Ohio Valley Society of ToxicologyAnnual Meeting Program October 28th 2016

Sponsored by:Givaudan Flavors

Eli Lilly and CompanyProcter & Gamble

Charles River LaboratorySociety of Toxicology

2016 Annual Meeting Agenda – October 28th, Eli Lilly Company

7:30 – 8:30: Registration (73 Main Lobby) and continental Breakfast

(93 Mezzanine)

8:30 – 8:40: Welcome by Dr. Lorrene Buckley, Sr. Research Fellow, Toxicology

(93 Auditorium)

8:40 – 8:55: Welcome and Logistics by Dan Petersen, OVSOT President

(93 Auditorium)

9:00 – 10:00: Keynote Address – James L. Stevens

(93 Auditorium)

10:00 – 11:00: Doctoral student platform presentations and judging

(93 Auditorium)

11:00 – 12:00: Poster viewing and judging

(76 Mezzanine)

12:00 – 1:15: Lunch/Lunch with an Expert/Poster viewing

(76 BDR)

1:15 – 2:15: Postdoctoral platform presentations and judging

(93 Auditorium)

2:15 – 3:15: Keynote Address – Dr. Nicole Kleinstreuer, NIEHS

(93 Auditorium)

3:15 – 3:30: Break & Refreshments

(93 Mezzanine)

3:30 – 4:00: Awards presentation and closing remarks (93 Auditorium)

Keynote Address

Weighted Gene Co-Expression Network Analysis (WGCNA): Systems biology approaches to understanding adverse outcomes.

James L. Stevens, Distinguished Research Fellow, Lilly Research Laboratories

Dr. Stevens is a Distinguished Research Fellow at Lilly Research Laboratories. For over four decades in government, academis and industry he has studied molecular and cellular responses to the metabolism and toxicity of drugs and xenobiotics. His current research focuses on application of systems biology approaches to improving drug safety assessment and elucidating mechanisms of drug toxicity. Dr. Stevens received his Ph.D. in Pharmacology from the University of Minnesota in 1980. Prior to joining Lilly Research Laboratory in 2000, he held positions at the National Institutes of Health, the Food and Drug Administration, the University of Vermont and the W. Alton Jones Cell Science Center, where he was Executive Director. He has served on a variety of national advisory committees including the HESI Board of Trustees, National Advisory General Medical Sciences at NIH, National Toxicology Program Board of Scientific Councilors (BOSC), the EPA BOSC Subcommittee on Chemical Safety for Sustainability, as well as the Boards of Directors for Argonex Pharmaceuticals, Inc., and Upstate Biotechnology, Inc. He received the Achievement Award from the Society of Toxicology in 1994 and was elected a Fellow of the American Association for the Advancement of Sciences in 1996.

Keynote Address Abstract

Weighted Gene Co-Expression Network Analysis (WGCNA): Systems biology approaches to understanding adverse outcomes.

James L. Stevens, Distinguished Research Fellow, Lilly Research Laboratories

Cells and organs are modular with regard to biological functions.  Defining co-expression

networks is an attractive approach to using gene expression data to capture the modular nature

of individual biological processes. Using weighted gene co-expression network analysis

(WGCNA) and rat liver gene expression data we defined 415 co-expression modules organized

in an interconnected hierarchy to yield a visual representation of the transcriptome (the “TXG-

MAP”). Some co-expression modules represent known biological and stress response

pathways, e.g. Nrf2 oxidative stress, ribosomal biogenesis, lipid metabolism, endoplasmic

reticulum stress, etc., while others do not have obvious pathway associations. Specific patterns

of modules show high association with the emergence of adverse liver histology after drug and

chemical treatment. Thus, co-expression approaches provide an attractive approach to

defining known and novel mechanisms for complex pathologies and simplifying analysis of gene

expression data.

