Central States Regional Chapter of

The Society of Toxicology

Annual Fall Meeting

September 21-22, 2017

Gateway Center and Hotel, Ames, Iowa

Global Toxicology

SPONSORS

Office of Biotechnology at Iowa State University: http://www.biotech.iastate.edu/ The Office of Biotechnology facilitates and advances programs in research, education, and outreach that contribute to the goals of Iowa State University’s Strategic Plan in the area of Biotechnology.

Interdepartmental Toxicology: http://www.toxicology.iastate.edu Ph.D. and M.S. graduate program at Iowa State

College of Veterinary Medicine: http://vetmed.iastate.edu/ First public veterinary school in the United States; founded in 1879. Strong tradition of leadership in teaching, learning, research and service in veterinary medicine. College of Agriculture and Life Sciences: http://www.cals.iastate.edu/ Our college truly is one of the best schools of agriculture and life sciences in the world. The preparation our students receive not only makes them leaders in their chosen fields, but also in society. Iowa State University Graduate College: https://www.grad-college.iastate.edu/ Over 120 graduate majors are available. Find a professor with your research interests or find by specific graduate programs opportunities for advanced degrees. https://www.grad-college.iastate.edu/academics/programs/apprograms.php

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Photo credit: Adhithiya Manohar-Charle:. Red Tail Hawk. Gharial Crocodile. Slow Shutter. Pixabay: Earth. Chemistry.

Central States Society of Toxicology 2017 Program

Officers: President: Richard J. Martin, Iowa State University President-Elect: Thu Annelise Nguyen Past President: Andrea Adamcakova-Dodd Secretary/Treasurer: Hans-Joachim Lehmler, University of Iowa Councilors: Deven Dandekar James Sacco Post-doc Representative: Student Representative: Eric Uwimana Meeting Moderators: Richard Martin, Iowa State University Colin Wong, Iowa State University Aileen Keating, Iowa State University Caleb Corona, Iowa State University Niranjana Krishnan, Iowa State University Dan Luo, Iowa State University Judges: Suzanne Hendrich, Iowa State University (oral, graduate) Larry Robertson, University of Iowa (oral, graduate) Joel Coats, Iowa State University (oral, post doc) Audio/Visual Support: Edmund Norris, Iowa State University Registration: Melanie Abongwa, Iowa State University Laura Burns, Iowa State University Judges: Post Doc: Graduate Student: Place: Gateway Hotel and Conference Center Dates: September 21-22, 2017

PROGRAM—AT A GLANCE Central States Regional Chapter of the Society of Toxicology Annual Meeting September 21-22, 2017. Gateway Hotel and Center, Ames, Iowa. Host: Iowa State University Global Toxicology Thursday, September 21, 2017, Molecular Biology Bulding, on ISU campus 5:30 p.m. Social. Atrium, Molecular Biology Building, Iowa State University campus, 2437

Pammel Drive. 6:30 p.m. Pizza and salad (self serve). Bar drinks available for sale. 7:30 p.m. Poster Pitch 8:00 p.m. John Doull Award and Lecture 9:00 p.m. Graduate Student and postdocs leave for activities at Perfect Games 9:00 p.m. Faculty, industry, others, meet at Gateway Hotel Bar for additional conversation Friday, September 22, 2017 Gateway Hotel and Conference Center 8:00 a.m. Central Prairie--continental breakfast 8:00 a.m. Outside of North Prairie--Registration (pick up packets and programs) 8:00 a.m. Central Prairie--Set up posters 8:15 a.m. Conference Room #2--CSSOT Officer Meeting 8:55 a.m. North Prairie--Program 9:00 a.m. Welcome – Wendy Wintersteen, Dean, College of Agriculture Life Sciences 9:10 a.m. Welcome – Richard J. Martin, CSSOT President, Iowa State University 9:15 a.m. Gary W. Miller, Dept of Environmental Health, Emory University

“A Global Approach to Environmental Challenges” 10:15 a.m. Ana Maria Velez Arango, Department of Entomology, U Nebraska-Lincoln

“RNAi for Insect Pest Management” 11:15 a.m. Break and Poster Session. Participants may vote for their poster selection. 12:15 a.m. Central Prairie: Lunch (_________ or Vegetable Pad Thai) 1:15 p.m. North Prairie – Program continues 1:30 p.m. Vanessa A. Fitsanakis, Pharmaceutical Sciences, Northeast Ohio Medical U.

“Neurotoxicity Associated with the Manganese-Containing Fungicide Mancozeb”

2:30 p.m. Post Doctoral Fellow Oral Presentations (2) 3:00 p.m. Break—complete voting for poster selection. Votes will be counted. 3:15 p.m. Graduate Student Oral Presentations (4) 4:15 p.m. Break 4:30 p.m. Student and Post-Doctoral Awards 5:00 p.m. Meeting adjourned.

Global Toxicology Thursday, September 21, 2017, Molecular Biology Building, on ISU campus 5:30 p.m. Social. Atrium, Molecular Biology Building, Iowa State University campus, 2437 Pammel Drive. 6:30 p.m. Pizza and salad (self serve). Bar drinks available for sale. 7:30 p.m. Poster Pitch Presenters have one minute to present their research. Allowed one slide. Participants decide on best presentation taking into account the slide and the oral presentation. Use tear off ticket below Award: $100 for best pitch 8:00 p.m. John Doull Award and Lecture 9:00 p.m. Graduate Student and postdocs leave for activities at Perfect Games http://perfectgamesinc.com/PG/index.php/en/ bowling, laser tag, arcade 9:00 p.m. Faculty, industry, others, meet at Gateway Hotel and Conference Bar for \ additional conversation

My Choice for One Minute Best Pitch is: _________________________________________

Global Toxicology FRIDAY, September 22, 2017, GATEWAY HOTEL AND CONFERNCE CENTER

8:15 to 8:45: CSSOT Officer Meeting

Conference Room #2, Gateway Hotel and Conference Center

8:00 to 9:00 a.m. in North Prairie: Computer person, Ed Norris (Entomology/Coats Lab) will help folks load their talks and presentations

8:00 to 9:00 a.m. Central Prairie: Registration, Continent Breakfast, Poster setup.

Assisting with registration: Kayla Cappelle (Agronomy/Wolt Lab) and Melanie Abongwa (Post doc)

Poster Judging. Every registered CSSOT member will have a ballot to nominate two posters of their choice by 3:15 p.m. for travel awards to SOT. The top 4 poster "ticket" winners will each receive $200 towards reimbursement for travel to SOT in the spring of 2018

9:00: Welcome

9:00: Wendy Wintersteen, Dean, College of Agriculture and Life Sciences.

9:10: Richard Martin, Chair, Interdepartmental Toxicology

9:15: Introduction: Aileen Keating, Animal Science.

9:15: Gary W. Miller, Department of Environmental Health, Rollins School of Public Health, Emory University, GA.

"A Global Approach to Environmental Challenges"

10:15: Introduction: Colin Wong, Entomology, graduate student in Joel Coats Lab

10:15: Ana Maria Velez Arango, Department of Entomology, University of Nebraska-Lincoln

"RNAi for Insect Pest Management"

11:15: Break and Poster Presentations, Voting begins for poster presentations.

12:15: Lunch. Join presenters at tables. Room: Central Prairie

_________________ or Vegetable Pad Thai with salad, vegetable, starch, dessert

1:15: Return to Room: North Prairie

1:30: Introduction: Caleb Corona, Entomology, graduate student in Joel Coats Lab

1:30: Vanessa A. Fitsanakis, Pharmaceutical Sciences, Northeast Ohio Medical University.

"Neurotoxicity Associated with the Manganese-Containing Fungicide Mancozeb."

2:30: Two Post-Doctoral Fellow Oral Presentations (15 minutes each) Melanie Abongwa, Iowa State University Ahmed A. Ismail, University of Iowa Monitor: Niranjana Krishnan, Entomology, graduate student in the Bradbury Lab

Judge for post doc presentation is: Joel Coats, Iowa State University

1 Travel Award to SOT ($300): Sponsored by: Charles River

3:00: Break

3:15: All votes should be cast by 3:15 for choice of poster awards. Box in Central Prairie

Individuals who will count votes:

3:15: Four Graduate Student Oral Presentations (15 minutes each) Niranjana Krishnan, Iowa State University

Nyzil Massey, Iowa State University Eric Uwimana, University of Iowa Colin Wong, Iowa State University

Monitor: Dan Luo, Biomedical Sciences, graduate student in the Anu. Kanthasamy Lab

Judges for graduate student presentations are: Suzanne Hendrich, Iowa State University and Larry Robertson, University of Iowa

1 Travel Award to SOT ($300); Sponsored by: CSSOT

4:15: Break

4:30: Graduate Student and Post-Doctoral Awards

Presenter: Richard Martin, President, CSSOT

5:00: Meeting Adjourned

POSTER PRESENTATION BALLOT (choose 2)

P_____ Presenter: _________________________________

P _____ Presenter: __________________________________

What are the main points to consider when judging a poster

1. Quality of the science 2. Knowledge and enthusiasm of presenter 3. Visual clarity of the poster

Gary W. Miller, Professor, Environmental Health, Asa Griggs Candler Professor, Rollins School of Public Health, Atlanta GA

Gary.miller@emory.edu

Dr. Miller completed his doctoral training in Pharmacology and Toxicology and postdoctoral training in Molecular Neuroscience. His research has focused on environmental factors involved in the development of neurodegenerative conditions, such as Parkinson's disease. His laboratory works at the interface of neuroscience and toxicology, using a wide

variety of experimental techniques. Dr. Miller is Director of the Emory HERCULES center, an NIEHS-funded center focused on the exposome, the environmental analogue to the genome. He also serves as Director of Emory's CHEAR U2C Center and Emory's NIEHS-funded T32 Training Grant in Environmental Health Sciences and Toxicology.

Dr. Miller is a Georgia Research Alliance Distinguished Investigator and received the Achievement Award from the Society of Toxicology. He currently serves as Editor-in-Chief of Toxicological Sciences, the official journal of the Society of Toxicology.

A Global Approach to Environmental Challenges

Ana María Vélez, Assistant Professor, Department of Entomology, University of Nebraska-Lincoln.

