated Live-Cell Assay to Assess 87 Developmental Neurotoxicants Lindsey St.Mary, Lisa Truong, Robyn L. Tanguay, Andrew A. Bieberich, Raymond O. Fatig III Oregon State University, AsedaSciences Inc. The demand and ability for increased screening efficiency to assess chemical toxicity has increased with development of high-throughput toxicological assays involving alternative models, automation, and machine learning (ML). The benefit of comparing across species and assays allows investigators to prioritize chemicals and understand how different model systems might complement one another. The study objective was to assess performance similarity between a developmental zebrafish model screen and an automated live-cell assay for a set of 87 compounds. The AsedaSciences SYSTEMETRIC® Cell Health Screen was developed to estimate acute toxicity risk. This platform uses supervised ML to classify cell stress phenotypes in HL60 cells following a 4-h exposure to a chemical, while the early life stage zebrafish provides a whole systems approach to assess toxicity. In this study, chemical hits for abnormalities in zebrafish morphology and mortality were compared with chemicals generating values > 0.5 for the Cell Health Index (CHI™), which is an outcome class probability from the ML classifier using 12 parameters simultaneously from the cell-based screen (CHI=1: highest possible toxicity risk). The concordance between both models was 13 chemicals. Zebrafish were more sensitive, with 36 bioactivity hits, while 22 induced adverse effects in HL60 cells. Behavioral analyses of endpoints within the zebrafish screen, assessing embryo, larval, and brain morphology, were performed to confirm correlations with embryonic photomotor response (EPR) as a predictor of brain and behavioral abnormalities found in 120 hpf larvae. Finally, several chemicals that induced morphological effects in zebrafish and high risk in the Cell Health Screen also produced morphological hits in TOXcast screenings that utilized diverse data streams collected using in vitro and in vivo systems. Collectively, these results support combining zebrafish and the Cell Health Screen for rapid hazard assessments.


Postdoctoral Fellow Flash Talk Abstracts 4. Primary Rat Hepatocytes as a Model System for Investigating Xenobiotic-Induced Alternative Splicing Jacki L. Coburn, Aurea M. Flores, Andrew J. Annalora, Craig B. Marcus Oregon State University, Purdue University Primary rat hepatocytes (PRHs) offer the possibility of a more humane alternative to in vivo xenobiotic exposures that reduces the number of animals used in a study coupled with an advantage over immortalized hepatocyte lines in enzyme induction studies. However, the conditions under which the PRHs are cultured may greatly affect baseline enzyme levels and enzyme induction when compared to the in vivo system. These conditions may also affect not only the expression levels of the enzymes, but also affect the location splice sites on pre-mRNA. Due to mechanisms that have yet to be fully elucidated, environmental exposures may induce splice variants in mRNA transcripts. The induction of known splice variants may be indicative of alternative splicing events throughout the transcriptome, and full RNA sequencing comparing splicing profiles of vehicle control to xenobiotic treated hepatocytes will be carried out once reproducible splicing is achieved. Previously, in vivo and ex vivo studies in a male Sprague-Dawley rat model showed that exposure to certain xenobiotics induced the expression of an alternatively spliced CYP2B isozyme. This variant has been detected via multiplexed endpoint PCR in cryopreserved PRHs; however, high vehicle control expression of the splice variant and variability between replicates within treatments groups has made determination of splice variant induction challenging. Here we outline some of the culture conditions of the PRHs as well as show some preliminary data obtained from the PRH experiments


Postdoctoral Fellow Flash Talk Abstracts

5. Chronic Exposure to Ambient Traffic-related Air Pollution (TRAP) Alters Gut Microbial Abundance and Bile Acid Metabolism in a Transgenic Rat Model of Alzheimer’s Disease

Moumita Dutta1, Kris M. Weigel1, Kelley T. Patten2, Anthony E. Valenzuela2, Christopher Wallis3, Keith J. Bein3,4, Anthony S. Wexler3,5, Pamela J. Lein2, Julia Yue Cui1 1Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA, 2Department of Molecular Biosciences, University of California Davis (UC Davis) School of Veterinary Medicine, Davis, California, USA, 3Air Quality Research Center, UC Davis, Davis, California, USA

Background. Traffic-related air pollution (TRAP) is linked to increased risk for age-related dementia, including Alzheimer’s disease (AD). The gut microbiome is posited to influence AD risk, and an increase in microbial-derived secondary bile acids (BAs) is observed in AD patients. We recently reported that chronic exposure to ambient TRAP modified AD risk in a sex-dependent manner in TgF344 AD (TG) rat.