Graduate Student Platform Presentation Abstracts

The Effects of Age and Cancer In A Clinically-Relevant Mouse Model Of Cisplatin-Induced Kidney Injury

Cierra Sharp, Mark Doll, Tess Dupre, Levi Beverly, Leah Siskind

University of Louisville, Department of Pharmacology and Toxicology, 505 S Hancock Street, 252 G, Louisville, KY 40202

Acute kidney injury (AKI), the rapid loss of kidney function, occurs in 30% of patients treated with cisplatin. Cisplatin is a chemotherapeutic used in the treatment of many solid tumor cancers. Longitudinal studies have indicated that the severity and number of episodes of AKI in a patient's life puts them at higher risk of developing chronic kidney disease (CKD). In fact, patients that have AKI are 30 times more likely to develop CKD, which is characterized by fibrosis. We developed a more clinically relevant mouse model of cisplatin-induced kidney injury that involves repeated low doses of cisplatin, leading to interstitial fibrosis. To increase clinical relevance, we incorporated two common comorbidities- advanced age and cancer. The age range of cancer diagnosis is 55-70 years old, and older adults are more susceptible to developing AKI. While only patients with cancer receive cisplatin, cancer is rarely incorporated into animal models used to study AKI. Our objective was to determine if old mice treated with our repeated cisplatin dosing regimen would have an exacerbated decline in kidney function and worsened injury. We also wanted to determine if cancer led to worsened overall kidney outcomes when tumor-bearing mice were treated with cisplatin. In order to do so, forty week old FVB mice were treated with our repeated dosing cisplatin regimen (7 mg/kg cisplatin i.p. once a week for 4 weeks; mice were sacrificed 72 hrs after the final dose) and compared to treated 8 wk old FVB mice. To incorporate cancer into our model, we treated 40 wk old mice harboring mutant Kras-driven lung adenocarcinomas with our cisplatin dosing regimen. Kidney function and injury were assessed via BUN and NGAL assays. Real-time qRT-PCR was used to determine mRNA expression levels of inflammatory cytokines. Kidney immune cell types were quantified via flow cytometry. Western blot analyses were performed to determine levels of fibrotic proteins. Finally, collagen deposition was assessed via Sirius red/ Fast green staining, and presence of myofibroblasts was determined by IHC. We found no significant differences in kidney function, pathological mechanism, or fibrosis between 8 wk and 40 wk old treated mice, despite significantly reduced infiltrating immune cells in 40 wk old mice. Mice harboring lung tumors had reduced survival rates (100% to 25%). Mice harboring lung tumors that survived cisplatin had no changes in inflammatory cytokines or chemokines. While injury did not appear to be worse in the surviving mice at Day 24, NGAL levels peaked after Dose 2 of cisplatin, indicating mice with lung tumors sustained injury early on. Furthermore, fibrosis was significantly increased as compared to age-matched, non-cancer treated mice. Finally, EGFR protein levels and activation of EGFR were greatly increased in kidneys of lung tumor-bearing treated mice, but not in age-matched, non-cancer treated mice. We concluded that age as a factor alone did not lead to declined kidney function or worsened injury. Cancer, however, led to a decrease in overall survival, an earlier peak in injury, and worsened fibrosis. EGFR and its activation are key regulators of kidney fibrosis and may be a mechanism of action for worsened kidney fibrosis in the lung tumor-bearing

mice treated with cisplatin. Thus, the comorbidity of cancer should be included in animal models of kidney injury.

Assessing the effects of Cr(VI) on dynamic nucleosomes and its potential for dysregulating transcription

Andrew VonHandorf1, Francisco J. Sánchez-Martín2, Jacek Biesiada1, Mario Medvedovic1, Alvaro Puga1

1Environmental Health, University of Cincinnati, Cincinnati, OH, 2Molecular Cancer Therapeutics, IMIM Hospital Del Mar Medical Research Institute, Barcelona, Spain