Avelezarango2@unl.edu

Ana María Vélez received her Ph.D. in Entomology from the University of Nebraska-Lincoln, an M.S. in Entomology from the National University of Colombia and a B.S in Biology from the Pontificia Universidad Javeriana, Colombia. She is currently an Assistant Professor of Entomology at the University of Nebraska-Lincoln with a lab that focuses on current and emerging technologies for insect pest management. Specific goals are to

understand how pests and non-target insects respond and adapt to existing and newly developing technologies. Over her career, Ana has developed an expertise in the mode of action of plants expressing proteins from Bacillus thuringiensis, insect resistance to these toxins, and different aspects of RNAi as a pest management tool, including risk assessment.

RNAi for Insect Pest Management

RNA interference (RNAi) involves the downregulation of gene expression by double stranded RNA molecules (dsRNA). RNAi has been used for over a decade to study gene function and more recently being explored as a pest management tool. The first species to be targeted by a commercial RNAi product is the western corn rootworm, Diabrotica virgifera virgifera, a key pest of corn in the United States. In June 2017, the EPA registered the first plant product expressing dvSnf7 dsRNA targeting this pest. This presentation will explain how RNAi works, describe current targets for western corn rootworms, strategies for dsRNA delivery other than plants, and risk assessment implications.

Vanessa Fitsanakis, Ph.D., Associate Professor of Pharmaceutical Sciences, Northeast Ohio Medical University.

vfitsanakis@neomed.edu

Dr Vanessa A. Fitsanakis received her PhD in Neuroscience from Vanderbilt University in 2003. She was a postdoctoral fellow in the laboratory of Michael Aschner, PhD, in the Department of Physiology and Pharmacology at Wake Forest University from 2003 till 2005. In 2005, she returned to Vanderbilt University with Dr Aschner and joined the Division of Neurotoxicology in the Department of Pediatrics as

a Research Instructor. In 2006 she moved to the Department of Biology at King University in Bristol, TN (USA), and is currently in her seventh year as chair of the department. She teaches Mammalian Toxicology, Neuroscience, Anatomy & Physiology, Research Methods in Biology, and Biochemistry. She is funded by the NIEHS and has an active undergraduate research lab. In her short time at King College, she has trained seven students. Dr. Fitsanakis currently serves as an Ad Hoc reviewer for ToxSci, Neurotoxicology, Brain Research and Human and Experimental Toxicology. She has been a member of the Society of Toxicology since 2000. Her research interests include mechanisms of neurotoxicity of heavy metals, particularly depleted uranium and manganese, and in the potential role that environmental toxicants, such as pesticides, may play in the etiology of idiopathic Parkinson's disease.

Neurotoxicity Associated with the Manganese-Containing

Fungicide Mancozeb

John Doull, Ph.D. and M.D., recipient of the first award of CSSOT, was a Professor Emeritus of Pharmacology and Toxicology in the Department of Pharmacology, Toxicology and Therapeutics at the University of Kansas Medical Center. He is an author of the acclaimed reference book Cassarett & Doull’s Toxicology. September 13, 1922 to March 24, 2017.

JOHN DOULL AWARD 2017

Joel Coats, Distinguished Professor, Pesticide Toxicology Lab, Department of Entomology, Iowa State University.

Dr. Joel Coats served the Central States Chapter of the Society of Toxicology as a Counselor in 1986, and as President-Elect in 1987-1988. He also served as Program Chair for the Annual Meeting that year and organized and hosted that meeting in Ames, Iowa. He was President of the Central States Chapter for 1988-1989, and Past President the following year. At the 2013 CS-SOT Annual Meeting he gave the keynote presentation on “Green Chemistry Approaches to Pesticides.” He has trained MS and PhD graduate students in toxicology with a focus on insecticidal toxicology and taught toxicology to graduate students. Joel’s research program includes two main areas: (1) insect toxicology and (2) environmental toxicology and environmental chemistry of agrochemicals. His research in the insect toxicology area is focused primarily on natural products as insecticides and insect repellents, including investigations of their spectrum of activity, mechanisms of action, metabolism, synthesis of biorational derivatives and analogs, quantitative structure-activity relationships. His scientific publications include 10 books, 7 review articles, 41 book chapters, and 142 peer-reviewed journal articles, and he holds 9 patents. Dr Coats has been recognized for his research as a Charles F. Curtis Distinguished Professor at Iowa State University. He has received the International Award for Research in Agrochemicals from the American Chemical Society Agrochemical Division, and he is a Fellow of the American Association for the Advancement of Science (AAAS), and a Fellow of the Entomological Society of America and the Agrochemicals Division of ACS. In October 2013 he received the Alumni Achievement Award from the University of Illinois College of Liberal Arts and Sciences.

EDUCATION: Ph.D.-Entomology (Insecticide Toxicology), Minor in Chemistry, University of Illinois, Urbana-Champaign, IL M.S.-Entomology, University of Illinois, Urbana-Champaign, IL B.S.-Zoology (with distinction), Minor in Chemistry, Arizona State Univ., Tempe, AZ

Oral Presentation Abstracts

Post-Doctoral Students

O1 Monepantel is a non-competitive antagonist of nicotinic acetylcholine receptors from Ascaris suum and Oesophagostomum dentatum Melanie Abongwaa,#, Djorje Marianovicb,#, James G. Tiptona, Fudan Zhenga, Richard J. Martina, Sasa Trailovicb, Alan P. Robertsona, a Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA. b Department of Pharmacology and Toxicology, College of Veterinary Medicine, University of Belgrade, Belgrade, Serbia. # Contributed equally Abstract Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype comprising of ACR-16, a pyrantel/tribendimidine preferring heteromeric subtype comprising UNC-29, UNC-38 and UNC-63 subunits, and a levamisole preferring subtype comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6 ± 3.1 nM and 0.2 ± 2.3 µM). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the antagonism was non-competitive in nature. Our findings suggest that the mode of action of monepantel is more complex than previously described.

O2 RECOVERY OF BIOMARKERS AND CHRONICITY OF NEUROBEHAVIORAL PERFORMANCE AMONG ADOLESCENT PESTICIDE APPLICATORS

Ahmed A. Ismail1, James R. Olson2,3, Matthew R. Bonner3, Kai Wang4, Olfat Hendy5, Gaafar Abdel Rasoul6, Diane S. Rohlman1

1Occupational and Environmental Health, University of Iowa, Iowa City, IA, 2Pharmacology and Toxicology, State University of New York, Buffalo, NY, 3Epidemiology and Environmental Health, State University of New York, Buffalo, NY, 4Biostatistics, University of Iowa, Iowa City, IA, 5National Liver Institute, Menoufia University, Shebin El-Kom, Egypt, 6Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt

Egyptian adolescents are hired as seasonal workers to apply pesticides to the cotton crop and may work as applicators for several years. However, few studies have examined the effects of repeated pesticide exposure on health outcomes. The goal of this study was to determine the impact of repeated pesticide exposure on neurobehavioral (NB) performance and biomarkers of exposure (urinary metabolite) and effect (cholinesterase activity). Eighty-four adolescents from two field stations in Menoufia, Egypt, were examined at four time points: before and during pesticide application season in 2010 and again before and during application season in 2011. At each of the four time points, participants completed a questionnaire and an NB test battery, and were assessed for urinary levels of the chlorpyrifos metabolite TCPy (3,5,6-trichloro-2-pyridinol) and blood cholinesterase activity. Following the study cohort over two consecutive pesticide application seasons revealed that TCPy levels significantly increased following exposure, and returned to baseline levels following the end of the application season Blood butyryl cholinesterase activity exhibited a similar pattern. Although NB outcomes displayed learning and practice effects over time, deficits in performance were significantly associated with increased TCPy levels with reduction in number of NB measures showing improvement over time. Biomarkers of exposure and effect demonstrated changes associated with pesticide application and recovery after application ended. Deficits in NB performance were correlated with elevated pesticide exposure. Data demonstrated that repeated pesticide exposure may exert a long-term adverse impact on human health. (NIH grants: R21 ES017223 and R01 ES022163 (Rohlman, PI)).

Oral Presentation Abstracts

Graduate Students

O3 RISK ASSESSMENT OF FOLIAR INSECTICIDES COMMONLY USED IN CORN AND SOYBEAN PRODUCTION ON MONARCH BUTTERFLY (DANAUS PLEXIPPUS) LARVAE

Niranjana Krishnanⁱ, Keith Bidneⁱⁱ, Richard Hellmichⁱⁱ, Joel Coatsⁱ and Steven Bradburyⁱ ⁱIowa State University, Department of Entomology, Ames, Iowa and ⁱⁱUSDA-ARS, Ames, Iowa Over the last two decades populations of monarch butterflies in North America have declined significantly. Conservation efforts in the U.S. Midwest are focused on restoring milkweed (Asclepias species), the sole food source of monarch larvae. Restored milkweed habitat could be placed in close proximity of corn and soybean fields, where insecticides are often used for pest management. Risks of monarch larvae exposure to these insecticides at the individual habitat patch and landscape scales are unknown. Larvae could be exposed through the cuticle

by spray drift from foliar applications. Dietary exposure also could occur if monarch larvae ingest milkweed leaves with insecticide residues from spray drift deposition or systemic uptake from treated seeds. Cuticular toxicity studies were undertaken with beta-cyfluthrin, chlorantraniliprole, chlorpyrifos, imidacloprid and thiamethoxam and percent mortality was calculated at different distances from treated fields (0, 50, 100 and 125 feet) using AgDRIFT, a spray drift model. For aerial applications, predicted percent mortality for 1st instars ranged from 100% to 64%, 100% to 16% and 100% to 7% at the edge of field, 50 feet and 100 feet away from treated fields, respectively. For 5th instars, percent mortality ranged from 100% to 0%, 95% to 0% and 86% to 0%. In general, predicted mortality rates were higher for beta-cyfluthrin and chlorantraniliprole and lower for thiamethoxam and imidacloprid. On-going dietary studies are addressing acute, subchronic and chronic effects. Neonicotinoid-treated 5th instar larvae exhibited arrested pupal development wherein splitting of the cuticle along the ecdysial lines ceased during pupation. This phenomenon occurred less frequently with the other insecticides. The estimated larval survival rates obtained from the deterministic risk assessments are being incorporated into a projection model to predict population responses at the landscape level. These analyses will inform conservation costs and benefits of establishing habitat in areas potentially exposed to insecticides.

