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Center of Excellence in Environmental Toxicology (CEET)

ELEVENTH ANNUAL SYMPOSIUM

Exposure Biology: Population-based to Personal

Arthur H. Rubenstein AuditoriumSmilow Center for Translational Research

Perelman School of Medicine at the University of Pennsylvania

June 15, 2016

Schedule 1 – 2

Welcome and Keynote Speaker 3

Mission and Vision Statement 4

New in CEET 5 – 6

Members of CEET 7 – 8

Poster Abstracts

Lung and Airway Disease 9 – 10

Oxidative Stress and Oxidative Stress Injury 10

Reproduction, Endocrinology, and Development 11 – 18

Gene-Environment Interactions 19

Translational Biomarker Core 19 – 20

Community Outreach and Engagement Core 21 – 24

Superfund Research Program 24 – 29

Acknowledgements 30

Contents

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7:30 – 8:30 A.M. CONTINENTAL BREAKFAST AND REGISTRATION

8:30 – 8:45 A.M. Welcome Trevor M. Penning, PhD The Thelma Brown and Henry Charles Molinoff Professor of Pharmacology Professor of Biochemistry & Biophysics and OB/GYN Director of Center of Excellence in Environmental Toxicology

POPULATION-BASED EXPOSURE BIOLOGY

GIS-TOOLS Moderator: Edward A. Emmett, MD, Director of Community Engagement Core, Penn-SRP

8:45 – 9:15 A.M. Critiquing Geographic Research and the Use of Maps Charles Branas, PhD Professor of Epidemiology and Director of the Cartographic Modeling Laboratory University of Pennsylvania

9:15 – 9:45 A.M. A Study of People – The Ambler Community, Types of Exposure to Asbestos, and Mesothelioma Douglas Wiebe, PhD Associate Professor of Epidemiology in Biostatistics and Epidemiology University of Pennsylvania

9:45 – 10:15 A.M. Mapping of Waste Products from Unconventional Gas Drilling in the Marcellus Shale Region of Pennsylvania George Gerton, PhD Research Professor of Reproductive Biology in Obstetrics and Gynecology University of Pennsylvania

10:15 – 10:45 A.M. BREAK

ENVIRONMENTAL SAMPLING Moderator: Rebecca Simmons, MD, Deputy Director of CEET

10:45 – 11:15 A.M. Indoor and Outdoor Air Pollution and COPD Nadia Hansel, MD, MPH Associate Professor of Medicine Pulmonary and Critical Care Medicine Director of Center of Excellence on Health Disparities Research Johns Hopkins University, School of Medicine

11:15 – 11:45 A.M. Water Pollution and the Challenge of Harmful Algal Blooms Ellen Gilinsky, PhD Senior Policy Adviser, Office of Water US EPA

COMMUNITY OUTREACH & ENGAGEMENT

11:45 – 12:15 P.M. Evaluating the Community Exposome Marilyn Howarth, MD Director of Community Outreach and Engagement Core, CEET

12:15 – 1:30 P.M. LUNCH

Eleventh Annual CEET Symposium

Exposure Biology: Population-based to PersonalWednesday, June 15, 2016

Smilow Center for Translational Research

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1:30 – 2:30 P.M. KEYNOTE Introduction: Trevor M. Penning, PhD

“Exposomics” Martyn T. Smith, PhD Professor of Toxicology & Kenneth and Marjorie Kaiser Chair of Cancer Epidemiology, Division of Environmental Health Sciences, School of Public Health, University of California Berkeley

Director of the Superfund Research Center, University of California, Berkeley

PERSONAL-BASED EXPOSURE BIOLOGY

PERSONALIZED SYSTEMS BIOLOGY Moderator: Ian A. Blair, PhD, Director of Penn-SRP and Director of Translational Biomarker Core, CEET

2:30 – 2:50 P.M. Genomic and Environmental Approaches to Study Asthma Blanca Himes, PhD Assistant Professor of Epidemiology in Biostatistics and Epidemiology University of Pennsylvania

2:50 – 3:10 P.M. Targeted and Untargeted Biomarkers of Environmental Exposures Clementina Mesaros, PhD Technical Director of Translational Biomarker Core, CEET

3:10 – 3:30 P.M. Exposure Biology Informatics Jason Moore, PhD Director of Exposure Biology Informatics Core, CEET

3:30 – 4:30 P.M. POSTER VIEWING AND BREAK

BIOMONITORING/ BIOSENSORS Moderator: George L. Gerton, PhD, Leader of Affinity Group Reproduction, Endocrinology & Development, CEET

4:30 – 4:50 P.M. Biosensors Based on Graphene and Carbon Nanotubes Charlie Johnson, PhD Professor of Physics, and Materials Science and Engineering, Electrical and Systems Engineering Director of Nano/Bio Interface Center University of Pennsylvania

4:50 – 5:10 P.M. Using Wearable Devices to Change Health Behaviors Mitesh Patel, MD, MBA, MS Assistant Professor of Medicine and Health Care Management University of Pennsylvania

5:10 – 6:30 P.M. RECEPTION

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Martyn Smith received his PhD in Biochemistry from St. Bartholomew’s Hospital in London and did postdoctoral training in toxicology at the Karolinska Institute in Stockholm. Dr. Smith is a laboratory scientist with expertise in molecular epidemiology, toxicology and genomics, and his research is aimed at finding the causes of chronic diseases, including cancer and diabetes.

He currently teaches toxicology and mentors graduate students and postdoctoral scholars in the Molecular Toxicology, Epidemiology and Environmental Health Science programs. He is also Director of the Superfund Research Center at Berkeley, a position he has held continuously since 1987.

Dr. Smith is a Fellow of the American Association for the Advancement of Science. He received the 2010 Children’s Environmental Health Network Award, became an Elected Fellow of the Collegium Ramazzini in 2012, and received the 2014 Alexander Hollaender Award from the Environmental Mutagenesis and Genomics Society.

Martyn T. Smith, PhDProfessor of Toxicology and Kenneth and Marjorie Kaiser Chair of Cancer Epidemiology, Division of Environmental Health Sciences, School of Public Health, University of California Berkeley

Director of the Superfund Research Center University of California Berkeley

Keynote Speaker

It is with enormous pride that I welcome you to the 11th Annual Symposium of the Center of Excellence in Environmental Toxicology (CEET), the University of Pennsylvania Environmental Health Sciences Core Center. Every year we choose a theme to embrace so that we can learn more about a field and how it might align with current and future directions of the CEET. This year’s theme is no exception. Exposure Biology: Population-Based to Personal embraces the expansive science on how to monitor exposures and human response. It spans Geographic Information Systems, Environmental Sampling, the Community Exposome, Personalized Systems Biology, and Biomonitoring and Biosensors.

We are particularly pleased that Dr. Martyn Smith, Professor of Toxicology, Director-Superfund Research Program, University of California Berkeley will be the keynote speaker, and will address the topic “Exposomics”.

– Trevor PenningDirector, CEET

11TH ANNUAL SYMPOSIUM

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Mission and Vision Statement

The Center of Excellence in Environmental Toxicology (CEET) was launched in 2005 and receives grant sup-port from the National Institute of Environmental Health Sciences (NIEHS). It is one of only twenty designated Environmental Health Sciences Core Centers in the nation; it is the only one in the Commonwealth of Penn-sylvania, and the only one in US EPA Region III (PA, DE, MD, WV, VA and Washington, DC). As such it is a regional and national resource.

The CEET elucidates the mechanistic links between environmental exposures and human disease and trans-lates its findings into action to improve the health of vulnerable individuals, and local, national and global communities.

The CEET marries its relevant research excellence to tackle the environmental challenges that may present an assault on our public health. Many of these challenges have their origins in community-based concerns. Examples include the hazard presented by petrogenic polycyclic aromatic hydrocarbons from the Deepwater Horizon oil spill in the Gulf of Mexico; the fate, transport, remediation and adverse health effects of asbestos exposure in the Ambler Community in SE. Pennsylvania (which is home to one of the largest Superfund As-bestos hazardous waste sites in the country); and natural gas drilling operations in the Marcellus Shale, where citizens are concerned about the effects of air pollution and water contamination on their health. These com-munity public health concerns are often identified by our Community Outreach and Engagement Core which has a history in conducting community-based participatory research, wherein research findings are translated back to the affected community using a “community-first communication model”.

The CEET has research excellence in themes that are related to environmental health that exist in our imme-diate area. Its Affinity Group in Lung and Airway Disease examines the relationship between poor air quality and air pollution in our region (ozone, fine particulate matter, allergens, SO2, NO2 and CO emissions) and dis-ease (asthma, lung cancer, mesothelioma and COPD); Its Affinity Group in Reproduction, Endocrinology, and Development examines the relationship between exposures in windows of susceptibility and resultant health outcomes. Investigators explore the association between in utero exposures, epigenetic imprinting and the developmental basis of adult disease. These organ-based themes are linked to our Affinity Groups in disease mechanism, which include Oxidative Stress and Oxidative Stress Injury and Gene-Environment Interactions.

The CEET enables its investigators to conduct exposure science using its Translational Biomarker Core, which uses sophisticated liquid chromatography mass spectrometry methods to identify and develop assays of bio-markers of exposure and effect. The Core measures changes in targeted and unbiased metabolomes following response to exposures and during disease onset and progression. The CEET maintains an Exposure Biology Informatics core so that large siloed data bases in genomics, proteomics, metabolomics and exposomics can be merged as predictors of responses and adaptation to environmental exposure that account for the phenome. The Integrated Health Sciences Facility Core (IHSFC) of the CEET provides assistance with a broad range of trans disciplinary services including study design, enrollment of research subjects, population and community exposures, access to biospecimens via a CEET biorepository and data management, and genetic and non-ge-netic biostatistical analyses.

The CEET engages six communities in S.E. Pennsylvania to empower them with new knowledge so that they are better informed to tackle issues of environmental health threats, health disparities and environmental justice. To improve the environmental health of these and similar affected communities, the CEET is actively involved in the education of health care professionals (Residency Program in Occupational and Environmental Health, Nursing concentration in Occupational and Environmental Health, and Masters of Public Health Programs).

The CEET also disseminates findings to all stakeholders including community organizations, local, state and federal officials and agencies (Pennsylvania Department of Health, Pennsylvania Department of Environmental Protection, Environmental Protection Agency) to affect change in environmental health and public health policies.

The CEET is a flexible entity that marshals excellence in basic, translational, patient-oriented and popula-tion-based research in the School of Medicine and Children’s Hospital of Philadelphia. Although primarily housed in the School of Medicine, the 70 CEET members belong to 19 departments and 6 schools at the Uni-versity of Pennsylvania.