Objectives. In this study, we used samples from the same cohort to test our hypothesis that TRAP sex-dependently produces gut dysbiosis and increases secondary BAs to a larger extent in the TG rat relative to wild type (WT) controls.

Methods. Male and female TG and age-matched WT rats were exposed to either filtered air (FA) or TRAP from 28 days up to 15 months of age (n= 5-6). Samples were collected after 9 or 14months of exposure.

Results. At 10 months of age, TRAP tended to decrease the alpha diversity as well as the beneficial taxa Lactobacillus and Ruminococcus flavefaciens uniquely in male TG rats as determined by 16S rDNA sequencing. A basal decrease in Firmicutes/Bacteroidetes (F/B) ratio was also noted in TG rats at 10 months. At 15 months of age, TRAP altered more inflammation-related bacteria in the gut of female rats from both genotypes. BAs in females were more affected by chronic TRAP exposure, with a general trend of increase in host-produced unconjugated primary and microbiota-produced secondary BAs. Most of the mRNAs of the hepatic BA-processing genes were not altered by TRAP, except for a down-regulation of the BA-uptake transporter Ntcp in males.

Conclusion. In conclusion, chronic TRAP exposure produced distinct gut dysbiosis and altered BA homeostasis in a sex and host genotype-specific manner.


William M. Baird Travel Endowment Fund

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Seeking permanent funding in 2021! The fund was established to acknowledge the pioneering work and contributions of Dr. Baird to the

field of polycyclic aromatic hydrocarbon toxicology and carcinogenesis and to provide funds to enable deserving students to attend the annual SOT meeting and other toxicology-oriented meetings.

With the global COVID-19 pandemic and the inability to host in person meetings we lost one of our

main fundraising venues for the Baird Endowment to meet permanent status. In 2021 we need to show continued progress to support funding of the Baird Endowment and reach our goal of permanent endowment status so as in person meetings resume we can begin granting awards for deserving

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Thank you to the generous donations from:

• Elizabeth Baird in honor of William Baird • Tod Harper & Leah Wehmas • Brinda Mahadevan • Nadia Moore • Miriam C. Poirier • Bob Roth and Patti Ganey • David E. Williams in honor of Dr. William Baird

Dr. William M. Baird, PhD, Emeritus Professor, Department of Environmental & Molecular Toxicology and Professor, Department of Biochemistry & Biophysics at Oregon State University, was born in 1944 in Philadelphia PA. He received his B.S. Chemistry from Lehigh University and his Ph.D. in Oncology from the University of Wisconsin-Madison. Dr. Baird has previously been appointed as Glenn L. Jenkins Professor of Medicinal Chemistry and Cancer Center Director at Purdue University, and Director, Environmental Health Sciences Center at Oregon State University. Dr. Baird has pursued a highly distinguished and internationally recognized career based on developing a molecular understanding of the mechanisms of carcinogenesis by environmental Polycyclic Aromatic Hydrocarbons (PAHs), with an emphasis on the role of metabolism by cytochrome P450 isoforms inactivation and detoxification of PAHs; the interactions of carcinogenic chemicals with nucleic acids and chromatin; chemoprevention and the mechanism of action of chemopreventive agents; and the role of PAHs in tumor induction and formation of PAH-DNA adducts as biomarkers for exposure. Dr. Baird has published over 140 peer-reviewed manuscripts, been the recipient of continuous major funding from NIH and other extramural research funding agencies, and received numerous awards and recognitions. Dr. Baird has been a leader and mentor in this field for over 40 years, training students and early career scientists and providing critical new information relating directly to our understanding the mechanism of action of carcinogenic chemicals to which large numbers of humans are exposed and are important for risk assessment and chemoprevention.

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Interaction of the Microbiome and the Immune System

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