O4 SWINE BARN DUST EXPOSURE ACTIVATES MICROGLIA THROUGH INDUCTION OF OXIDATIVE STRESS AND THE RESULTANT NEUROINFLAMMATION APPEARS TO INVOLVE HMGB1-RAGE SIGNALING.

Nyzil Massey1, Sreekanth Puttachary2, Sanjana Mahadev-Bhat1, Denusha Shrestha1, Anumantha G. Kanthasamy1, Chandrashekhar Charavaryamath1. 1Biomedical Sciences, Iowa State University, Ames, IA and 2Oregon State University, Corvallis, OR

Swine production in the US is a major animal production activity with 550,000 jobs and $39 billion added to GDP. Large swine farms employ full time workers who are exposed to occupational contaminants including organic dust, lipopolysaccharide (LPS), peptidoglycan (PGN) and gases. Exposed workers mainly report bronchitis, asthma and loss of lung function. Most studies have focused on mechanisms of inflammation underlying the respiratory symptoms. But effect of barn exposure on other vital organs such as brain remain largely unknown. Brain microglial cells are similar to macrophages of other organs in immune activation and microglial activation is known to cause neuroinflammation. Therefore, we tested a hypothesis that swine barn dust extract (SBDE) activates microglial cells of the brain using an in vitro model.

Settled dust from commercial swine operations was collected, buffer extract prepared and filter sterilized. Mouse (C57BL6) microglial cell line were treated with SBDE or LPS or PGN or medium and stained for the expression of Iba1 (microglial activation), gp91 phox (reactive oxygen

species (ROS)), HMGB1 (high-mobility group box-1), RAGE (receptor for advanced glycation end products) and DAPI at 0, 6, 24 and 48 h post treatment. Along with SBDE or LPS or PGN, microglia were co-treated with Ethyl Pyruvate (EP) or Mito-Apocynin (Mito-Apo) and HMGB1 translocation or 3-NT (reactive nitrogen species (RNS) marker) expression were measured respectively.

Microglia morphologically appeared reactive with an upregulation of Iba1 and gp91 phox by 24 hours post PGN or SBDE treatment. At 48 hours, SBDE treated microglia demonstrated nuclear to cytoplasmic translocation of HMGB1 and co-localization with RAGE in the cytoplasm. EP treatment reduced HMGB1 and RAGE expression while Mito-Apo treatment reduced the expression of 3-NT.

Our data indicate that, SBDE induces microglial activation, ROS production, translocation of HMGB1 and co-localization of HMGB1 and RAGE to indicate neuroinflammatory signaling (Iowa State University).

O5 ENANTIOSELECTIVELY FORMATION OF HYDROXYLATED 2,2',3,3',4,6'-HEXACHLOROBIPHENYL (PCB 132) METABOLITES BY HUMAN LIVER MICROSOMES Eric Uwimana1 and Hans-Joachim Lehmler1 1Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa 1Iowa Superfund Research Program, Synthesis Core, Projects 3 & 5 Persistent organic pollutants such as polychlorinated biphenyls (PCBs) affect human health because of their presence in the human diet and air. PCB congeners with 3 or 4 chlorine substituents in ortho position have been associated with neurodevelopmental disorders. Many of these neurotoxic congeners are axially chiral and exist as two stable rotational isomers, called atropisomers, which are non-superimposable mirror images of each other. Hydroxylated metabolites (OH-PCBs) of these PCBs are also chiral and potentially toxic to the developing brain. We hypothesized that the oxidation of PCB 132 by human liver microsomes is enantioselective and varies from individual to individual. Racemic PCB 132 (50 µM) was incubated with pooled (pHLM) or individual human liver microsomes (iHLM) for 10 or 30 min at 37 °C. In addition, racemic PCB 132 (5 µM) was incubated with pooled (pHLM) for 2 h at 37°C. Levels of 2,2',3,4,4',6'-hexachlorobiphenyl-3'-ol (3'-140; 1,2 shift product), 2,2',3,3',4,6'-hexachlorobiphenyl-5'-ol (5'-132) and 2,2',3,3',4,6'-hexachlorobiphenyl-4'-ol (4'-132) as well as enantiomeric fractions (EF) of PCB 132, 3'-140 and 5'-132 were determined. Depending on the iHLM preparation, the major metabolite formed was either 3'-140 or 5'-132. 4'-132 was only a minor metabolite of PCB 132. The second eluting atropisomer of PCB 132 was slightly enriched in 2 h incubations of pHLMs with the low PCB 132 concentration (5 µM). The formation of the first eluting atropisomer of 3'-140 was nearly enantiospecific (EF > 0.8). The second eluting atropisomer of 5'-132 was enriched in all microsomal preparations investigated. EF values differed between iHLM preparations and ranged from 0.84 to 0.96 for 3'-140 and 0.12 to 0.19 for 5'-132. These findings suggest that there are inter-individual differences in the

enantioselective biotransformation of PCB 132 to OH-PCBs in humans. Studies of the atropselective toxicity of PCB 132 and its hydroxylated metabolites are needed to determine the role of atropselective metabolism in PCB-mediated developmental neurotoxicity in at-risk populations.

O6 ANALYSIS OF ACTIVITY OF MONOTERPENOID PLANT COMPOUNDS ON NEMATODE ACETYLCHOLINE RECEPTORS Colin Wong1, Melonie Abongwa1, Shivani Choudhary2, Alan Robertson2, Richard Martin2, Joel Coats1. 1Toxicology, Iowa State University, Ames IA 2Biomedical Science, Iowa State University, Ames IA Purpose: Monoterpenoids found in natural plant products have been shown to have biocidal activity against agricultural, human health and veterinary pests. Specific neurological activity has been found to be highly variable between different monoterpenoids acting on different organisms and receptors. This research follows findings that carvacrol can act as an antagonist against the ACR-16 nicotinic acetylcholine receptor from Ascaris suum. We tested multiple plant derived monoterpenoids with the ACR-16 receptor. Methods: Receptors were expressed in oocytes from Xenopus lavis. We used two electrode voltage clamp to test the monoterpenoids. Initial screens were done at 100 µM for each compound. Further characterization used 100 µM and 10 µM concentrations. Results: Several compounds were shown to have significantly greater activity from Carvacrol (11.0% inhibition at 100 µM) on the ACR-16 receptor, including menthyl acetate (20.0% inhibition at 100 µM). No monoterpenoids showed agonist activity on the ACR-16 receptor. Concentration response curves showed that the monoterpenoids tested had no significant effect on the EC50 values of the acetylcholine response. Several monoterpenoids gave significantly lower maximal currents than the control. Conclusions: This research confirms previous findings that monoterpenoids as a class of compounds have varied but noticeable neurotoxic effects at a nicotinic acetylcholine receptor. We plan to characterize those compounds most active at our chosen target site.

Poster Presentation Abstracts

Graduate Students

P1 RISK ASSESSMENT OF FOLIAR INSECTICIDES COMMONLY USED IN CORN AND SOYBEAN PRODUCTION ON MONARCH BUTTERFLY (DANAUS PLEXIPPUS) LARVAE

Niranjana Krishnanⁱ, Keith Bidneⁱⁱ, Richard Hellmichⁱⁱ, Joel Coatsⁱ and Steven Bradburyⁱ ⁱIowa State University, Department of Entomology, Ames, Iowa and ⁱⁱUSDA-ARS, Ames, Iowa

Over the last two decades populations of monarch butterflies in North America have declined significantly. Conservation efforts in the U.S. Midwest are focused on restoring milkweed (Asclepias species), the sole food source of monarch larvae. Restored milkweed habitat could be placed in close proximity of corn and soybean fields, where insecticides are often used for pest management. Risks of monarch larvae exposure to these insecticides at the individual habitat patch and landscape scales are unknown. Larvae could be exposed through the cuticle by spray drift from foliar applications. Dietary exposure also could occur if monarch larvae ingest milkweed leaves with insecticide residues from spray drift deposition or systemic uptake from treated seeds. Cuticular toxicity studies were undertaken with beta-cyfluthrin, chlorantraniliprole, chlorpyrifos, imidacloprid and thiamethoxam and percent mortality was calculated at different distances from treated fields (0, 50, 100 and 125 feet) using AgDRIFT, a spray drift model. For aerial applications, predicted percent mortality for 1st instars ranged from 100% to 64%, 100% to 16% and 100% to 7% at the edge of field, 50 feet and 100 feet away from treated fields, respectively. For 5th instars, percent mortality ranged from 100% to 0%, 95% to 0% and 86% to 0%. In general, predicted mortality rates were higher for beta-cyfluthrin and chlorantraniliprole and lower for thiamethoxam and imidacloprid. On-going dietary studies are addressing acute, subchronic and chronic effects. Neonicotinoid-treated 5th instar larvae exhibited arrested pupal development wherein splitting of the cuticle along the ecdysial lines ceased during pupation. This phenomenon occurred less frequently with the other insecticides. The estimated larval survival rates obtained from the deterministic risk assessments are being incorporated into a projection model to predict population responses at the landscape level. These analyses will inform conservation costs and benefits of establishing habitat in areas potentially exposed to insecticides. P2 IMPAIRED LEARNING AND MEMORY IN RATS AFTER SUBCHRONIC INHALATION EXPOSURE TO AN INDOOR SCHOOL AIR MIXTURE OF PCBs Hui Wang1, Andrea Adamcakova-Dodd1, Benjamin R. Steines1, Ezazul Haque2, Xueshu Li1, Hans-Joachim Lehmler1,2, Peter S. Thorne1,2

1Department of Occupational & Environmental Health, College of Public Health, University of Iowa 2Interdisciplinary Graduate Program in Human Toxicology, Graduate College, University of Iowa Polychlorinated biphenyls (PCBs) are persistent, bioaccumulative, toxic pollutants, which are still present in the ambient environment and indoor buildings despite their production being ceased since the 1970s. Exposure to PCBs, especially during fetal and childhood developmental period, can affect IQ, learning and memory abilities. In this study we generated a PCB School Air Mixture (SAM) resembling the profile of East Chicago indoor school air in order to evaluate the effects of PCBs on learning and memory. Female Sprague-Dawley rats were exposed simultaneously to either SAM vapor (n = 8) or clean air (n =