Hexavalent chromium, Cr(VI), is well-established as a carcinogen with prevalent use in many industrial processes; however, the mechanisms associated with DNA damage, disruption of normal transcriptomic profiles, and carcinogenesis require further elucidation. We have shown in previous research using FAIRE-Seq that Cr(VI) elicits a spectrum of chromatin accessibility changes in regulatory elements dependent on the dose and length of exposure in Hepa-1c1c7 cells (mouse hepatoma) that correlates with changes in the transcriptome. Our current research sought to investigate these findings further using ATAC-Seq to measure genome-wide differential chromatin accessibility in Hepa-1c1c7 cells treated with 0.1, 0.5, or 1 μM Cr(VI) for three days. We found that all Cr concentrations demonstrated a moderate degree of overlap while exhibiting treatment-specific profiles as well. Despite differences in timing and sample preparation conditions, the comparable low-dose 0.5 μM Cr(VI) treatment in both FAIRE and ATAC exhibited a large degree of similarity in differentially enriched motifs, of which the CTCF motif and its paralog, BORIS, were consistently ranked in the upper tiers of enrichment for all conditions. Interestingly, CTCF has been shown to regulate transcription through the organization of nucleosomes, insulation of epigenomic signals, and demarcation of topologically associated domains via spatial interactions. Therefore, we chose to investigate the impact of a low dose (2 μM) Cr(VI) treatment on dynamic nucleosome positioning using the DANPOS2 algorithm to identify changes in occupancy, shift, or “fuzziness”. Indeed, the occupancy of nucleosomes in promoter regions exhibited differential enrichment for the CTCF/BORIS motifs, suggesting altered motif accessibility in regions crucial for transcription. Independently, a preliminary ChIP-Seq experiment identified approximately 700 conservative peaks denoting differential binding of CTCF in cells treated with 25 μM Cr(VI) compared to control. The CTCF motif was identified and centered in 61 of these peaks, of which a substantial number exhibited overlap with differential peaks called in the ATAC samples. Evidence from other labs suggests that carcinogenesis may disrupt the normal patterns of CTCF binding, potentially altering the functional transcriptome through the modulation of local chromatin topology; our findings using FAIRE, ATAC, and Nucleosome Positioning identify changes in differentially accessible chromatin that are enriched for the CTCF motif, supporting the notion that Cr(VI) exposure results in broad structural chromatin remodeling. It is possible, therefore, that Cr(VI) may disrupt the normal patterns of CTCF binding, affecting the cell’s ability to maintain the stability of its transcriptome and respond appropriately to insult by breaking the local chromatin contacts associated with proper spatial orientation. Supported by NIEHS R01ES010807 and NIH 2T32ES007250-26.

Acute-on-chronic alcohol exposure using the “NIAAA Model” concomitantly damages the liver and lung

Lauren G. Poole1, Juliane I. Beier1, Edilson Torres-Gonzáles2, Anwar Anwar-Mohamed1, Nikole L. Warner3, Christine E. Dolin1, Calvin T. Nguyen-Ho1, Jesse Roman3, Gavin E.

Arteel1

1University of Louisville Department of Pharmacology and Toxicology, 2University of Louisville Department of Medicine, 3University of Louisville Department of Microbiology and

Immunology

The liver is a primary target of alcohol toxicity, but chronic alcohol abuse can damage several other organs, including the lung. In fact, previous studies have shown that chronic ethanol exposure increases the incidence, severity, and mortality of sepsis-induced acute lung injury (ALI), and mortality is even higher in patients with underlying liver disease. Furthermore, recent work by this group has indicated that liver injury caused by alcohol may contribute to lung pathology. The ‘NIAAA’ model of acute-on-chronic alcohol exposure is a recently developed model that appears to better recapitulate alcoholic (steato)hepatitis. The impacts of this liver model on lung pathology have not been explored; the goal of this study was therefore to characterize simultaneously the effects of acute-on-chronic alcohol on the liver and lung. Methods. 10 W male C57Bl6/J mice were exposed to alcohol on the NIAAA protocol: 10 days liquid diet (5% EtOH), followed by a bolus gavage (5 g/kg). Animals were sacrificed 9 or 24 hours after the gavage, and liver and lung tissue, plasma, and bronchoalveolar lavage fluid (BALF) were collected for analysis. Additionally, some animals received an intratracheal administration of lipopolysaccharide (LPS, 1 mg/kg) Injury was determined histologically or biochemically using qRT-PCR and ELISA. Results. As expected, acute-on-chronic alcohol feeding caused significant steatohepatitis, characterized by increased plasma AST/ALT, as well as fat and neutrophil accumulation. Interestingly, acute-on-chronic alcohol exposure also caused transient lung injury. Similar to liver, this injury was characterized by a predominantly neutrophilic inflammatory response in lung tissue and BALF. Indices of the pulmonary inflammasome were also elevated by acute-on-chronic alcohol in the lung, analogous to previous findings in the liver. Additionally, acute-on-chronic alcohol pre-exposure exacerbated inflammatory cell infiltration into the airway space after LPS instillation.  Conclusions. Taken together, these data indicate that the newly developed NIAAA model of alcoholic (steato)hepatitis also develops concomitant lung damage. These results indicate, for the first time, that alcohol exposure is sufficient to induce pulmonary inflammatory injury and emphasize the parallel (and potentially interdependent) damage between the liver and the lung after alcohol exposure.  This work was supported, in part by NIH grants R01AA021978 (GEA, PI) and R01AA013353 (JR, PI) and by an internal grant from the University of Louisville. 