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New in CEET

Dr. Simmons is the Hallam Hurt Professor of Pediatrics at the University of Penn-sylvania and an attending neonatologist at the Children’s Hospital Philadelphia (CHOP) and the Hospital of the University of Pennsylvania. As Deputy Director of the CEET, she continues to direct the Pilot Project Program and is co-director of the Career Development Core. Her appointment at CHOP positions the CEET to conduct environmental health science research at the pediatric-adult divide.

The principal goal of her research program is to determine the underlying molecu-lar mechanisms that link an abnormal intrauterine milieu to the later development of diseases such as obesity and type 2 diabetes after birth. She has established rodent animal models of intrauterine growth restriction, maternal obesity, and ma-ternal exposure to endocrine disruptors. All of these animal models induce obesity, beta-cell and hepatic dysfunction in the offspring, ultimately leading to the devel-opment of obesity and diabetes. Using these animal models her lab is elucidat-ing the role of epigenetic mechanisms (DNA methylation, histone modifications, non-coding RNAs) in the development of an abnormal phenotype in the offspring in the ß-cell, liver, fat, and brain. Using next generation sequencing, she is assess-ing how an altered maternal milieu impacts the epigenome and the transcriptome and the underlying molecular mechanisms that induce genome-wide epigenetic modifications in the very early embryo and placenta as well as in offspring later in life. She has extended these studies to investigate the transgenerational effects of the endocrine disruptor, BPA on the ß-cell (funded by RO1ES023284) as well as to study the effects of lead exposure during intrauterine and early postnatal life on circadian rhythm and sleep behaviors in the offspring. Both of the projects were developed using pilot project funds from the CEET.

She has been a member of the P30 Environmental Health Sciences Core Center at PENN, CEET, since 2006. During this time, Dr. Simmons assumed various leader-ship positions within the CEET. As co-director of the CDC, she established a Fron-tiers Course in Environmental Health for fourth year medical students to promote their interest in the discipline.

She is a co-Project leader on the Penn Superfund Research and Training Program P42 ES023720 to determine the epigenetic initiators of Mesothelioma in human and animal models.

There have been a number of important changes in the CEET structure that will help propel the mission of the center further forward. To emphasize our theme of translational environmental health sciences, we have continued in our tradition of appointing a clinician scientist as Deputy Director. Rebecca Simmons MD, the Hallam Hurt Professor of Pediatrics assumes this role. Dr. Simmons has held many leadership positions in the CEET, including acting as co-director of the Career Development Core and Director of the Pilot Project Program. She has a portfolio of grant support from NIEHS including studies on the trans-genera-tional effects of endocrine disrupting chemicals.

The facility core structure of the CEET also has new leadership. We welcome Anil Vachani MD, MSCE as the new director of the Integrative Health Sci-

ences Facility Core. Dr. Vachani has a long interest in working with the CEET. He was co-investigator on the PA-DOH Gene-Environment Interactions in Lung Cancer program; and works with the Translational Biomarker Core to identify new biomarkers of expo-sure and effect and disease onset. His own research is related to the development of new biomarkers for the early detection of thoracic malignancies.

We also welcome Jason Moore, PhD as the new director of the Exposure Biology Informatics Core. Dr. Moore is an expert in the integration of large scale-“omics” data for determining disease suscep-tibility. He has established machine-learning tools to develop predictive models of human health and disease.

Rebecca A. Simmons, MD Deputy Director of CEET

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Dr. Vachani is Associate Professor of Medicine in the Division of Pulmonary, Aller-gy and Critical Care and continues the tradition of excellence in the IHSFC with its historical focus on pulmonary disease. He is Chief of Clinical Research, Section of Interventional Pulmonology and Thoracic Oncology, Department of Medicine, University of Pennsylvania Medical Center, Director, Lung Nodule Program, Uni-versity of Pennsylvania Medical Center, Director of Bronchoscopy, Philadelphia VA Medical Center, and Chair, Institutional Review Board 2 & 9, University of Pennsylvania. He has been a member of the CEET since 2006. He is a co-inves-tigator on the Penn Superfund and Research Training Program – Asbestos Fate, Exposure, Remediation, and Adverse Health Effects (P42 ES023720) on Project 6, which aims to identify serum biomarkers to distinguish between asbestos expo-sure and mesothelioma led by Dr. Ian A. Blair.

Dr. Moore is the Edward Rose Professor of Informatics and Director Institute for Bioinformatics. His background and expertise is at the intersection of computer science, statistics, and the biological sciences with a focus on genetics and ge-nomics. He leads the NIH-funded Computational Genetics Laboratory that de-velops innovative computational methods for solving complex problems across the biomedical sciences using artificial intelligence, machine learning, and visual analytics. He also serves as director of the Penn Institute for Biomedical Informat-ics (IBI). The research goal of IBI is to develop, evaluate and apply novel compu-tational methods and open-source software for identifying genetic and genomic biomarkers associated with disease susceptibility. The IBI focus is on methods that embrace, rather than ignore, the complexity of the genotype-to-phenotype mapping relationship due to phenomena such as epistasis and plastic reaction norms. Areas of interest include artificial intelligence, bioinformatics, biomedical informatics, complex systems, computational biology, genetic epidemiology, ge-nomics, human genetics, machine learning, and visual analytics.

Anil Vachani, PhD

Director of the Integrative Health Sciences Facility Core (IHSFC)

Jason H. Moore, Ph.D.

Scientific Director and Principal Investigator of Exposure Biology and Informatics Core

New in CEET

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Affinity Group ILUNG AND AIRWAY DISEASE

Director: Michael Beers, M.D

Steve Albelda, MDAndrea Apter, MD, MSc Jason Christie, MD, MSCEMelpo Christofidou-Solomidou, PhD Peter DeCarlo, PhD*Dongeun (Dan) Huh, PhD Marcelo Kazanietz, PhD Vera Krymskaya, PhD Edward Morrisey, PhDTrevor Penning, PhD Joseph Testa, MD*Anil Vachani, MD, MSCE

Affinity Group IIOXIDATIVE STRESS AND OXIDATIVE STRESS INJURY

Director: Ian Blair, PhD

Paul Axelsen, MDJoseph Baur, PhDMichael Beers, MDBrenda Casper, PhDJeffrey Field, PhD Aron Fisher, MD Garret FitzGerald, MDReto Gieré, PhDHarry Ischiropoulos, PhD Douglas Jerolmack, PhDKelly Jordan-Sciutto, PhD Vladimir Muzykantov, MD, PhD Trevor Penning, PhDRebecca Simmons, MD Andrew Strasser, PhD

Affinity Group IIIREPRODUCTION, ENDOCRINOLOGY, AND DEVELOPMENT (READ)

Director: George Gerton, PhD

Marisa Bartolomei, PhD Shelley Berger, PhDSamantha Butts, MD, MSCEChristos Coutifaris, MD, PhDTed Emmett, MD, MSStruan Grant, PhDKaren Knudsen, PhD*Michael Levine, MDJianghong Liu, PhD, RNSarah Millar, PhD Katherine Nathanson, MDSam Parry, MD Trevor Penning, PhD Sara Pinney, MD, MSRebecca Simmons, MD Nate Snyder, PhD*Virginia Stallings, MDSigrid Veasey, MDJeremy Wang, MD/PhD

Affinity Group IVGENE-ENVIRONMENT INTERACTIONS

Director: Marisa Bartolomei, PhD

Shelley Berger, PhD Ian Blair, PhD Jinbo Chen, PhDYouhai Chen, MD, PhD Jason Christie, MD, MSCE Struan Grant, PhDHakon Hakonarson, MD, PhD Blanca Himes, PhDHongzhe Li, PhD Sarah Millar, PhD Jason Moore, PhD Katherine Nathanson, MDJennifer Pinto-Martin, PhD, MPH Trevor Penning, PhDVirginia Stallings, MDSarah Tishkoff, PhDAalim Weljie, PhD

CENTER OF EXCELLENCE IN ENVIRONMENTAL TOXICOLOGYPerelman School of Medicine at the University of Pennsylvania

ADMINISTRATIVE COREDirector: Trevor Penning, PhD

Deputy Director: Rebecca Simmons, MD

*Adjunct Member

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CENTER OF EXCELLENCE IN ENVIRONMENTAL TOXICOLOGYPerelman School of Medicine at the University of Pennsylvania

EXPOSURE BIOLOGY INFORMATICS CORE

Director: Jason Moore, PhD Technical Director: Zhiping (Paul) Wang, PhD

TRANSLATIONAL BIOMARKER CORE

Director: Ian Blair, PhD Technical Director: Clementina Mesaros, PhD

INTEGRATED HEALTH SCIENCES FACILITY CORE

Director: Anil Vachani, MD, MSCE

Human Studies Design and Performance Services Director: Anil Vachani, MD, MSCE

Population Exposure Services Associate Director: Ted Emmett, MD, MS

Virtual Biorepositories Associate Director: Anil Vachani, MD, MSCE

Biostatistics Associate Director: Kathleen Propert, ScD Genetics Statistician: Mingyao Li, PhD Statistician: Wei-ting Hwang, PhD

COMMUNITY OUTREACH AND ENGAGEMENT CORE

Co-Director: Marilyn Howarth, MD Co-Director: Richard Pepino, MS

Maria Andrews, MSAndrea Apter, MD, MScFran Barg, PhD Charles Branas, PhD Pamela Dalton, PhD Jeff Field, PhD Ira Harkavy, PhD Michael Z. Levy, PhD Jianghong Liu, PhD, RN Judith McKenzie, MD, MPH Kevin Osterhoudt, MD, MSCEPouné Saberi, MD, MPH

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Lung and Airway Disease

POSTER ABSTRACTS

L1 Estrogen Receptor-mediated Shuttling of Genotoxic PAH Ortho-quinones into the Nucleus in Lung Cancer

Isabelle G. Lee and Trevor M. Penning

Department of Systems Pharmacology and Translational Therapeutics, Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA

Email: [email protected]

Polycyclic aromatic hydrocarbons (PAHs) are a diverse class of environmental toxicants with two or more fused benzene rings; these compounds are common byproducts of incomplete combustion of fossil fuels, and they are suspect human carcinogens. Even though they have natural sources such as forest fires and volcanoes, most PAHs in ambient air are anthropogenic. This class of compounds can be found in diesel exhaust, smoked or barbecued foods and cigarette smoke. Cigarette smoke and tobacco products account for 90% of human lung cancers in the United States. PAHs have to be metabolically activated into reac-tive genotoxins in order to cause their mutagenic and carcinogenic effects. One of the pathways of PAH activation involves the formation of PAH o-quinones by aldo-keto reductases, and these o-quinones are ligands for the aryl hydrocarbon receptor (AhR). Previous work in our laboratory has provided evidence that the AhR acts as a carrier involved in the shuttling and concentration of the representative PAH o-quinone, benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) to the nucleus (Park et al, 2009). This process enhances PAH o-quinone-mediated oxidative DNA damage in the form of DNA strand breaks and generation of mutagenic 8-oxo-7,8-dihydro-2-deoxyguanosin (8-oxo-dG) lesions. Given the similarity between the planar B[a]P-7,8-dione to the estrogen quinones and evidence by Wang et al that the estro-gen receptor (ER) acts as a “Trojan Horse” to shuttle these genotoxic estrogen metabolites to the nucleus to promote oxidative DNA damage, we hypothesize that PAH o-quinones can be translocated into the nucleus by the estrogen receptor in a similar fashion, to enhance oxidative DNA damage. We have used Ishikawa cells, a human endometrial adenocarcinoma cell-line, and alkaline phosphatase activity as the read-out for ER activation to determine whether B[a]P-7,8-dione activates the estrogen receptor. Thus far, we have demonstrated that B[a]P-7,8-dione is a ligand for the ER and seek to determine the cross talk between the ER and AhR in mediating the genotoxic effects of PAH in human lung cells.