8) through a nose-only exposure system. The exposure was 4 h/day, 7 days/week for 4 weeks. Positive control rats (n = 6) for assessing neurological behavior were treated with 1-bromopropane (1-BP, 800 mg/kg bw, by gavage) for 12 days. Sentinel rats (n = 2) were kept in vivarium for health surveillance. Morris Water Maze (MWM) was performed for 6 days to evaluate the learning and memory abilities. Oxidative stress in supernatants of brain homogenates was assessed by: determination of reactive oxygen and nitrogen species, detection of 4-hydroxynonenal (HNE) protein adducts and formation of malondialdehyde (MDA). Significantly increased escape latency was found in 1-BP group at 4th and 5th day, compared with control group (p<0.01, p<0.01 respectively). In contrast, no significant differences were found between SAM exposure group and control group, but from the learning efficiency, SAM group was slightly lower than control. Additionally, significant decreases of time spent in target quadrant and number of crossing platform were observed in 1-BP group, compared with control group (p<0.01, p<0.01, respectively); similar decrease of number of crossing platform was found in SAM exposure group (p<0.05). None of the oxidative stress tests showed a statistical significance when SAM group was compared with controls. Results suggest the impairment of memory and learning after SAM exposure might not result from oxidative stress. Further investigation is needed to uncover the mechanism of the learning and memory deficit caused by PCBs. (Supported by NIH P30ES005605 and NIH P42ES013661)

P3 Manganese Exposure Impairs Endosomal and Exosomal Recycling Machinery to Enhance the Release of Misfolded α-Synuclein Dharmin Rokad, Dilshan S. Harischandra, Vivek Lawana, Dan Luo, Huajun Jin, Vellareddy Anantharam, Arthi Kanthasamy, Anumantha G. Kanthasamy Biomedical Sciences, Iowa State University, Ames, IA Environmental exposure to excessive manganese (Mn) has been shown to increase the risk for chronic neurological diseases like Parkinson’s disease (PD). The aggregation of α-synuclein (α-syn) is considered a key pathophysiological feature of PD. Recently, it has been shown that oligomeric α-syn can be released from neurons via exosomes, enabling misfolded α-syn to propagate to neighboring cells. Our recent study revealed that Mn exposure greatly increases the release of aggregated α-syn-containing exosomes from dopaminergic neurons. However, the cellular and molecular signaling mechanisms driving Mn-induced exosome release remain unknown. Herein, we examined how Mn modulates endosomal protein trafficking and α-syn degradation to promote exosomal release. MN9D dopaminergic neuronal cells stably expressing human wild-type α-syn were exposed to Mn (300 μM) for 24 h and expression of the key Rab GTPases involved in endosomal trafficking, Rab11a and Rab27a, were examined by Western blot and immunocytochemistry (ICC). Mn significantly suppressed Rab11a expression both at the protein and mRNA levels, suggesting that the metal disrupts endosomal-recycling mechanisms, thereby forcing endosomes to mature into multivesicular bodies (MVBs). Next, we examined whether Mn alters Rab27a, a signaling molecule that regulates exosome release through fusion of MVBs with the plasma membrane. Interestingly qRTPCR, western blot and ICC analyses revealed that Mn exposure upregulated the mRNA and protein expression of Rab27a, suggesting that Rab27a upregulation contributes to Mn-induced exosomal release. Since Mn increases α-syn load due to the intracellular accumulation of MVBs, we then examined the effect of Mn on α-syn clearance via autophagic/lysosomal degradation. Western blot analysis revealed that Mn upregulated the expression of the autophagosomal markers LC3-II and Beclin-1, whereas the lysosomal marker LAMP2 was downregulated in Mn-treated α-syn expressing cells. Taken together, these novel findings suggest that

Mn compromises endosomal trafficking, leading to autophagic/lysosomal impairment, thereby promoting MVB formation and the exosomal release of misfolded α-syn. (NIH grants ES19267, ES26892, and Linda Lloyd Endowed Chair)

P4 DEVELOPMENT OF A MRM METHOD TO DETECT OH-PCBS IN ENVIRONMENT SAMPLES. Panithi Saktrakulkla1, Keri C. Hornbuckle1,2 1Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, Iowa 52242, United States 2Department of Civil & Environmental Engineering, and IIHR-Hydroscience and Engineering, The University of Iowa, Iowa City, Iowa 52242, United States Hydroxylated polychlorinated biphenyls (OH-PCBs) are oxidative metabolites of PCBs in humans and other organisms and also found as residuals in original Monsanto Aroclors. However, OH-PCBs are usually found at lower quantities than their parent PCBs in either tissues or environmental matrices. Detection of OH-PCBs is therefore challenging. Here we present a method for detection and quantification of OH-PCBs using gas chromatography with tandem mass spectrometer (GC-MS/MS) in multiple reaction monitoring (MRM) mode. This novel method required optimization of instrumental parameters including congener-specific determination of product ions and collision cell energies. The new method is composed of 37 acquisition time segments during a typical chromatographic run time of 70.3 minutes in Agilent J&W DB-1701 MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness), when the published method contains only 5 time segments. The method was tested on a calibration solution set of 72 OH-PCB congeners. The result was compared to the published method and exhibited a relative peak response for a 50 ng/mL concentration up to 119 times greater: 72 congeners exhibited higher responses compared to the published method (68 is statistically different); 6 congeners were equal; and 3 congeners were lower, but less than 10% (2 is statistically different). The effectiveness of the method was examined for previously published OH-PCBs in environmental sample extracts. Initial findings suggest that the method will enable detection and quantification of more OH-PCB congeners at lower concentrations in human serum, sediments, water, and air. (Supported by NIH grant P42 ES013661) P5 TOXICITY ASSESSMENT FOLLOWING AEROSOLIZATION OF SILVER SUPPORTED ON SILLICA NANOPARTICLES USING IN VITRO AND IN VIVO METHODS.

Wang Y., Adamcakova-Dodd A., Steines B.R., Givens B.E., Areecheewakul S., Altmaier R., O’Shaughnessy P.T., Salem A.K., Thorne P.S. University of Iowa, Iowa City, IA Silver (~7 nm) supported on silica (~10 nm) nanoparticles (10%Ag@SiO2) were provided by HSPH-NIEHS Nanosafety Center with full physicochemical and morphological characterization (10%AG-SIO2-JB20161109-1:T10). Nanoparticles were synthesized using Flame spray pyrolysis. Nanomaterial was used as supplied without further modification. Toxicity assessment was done using in vitro submersed (50, 100 and 200 µg/mL) and air liquid interface (Vitrocell Systems) conditions using A549 lung epithelium cells as well as in vivo sub-acute (4 hr/day, 5 days/week for 2 weeks) study. A549 cells were exposed to airborne Au NPs at a constant flow rate of 5

mL/min for 4h periods. Female mice (C57Bl/6) were exposed by a nose-only inhalation system (inExpose, Scireq, Emka Technologies). Aerosol was generated using BGI Collison nebulizer. Generated mass aerosol concentrations measured gravimetrically were 4 mg/m3 (in vitro study, ALI) and 3.5 ± 1.1 mg/m3 (sub-acute study). Particle size distribution of the aerosolized ENMs yielded a geometric mean mobility diameter of 91 nm. Shams (controls) were exposed to HEPA filtered laboratory air. Mice were necropsied immediately (0 wk) or 3 weeks (3 wks) later after the last exposure. In vitro exposures in submersed conditions showed dose-dependent toxicity (MTS assay). Exposure of A549 cells at air liquid interface decreased cell viability to 75.7% compared to controls and significantly (p < 0.001) elevated levels of lactate dehydrogenase and interlukin-8. Sub-acute in vivo exposure caused a significant (p<0.0001) increase of total number cells recruitment into the lungs (measured in bronchoalveolar lavage [BAL] fluid) at 0 wk post exposure (1,756 x 103 cells/mouse) compared to shams (47 x 103 cells/mouse). At 3 wks post exposure, the cell numbers returned to normal (40 x 103 cells/mouse). The percentage of neutrophils in BAL fluid was 51% at 0 wk, 7% at 3 wk post exposure and 1.6% in shams. Analyses of total protein, LDH, ROS and cytokines in BAL fluid were performed. Pulmonary mechanics after increasing methacholine challenge and histopathology of the lungs after H&E and Masson Trichrome staining were evaluated. (NIH U01 ES027252 and NIH P30 ES005605)

P6 INVESTIGATION OF THE EFFECTS OF ALPHA-CYPERMETHRIN AND PRENATAL STRESS ON EMBRYONIC BRAIN DEVELOPMENT

Benjamin Elser1, Hans-Joachim Lehmler1,2, Hanna Stevens1,3 1Interdisciplinary Program in Human Toxicology, University of Iowa, Iowa City, Iowa 2Department of Occupational and Environmental Health, University of Iowa, Iowa City, Iowa 3Department of Psychiatry, University of Iowa, Iowa City, Iowa

Alpha-cypermethrin is a type II pyrethroid insecticide that has been recommended for use by pregnant women to prevent transmission of the Zika virus. As such, there is a high probability for prenatal exposure to the developing embryo, which has previously been demonstrated to be particularly susceptible to the toxicity of pyrethroids. Our lab has also previously demonstrated that prenatal stress is a risk factor for embryonic brain development through interference with the migration of GABAergic progenitor cells in the developing telencephalon. Furthermore, several studies have suggested that psychological stress can have a variety of effects on drug metabolism and tissue distribution, as well as compromise the placenta’s ability to prevent xenobiotics from entering fetal circulation. As such, we aimed to investigate the effects of prenatal exposure to alpha-cypermethrin on embryonic brain development, alone and in combination with restraint stress. To accomplish this, female CD1 mice were administered alpha-cypermethrin in peanut butter at doses of 0 mg/kg, 3 mg/kg, or 10 mg/kg b.w. throughout the first 14 days of pregnancy. In addition, half of the mice were subjected to restraint stress (3x45 min) on the 12th and 13th days of gestation (E12-E13). Mice were sacrificed on E14 and embryonic brains were analyzed for alterations in GABAergic progenitor migration; as well as for alterations in forebrain volume and proliferative zone size in the ganglionic eminence. No overt signs of maternal toxicity (decreased body/organ weights) were observed in the cypermethrin treated mothers.