OVSOT 2016 Annual Meeting Poster Abstracts

Undergraduate Students

Water Contaminated with Atrazine Alters Expression of TPD52L1 with the Zebrafish Model

Devang H. Thanki, Jinyoung Lee, Sara E. Wirbisky, and Jennifer L. Freeman

School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, IN 47907

Atrazine is a commonly used herbicide in the Midwestern United States. Currently, Indiana uses about 26 percent of its land to grow corn, and atrazine is used in ample amounts, resulting in runoff that leads to contamination of drinking water. The Environmental Protection Agency has set the Maximum Contaminant Level (MCL) at 3 parts per billion (ppb) in drinking water but the herbicide is often found at higher levels. However, data suggests that atrazine can be harmful even at 3 ppb. Previous studies from our laboratory have shown atrazine alters gene expression associated with neuroendocrine and reproductive system function, carcinogenesis, and cell cycle regulation following developmental atrazine exposure at 0.3, 3, or 30 ppb. From these studies, it was shown that at 72 hours post fertilization (hpf), atrazine elicits alterations of the gene, tumor protein D52 (TPD52L1) in the zebrafish. Previous studies have identified TPD52L1 in cell proliferation and calcium signaling, along with regulating expression at the G2-M transition in breast cancer cells. The goal of this project was to characterize the expression of TPD52L1 during development of the zebrafish, as well as determine genetic alterations caused by developmental atrazine exposure. To characterize the expression of TPD52L1 throughout embryogenesis, zebrafish embryos were bred and embryos were collected at 24, 36, 48, 60, and 72 hpf. In addition, alteration in TDP52L1 gene expression following atrazine exposure was assessed at the same developmental time points. Analysis showed consistent levels of gene expression throughout embryonic development, a significant increase at 36 hpf in the 0.3 and 3 ppb treatments and significant increase at 60 hpf in all three treatments. This indicates that TPD52L1 expressions are induced by atrazine exposure at specific developmental time points. Western blot analysis is now being completed to determine if the gene expression changes are translated to protein alterations.

Differential Dose Effects from Chronic Taurine Exposure in Adolescent C57BL/6J Mice

Jamie Weimer, Lisa Massie, Yislain Villalona, Cecile Marczinski, and Christine Perdan Curran

Northern Kentucky University

Taurine is an amino acid found in high concentration in energy drinks and has shown some benefit as a neuroprotectant in the aged brain. However, our previous work and the work of others suggest that high-dose taurine can be neurotoxic. In our initial studies, we found sex differences with males have the greatest deficits in spatial and non-spatial learning and memory. We followed up those findings with a dose response study: providing 0, 0.06% or 0.12% taurine in drinking water beginning at P28 until early adulthood. Here we report findings from tests of anxiety, social recognition and alcohol preference, because human studies have shown that energy drinks can increase alcohol consumptions. Both groups of taurine mice buried more marbles than control mice, which is a general indicator of anxiety. However, the differences were not statistically significant. In a standard 3-chambered test of socialization and social recognition, the low-dose taurine mice spent the shortest amount of time in the chamber with a stranger mouse (P = 0.099). But the high-dose taurine mice spent the least time exploring the chamber with a new stranger mouse in the second phase of the test. We used a standard alcohol preference test with the concentration of alcohol increasing over several days. Low-dose taurine mice drank significantly more alcohol than control mice in the first phase (P= 0.44) and there was a trend for higher consumption in the second phase (P = 0.78). Both taurine-treated groups consumed more alcohol than controls in the final phase, but the differences were not statistically significant (P =0.105). Supported by ES020053 and GM103436.