Supported by NIEHS T32 ES019851

L2 Metabolic Activation of Nitroarenes by Human Aldo-Keto Reductases (AKR1C1-AKR1C3)

Jessica Murray1, Meng Huang1, Clementina Mesaros2, Volker Arlt3, Karam El-Bayoumy4, Ian Blair1,2, and Trevor M. Penning1,2

1Center of Excellence in Environmental Toxicology and 2Center for Cancer Pharmacology, University of Pennsylvania, Philadelphia, PA, United States; 3Department of Environmental and Genetic Toxicology, King’s College London, London, United Kingdom; and 4Department of Biochemistry & Molecular Biology, Penn State College of Medicine, Hershey, PA, United States


Diesel engine exhaust (DEE) is listed as a Group 1 Carcinogen by the International Agency for Research on Cancer (IARC) and contributes to occupational and environmental causes of lung cancer. Nitrat-ed-polycyclic aromatic hydrocarbons, or nitroarenes, are major constituents of DEE and are detected in ambient air pollution. Nitroarenes require metabolic activation to exert their mutagenic and tumorigenic effects. Metabolic activation of nitroarenes involves three successive two-electron reductions, leading to the formation of nitroso-, hydroxylamino-, and amine derivatives. The hydroxylamino-derivatives

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Lung and Airway Disease

POSTER ABSTRACTS

can give rise to DNA-adducts following sulfonation or acetylation. Identification of human enzymes involved in this process will be important for the assessment of individual susceptibilities. NADPH: Quinone Oxidoreductase (NQO1) is considered the primary nitroreductase in the metabolic activation of nitroarenes. However, AKR1C3 displays nitroreductase activity towards the cancer chemotherapeutic agent PR-104A and so we sought to determine whether AKR1C subfamily members could also con-tribute to toxification of nitroarenes. We have determined that AKR1C1-AKR1C3 catalyze the nitrore-duction of 3-nitrobenzanthrone (3-NBA), a representative nitroarene, using discontinuous UV-HPLC assays. Evidence for the formation of the nitroso-, hydroxylamino-, and amine- products was obtained by UPLC-HRMS/MS. Specific activities for 3-aminobenzanthrone formation were compared with those for recombinant NQO1. Another representative nitroarene, 6-nitrochrysene (6-NC), has the unique charac-teristic that it can be activated by both monooxygenation and nitroreduction. Here we demonstrate that AKR1C1-AKR1C3 display dihydrodiol dehydrogenase and nitroreductase activity towards 6-nitrochry-sene-1,2-dihydrodiol. Reaction products were characterized by LC-ion-trap mass spectrometry. The nit-roreduction of diverse nitroarenes by AKR1C enzymes suggest that they may play a role in the activation of these diesel exhaust carcinogens. Notably both NQO1 and the AKR1C genes are highly induced by Nrf2-Keap1-ARE signaling, suggesting that the antioxidant response may not be entirely protective in the context of DEE exposures.

Supported by P30-ES013508 to TMP and T32 019581 to JRM

Oxidative Stress and Oxidative Stress Injury

OS1 Biliary Toxin Biliatresone Depletes Hepatic Glutathione and Forms Glutathione Adducts in Larval Zebrafish

Xiao Zhao1, Kevin P. Gillespie2, Dylan M. Marchione2, Ian A. Blair2,3, Michael A. Pack1,3

1Division of Gastroenterology, Department of Medicine, 2Department of Systems Pharmacology and Transla-tional Therapeutics, and 3Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Biliary atresia is a congenital extrahepatic cholangiopathy whose cause is unknown. The plant toxin biliatresone has been used with animal models to induce the biliary atresia phenotype and investigate the underlying disease mechanisms. Liquid chromatography – mass spectrometry measurements of the antioxidant glutathione revealed that biliatresone ingestion by larval zebrafish results in dramatic depletion of reduced glutathione in the liver within 4 hours, despite no detectable impact on glutathione levels in other tissues. Subsequent recovery of hepatic glutathione levels observed after 12 hours of biliatresone treatment was investigated with 13C

3-L-cysteine treatment, which demonstrated that new glutathione

is synthesized from its component amino acids following biliatresone-induced glutathione depletion. The formation of a biliatresone-glutathione adduct, as previously identified in vitro, was detected in trace amounts in biliatresone-treated cell culture and larval zebrafish. The disparity between glutathione depletion and adduct formation may be due to the transient nature of the adduct in vivo, instability of the adduct in sample processing, or alternative mechanisms of biliatresone-induced glutathione depletion.

Supported by NIH/NIDDK R01-DK 092111

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Reproduction, Endocrinology, and Development (READ)

R1 The Development of (R)-(2-(6-methoxynaphthalen-2-yl)butanoic Naproxen, A Potent and Selective Inhibitor for AKR1C3 for the Treatment of Castrate-resistant Prostate Cancer

Adegoke Adeniji1, Daniel Tamae1, Beau Wangtrakuldee1, Lawrence J. Marnett2, Trevor M. Penning1

1Department of Systems Pharmacology and Translational Therapeutics and the Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 2Departments of Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN


Androgen deprivation therapy (ADT) is a cornerstone in prostate cancer treatment. Patients undergoing ADT often progress to castrate resistant prostate cancer (CRPC) and succumb to the disease despite cas-trate levels of serum testosterone (T) and 5a-dihydrotestosterone (5a-DHT). CRPC is driven by adaptive intratumoral androgen biosynthesis and increased androgen receptor (AR) signalling. The up-regulation of a key androgen biosynthetic enzyme, type 5 17b-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) has been implicated in driving CRPC tumors. AKR1C3 catalyzes the conversion of the precursors, ∆4-androstene-3,17-dione and 5a-androstane-3,17-dione to T and 5a-DHT, respectively. In addition to its catalytic activity, AKR1C3 functions as an AR specific co-activator and it is up-regulated by TMPRSS2-ERG upon androgen deprivation (Powell, K. et al., Clin Cancer Res, 2015) and in enzalut-amide-resistant tumors (Liu C. et al., Cancer Res, 2015). Enzalutamide resistance can be surmounted by the non-steroidal anti-inflammatory drug (NSAID) indomethacin. NSAIDs are non-selective inhibitors of the AKR1C enzymes and we are repurposing NSAIDs so that they are selective for AKR1C3 but no longer inhibit their COX-1 and COX-2 targets. We now find that R- and S-naproxen inhibit AKR1C3 with mid-nanomolar potency but that the R-isomer is devoid of activity on COX isozymes. Only the R-isomer was selective for inhibition of AKR1C3 while the S-isomer will inhibit both AKR1C3 and AKR1C2. Exploiting this stereochemical preference we identified (R)-2-(6-methoxynaphthalen-2-yl)bu-tanoic naproxen as a potent and competitive AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound blocked AKR1C3 mediated production of T and induction of PSA in a CRPC model cell line stably transfected to over-express AKR1C3 (LNCaP-AKR1C3). This compound was devoid of inhibitory activity on COX isozymes. These studies identify R-profens as a new class of repurposed NSAIDs that can target AKR1C3 in CRPC patients.

Supported by NCI 1P01-CA 0163227

R2 Increased miRNA Expression in Second Trimester Amniotic Fluid from Women Subsequently Diagnosed with Diabetes During Pregnancy

Rikka Azuma1 Jacqueline Alexander1, Sara E Pinney1,2,3

1Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia; 2Department of Pedi-atrics, University of Pennsylvania, Philadelphia, PA; 3Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Background: Diabetic pregnancy induces marked abnormalities in fetal development that can result in abnormal fetal growth. Alterations in miRNA expression in amniotic fluid (AF) from women with dia-betes during pregnancy (DDP) and the effect on fetal development have not been defined.

Objective: To identify miRNAs in second trimester AF that correlate with the subsequent diagnosis of DDP.

POSTER ABSTRACTS

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POSTER ABSTRACTS

Methods: AF was collected at gestational age 16-18 weeks from 20 women with DDP who delivered at term and 20 healthy term pregnancies. DDP was characterized based on outcome survey data and confirmed by AF c-peptide measurements. MiRNA expression was measured with a circulating miRNA assay (Firefly Bioworks, Abcam) and normalized to the geometric mean. 88 miRNAs were selected based on references to gestational diabetes, diabetes, preeclampsia and fetal development. MiRNA gene targets were determined from TargetScan and pathway enrichment was analyzed via Reactome.

Results: DDP samples had significant increases in hsa-miR-199a-3p, hsa-miR-503-5p, and hsa-miR-1268a (fold change: 1.77, 1.50, 1.77 respectively) (p ≤0.05). Target pathway enrichment analysis iden-tified IGF2 Binding Proteins pathway for hsa-miR-199a-3p, Mitotic G1-G1/S Phase pathway for hsa-miR-503-5p, and Oncogene Induced Senescence pathway for hsa-miR-1268a.

When only female offspring samples were considered (10 DDP/ 10 Control), DDP samples had signifi-cant increases in hsa-miR-378a-3p, hsa-miR-885-5p, and hsa-miR-7-1-3p (fold change: 1.50, 1.64, 1.62 respectively) (p ≤0.05). Target pathway analysis identified VPR Mediated Apoptosis by Mitochondrial Outer Membrane Permeabilization, Golgi to ER Retrograde Transport, COPI Mediated Transport, and Regulation of RAS by GAPs as enriched pathways for hsa-miR-378a-3p. The DNA Replication CDC6 Association with ORC:origin Complex pathway was identified for hsa-miR-885-5p. Apoptotic Cleavage of Cell Adhesion Proteins pathway was identified for hsa-miR-7-1-3p.