Furthermore, embryonic body and placenta weights were unchanged between all treatments examined. At odds with our previous studies, migration of GABAergic progenitors was found not to be significantly decreased from controls for either prenatal stress or cypermethrin, alone or in combination. This suggests a potential unintended stress buffer mechanism in our experimental method. However, our results suggest that cypermethrin may be capable of inducing several unique teratogenic effects. These include what appears to be an extreme case of abnormal brain development resembling exencephaly in one embryo exposed to 10mg/kg alpha-cypermethrin, as well as several cases of abnormal development in other embryos prenatally exposed to both cypermethrin alone and in combination with prenatal stress. In addition, two separate cases of monochorionic twins with shared placentas, a rare occurrence, were identified in cypermethrin treated litters; one of which demonstrating a case previously described in the literature as “twin-twin transfusion syndrome”. No noticeable teratogenic abnormalities were observed in mice fed control peanut butter alone or in combination with prenatal stress, suggesting that these outcomes may be a result of prenatal exposure to cypermethrin. Taken together, these findings suggest potential adverse outcomes on embryonic development as a consequence of prenatal cypermethrin exposure at doses lower than those which produce maternal toxicity. As such, more research will be done to confirm these teratogenic effects and identify mechanisms by which prenatal cypermethrin exposure can impact embryonic brain development.

P7 SELENIUM: A CRITICAL TRACE ELEMENT FOR REDOX TOXICOLOGY

Jeffrey M. Stolwijk1, Weipeng Bian1, Juan Du1, Brett A. Wagner1, Yousef Zakharia1 and Garry R. Buettner1 1University of Iowa, Iowa City, IA

Selenium is an essential trace element for optimal human health. However, selenium intake must be within a relatively narrow range to prevent deficiency or cause toxicity. The RDA is on the order of 55 µg d-1. Insufficient selenium intake affects an estimated 1 billion people around the world. The dietary availability of selenium depends on the content of the soils and water of our food sources.

Currently there are 25 known selenoproteins. A set of these selenoenzymes, such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR) have an important role in the redox biology of cells and tissues. Exposure to xenobiotics can lead to an increased and potentially detrimental flux of ROS, e.g. superoxide and H2O2. These selenoenzyme families play an important role in the system that removes these species to protect against inappropriate oxidations. In cell culture studies, appropriate expression of these enzymes is required to gain the best biochemical and toxicological information. It has been appreciated for over two decades that cell culture media is typically deficient in Se. This can lead to low activity for GPx and TrxR. Although some laboratories supplement cell culture media with Se, sound, information on what is the ideal level to achieve is maximal activity and not produce any detrimental effects is lacking. Here we present data on the dose-response of Se-enzyme activity vs. available Se in cell culture media. Our first data suggest that Se supplementation of about 100 nM maximizes the activity of GPx1, our sentinel Se-enzyme. As expected Se, as selenite is very toxic at higher levels. However, Se as selenomethionine shows little toxicity, even at very high levels.

Because of the low toxicity of selenomethionine, it is also used in concentrations orders of magnitude higher than the RDA. These high doses of selenomethionine target hypoxia inducible factor-1

(HIF-1α) in cancer patients with the von Hippel-Lindau syndrome. Where HIF-1α plays a key role in this form of cancer. The treatment is conducted in a clinical trial and shows promising results. (This work was supported by the National Institutes of Health (NIH), grants R01 CA169046, R01 GM073929, P42 ES013661, P30 ES005605, and P30 CA086862.)

P8 Exploring the relationship between PaOA1 receptor modulation and the insecticidal character of various terpenoids Edmund Norris1, Aaron Gross2, Michael Kimber3, Lyric Bartholomay4, Joel Coats1

1Pesticide Toxicology Laboratory, Department of Entomology, Iowa State University of Science and Technology, Ames, IA, USA, 50011 2Emerging Pathogens Institute, University of Florida, Gainesvile, FL, USA, 32611 3Department of Biomedical Science, Iowa State University of Science and Technology, Ames, IA, USA, 50011 4Department of Pathobiological Sciences, University of Wisconsin – Madison, Madison, WI, USA, 53706

Octopamine, a biogenic amine that is physiologically important in arthopods, has been implicated in numerous physiological roles, such as learning, memory, reproduction, and nerve stimulation, to name a few. Octopamine plays a limited role in mammalian systems, which suggests that octopamine receptors may be viable targets for the development of novel insecticides that possess little-to-no off-target effects in vertebrates. Previous studies have demonstrated that terpenoids are capable of acting on octopamine and tyramine receptors. To-date, we have developed a line of Chinese Hamster Ovary cells (CHO) that are stably expressing an α-adrenergic-like octopamine receptor that has been isolated from the American cockroach (Periplaneta Americana). This receptor was functionally characterized via a Fluo-4 NW calcium-mobilization assay (Invitrogen Technologies). Functional characterization demonstrated that octopamine is the preferred ligand compared to the structurally similar tyramine, with an EC50 value of 89.6 nM compared to the 463 nM of tyramine. Various monoterpenoids were screened as positive and negative modulators of this octopamine receptor. Numerous terpenoids were identified as either positive or negative modulators using a pre-incubation assay. To assess how positive or negative modulation were related to the insecticidal character of these terpenoids, select terpenoids were injected into American cockroach nymphs and mortality was recorded at 7 days after injection. Cockroach mortality was not strongly correlated to either positive or negative modulation of the octopamine receptor. These results suggest that terpenoids are capable of modulating a physiologically relevant receptor; however, percentage positive or negative modulation did not describe percentage mortality. This study may indicate that terpenoids are capable of acting upon octopaminergic systems within insects. Moreover, the fact that mortality was not correlated to octopamine receptor modulation demonstrates that toxicity of these terpenoids may be caused by activity at other physiological targets within the American cockroach.

P9 P7C3 PROTECTS RAT SPIRAL GANGLION NEURONS AFTER DEAFENING

Muhammad Taifur Rahman1,2, Bo Peng2, Catherine Kane2, Benjamin M Gansemer2, Steven H Green2 1. Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA 2. Department of Biology, The University of Iowa, Iowa City, IA

Background: Spiral ganglion neurons (SGNs) receive their sole input from hair cells, the auditory sensory cells and gradually die after destruction of hair cells with aminoglycosides (Alam et al. J Comp Neurol, 2007). P7C3, an aminopropyl carbazole has been shown to exert proneurogenic and neuroprotective roles in animal models of neurodegenerative disorders including Parkinson’s disease (De Jesús-Cortés et al. PNAS, 2012) and Amyotrophic Lateral Sclerosis (Tesla et al. PNAS, 2012) by activating the rate-limiting enzyme (NAMPT) in the NAD salvage pathway (Wang et al. Cell, 2014). We assessed the ability of an active analog of P7C3, P7C3A20, to protect SGNs after deafening. Methods: Sprague Dawley rats were injected intraperitoneally with kanamycin daily postnatal day 8 (P8)-P16 to destroy hair cells. Rats showing auditory brainstem response below 95 dB SPL were excluded from the experiment. From P22-P70, rats were injected daily intraperitoneally with P7C3A20, 20 mg/kg in vehicle (DMSO/corn oil) or, as a control, vehicle only. Rats were euthanized at P70 and cochleae fixed, cryosectioned parallel to the midmodiolar plane, labeled with myosin 6/7 antibodies to detect hair cells, and with NeuN and NF200 antibodies to detect, respectively, SGN nuclei and somata/axons. Only near-midmodiolar sections were used for analysis. Deafening was confirmed by absence of hair cells. Quantitation was done using the FIJI ImageJ package with some macros written in the lab. The outline of Rosenthal canal for each turn in the midmodiolar plane was manually traced for calculation of cross-sectional area. NeuN-labeled images were processed in ImageJ for automated counting of SGNs and calculation of SGN density in each turn. Results: Kanamycin injection resulted in profound deafness, confirmed by loss of outer and inner hair cells and absence of ABR. By P70, SGN density was significantly reduced in the basal region of the cochleae of deafened control vehicle-injected rats relative to hearing control animals. SGN loss at P70 in apical region of the cochleae was not significant. SGN in the basal region of the cochleae was significantly (P<0.05) reduced in deafened rats injected with P7C3. We conclude that P7C3 appears to be protective against SGN death after aminoglycoside deafening. This further suggests that dysregulation of NAD+ metabolism plays a role in the death of SGNs after deafening.

P10 SWINE BARN DUST EXTRACT INDUCES NUCLEAR TO CYTOPLASMIC TRANSLOCATION OF HMGB1 IN HUMAN AIRWAY EPITHELIAL CELL LINE BEAS-2B.

Sanjana Mahadev-Bhat1, Nyzil Massey1, Locke A. Karriker2, Baljit Singh3, Chandrashekhar Charavaryamath1. 1Biomedical Sciences, Iowa State University, Ames, IA, 2VDPAM, Iowa State University, Ames, IA and 3Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada Swine production in the US provides about 550,000 jobs and $39 billion to GDP. Swine barn dust (SBD) is rich in microbial pathogen associated molecular patterns (PAMPs) and exposed workers suffer from various respiratory symptoms and decline in lung function. Many researchers have examined the role of pattern recognition receptors (PRRs) in SBD-induced lung inflammation. However, the role of damage associated molecular patterns (DAMPs) is still largely unknown. Therefore, we tested a hypothesis that

High-mobility group box 1 (HMGB1), has a role in SBD-induced lung inflammation. HMGB1 is a nucleosome protein and undergoes a nuclear to cytoplasmic translocation under pathological conditions. Secreted HMGB1 interacts with TLRs and the receptor for advanced glycation end products (RAGE) to act as a DAMP.

First, we semi-quantified the cell specific expression of HMGB1 in barn exposed and control rats. Compared to controls, one, five and 20-day exposed rat lungs showed higher expression of HMGB1 in airway epithelium, septa and blood vessels. Next, settled swine barn dust was collected and a sterile extract (SBDE) was prepared. Human airway epithelial cell line (BEAS-2B) treated with SBDE showed nuclear to cytoplasmic translocation of HMGB1 and co-localization with RAGE in the cytoplasm to indicate possible HMGB1-RAGE signaling. Ethyl pyruvate (EP), an anti-inflammatory agent reduced the SBDE-induced HMGB1 translocation to cytoplasm. Taken together, our data indicate a role for HMGB1 in SBDE induced lung inflammation and potential for EP as a therapeutic agent (Iowa State University, Lung Association of Saskatchewan and CIHR-PHARE).