Finally, when only male offspring samples were considered (10 DDP/10 Control), DDP samples had a significant increase in hsa-miR-199a-3p (fold change: 2.20) (p≤0.05). Of note, this miRNA was also identified when samples of both offspring sex were considered (see above).

Conclusion: Expression of hsa-miR-199a-3p, hsa-miR-503-5p, hsa-miR-1268a is increased in second trimester AF approximately 10 weeks before the clinical diagnosis of gestational diabetes. Additional studies will determine the role these miRNAs play in fetal development in DDP.

Supported by NIH, R21 ES 011675

DDP Control Expression Expression Fold miRNA Target miRNA (arbitrary units) (arbitrary units) Change P value Pathway Enrichment

All Samples hsa-miR-199a-3p 19.25 10.86 1.77 0.01 IGF2 binding proteins(20 DDP / 20 Control) hsa-miR-503-5p 24.40 16.21 1.50 0.03 Mitotic G1-G1/S phases hsa-miR-1268a 41.55 23.86 1.77 0.03 Oncogene induced senescence

Female hsa-miR-378a-3p 363.49 241.69 1.50 0.02 VPR mediated apoptosis Offspring by mitochondrial outer Samples membrane permeabilization; (10 DDP / Golgi to ER retrograde 10 Control) transport; COPI mediated transport; Reg of RAS by GAPs

hsa-miR-885-5p 61.76 37.64 1.64 0.02 DNA replication - CDC6 association with ORC: origin complex hsa-miR-7-1-3p 172.33 106.53 1.62 0.004 Apoptotic cleavage of cell adhesion proteins

Male Offspring hsa-miR-199a-3p 21.88 9.93 2.20 0.01 IGF2 binding proteinsSamples (10 DDP/ 10 Control)

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POSTER ABSTRACTS

R3 Sex of Offspring Impacts DNA Methylation and Gene Expression in Placenta from Women with Diabetes During Pregnancy

Jacqueline Alexander1, April Teague2, Steven Chernausek2, Rebecca A. Simmons3,4,5, Sara E. Pinney1,4,5

1Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia; 2Pediatric Metabolic Re-search Unit, University of Oklahoma College of Medicine; 3Division of Neonatology, The Children’s Hospital of Philadelphia; 4Department of Pediatrics, University of Pennsylvania; 5Center of Excellence in Environ-mental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Background: Offspring of mothers with diabetes during pregnancy (DDP) are at increased risk for obe-sity and type 2 diabetes, which may be influenced by offspring sex. The mechanisms responsible for the phenomenon are unknown. We hypothesize that that DDP alters genome-wide DNA methylation re-sulting in differentially methylated loci of metabolically relevant genes and downstream changes in RNA and protein expression.

Methods: We mapped genome-wide DNA methylation with the Infinium 450K HumanMethylation BeadChip using a nested case-control design from a cohort of Native American and Hispanic women with DDP. Term fetal placentae were collected and matched 1:1 with controls based on maternal age, race/ethnicity, and offspring sex (n=17 pairs). Differential methylation (dm) was calculated as ∆b per pair and averaged across all samples. RNA and protein expression were assayed via RNA-Seq and Western Blot.

Results: For genome-wide DNA methylation, 465 CpGs had significant changes for male offspring, 247 for female offspring, and 277 for offspring of both sexes (p<0.001). In placentae from male offspring, CYBA, GSTM1, GSTM5, and RASSF2 showed decreased methylation in DDP samples compared to control and a corresponding increase in RNA and protein expression. In placentae from female offspring, KCNE1 and NXN showed increased methylation in DPP samples compared to control and a corre-sponding decrease in RNA and protein expression. PIWIL3 showed increased methylation in samples from women with DPP compared to control across both offspring sexes, and a corresponding decrease in RNA and protein expression.

Conclusions: In summary, DDP alters placental DNA methylation at metabolically relevant loci in a sex specific manner, with more probes affected in males. Many of these differentially methylated genes corresponded to changes in both RNA and protein expression. This finding may begin to explain the long-term metabolic effects of DDP on offspring.

Supported by NIH (RO1 DK 089034-03, K08 DK 0903-02), American Diabetes Association (1-10-CT-09), and the McCabe Foundation

R4 Metabolomic Profiling of Second Trimester Amniotic Fluid from Mothers with Diabetes During Pregnancy Shows Evidence of Altered Fetal Fuel Sources

Jacqueline Alexander1,2, Rikka Azuma1,2, Sara E. Pinney1,2,3

1Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia; 2Department of Pediat-rics; 3Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Background: Diabetes during pregnancy (DDP) induces marked abnormalities in fetal metabolism that can result in abnormal fetal growth.

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POSTER ABSTRACTS

Objective: To characterize the global metabolic signature of the developing fetus exposed to DDP by profiling changes in metabolomics in second trimester amniotic fluid (AF).

Design/Methods: AF from 20 women with DDP and 20 healthy controls was collected at GA 16-18 weeks. Analysis of 459 metabolites was performed at Metabolon, Inc. Statistical analyses included Welsh’s two sample and matched pairs t-tests, and FDR.

Results: Analysis revealed altered levels of 69 metabolites (28 increased, 41 decreased; p<0.05). Random Forest Plot analysis differentiated the DIP and control groups with a predictive accuracy of 73% consis-tent with profound metabolic profile differences. Principal Component Analysis showed a shift in the global metabolic profile that was more pronounced when separated by sex.

Conclusions: Metabolomic profiling of second trimester AF from women with DIP suggests that fetal metabolism may be altered weeks before gestational diabetes is diagnosed clinically.

Supported by NIH R21 ES011675 and the McCabe Foundation

R5 Paternal BPA Exposure Affects Offspring Glucose Tolerance: A Peek Through Exposure Windows Reveals that Timing is of the Essence

Amita Bansal1, Marisa S. Bartolomei2, Rebecca A. Simmons1,3

1Center for Research on Reproduction and Women’s Health, 2Department of Cell and Developmental Biology, and 3Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


As it is becoming increasingly accepted that maternal exposure to the ubiquitous environmental pollutant bisphenol A (BPA) predisposes offspring to metabolic impairments, paternal contribution in this con-text still remains unresolved. To investigate a relationship between paternal BPA exposure and offspring obesity and glucose tolerance, an accepted experimental mouse model was employed using dietary BPA exposure at doses comparable to human exposure levels. These doses were 7% corn oil diet (Control), 10 ug/mg/day (Low BPA), and 10 mg/kg/day (High BPA) with the low BPA and high BPA doses corre-

Fold P QPathway Metabolite Change Value Value

Impaired Glucose glucose 1.19 0.036 0.219

Metabolism 2-hydroxybutyrate 1.50 0.011 0.017 1,5 anhydroglucitol 0.73 0.022 0.182Gamma Glutamyl gamma-glutamyl tyrosine 0.50 0.021 0.182Amino Acids gamma-glutamyl valine 0.69 0.017 0.168

Fatty Acid Metabolism: palmitoleate (16:1n7) 3.32 0.022 0.182

Long Chain Fatty Acids stearate (18:0) 1.19 0.013 0.165 eicosenoate (20:1n9 or 11) 1.88 0.007 0.134 eicosapentaenoate (EPA; 20:5n3) 1.46 0.002 0.099 docosahexaenoate (DHA; 22:6n3) 1.45 0.004 0.115Fatty Acidy Metabolism: linoleate (18:2n6) 1.73 0.001 0.099Polyunsaturated Fatty Acids linolenate [alpha or gamma; (18:3n3 or 6)] 2.05 0.013 0.165(n3 and n6) dihomo-linolenate (20:3n3 or n6) 1.97 0.004 0.115 arachidonate (20:4n6) 1.61 0.001 0.099 docosapentaenoate (n6 DPA; 22:5n6) 1.45 0.038 0.226 glycerol 1.23 0.025 0.187Ketone Bodies 3-hydroxybutyrate (BHBA) 1.55 0.002 0.099Sphingolipid Metabolism stearoyl sphingomyelin 1.21 0.033 0.211

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POSTER ABSTRACTS

sponding to the NOEL and LOEL, respectively. Two exposure windows were investigated: 1) Exposure of male mice beginning at sexual maturation (5 weeks of age) and continuing for 12 weeks prior to mat-ing, and 2) in utero exposure, where dams were exposed beginning 2 weeks prior to mating and ending at weaning. The male offspring of the exposed dams were regarded as the in utero exposed F0 sires and mated to unexposed females at 8 weeks of age. F1 offspring from both exposure windows underwent body composition analysis via DEXA scan or NMR as well as glucose tolerance testing. Paternal BPA exposure during adulthood did not affect body weight, adiposity or glucose tolerance in offspring. How-ever, in utero exposed sires produced female offspring with impaired glucose tolerance. Body weight and adiposity were unaffected in either sex. These data demonstrate that while paternal BPA exposure after sexual maturity may be metabolically ineffectual, paternal BPA exposure during gestation and lactation precipitates sex-specific impairments in glucose tolerance. Further studies are underway to describe more precisely the glucose homeostatic impairment as well as elucidate epigenetic changes in sperm responsible for phenotypic transmission.

Supported by NICHD, NIEHS, T32 HD060556, R01 ES023284

R6 Mechanism Underlying Multigenerational Effects of Bisphenol A on Pancreatic Islets

Amita Bansal1,2,3, Rashid Cetewayo1,2,3, Frances Xin2,4, Changhong Li5, Marisa Bartolomei1,2,3,4, Rebecca Simmons1,2,3,4

1Center for Research on Reproduction and Women’s Health, 2Center of Excellence in Environmental Toxicol-ogy, 3Division of Neonatology, Department of Pediatrics, 4Department of Cell and Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, and 5Division of Endo-crinology and Metabolism, The Children’s Hospital of Philadelphia, Abramson Pediatric Research Center, Philadelphia, PA


Background: Exposure to an endocrine disruptor, Bisphenol A (BPA), is ubiquitous. BPA exposure is associated with increased risk of diabetes and obesity, yet the underlying mechanisms are unknown. We were the first to show that maternal BPA exposure alters metabolic health across multiple generations in mice. BPA mediates its action on islets via estrogen receptor (ER) alpha. Loss of ERalpha is associated with abnormal immune response. Immune response is necessary for normal pancreatic development. An important mechanism for beta cell-specific effects of BPA could be perturbed immune response. In the current study, we assessed the multigenerational effects of maternal BPA exposure on islets, and deter-mined whether these changes could be mediated via ERalpha and its consequent effects on islet immune response.

Methods: Islets were isolated from adult F1 and F2 offspring (n=5-6 litters per group) of F0 mothers exposed to relevant human exposure levels of 10 μg/kg/day (LowerB), 10 mg/kg/day (UpperB) BPA and 7% corn oil (Control) diets. Mitochondrial driven insulin secretion was determined by a-ketoiso-caproate (KIC) perifusion ramp study, and oxidative phosphorylation by high-resolution respirometry in intact islets using Oroboros. Cytosolic calcium [Ca2+]i responses in intact islets were measured by du-al-wavelength fluorescence microscopy. Beta cell mass was determined using the beta cell area calculated from triple immunofluorescent staining of insulin, glucagon and somatostatin proteins, and images were quantified using HALO image analysis software. Pancreatic sections were immunostained for CD3 for T-lymphocytes and F4/80 for macrophages. Cytokine profiling was assessed in total pancreatic lysates by Luminex assay. mRNA levels were determined by qPCR. Data were analysed by one-way ANOVA and p<0.05 was considered significant.

Results: LowerB males had reduced beta cell mass, whereas UpperB males had reduced islet mitochon-drial function. Our preliminary RNAseq analysis in F1 BPA treated vs Control mice islets revealed dif-ferential expression of key immune response genes. Immunostaining of F1 pancreatic sections confirmed

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POSTER ABSTRACTS

increased staining of CD3 and F4/80 in islets of LowerB and UpperB males compared to Controls. Luminex assay of F2 pancreatic lysates also indicated increased cytokine levels in LowerB and UpperB males. Both LowerB and UpperB males had reduced ERalpha mRNA levels across the two generations. No differences were reported in cytosolic calcium [Ca2+]i responses in islets.

Conclusion: Early life lower dose exposure is associated with reduced beta-cell mass, and upper dose exposure is associated with reduced islet mitochondrial function. We speculate that BPA exposure re-duces ERalpha expression, which in turn induces activation of different immune cell populations in beta-cells leading to beta-cell dysfunction. These speculations need to be confirmed by performing in depth immune profiling across the two generations, and conducting extensive genome editing studies to determine the role of ERalpha in mediating BPA induced beta cell-specific immune response.

Supported by NIEHS RO1 ES023284, T32 ES019851

R7 Optimization of Sex Chromosome Detection Throughout Mouse Development

Kristin M. Bircsak1,2, Andrew Prantner1, Teri Ord1, Matthew Moronta1, George, L Gerton1,2,3

1Center for Research on Reproduction and Women’s Health, 2Center of Excellence in Environmental Tox-icology, and 3Department of Obstetrics and Gynecology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Background: Paternal exposure to the persistent organic pollutant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with offspring sex ratio distortion towards a greater proportion of females than males. Sperm carrying either an X- or Y-chromosome determines offspring genotypic sex; however, the underlying mechanisms that may be perturbed following paternal TCDD exposure are unknown.

Objective: To optimize the methods used to detect sex chromosomes in mice at various stages of devel-opment (spermatid, embryo, fetus).

Methods: Single round spermatids were purified from adult mouse testes using enzymatic digestion and flow cytometry. Gestation day (GD) 4.5 blastocysts (embryo) were collected from superovulated female mice by uterine flushing, while GD 14.5 fetal tail tissue was obtained following a natural mating scheme. Real-time quantitative PCR (RT-qPCR) was performed with a primer pair that amplifies a gene on the X- (Kdm5c) and Y-chromosome (Kdm5d) to simultaneously detect the sex chromosome(s). Due to low amounts of DNA in single haploid round spermatids, nested primers were used in two rounds of PCR. DNA agarose gels were used to confirm the identity of amplified PCR products.

Results: RT-qPCR melting curve peaks revealed either a Kdm5c (X-chromosome, 83°C) or Kdm5d (Y-chromosome, 81°C) PCR product in 40% of individual round spermatids (n=16). A DNA agarose gel of the PCR products confirmed these findings with bands near 331 and 302 bp representative of Kd-m5c (X) and Kdm5d (Y), respectively. In blastocysts (GD 4.5), the presence of only Kdm5c PCR prod-ucts was indicative of female embryos (XX, n=3) while the presence of both Kdm5c and Kdm5d PCR products confirmed the presence of male embryos (XY, n=5). Of the six different fetal (GD 14.5) DNA isolation methods tested, the Viagen DirectPCR buffer yielded the expected Kdm5c/Kdm5d signals and is optimal for completing future high-throughput analyses of fetal genotypic sex.

Conclusions: The Kdm5c/Kdm5d primer set detected X- and Y-chromosome bearing individual round spermatids as well as the genotypic sex of gestation day 4.5 and 14.5 mouse embryos and fetuses, re-spectively. Future studies will employ these methods to help elucidate the mechanism by which paternal TCDD exposure distorts offspring sex ratio.

Supported by NIEHS R21 ES024527, P30 ES013508, and T32 ES019851

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POSTER ABSTRACTS

R8 Tracing Androgen Metabolism with Inhibition of Aromatase in Breast Cancer Lisa N. Bottalico1,2,3, Luis Gil de Gomez1,2,3, Alexander Frey4, Nathaniel W. Snyder4, Ian A Blair1,2,3

1Superfund Research Program, 2Center of Excellence in Environmental Toxicology, and 3Department of Sys-tems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA; 4Drexel University, Philadelphia, PA


Inhibition of aromatase in breast cancer has proven to be a remarkably effective therapeutic strategy to combat estrogen receptor (ER) positive breast cancers. The enzyme aromatase (CYP19A1) catalyzes the conversion of androgens to estrogens, and this conversion constitutes the primary source of estrogens in women after menopause. Aromatase inhibitors (AI) are a first line of therapy against ER-positive breast cancer, but acquired resistance is a significant problem. While the hallmark of acquired resistance is considered to be activation of growth signaling pathways capable of driving proliferation independent of ER, little is known about changes in estrogen or androgen metabolism that may occur locally to help the breast tumor adapt to growth in an estrogen-depleted environment. This study aims to investigate androgen metabolism and estrogen formation from androgen precursors in in vitro models of aromatase inhibitor therapy and resistance. LC-MS/MS methods developed in our laboratory allow sensitive quan-tification of a panel of estrogen and androgen metabolites in human biological samples. This study rep-resents an application of these methods to trace metabolism of 4-androstenedione and testosterone with and without inhibition of aromatase. Cell models include MCF-7Aro, an ER+ MCF-7 cell line in which aromatase is overexpressed and LTED-Aro, a derivative of MCF-7Aro which has adapted to growth in the absence of estradiol and is a model of late-stage endocrine therapy resistance. LC-MS studies demon-strate that among the third-generation AI, letrozole is consistently found to have the greatest potency for inhibiting aromatase in vitro. In MCF-7Aro, altered metabolism of 4-androstenedione is observed in the presence of AI. LTEDAro is observed to have enhanced aromatase activity, to the extent that testosterone and 4-androstenedione continue to be metabolized in the presence of AI. Current work is aimed at quan-tifying estrogens and androgens in serum of women on AI to trace individual therapeutic response.

Supported by NIH/NHLBI U54 HL117798

R9 A Novel Treatment for Castration-resistant Prostate Cancer using Inhibitors of AKR1C3 with AR Antagonist Activity

Phumvadee Wangtrakuldee1, Adegoke O. Adeniji1, Daniel H. Tamae1,2, Tianzhu Zang1,2, Trevor M. Penning1,2 1Department of Systems Pharmacology and Translational Therapeutics and 2Center of Excellence in Environ-mental Toxicology, Perelman School of Medicine at the University of Pennsylvania


Castration-resistant prostate cancer (CRPC) is a commonly observed fatal metastasized form of prostate cancer that arises despite androgen-deprivation therapy treatment. Current treatments using Abiraterone acetate or Enzalutamide have been clinically shown to be short-lived due to the emergence of drug re-sistance. Type 5 17-b-hydroxysteroid dehydrogenase (aldo-keto reductase 1C3 or AKR1C3) catalyzes NADPH-driven reactions of ∆4-androstene-3,17-dione (∆4AD) to produce testosterone (T) and the re-duction of 5a-androstane-3,17-dione to form 5a-dihydrotestosterone (DHT) in the prostate. AKR1C3 is the major enzyme involved in the formation of intratumoral androgens that activate the androgen receptor (AR) in CRPC. Compound 1 (3-((4-nitronaphthalen-1-yl)amino)benzoic acid) developed from a library of synthetic N-phenylanthranilic acids was shown to selectively inhibit AKR1C3 activity com-pared to other AKR1C isoforms and antagonize the AR. Structure activity relationships of newly synthe-

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POSTER ABSTRACTS

sized derivatives of compound 1, strategically aimed to improve AKR1C3 inhibitor selectivity and AR antagonism, will be discussed. Two analogs exhibit greater than 300-fold selectivity for AKR1C3 over other AKR1C isoforms with IC50 values lower than 60 nanommolar. We also found that R-bicalut-amide, a clinically used AR antagonist, was a weak inhibitor of AKR1C3 yielding an IC50 value = 10 micromolar. To elucidate the pharmacophore shared by AKR1C3 inhibitors and AR antagonists molecu-lar docking was performed using the lead compounds and R-bicalutamide to bind to the AKR1C3 active site and the ligand binding domain of AR. This information will be exploited to design compounds that have nanomolar potency for AKR1C3 and AR, and such agents could provide an advance for CRPC treatment.

Supported by RO1 CA90744 , PO1 CA163227 awarded to TMP

R10 Abnormal Phenotypes Following Exposure to Endocrine Disrupting Com-pounds

Frances Xin1,2, Tre Artis1, Erin Fischer1, Martha Stefaniak1, Marisa S. Bartolomei1,2

1Department of Cell and Developmental Biology and 2Center of Excellence in Environmental Toxicology Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Fetal exposure to endocrine disrupting compounds (EDCs) results in aberrant developmental outcomes and increased disease susceptibility in adult life. Although the precise mechanisms by which these com-pounds act remain to be elucidated, it has been proposed that epigenetic pathways mediate their effects. Exposure to the ubiquitous EDCs bisphenol A (BPA) and di(2-ethylhexyl)phthalate (DEHP) have been shown to alter DNA methylation, an epigenetic regulatory mechanism critical for proper development. Previous work from our lab has demonstrated a role for misregulation of the imprinted Igf2 gene in glu-cose intolerant adult males following early life exposure to BPA. In our mouse model, fetal exposure to DEHP (50 μg, 1 mg, 10 mg, 100 mg per kilogram body weight per day) is associated with non-mono-tonic changes in global DNA methylation in the mid-gestation embryo. Although loss of imprinting at the Igf2 locus in the mid-gestation embryo is not observed in the DEHP exposure model, total mRNA expression remains to be assessed. In adulthood, metabolic parameters (ex. body weight, body fat, glucose tolerance) are altered in a sex-specific manner. Tissue-specific expression and DNA methylation studies are ongoing. Because humans are rarely exposed to a single EDC at once, future work will also include assessing the synergistic and/or antagonistic effects of combinatorial exposures.