P11 SWINE BARN DUST EXPOSURE ACTIVATES MICROGLIA THROUGH INDUCTION OF OXIDATIVE STRESS AND THE RESULTANT NEUROINFLAMMATION APPEARS TO INVOLVE HMGB1-RAGE SIGNALING.

Nyzil Massey1, Sreekanth Puttachary2, Sanjana Mahadev-Bhat1, Denusha Shrestha1, Anumantha G. Kanthasamy1, Chandrashekhar Charavaryamath1. 1Biomedical Sciences, Iowa State University, Ames, IA and 2Oregon State University, Corvallis, OR

Swine production in the US is a major animal production activity with 550,000 jobs and $39 billion added to GDP. Large swine farms employ full time workers who are exposed to occupational contaminants including organic dust, lipopolysaccharide (LPS), peptidoglycan (PGN) and gases. Exposed workers mainly report bronchitis, asthma and loss of lung function. Most studies have focused on mechanisms of inflammation underlying the respiratory symptoms. But effect of barn exposure on other vital organs such as brain remain largely unknown. Brain microglial cells are similar to macrophages of other organs in immune activation and microglial activation is known to cause neuroinflammation. Therefore, we tested a hypothesis that swine barn dust extract (SBDE) activates microglial cells of the brain using an in vitro model.

Settled dust from commercial swine operations was collected, buffer extract prepared and filter sterilized. Mouse (C57BL6) microglial cell line were treated with SBDE or LPS or PGN or medium and stained for the expression of Iba1 (microglial activation), gp91 phox (reactive oxygen species (ROS)), HMGB1 (high-mobility group box-1), RAGE (receptor for advanced glycation end products) and DAPI at 0, 6, 24 and 48 h post treatment. Along with SBDE or LPS or PGN, microglia were co-treated with Ethyl Pyruvate (EP) or Mito-Apocynin (Mito-Apo) and HMGB1 translocation or 3-NT (reactive nitrogen species (RNS) marker) expression were measured respectively.

Microglia morphologically appeared reactive with an upregulation of Iba1 and gp91 phox by 24 hours post PGN or SBDE treatment. At 48 hours, SBDE treated microglia demonstrated nuclear to cytoplasmic translocation of HMGB1 and co-localization with RAGE in the cytoplasm. EP treatment reduced HMGB1 and RAGE expression while Mito-Apo treatment reduced the expression of 3-NT.

Our data indicate that, SBDE induces microglial activation, ROS production, translocation of HMGB1 and co-localization of HMGB1 and RAGE to indicate neuroinflammatory signaling (Iowa State University).

P12 SWINE BARN DUST-INDUCED LUNG INFLAMMATION AND EXPRESSION OF TRANSIENT RECEPTOR POTENTIAL ANKYRIN 1 (TRPA1).

Denusha Shrestha1, Sanjana Mahadev-Bhat1, Nyzil Massey1, Locke A. Karriker2, Baljit Singh3, Chandrashekhar Charavaryamath1. 1Biomedical Sciences, Iowa State University, Ames, IA, 2VDPAM, Iowa State University, Ames, IA and 3Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada Swine production in the US is a major animal production industry that adds 550,000 jobs and $39 billion to GDP. Swine barn dust (SBD) is rich in microbial products, odorants and gases and exposed workers report various respiratory symptoms and decline in lung function. Several researchers have investigated mechanisms of SBD-induced lung inflammation. However, mechanism of airway sensitization remains largely unknown. Transient receptor potential ankyrin 1 (TRPA1) is a nociceptive, calcium-permeable cation channel expressed on capsaicin-sensitive sensory neurons, endothelial and inflammatory cells. TRPA1 channels are considered as polymodal sensors of airway irritants and represent potential targets to reduce airway inflammation. Therefore, we tested a hypothesis that swine barn exposure induces increased expression of TRPA1 in the lung.

Initially, we semi-quantified immunohistochemical expression of TRPA1 in a rat model of barn exposure. Compared to controls, barn exposed rat lungs showed increased expression of TRPA1 in endothelial cells, bronchi, alveolar septa and type II alveolar epithelial cells. Next, we cultured human airway epithelial cells (BEAS-2B) and demonstrated TRPA1 protein expression in the plasma membrane with a molecular weight of 100 KDa (Western blot). Taken together, our data indicate an increased expression of TRPA1 in swine barn exposure induced lung inflammation (Iowa State University, Lung Association of Saskatchewan and CIHR-PHARE).

P13 ENANTIOSELECTIVELY FORMATION OF HYDROXYLATED 2,2',3,3',4,6'-HEXACHLOROBIPHENYL (PCB 132) METABOLITES BY HUMAN LIVER MICROSOMES Eric Uwimana1 and Hans-Joachim Lehmler1 1Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa 1Iowa Superfund Research Program, Synthesis Core, Projects 3 & 5 Persistent organic pollutants such as polychlorinated biphenyls (PCBs) affect human health because of their presence in the human diet and air. PCB congeners with 3 or 4 chlorine substituents in ortho position have been associated with neurodevelopmental disorders. Many of these neurotoxic congeners are axially chiral and exist as two stable rotational isomers, called atropisomers, which are non-superimposable mirror images of each other. Hydroxylated metabolites (OH-PCBs) of these PCBs are also chiral and potentially toxic to the developing brain. We hypothesized that the oxidation of PCB 132

by human liver microsomes is enantioselective and varies from individual to individual. Racemic PCB 132 (50 µM) was incubated with pooled (pHLM) or individual human liver microsomes (iHLM) for 10 or 30 min at 37 °C. In addition, racemic PCB 132 (5 µM) was incubated with pooled (pHLM) for 2 h at 37°C. Levels of 2,2',3,4,4',6'-hexachlorobiphenyl-3'-ol (3'-140; 1,2 shift product), 2,2',3,3',4,6'-hexachlorobiphenyl-5'-ol (5'-132) and 2,2',3,3',4,6'-hexachlorobiphenyl-4'-ol (4'-132) as well as enantiomeric fractions (EF) of PCB 132, 3'-140 and 5'-132 were determined. Depending on the iHLM preparation, the major metabolite formed was either 3'-140 or 5'-132. 4'-132 was only a minor metabolite of PCB 132. The second eluting atropisomer of PCB 132 was slightly enriched in 2 h incubations of pHLMs with the low PCB 132 concentration (5 µM). The formation of the first eluting atropisomer of 3'-140 was nearly enantiospecific (EF > 0.8). The second eluting atropisomer of 5'-132 was enriched in all microsomal preparations investigated. EF values differed between iHLM preparations and ranged from 0.84 to 0.96 for 3'-140 and 0.12 to 0.19 for 5'-132. These findings suggest that there are inter-individual differences in the enantioselective biotransformation of PCB 132 to OH-PCBs in humans. Studies of the atropselective toxicity of PCB 132 and its hydroxylated metabolites are needed to determine the role of atropselective metabolism in PCB-mediated developmental neurotoxicity in at-risk populations.

P14 USING BIORATIONAL INSECTICIDES AS COMPONENTS OF ATTRACTIVE TOXIC SUGAR BAITS TO CONTROL MOSQUITO POPULATIONS

Caleb Corona1, Edmund Norris1, Joel Coats1

1Insecticide Toxicology Lab, Department of Entomology, Iowa State University, Ames, IA Vector-borne diseases are one of the major issues impacting human health today. Controlling the arthropods that transmit them is a constantly evolving challenge. Commonly used classes of insecticides such as organophosphates, pyrethroids, and carbamates have been effective in the past, but are now being reconsidered due to the development of resistance among the target species and the potential for lethal non-target effects. This need for alternative insecticides has given rise to the field of natural products and their modified products (biorational products). Botanical essential oils made up of various monoterpenes and sesquiterpenes have been shown to possess insecticidal properties. Developing these natural products also requires the development of an assay method to evaluate the insecticidal qualities associated with these biorational products. Attractive Toxic Sugar Baits (ATSB) is a promising method to deploy biorational products for the control of mosquito populations. This vector control method uses the mosquito’s need to feed on sugar as a pathway to introduce the toxic agent into the mosquito’s diet. Our recent studies have indicated that when a biorational product is paired with the proper ATSB solution, it can function as an effective method to control Aedes aegypti mosquitoes, when compared to a starved control. This type of technology could be put into action in locations where any mosquito-borne diseases are present.

P15 THE INTERPLAY BETWEEN THE RESPONSE TO DNA DAMAGE AND CELLULAR BIOENERGETICS (ATP) AS SEEN UPON EXPOSURE TO A FLUX OF H2O2

Visarut Buranasudja1, Claire M. Doskey1, Brett A. Wagner2, Juan Du3, David J. Gordon4, Stacia Koppenhafer4, Joseph J. Cullen2,3,5, and Garry R. Buettner1,2 1Interdisciplinary Graduate Program in Human Toxicology, 2Free Radical and Radiation Biology Program, Department of Radiation Oncology, 3Department of Surgery, 4Department of Pediatrics, 5Veterans Affairs Medical Center, The University of Iowa, United States

Oxidative stress has been implicated in the molecular mechanism of toxicity of various xenobiotics. Such stress can lead to a wide range of harmful effects in cells, e.g. genome instability, cellular energy crisis, and even cell death. It is well established that H2O2 is a major contributor to oxidative stress. H2O2 can readily diffuse through the plasma membrane and attack fundamental targets, e.g. lipids, DNA, and proteins. In the current study, we take advantage of the basic chemical properties of ascorbate (vitamin C) to use it as a source for a flux of H2O2 in vitro. We have employed our powerful absolute quantitation approach to investigate the extent of oxidative DNA damage and downstream consequences, including changes in bioenergetics, on MIA-PaCa2 and PANC-1 cells upon exposure to a flux of H2O2. Using quantitative PCR-based measurements, we have found that the high flux of H2O2 produced by ascorbate induces both nuclear and mitochondrial DNA damage. In response to this DNA damage, we observed that poly (ADP-ribose) polymerase-1 (PARP-1) is activated, as measured by increased formation of poly (ADP-ribose) polymer. Using our unique absolute quantitation, we found that ascorbate-induced the activation of PARP-1 results in utilization of NAD+, and subsequently depletion of ATP (energy challenge) leading to mitotic cell death. The deleterious effects of ascorbate on DNA damage, reduction of NAD+ and availability of ATP were all abrogated by catalase, indicating the involvement of H2O2 in inflicting damage to DNA, and alterations of cellular bioenergetics. Time-course studies on MIA-PaCa2 cells showed that the level of NAD+ and ATP were reduced by 80% immediately after a 1-h exposure to ascorbate (4 mM; 14 pmol cell-1); both species returned to near basal levels within 24 h. In parallel with these metabolic and energetic restorations, the lesions in nuclear DNA were removed within 3 h; however, even after 24 h, lesions in mitochondrial DNA were only partially repaired. Hyperactivation of PARP-1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 Analyzer, we observed no changes in OCR or ECAR/PPR following treatment with ascorbate, indicating that the severe decrease in ATP is due solely to increased demand, not changes in the rate of production. Our quantitative approach clearly demonstrates the interconnection between the response to DNA damage and cellular bioenergetics during oxidative stress. We foresee that these new quantitative approaches could be very useful to the toxicology research community. These methods can provide more information leading to new insights as well as increased rigor and reproducibility in basic studies on xenobiotics. (Supported by NIH grants R01 CA169046, R01 CA184051, P30 CA086862, and P42 ES013661.)