Supported by NIEHS R01 ES023284 and T32 ES019851

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Gene-Environment Interactions

POSTER ABSTRACTS

GEI 1 Physiological Trait and Biomarker Variation among Sub-Saharan African Populations: The Impact of Gender, Subsistence, and Genetic Ancestry

Matthew E.B. Hansen1,2, Alessia Ranciaro1, Simon Thompson3, Jibril Hirbo4, William Beggs1, Sarah, A. Tishkoff1,2

1University of Pennsylvania and 2Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania; 3University College London; 4Vanderbilt University


The African continent is home to a diverse range of indigenous peoples that have adapted to a wide range of ecological environments and subsistence lifestyles. Many complex traits are expected to display variation between populations due to demographic history and/or natural selection to these diverse envi-ronments. In an effort to survey phenotypic variation in Africa and begin to understand the genetic and environmental factors that contribute to this variation, we have collected trait measurements on height (N=5,125), BMI (N=5,098), grip strength (N=1,968), systolic and diastolic blood pressure (N=2,002), pulse (N=2,008), and a panel of 9 blood biomarkers including LDL, HDL, triglycerides, glucose, and NEFA (N=1,490) from agricultural, pastoral, and hunter-gatherer communities across eastern and west-ern sub-Saharan Africa. We present the observed variation in these traits between sexes, across popula-tions, and across dietary categories. We find significant differences in trait values among these categories and significant differential enrichment in the distribution tails across dietary categories. A subset of nearly 2,200 individuals were genotyped on the Illumina 1M-Duo or the Illumina 5M Omni SNP array. We performed a GWAS using a linear mixed model approach on this subset. We tested the reproduc-ibility of associations for loci identified in non-African cohorts and find that height showed significant reproducibility while BMI does not. To assess the impact of genetic ancestry on trait variation, ancestry proportions were estimated using STRUCTURE and ADMIXTURE. Variance partitioning amongst subsistence categories, ancestry, and broad genetic relatedness were analyzed using GCTA, allowing us to estimate the impact of diet when controlling for genetic relatedness and ancestry. We discuss the degree to which genetic ancestry versus dietary practice impacts clinically relevant traits and the implications this has for clinical studies of human health in Africans.

Supported by NIH T32 ES019851-02

TBC1

Translational Biomarker Core (TBC)

Development and Validation of Stable Isotope Dilution LC-ESI-MS/MS Method for the Quantitation of Hydroxy-Androgens in Human Serum

Tianzhu Zang1,2, Daniel Tamae1,2, Clementina Mesaros1,3, Ian A. Blair1,3, Trevor M. Penning1,2

1Center for Excellence in Environmental Toxicology, 2Department of Systems Pharmacology and Translational Therapeutics, and 3Center for Cancer Pharmacology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104


The development of prostate cancer (CaP) is androgen-dependent, and localized advanced or metastatic disease can be treated by surgical or chemical castration. However, the recurrence of prostate cancer, also called castration resistant prostate cancer (CRPC), occurs despite castrate levels of circulating testosterone (T) and dihydrotesterone (DHT). CRPC can arise from the reactivation of the androgen receptor (AR) signaling pathway by adaptive intracrine androgen biosynthesis. Abiraterone acetate (a P450c17 inhibitor) which targets intracrine androgen synthesis has been approved by the FDA for the

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POSTER ABSTRACTS

TBC2

Translational Biomarker Core (TBC)

treatment of CRPC, but drug resistance eventually occurs. In order to better understand the mechanisms of drug resistance and develop the appropriate treatment for an individual patient, a comprehensive investigation of how androgen levels change in serum and prostate cancer tumors is imperative. Here, we report the development and validation of a stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometric (SID-LC-ESI-MS/MS) method to quantify nine human hydroxy-androgens as picolinates, simultaneously with the requisite specifcity and sensitivity. Our method allows us to interrogate the roles of 5-androstene-3b, 17b-diol and the backdoor pathway, which involves the conversion of 5a-androstane-3a, 17b-diol to DHT, in intratumoral androgen biosynthesis. Our method utilizes enzymatically synthesized 13C-5a-androstane-3a, 17b- and 3b, 17b-diols as internal standards. Sera of patients from the Total Androgen Pathway Suppression clinical trial were tested, and the levels of T and DHT obtained were comparable to those using a Girard-T derivatization procedure to quantify keto-androgens. Our method will provide reference values for these diols in adult male and female serum and when combined with the Girard-T method will enable the quantitation of the complete androgen metabolome to reveal mechanisms of drug resistance to hormone ablative therapy.

Supported by NCI 1P01-CA163227-01A1 awarded to TMP

Metabolism of a Representative Alkylated Petrogenic Polycyclic Aromatic Hydrocarbon (PAH) 6-ethyl-chrysene Associated with the Deepwater Horizon Oil Spill in Human Hepatoma (HepG2) Cells

Meng Huang, Ian A. Blair, Trevor M. Penning

Centers for Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Exposure to petrogenic polycyclic aromatic hydrocarbons (PAHs) in the food-chain is the major human health hazard associated with the Deepwater Horizon oil spill. Risk assessment is based on the assumption that petrogenic and pyrogenic PAHs have similar toxicological profiles yet petrogenic PAHs are either alkylated or oxygenated and information on their metabolism is lacking. We report the metabolic fate of 6-ethyl-chrysene as a representative alkylated petrogenic PAH in human HepG2 cells. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC-MS/MS as mono-phenols, O-sulfated mono-phenols, bis-phenols, O-sulfated bis-phenols, remote-quinones, hydroxylated remote-quinones, dihydrodiols, O-sulfated dihydrodiols, ortho-quinones, hydroxylated ortho-quinones, O-sulfated catechols, and O-methylated-O-sulfated catechols, suggesting similarity in the metabolic profile of 6-ethyl-chrysene and 5-methyl-chrysene which we reported earlier. The identification of dihydrodiols, ortho-quinones and catechol conjugates supports metabolic activation through P450 and AKR isozymes. Sulfated catechols can be used as biomarkers of human exposure to petrogenic PAH.

Supported by U19ES020676-03 to TMP

COEC2

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COEC2

Community Outreach and Engagement Core

POSTER ABSTRACTS

COEC1 Community Outreach: Short Term Educational Experiences for Research (STEER Grant) or Penn Undergraduate Environmental Health Scholars Program, Summer Program for Undergraduate Students

Jeffrey Field

Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA


Each summer, the Center of Excellence in Environmental Toxicology runs a community outreach education program for undergraduate students interested in environmental health science called the Penn Undergraduate Environmental Health Scholars Program through the Short Term Educational Experiences for Research (STEER) grant. STEER is funded by the National Institute of Health, and a $3500 stipend is provided to each student. Approximately 8 students are accepted each year into this 10-week program. The heart of the program is working one-on-one with a faculty member on an original research project. Projects address environmental health problems, but the specific topics vary greatly. The research can be laboratory research, field research, public health research, or a combination of these. Each student is matched up with a faculty mentor, most of whom are part of CEET, based on stated interests and career goals. The student-mentor relationship usually endures long after the program ends, with research often continuing into the school year. The program also involves a weekly “Cutting Edge” lecture. Most lectures are academic and are delivered by faculty mentors or other environmental health experts. Past lecture topics have included endocrine disruptors, environmental justice, integrated pest management, natural toxins, and tobacco exposure and PAHs. Additionally, a career discussion panel introduces students to future career opportunities and offers guidance, and a “responsible conduct of research” session emphasizes the importance of ethics in today’s research. STEER also includes a weekly field trip to environmental sites. Past locations have included superfund sites, environmental justice communities, wastewater treatment facilities, wildlife refuges, and local gardens. Field trips and lectures take place with the TREES high school students (STEER scholars serves as role models for them). At the end of the program, the STEER scholars present their work in an oral symposium.

Supported by R25 ES021649

Community Outreach: The Teen Research and Education in Environmental Science (TREES) Summer Program for High School Students

Jeffrey Field



In 2007, the Center of Excellence in Environmental Toxicology launched a community outreach education program for high school students called Teen Research and Education in Environmental Science (TREES) summer program. The eight students accepted each year into the eight-week summer program are introduced to laboratory science through unique hands-on research. Graduate student mentors, returning high school student mentors, and faculty members volunteer their time to guide the students one-on-one and as a group. TREES includes daily graduate student lectures on environmental issues and discussion sessions about related documentaries (e.g. An Inconvenient Truth, Thank You for Smoking). Special lectures focus on “survival” skills, such as laboratory safety, library and internet research, ethics, scientific writing, and presentation skills. A college admissions workshop, a library tour, and a formal campus tour are also included. Students also prepare reports on a natural product, such as a medicine,

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Cumulative Health Risks in Chester, PA Isabelle G. Lee, Marilyn V. Howarth

Center of Excellence in Environmental Toxicology and Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA


In the summer of 2015, I worked under the mentorship of Dr. Marilyn Howarth, Director of the COEC of the CEET, on a project assessing the cumulative health risks from exposure to industrial air toxics in Chester City Pennsylvania. Chester is small city with a predominantly low-income African American population. This community has a high concentration of industrial facilities, a number of waste-pro-cessing plants, substantial traffic from the I-95 interstate highway, and port traffic. The majority of solid waste for Delaware County (population 556,000) and surrounding areas is incinerated in Chester, and a large percentage of raw sewage and associated sludge is treated in Chester. In addition, many Chester residents live in an old housing stock which results in environmental exposures, such as poor indoor air quality, lead, mold, pesticides, and safety concerns. The members of this community express concerns about the health effects of living and working amid toxic substances, and complain about frequent illness, which they perceive as likely associated with environmental factors. I was able to collect data revealing the demographics, social determinants of health, the health outcomes, and air toxic exposures in Ches-ter. Our data show that indeed Chester, more than her neighboring communities and Pennsylvania as a whole, is overburdened by the emission of toxicants into the ambient air. Moreover, the risk for devel-oping cancers and respiratory illnesses is higher in Chester than her neighbors in Delaware county and Pennsylvania as a whole. Data from Pennsylvania’s Department of Health also show that Chester has higher cancer (lung, colorectal, breast and prostate) incidence rates when compared to the neighboring municipalities. Decreasing the emissions of air toxics from industries in Chester would alleviate some of the burden added by cumulative exposures.

Supported by T32 ES019851

COEC3


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that originated from an environmentally sensitive region of the world. TREES students also take part in two activities with STEER students, a weekly “cutting edge” faculty lecture and a weekly field trip to and environmental site. TREES begins with two weeks of structured laboratory exercises to teach basic lab techniques such as pipetting, weighing, microbial techniques, and several spectrophotometer level assays. The basic training leads to the most unique aspect of the program: an individually guided research project on a topic chosen by the student in consultation with the mentors. The students then present their individual research project results in a report, a poster, and oral symposium. Students are encouraged to develop projects in their communities based on their research and to present their work at their schools and local science fairs. Over the years, a diverse set of projects addressed environmental issues, including air, water, and food safety. TREES scholars have been highly successful in science fairs with most winning local awards, a number winning national honors, and several publishing their work. About 80% of the students major in STEM fields in college with about 25% majoring in environmental science, far above the national average. A half-dozen alumni are now in graduate school with three pursuing graduate degrees in environmental sciences.

Supported by R25 ES021649

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My Community, My Air Quality: The GAMP Student Ambassador Program Jessica R. Meeker1, Richard V. Pepino1,2

1Superfund Research Program Center and 2Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


It has been well documented that poor outdoor air quality is associated with an increased prevalence of asthma and related morbidities. In 2012 the Public Health Management Corporation’s Community Health Data Base estimated that 19.4% of adults in Philadelphia had asthma, a startling comparison to the 7.0% national prevalence. The South Philadelphia community in particular, faces a heavy burden of pollution from three major polluting sources: Philadelphia international airport, Philadelphia Energy Solutions refinery and I-95 and I-76, two major highways. In order to address this heavy burden of air pollution to the South Philadelphia community, we partnered with the local Girard Academic Music Program (GAMP) School to develop an air pollution curriculum with an emphasis on community out-reach. In collaboration with US-EPA region III and Philadelphia Air Management Services, we were able to develop an interactive curriculum that was integrated this school year into a tenth grade physical science course and a high school Environmental Science elective course. With a focus on dissemination to peers and the community, students worked on learning to translate air quality data into a variety of public service announcements and other presentations. These means of translation will be shared this summer at an outdoor event at the school, to educate community members on air quality and associated health risk information. This neighborhood and school-partnered event will be a step toward closing the information gap between a community overburdened by air pollution and government agencies with air quality data.

Supported by P3O ES013508

Evaluating Cancer Risks for Eastwick: Analysis of Air Toxics and the Clearview Landfill

Jessica R. Murray, Marilyn V. Howarth



Eastwick, PA is a community in southwest Philadelphia in close proximity to the oil refinery, a Super-fund site, major traffic routes I-95 and I-76, and the Philadelphia International airport. Residents are concerned about their cancer risk from these pollution sources. In 2011, the Pennsylvania Department of Health reported that Eastwick had 109% higher incidence of liver cancer than expected for Pennsyl-vania. In order to address community concerns, we performed a hypothesis-generating risk assessment to determine whether Eastwick had unique exposures that could account for high liver cancer incidence. We aimed to determine whether residents were exposed to known human carcinogens associated with liver cancer. The Agency for Toxic Substances and Disease Registry (ATSDR) and International Agency for Research on Cancer (IARC) monographs were used to determine whether local exposures were known human carcinogens implicated in liver cancer development. Exposures from proximity to the Superfund site and general air pollution in the region were considered. Web-based tools provided by the EPA {Toxic Release Inventory (TRI) Explorer; Environmental Risk Screening Indicators (RSEI) Model; Commu-nity-Focused Exposure and Risk Screening Tool (C-FERST); and Environmental Justice Screening and Mapping (EJScreen) tool} were used to identify and prioritize toxic releases in the area. Data from the 2005 National Air Toxics Assessment (NATA) were used to estimate exposure and cancer risks of air pollution for the region, and this model was supplemented by local data provided by the Philadelphia


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COEC4

COEC5

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Air Monitoring Network. The Remedial Investigation for the Lower Darby Creek Area Superfund was reviewed to analyze hazardous substances detected in surface soils in the neighborhood. We determined that air pollution exposures had decreased in the region during the past 10 years and liver carcinogens were no longer being detected by local air monitors. However, liver carcinogens have been detected in soil samples in the community and warrant further study to estimate whether Eastwick residents are at risk of exposure.

Supported by T32019581 to JRM


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S1 Understanding Diverse Perceptions of Environmental Toxicity and Their Ramifications for the Mandate of Community Engagement

Justin T. Clapp1, Jody A. Roberts2, Britt Dahlberg2, Edward A. Emmett1,3, Frances K. Barg1,3

1University of Pennsylvania, Philadelphia PA; 2Chemical Heritage Foundation; Philadelphia PA; 3Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA


Much of the research on lay understandings of environmental toxicity has focused on well-known cases of what has come to be known as popular epidemiology. This term describes concerted actions by groups of residents to gather information about a suspected local source of toxicity that is not being addressed by government, gain fluency in scientific methods and discourse, and lobby authorities to remediate this environmental hazard. Substantially less conceptual and empirical work has examined the diversity and uncertainty that often characterize community perceptions of toxicity. As such, this study used a social semiotic theory of how diverse perspectives on toxicity form to analyze oral history interviews conducted in 2013-14 with 24 individuals in the area of Ambler, Pennsylvania, USA. Ambler is a small town that was once a major center of asbestos manufacture and is home to two massive asbestos-containing waste sites, one of which was being remediated by the United States Environmental Protection Agency (EPA) at the time of the study. Interviews with local residents and EPA officials revealed that decades of lived experience had socialized them to particular conceptions of the asbestos waste, notions of the health risk it may present, and impressions of the scientific and governmental authorities tasked with addressing it. While the EPA has in recent years attempted to enhance its engagement with the communities where it implements cleanups, the knowledge of Ambler-area residents was found to be in certain ways funda-mentally incompatible with the epistemology underlying the EPA’s remediation approach. This research explains the nature of this incompatibly and suggests alternative community engagement approaches that better acknowledge the diversity of perspectives on toxicity and health.

Supported by NIH R25 OD010521, P42 ES023720

Superfund Research Program

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S2 Is Phytoremediation a Good Solution for Remediation of Asbestos Contaminated Sites?

Cedric Gonneau1, Sanjay Mohanty2, Jane Willenbring2,3, Brenda Casper1,3

1Departments of Biology and 2Earth and Environmental Science, University of Pennsylvania, Philadelphia, PA; 3Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Asbestos is a colloquial term encompassing several fibrous silicates and cause mesothelioma and cancer of the lung. In the US, there are 1312 asbestos contaminated sites including superfund, Brownfield or naturally occurring asbestos sites. Currently, the EPA protocol for treatment of asbestos contaminated sites is to move it and/or cap it. However, capped piles containing fibers pose threats to nearby human populations. Indeed, new remediation strategies should be explored mainly phytoremediation.

Three locations with different asbestiform minerals were studied here: BoRit, a superfund site with two areas: sediments and stream banks. Nottingham Park, a serpentine site with several mines such as the Chromite mine.

To determine feasibility of phytoremediaton of asbestos contaminated soils, we evaluated:

•fertility using Fertility Capability Classification (Texture and 12 modifiers) and determined soils properties (pH, CEC, Phosphorus, total and bioavailable concentrations of heavy metals).

• toxicitybyanecotoxicologicaltest.Wedeterminedpercentageofinhibitionofseedgerminationand root growth on three species (one Poaceae and two Brassicaceae).

All locations presented significantly different soil parameters. Among 12 modifiers and 23 soil parame-ters, limiting factors of fertility were percentage of gravel, pH, P, K, and total concentrations of Ni and Cr but their bioavailability was low. In an ecotoxicological test, a lower effect on seed germination was observed (5-30% of control). Concerning root growth, variability between three species was found to be 2-50 % of the inhibition of control, especially for Poaceae.

Our results showed that it is important to take into account all soil limiting parameters – not only pollut-ants. Moreover, utilization of compost will decrease pH and increase soil fertility but could also increase bioavailability of trace elements (Ni or Cr). Indeed, serpentinophytes seem to be an adequate species for phytoremediation. Our research proposes and validates a novel approach to the remediation of asbes-tos-polluted soil.

Supported by NIH/NIEHS, P42 ES023720 Penn Superfund Research Program Center Grant

S3 Animal Models of Malignant Mesothelioma Craig W. Menges1, Mitchell Cheung1, Yuwaraj Kadariya1, Ralph Pietrofesa2, Melpo Christofidou-

Solomidou2,3

1Fox Chase Cancer Center, Philadelphia, PA; 2Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 3Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Malignant mesothelioma (MM) is a highly aggressive, notoriously treatment-resistant cancer usually caused by exposure to asbestos fibers. With estimates of >20 million individuals at risk worldwide, new approaches in disease management and prevention are badly needed. In Ambler, Pennsylvania, there is an elevated incidence of MM linked to decades of asbestos manufacturing, and the presence of an asbestos-contaminated waste site continues to jeopardize the health of residents living in the vicinity.

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The genetic basis for MM has historically focused on somatic mutations of the tumor suppressor genes CDKN2A and NF2 as key alterations influencing initiation and progression. More recently, BAP1 has been strongly implicated as a major player in MM based on the discovery of germline BAP1 mutations in two families with a high incidence of MM and other cancers. Moreover, BAP1 mutations are common in sporadic (non-familial) cases of MM, as well. Previous in vivo carcinogenicity studies with crocido-lite-exposed heterozygous (+/mut) Nf2;Cdkn2a and Bap1 knockout mice have revealed that induction of MM is accelerated in these mice compared to that of similarly exposed wild-type littermates. RNA extracted from the peritoneal cavity of crocidolite-exposed Bap1+mut mice are being used to identify temporal expression changes connected with asbestos exposure. In an ongoing collaborative chemopre-vention study, our Nf2+/-;Cdkn2a+/- mouse model is being used to determine if flaxseed lignan can delay or lower MM incidence. Finally, we will validate if asbestos remediated via iron chelation results in reduced carcinogenicity in asbestos-exposed mice. These studies represent a comprehensive approach bringing together tumor biology/genetics with epigenetic regulation, which will yield basic insights into mechanisms/interactions that drive MM development and progression, with translational implications for understanding tumor susceptibility and prevention.