P16 ASSESSMENT OF TRACE METALS IN SURFICIAL SOIL AND BLOOD OF PARTICIPANTS IN THE AESOP STUDY COHORT IN EAST CHICAGO

Ezazul Haque1, Jeanne DeWall2, Andrea Adamcakova-Dodd2, Drew E. Latta3, Benjamin C. Bostick4, Peter S. Thorne1,2

1Interdisciplinary Graduate Program in Human Toxicology, Graduate College, University of Iowa 2Department of Occupational & Environmental Health, College of Public Health, University of Iowa 3Department of Civil & Environmental Engineering, College of Engineering, University of Iowa 4Lamont-Doehrty Earth Observatory, Columbia University in the City of New York

Polychlorinated biphenyls (PCBs) are mixtures of persistent organic pollutants comprised of 209 congeners depending on the number and position of chlorine atoms. Despite being banned from production in 1979 in the U.S., their physicochemical properties have kept them prevalent in the environment today and for the future. PCBs are commonly found in areas with industrial activities, which tend to also have presence of other toxic, bioaccumulative environmental pollutants such as trace metals. Recently, there has been a rise in concern for potential adverse effects from co-exposure to PCBs and trace metals. For this study, we assessed concentrations of trace metals in sidewalk surficial soils in proximity to a cohort in the Airborne Exposure to Semivolatile Organic Pollutants (AESOP) Study which has been following mothers and their children in East Chicago, IN and Columbus Junction, IA since 2008. Surficial soil samples (n=200) from sidewalks were collected in accordance to U.S. EPA Soil Sampling Guidelines (Method #SESDPROC-300-Rl) using a soil sampling core which were then analyzed in situ using X-ray fluorescence spectroscopy (XRF) for Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Zr, Mo, Ag, Cd, Sn, Sb, Ba, Hg, and Pb. Concentration of lead in East Chicago was found to be significantly greater than Columbus Junction (p<0.01). Furthermore, lead in soil was found to be greater than the EPA maximum contaminant level (MCL) of 400 ppm in 40% and 0% of the samples from East Chicago and Columbus Junction, respectively. Trace metals in clotted blood were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS) using an acid digestion protocol from the CDC (Method 8005). Results from analysis of a subset of blood clot samples indicate the presence of trace metals in blood of AESOP Study participants in East Chicago. These preliminary results indicate the presence of aforementioned pollutants in East Chicago soil environment and blood and in some cases significantly above the regulatory limits. Ongoing work seeks to better understand the mechanisms responsible for synergistic toxicity resulting from co-exposure to PCBs and trace metals.

Post-Doctoral Students

P17 RECOVERY OF BIOMARKERS AND CHRONICITY OF NEUROBEHAVIORAL PERFORMANCE AMONG ADOLESCENT PESTICIDE APPLICATORS Ahmed A. Ismail1, James R. Olson2,3, Matthew R. Bonner3, Kai Wang4, Olfat Hendy5, Gaafar Abdel Rasoul6, Diane S. Rohlman1

1Occupational and Environmental Health, University of Iowa, Iowa City, IA, 2Pharmacology and Toxicology, State University of New York, Buffalo, NY, 3Epidemiology and Environmental Health, State University of New York, Buffalo, NY, 4Biostatistics, University of Iowa, Iowa City, IA, 5National Liver Institute, Menoufia University, Shebin El-Kom, Egypt, 6Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt Egyptian adolescents are hired as seasonal workers to apply pesticides to the cotton crop and may work as applicators for several years. However, few studies have examined the effects of repeated pesticide exposure on health outcomes. The goal of this study was to determine the impact of repeated pesticide exposure on neurobehavioral (NB) performance and biomarkers of exposure (urinary metabolite) and effect (cholinesterase activity). Eighty-four adolescents from two field stations in Menoufia, Egypt, were examined at four time points: before and during pesticide application season in 2010 and again before and during application season in 2011. At each of the four time points, participants completed a questionnaire and an NB test battery, and were assessed for urinary levels of the chlorpyrifos metabolite TCPy (3,5,6-trichloro-2-pyridinol) and blood cholinesterase activity. Following the study cohort over two consecutive pesticide application seasons revealed that TCPy levels significantly increased following exposure, and returned to baseline levels following the end of the application season Blood butyryl cholinesterase activity exhibited a similar pattern. Although NB outcomes displayed learning and practice effects over time, deficits in performance were significantly associated with increased TCPy levels with reduction in number of NB measures showing improvement over time. Biomarkers of exposure and effect demonstrated changes associated with pesticide application and recovery after application ended. Deficits in NB performance were correlated with elevated pesticide exposure. Data demonstrated that repeated pesticide exposure may exert a long-term adverse impact on human health. (NIH grants: R21 ES017223 and R01 ES022163 (Rohlman, PI)). P18 Impact of glyphosate on ovarian signaling pathways regulating folliculogenesis and steroidogenesis. Ganesan, S., Nteeba, J. and Keating, A.F. Department of Animal Science, Iowa State University, Ames, IA 50011 Representing 50% of U.S. herbicide usage at approximately 155 million lbs (70.5 Kg) per annum, environmental glyphosate (GLY) abundance is extensive. In vitro GLY exposure reduces the abundance of steroidogenic acute regulatory protein (STAR) and cytochrome P450 19A (CYP19A1) - ovarian proteins that catalyze the first and last steps, respectively, of cholesterol conversion to 17β-estradiol. This study investigated the hypothesis that GLY alters ovarian signaling pathways involved in steroidogenesis and folliculogenesis in vivo. Wild type non-agouti (a/a) mice were exposed to GLY (2 mg/kg/day) at PND42 for five days per week over 20 weeks and were euthanized in the pro-estrus phase of their estrous cycle. GLY exposure did not impact (P > 0.05) body weight or weights of the ovary, uterus, kidney, or spleen. Hepatic weight was, however, increased (P < 0.05) by GLY exposure. GLY exposure depleted (P < 0.05) primordial follicle number but no effect (P > 0.05) on primary, secondary, antral follicles or corpora lutea numbers was observed. The phosphatidylinositol-3 kinase (PIK3) pathway is critical for oocyte viability as well as activation of primordial follicles into the growing pool. GLY exposure did not impact (P > 0.05) mRNA encoding kit ligand (Kitl), KIT proto-oncogene receptor tyrosine kinase (Kit), insulin receptor (Insr) or insulin receptor substrate 2 (Irs2). Irs2 mRNA abundance was reduced (P < 0.05) by GLY exposure. Thymoma viral proto-oncogene 1 (AKT) is a central mediator of PIK3 signaling and while total AKT protein was unaffected (P > 0.05) by GLY exposure, phosphorylated AKT protein was increased (P <

0.05). The PIK3 pathway is also involved in regulating ovarian steroidogenesis, thus, we investigated impacts of GLY on steroidogenic signaling. Increased (P < 0.05) mRNA encoding Star, Cyp11a1, but reduced (P < 0.05) abundance of Cyp19a1 and Esr2 were observed in GLY exposed mice relative to control mice. STAR protein abundance was reduced, CYP11A1 unaffected, HSD3B increased and CYP19A1 decreased by GLY exposure (P < 0.05). These data support that GLY affects signaling components in the exposed mouse ovary that are essential for proper ovarian function, specifically those involved in regulation of folliculogenesis, viability of the female gamete and steroid hormone synthesis.

P19 TRANSCRIPTOMIC PROFILE ANALYSIS IN BRAIN INFERIOR COLLICOLUS FOLLOWING ACUTE HYDROGEN SULFIDE EXPOSURE

Dong-Suk Kim1, Poojya Anantharam1, Elizabeth M. Whitley2, Wilson K. Rumbeiha1

1Veterinary Diagnostic & Production Animal Medicine, Iowa State University, Ames, IA and 2Pathogenesis LLC, Gainesville, FL Hydrogen sulfide (H2S) is a gaseous molecule produced endogenously and in the environment. Endogenously, it plays important roles in many physiological functions. Nonetheless, acute exposure to high concentration of H2S causes severe brain damage and induces long-term neurological disorders. The cellular and molecular mechanisms underlying the H2S-induced dysfunction of central nervous system have not yet been clearly elucidated. To better understand the mechanisms of H2S-induced neurodegeneration, we performed RNA sequencing analysis to identify differentially expressed genes (DEG) and pathways that contribute to H2S-induced neurotoxicity. C57BL/6j black mice were exposed by whole body inhalation to 765 ppm of H2S for 1, 2 or 4 days for 40 min on the first day and 15 min on subsequent days. The H2S-treated group showed behavioral motor deficits and developed lesions in inferior colliculus (IC), among other regions. The IC was dissected at 2 hr post H2S exposure for each group and used for the transcriptomic analysis. RNA-Seq libraries were prepared using poly (A) enrichment and mRNAs were sequenced at 20 million reads per sample on an Illumina HiSeq2000 system. Acute exposure to H2S induced 283, 193 and 296 DEG (q-value < 0.05, fold-change > 1.5) for day 1, 2 and 4, respectively. Analyses of biological pathways using Ingenuity Pathway Analysis system revealed that multiple biological pathways were dysregulated during H2S exposure, including the p-53 and MAPK signaling pathways. These results were further validated by quantitative real-time PCR revealing that expressions of Ccl2, p21, Edn1, Fosb, Gfap, and IL-6 were upregulated. Enzyme-linked immunosorbent assay also showed increase of TNFα and IL-1b cytokines in IC during H2S exposure. These results indicate that exposure to H2S induces dysregulation of multiple pathways leading to activation of the inflammation response. The obtained transcriptomic data may provide important clues to elucidate the mechanisms of H2S-induced neurotoxicity, neurodegeneration, and neurological disorders.

P20 Monepantel is a non-competitive antagonist of nicotinic acetylcholine receptors from Ascaris suum and Oesophagostomum dentatum

Melanie Abongwaa,#, Djorje Marianovicb,#, James G. Tiptona, Fudan Zhenga, Richard J. Martina, Sasa Trailovicb, Alan P. Robertsona, a Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA. b Department of Pharmacology and Toxicology, College of Veterinary Medicine, University of Belgrade, Belgrade, Serbia. # Contributed equally Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype comprising of ACR-16, a pyrantel/tribendimidine preferring heteromeric subtype comprising UNC-29, UNC-38 and UNC-63 subunits, and a levamisole preferring subtype comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6 ± 3.1 nM and 0.2 ± 2.3 µM). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the antagonism was non-competitive in nature. Our findings suggest that the mode of action of monepantel is more complex than previously described.

UNDERGRADUATES

P21 SINGLE NUCLEOTIDE POLYMORPHISMS AND MICROSATELLITES IN THE CANINE GLUTATHIONE S-TRANSFERASE PI 1 (GSTP1) GENE PROMOTER

Anastasia Yablochkin, Sarah Mann, James Sacco Ellis Pharmacogenomics Laboratory, College of Pharmacy and Health Sciences, Drake University, Des Moines, IA

Background. Genetic polymorphisms within the Glutathione S-transferase P1 (GSTP1) gene affect the elimination of toxic xenobiotics by the GSTP1 enzyme. In dogs, exposure to environmental chemicals that may be GSTP1 substrates is associated with cancer. The objectives of this study were to investigate the genetic variability in the GSTP1 promoter in a diverse population of 278 purebred dogs, compare the incidence of any variants found between select breeds, and predict their effects on gene expression. To provide information on ancestral alleles, a number of wolves, coyotes, and foxes were also sequenced. Results. Fifteen single nucleotide polymorphisms (SNPs) and two microsatellites were discovered. Three of these loci were only polymorphic in dogs while three other SNPs were unique to wolves and coyotes.

The major allele at c.-46 is T in dogs but is C in the wild canids. The c.-185 delT variant was unique to dogs. The microsatellite located in the 5’ untranslated region (5’UTR), was a highly polymorphic GCC tandem repeat, consisting of simple and compound alleles that varied in size from 10 to 22-repeat units. The most common alleles consisted of 11, 16, and 17-repeats. The 11-repeat allele was found in 10% of dogs but not in the other species. Unequal recombination between similar and distinct alleles may be the mechanism for the multiple microsatellites observed. Twenty-eight haplotypes were constructed in the dog and an additional 8 were observed in wolves and coyotes. While the most common haplotype across broad breed groups was the wild-type *1A(17), other prevalent haplotypes included *3A(11) for Greyhounds, *6A(16) in Labrador Retrievers, *9A(16) in Golden Retrievers, and *8A(19) in Standard Poodles. Boxers and Siberian Huskies exhibited minimal haplotypic diversity. Compared to the simple 16*1 allele, the compound 16*2 allele (found in 12% of dogs) may interfere with transcription factor binding and/or the stability of the GSTP1 transcript. Conclusions. Dogs and other canids exhibit extensive variation in the GSTP1 promoter. Distinct haplotypes were prevalent in certain breeds. Unequal crossing-over explains most of the multiple 5’UTR microsatellites observed. Certain variants may affect gene expression and are currently being investigated via promoter characterization studies. (Drake Research Grant, 100000-202 PRsAGX).

FACULTY OR RESEARCHER

P22 UTILIZING RNAi TOXICITY TO IDENTIFY ANTHELMINTIC TARGETS IN NEMATODES .

Verma Saurabh, Kashyap Sudhanva, Robertson Alan P, and Martin Richard J Department of Biomedical Sciences, College of Vet Med, Iowa State University, Ames, IA Insect transmitted filarial infections are serious human and animal health problems in tropical and subtropical locations. They are caused by different nematode species (e.g. Brugia malayi, Dirofilaria immitis, Onchocerca spp., loa loa). Filarial infection in animal are usually life threatening. Prevention of these infections relies on controlling the insect population and prophylaxis. Few available adulticides has limited the treatment to prevention and treatment of larval stages only. Our studies on nicotinic acetylcholine receptors (nAChRs) are focused on identifying the predicted targets of existing cholinergic anthelmintics and identifying if some of the classic and newer anthelmintics may be effective on adults. We have used a new technique of single muscle cell PCR assays to identify the nAChR subunits in the adult worms and whole cell patch-clamp recordings to characterize filarial muscle nAChR responses to different cholinergic anthelmintics. The most potent compound was pyrantel, which generated the largest current responses (EC50 63.5nM) but acetylcholine (EC50, 2.36 µM), levamisole (EC50, 3.35 µM), nicotine (EC50, 30.5 µM) and bephenium (EC50, 4.24 µM), also produced currents. We have also used RNA interference (dsRNA) as a reverse genetic tool for pharmacological and phenotypic characterization of the nAChRs. We were able to disrupt nAChR formation using dsRNA directed against unc-29 & unc-38. The resulting phenotype showed more than a 50% reduction in motility. qPCR confirmed that there was a mean reduction of 91% of unc-29 and 88% reduction of unc-38 transcription. Our patch-clamp recordings from the dsRNA treated worms revealed that levamisole responses were reduced to a

greater extent than the nicotine suggesting the specific knockdown of levamisole receptors. Characterizing the pharmacological properties of the nAChR’s of these parasites will help to identify novel drug targets, which can be utilized for the treatment of other filarial species, opening up the possibility of designing combination therapies with other anthelmintics like ivermectin and emodepside to counter the development of resistance and potential to treat adult worms. Supported by NIH grants: NIH R01 A1047194-12 to RJM; R21 AI092185-02 to APR

List of Participants

Last Name First Name EMAIL Affiliation

Abongwa Melanie mabongwa@iastate.edu Iowa State University Bradbury Steven spbrad@iastate.edu Iowa State University Buranasudja Visarut visarut-buranasudja@uiowa.edu U of Iowa Burns Laura lburns@iastate.edu Iowa State University Cappelle Kayla kmneff@iastate.edu Iowa State University Casillas Robert rcasillas@mriglobal.org MRIGlobal Charavaryamath Chandru chandru@iastate.edu Iowa State University Charli Adhithiya adhicha@iastate.edu Iowa State University Choudhary Shivani Shivani@iastate.edu Iowa State University Coats Joel jcoats@iastate.edu Iowa State University Corona Caleb clcorona@iastate.edu Iowa State University Croutch Claire ccroutch@mriglobal.org MRIGlobal Denusha Shrestha Denusha@iastate.edu Iowa State University Fitsanakis Vanessa vafitsankis@neomed.edu NE Ohio Medical U. Fredericks Jorrell jorrellf@iastate.edu Iowa State University Ganesan Shanthi shanthig@iastate.edu Iowa State University Stolwijk Jeffrey Ezazul-haque@uiowa.edu University of Iowa Hendrich Suzanne shendric@iastate.edu Iowa State University Ismail Ahmed ahmed-a-ismail@uiowa.edu University of Iowa Joachim-Lehmler Hans hans-joachim-lehmler@uiowa.edu U of Iowa Kanthasamy Anumantha akanthas@iastate.edu Iowa State University Kanthasamy Arthi arthik@iastate.edu Iowa State University Kasturi Partha pkasturi@kumc.edu Kansas Univ Med Cnt Keating Aileen akeating@iastate.edu Iowa State University Klimavicz James jamesk1@iastate.edu Iowa State University Krishnan Niranjana nkrish@iastate.edu Iowa State University Lawana Vivek vjlawana@iastate.edu Iowa State University Luo Jie vxl1008@iastate.edu Iowa State University Mahadev Bhat Sanjana smbhat@iastaete.edu Iowa State University Manne Sireesha manne@iastate.edu Iowa State University Martin Richard rjmartin@iastate.edu Iowa State University Massey Nyzil Nyzil@iastate.edu Iowa State University Miller Gary gary.miller@emory.edu Emory University

Nguyen, Thu Annelise tnguyen@vet.k-state.edu Kansas State Univ. Norris Edmund ejnorris@iastate.edu Iowa State University Radke Scott slradke@iastate.edu Iowa State University Robertson Larry larry-robertson@uiowa.edu University of Iowa Rokad Dharmin rokaddd@iastate.edu Iowa State University Rumbeiha Wilson Rumbeiha@iastate.edu Iowa State University Sacco James james.sacco@drake.edu Drake University Saktrakulkla Panithi Panithi_saktrakulkla@uiowa.edu U. of Iowa Shah Nidhi nidhi9@iastate.edu Iowa State University Souvarish Sarkar rakras26@iastate.edu Iowa State University Stolwijk Jeffrey Jeffrey-stolwijk@uiowa.edu University of Iowa Uwimana Eric eric-uwimana@uiowa.edu University of Iowa Velez Ana avelezarango2@unl.edu U Nebraska-Lincoln Verma Saurabh sverma@iastate.edu Iowa State University Wang Hui hui-wang-1@uiowa.edu Iowa State University Wild Linda lmwild@iastate.edu Iowa State University Wolday Mengisteab mtwolday@iastate.edu Iowa State University Wong Colin cwong1@iastate.edu Iowa State University Yablochkin Anastasia Anastasia.yablochkin@drake.edu Drake University

Index of Abstract Authors Name (alpha) Page in this booklet Ganesan, Shanthi Kim, Dongsuk Krishman, Niranjana