Supported by NIH/NIEHS P42 ES023720

S4 Asbestos Fiber Mobility via Groundwater Flow: A Paradigm Shift Sanjay Mohanty1, Ashkan Salamatipour1, Douglas, J. Jerolmack1,2, Jane, K. Willenbring1,2

1University of Pennsylvania, Philadelphia PA; 2Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA


Asbestos buried underground is typically considered safe because asbestos fibers are assumed to strongly attach to soil without moving. This long-standing assumption stems from the theory that asbestos fibers, owing to their shape, size and positive surface charge, would be retained at pores or attached to soil, which typically has negative surface charge. However, we show that asbestos fibers can move in soil under natural conditions: in the presence of natural organic matter (NOM). NOM is produced from decayed biomass and typically consists of fulvic acid, humid acid, and humin. By injecting a chrysotile asbestos-fi-ber suspension through saturated sand and soil columns with or without NOM, we show that asbestos fibers do move through soil and sand in the presence of NOM. Without NOM, asbestos fibers are not mobile, even at high pH and low ionic strength – the geochemical conditions that are typically expected to enhance the mobility of particles. The enhanced mobility of asbestos fibers in the presence of NOM is attributed to charge reversal of the fibers (from positive to negative), which lowers their attachment to sand or soil grains. This result challenges the long-standing assumption that asbestos fibers are immobile below ground and highlights the need to evaluate the long-term effectiveness of current remediation strategies that do not account for possible asbestos exposure via groundwater pathways.

Supported by NIEHS P42 ES023720

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S5 Asbestos Induces Oxidative Stress and Activation of Nrf2 Signaling in Murine Macrophages: Chemopreventive Role of the Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605)

Ralph A. Pietrofesa1, Anastasia Velalopoulou1, Steven M. Albelda1,2, Melpo Christofidou-Solomidou1,2

1Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, and 2Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Background: The interaction of asbestos fibers with macrophages generates harmful reactive oxygen spe-cies (ROS) and subsequent oxidative cell damage that are key processes linked to malignancy. Secoisolari-ciresinol diglucoside (SDG) is a non-toxic, flaxseed-derived pluripotent compound that has antioxidant properties and may thus function as a chemopreventive agent for asbestos-induced mesothelioma. We thus evaluated synthetic SDG (LGM2605) in asbestos-exposed, elicited murine peritoneal macrophages as an in vitro model of tissue phagocytic response to the presence of asbestos in the pleural space.

Methods: Murine peritoneal macrophages (MFs) were exposed to crocidolite asbestos fibers (20 μg/cm2) and evaluated at various times post-exposure for cytotoxicity, ROS generation, malondialdehyde (MDA), and levels of 8-iso Prostaglandin F2a (8-isoP). We then evaluated the ability of LGM2605 to mitigate as-bestos-induced oxidative stress by administering LGM2605 (50 μM) 4-hours prior to asbestos exposure.

Results: We observed a significant (p<0.0001), time-dependent increase in asbestos-induced cytotoxicity, ROS generation, and the release of MDA and 8-iso Prostaglandin F2a, markers of lipid peroxidation, which increased linearly over time. LGM2605 treatment significantly (p<0.0001) reduced asbestos-in-duced cytotoxicity and ROS generation, while decreasing levels of MDA and 8-isoP by 71-88% and 41-73%, respectively. Importantly, exposure to asbestos fibers induced cell protective defenses, such as cellular Nrf2 activation and the expression of phase II antioxidant enzymes, HO-1 and Nqo1 that were further enhanced by LGM2605 treatment.

Conclusions: LGM2605 boosted antioxidant defenses, as well as reduced asbestos-induced ROS gener-ation and markers of oxidative stress in murine peritoneal macrophages, supporting its possible use as a chemoprevention agent in the development of asbestos-induced malignant mesothelioma.

Supported by NIEHS/NIH, P42 ES023720 Penn Superfund Research Program Center Grant

S6 A Comparison of Statistical Methods for Detection of Serum Lipid Biomark-ers for Mesothelioma and Asbestos Exposure

Rengyi (Emily) Xu1, Clementina Mesaros2,3, Liwei Weng2,3, Anil Vachani4, Ian A. Blair2,3, Wei-Ting Hwang1

1Department of Biostatistics and Epidemiology, 2Centers for Cancer Pharmacology and Excellence in Envi-ronmental Toxicology, and 3Department of Pharmacology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; 4Pulmonary, Allergy, and Critical Care Division, Hospital of the University of Pennsylvania, Philadelphia, PA


Objective: To determine which serum lipid or combination of serum lipids can be useful to distinguish patients with mesothelioma and individuals previously exposed to asbestos from controls.

Methods: Serum samples from patients diagnosed with mesothelioma (n=40), asbestos- exposed sub-jects (n=40), and controls (n=40) were analyzed by the XCMC differential analysis and SIEVE software (Thermo Scientific). Thirty-five serum candidate lipids were identified for further statistical analysis. In

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this study, we compared three statistical models: univariate logistics regression analysis then selected the top five performed lipids, stepwise logistic regression, and least absolute shrinkage and selection operator (LASSO) logistic regression. In addition, two sets of predictors are considered: (1) the area ratios that measure the abundance of each lipid relative to the internal standards separately and (2) the correspond-ing principal components. The use of latter was to lessen the collinearity associated with high correlations observed in the original area ratios measurements. Cross-validated AUC is used to compare models; difference and similarity on the selected lipids across different methods are examined.

Results: For mesothelioma versus controls, all three approaches that use the original area ratios selected the lipid with m/z ratio of 372.31. Lipids with m/z ratio 379.28, 800.58, and 374.33 were identified only by univariate approach and lipids with m/z ratio 782.57, 244.23, and 494.32 were identified only by LASSO regression. Using the computed principal components as predictors, all three models selected PC1, which loads heavily on lipid with m/z ratio of 782.57, and PC6, which loads mostly on lipids with m/z ratio of 372.31 and 374.33. Cross-validated AUC for the models ranges from 0.988 to 0.993. For asbestos exposed versus controls, lipids with m/z ratio of 1464.80 and 329.21 are selected by both uni-variate and stepwise regression if the original area ratio levels were used as predictors. Using the principal components, all three approaches identified PC3 (loading heavily on lipid with m/z ratio of 820.36), PC10 (loading heavily on lipid with m/z ratio of 244.23) and PC8 (loading heavily on lipid with m/z ratio of 329.21). Cross-validated AUC for the models ranges from 0.914 to 0.999.

Conclusions: Different statistical models have selected different but equivalent sets of lipids using differ-ent search algorithms. Multiple biomarkers provide a better prediction than single biomarkers. Results of the current investigation will warrant additional validation using independent samples.

Supported by P42 ES023720 Penn Superfund Research Program Center Grant

S7 The Effect of Asbestos Exposure on Mitochondrial Metabolites in Lung Cells Liwei Weng, Clementina Mesaros, Dominic Ciccimaro, Ian Blair

Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA


Pronounced asbestos exposure in the community of Ambler, PA has resulted in high prevalence of asbes-tos related diseases including lung cancer and mesothelioma. Unfortunately, the pathogenic connections between asbestos exposure and the related diseases have not been well characterized. Various in vivo and in vitro studies suggest that oxidative stress induced by asbestos plays crucial role in developing asbes-tos-related diseases. Oxidative stress can result in a series effects on metabolites, especially the metabolites produced in mitochondrial. The aim of this study is to monitor the change in the mitochondrial metab-olites induced by different types of asbestos including chrysotile and crocidolite, the well-characterized amphibole fiber. Mitochondrial metabolites including metabolites involved in the Krebs cycle play signif-icant roles in a variety of essential cellular processes including ATP generation. Coenzyme A (CoA-SH) functions as a carrier to transfer fatty acids to mitochondria. Acyl-CoAs, the downstream metabolites of CoA-SH are not only involved in the metabolism of fatty acids, but also play important roles in epi-genetics and steroid synthesis.

Herein, we quantified cellular acyl-CoAs by liquid chromatography/multiple reaction monitoring mass spectrometry (LC-MRS-MS) and measured the change in the level of the metabolites induced by asbestos treatment. Asbestos exposure decreased cellular CoA-SH levels as well as other acyl-CoA levels including Ac-CoA, HMG-CoA, bHB-CoA, and succinyl-CoA. Given that Ac-CoA is the most important substrate for the histone acetylation, decreased Ac-CoA levels could result in deleterious effects on regulating gene

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expression. In addition, HMG-CoA is involved in steroid synthesis. The level of change in HMG-CoA could affect cellular signaling. We expect the effects of asbestos exposure on human tissues are chronic and accumulative. Thus, we also carried out time-course experiments to monitor the change in these metabolites, which can shed light on the mechanism by which asbestos induces tissue damage and meso-thelioma.

Supported by NIEHS P42 ES023720 T32 ES019851

S8 Dynamics and Mechanisms of Asbestos-fiber Aggregate Growth in Water

Lei Wu1, Carlos Ortiz1, Douglas Jerolmack1,2

1Department of Earth and Environmental Science, University of Pennsylvania, Philadelphia, PA; 2Center of Excellence in Environmental Toxicology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA


Most colloidal particles including asbestos fibers form aggregates in water, when solution chemistry provides favorable conditions. To date, the growth of colloidal aggregates has been observed in many model systems under optical and scanning electron microscopy; however, all of these studies have used near-spherical particles. The highly elongated nature of asbestos fibers may cause anomalous aggregate growth and morphology, but this has never been examined. Although the exposure pathway of concern for asbestos is through the air, asbestos particles typically reside in soil that is at least partially saturated, and aggregates formed in the aqueous phase may influence the mobility of particles in the environment. Here we study solution-phase aggregation kinetics of asbestos fibers using a liquid-cell by in situ mi-croscopy, over micron to centimeter length scales and from a tenth of a second to hours. We employ an elliptical particle tracking technique to determine particle trajectories and to quantify diffusivity. Experi-ments reveal that diffusing fibers join by cross linking, but that such linking is sometimes reversible. The resulting aggregates are very sparse and non-compact, with a fractal dimension that is lower than any pre-viously reported value. Their morphology, growth rate and particle size distribution exhibit non-classical behavior that deviates significantly from observations of aggregates composed of near-spherical particles. We also perform experiments using synthetic colloidal particles, and compare these to asbestos in order to separate the controls of particle shape vs. material properties. We have found that altering water pH influences the growth rate of chrysotile aggregates, but not their structure. This direct method for quan-titatively observing aggregate growth is a first step toward predicting asbestos-fiber aggregate size distri-butions in the environment. Moreover, many emerging environmental contaminants – such as carbon nanotubes – are elongated colloids, and our work suggests that theories for aggregate growth may need to be modified in order to model these particles. These results also suggest a large but simple control of water chemistry on aggregate dynamics. Our findings may improve predictions for aggregate size in different environments, and may lead to a new strategy for asbestos containment in disposal sites by manipulating soil pH to encourage aggregation and immobilization.

Supported by NIEHS P42 ES023720

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The Center of Excellence in Environmental Toxicology would like to thank all those who made this Symposium possible including: Perelman School of Medicine at the University of Pennsylvania, the National Institute of Environmental Health Sciences, and the invited speakers.

ACKNOWLEDGEMENTS

http://ceet.upenn.edu

Design and artwork: Mary A. Leonard Biomedical Art & Design, Perelman School of Medicine at the University of Pennsylvania

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