Methods Development for Microbiome Analysis of PCB-treated Mice

Shelby Caudill, Nicholas Brinkman and Christine Perdan Curran

Northern Kentucky University

Polychlorinated biphenyls (PCBs) are widespread pollutants found in the human food supply that cause cancer, birth defects, and deficits in learning and memory. The microbiome is a term used to describe the microbes that live in and on the body. Recent reports indicate that pollutants in our food can change the intestinal microbiome and harm human health, including the developing brain. PCBs are among the pollutants suspected of changing the microbiome, but the majority of work has been done in animal studies using adult rodents. We are developing a mouse model to examine the effects of PCB exposure during gestation and lactation, because the developing fetus and newborns are most susceptible to PCB effects. Due to the smaller sample sizes in a mouse model, we compared three methods for extracting DNA from feces and intestinal contents of dams and pups. We concluded that a commercial soil DNA extraction kit was most effective in terms of both quantity and quality of DNA. Future work will include a metagenomics 16S rRNA analysis to identify the major microflora in PCB-treated and control dams and pups. Supported by ES020053 and GM103436.

The cognitive effects of chronic taurine exposure in C57BL/6J mice: a dose-response study

Lisa Massie, Jamie Weimer, Yislain Villalona, Cecile Marczinski, and Christine Perdan Curran

Northern Kentucky University

The amino acid taurine has shown positive effects on the cardiovascular system, nervous system, and immune system when absorbed from a normal diet; however, energy drinks often contain high levels of taurine that could exceed recommended limits. While little research has been conducted on the effects of taurine supplements, our previous studies have shown learning and memory deficits in adolescent mice chronically treated with 0.12% taurine in drinking water. The greatest deficits were seen in male mice. To follow up on these studies, we conducted a dose-response study using 0, 0.06, and 0.12% taurine in male and female C57BL/6J mice. Mice were treated from postnatal day 28 (adolescence) through completion of behavioral testing (early adulthood). Our behavioral battery includes three tests of behavior and three tests of learning and memory. Here we report results of tests of spatial and non-spatial learning and memory. We found significant deficits in non-spatial learning and memory (P < 0.05) in the Novel Object Recognition test at both doses used. In the Morris Water Maze, we found a significant sex * treatment interaction in the Reverse Phase, but the trend was the opposite of our original findings. Males on high-dose taurine out-performed low-dose taurine mice and controls. The high-dose males also performed significantly better than high-dose females (P < 0.01). Together, both studies suggest caution when consuming high quantities of energy drinks during adolescence and early adulthood despite improved performance on some cognitive tests. Supported by ES020053 and GM103436.

Copper (Cu) Distributions in different Organs of Cu-overload and Cu-deficient Rats

Vivien Lai, Sherleen Fu, and Wei Zheng

School of Health Sciences, Purdue University, West Lafayette IN 47907, 915 W State St B427, West Lafayette, IN 47906

As an essential metal in the human body, Cu serves as an indispensable cofactor for diverse enzymes and proteins.  A balanced Cu homeostasis plays an important role in maintain the normal development and function of the human body. Either excessive or deficient of Cu can cause severe health issues, particularly the brain damages such as Wilson’s disease with Cu overload (CuO) and Menkes disease due to Cu deficiency (CuD). In the modern daily life, over 90% of the water piping is made of Cu, it is a potential risk for people to acquire excessive Cu from the drinking water. The dairy-rich diet is considered as a major reason to cause Cu deficiency in the population. To understand the mechanisms by which CuO or CuD causes neurological disorders, it is essential to establish animal models to study the Cu homeostasis in the body. This study was designed to generate the CuO and CuD models in Sprague-Dawley rats by ip injections with 3 mg Cu/kg as CuCl2 and 10 mg/kg of ammonium tetrathiomolybdate (Cu chelator), respectively, for 12 days. The control and CuO groups were fed with the normal AIN-93A diet, while animals of the CuD group were fed with the AIN-76A Cu deficient diet. Deionized water without Cu was provided as drinking water for all animals. At the end of the treatment, the organs of liver, brain, heart, lung, spleen, and kidney were collected for the measurement of Cu level using atomic absorption spectroscopy. By comparison with the Cu level in control livers, the Cu content in the CuD liver was significantly decreased by 3.2 fold (4.418± 0.245 ug Cu/g tissue in controls vs. 1.379± 1.212 ug/g in CuD; n=7/group, p<0.0001). In CuO studies, a significant increase of liver Cu level was detected in the CuO animals (430.8± 187.8 ug Cu/g tissue) when compared to the control (4.418± 0.245 ug Cu/g tissue; n=7, p<0.01). These data indicate that the CuO and CuD treatments significantly alter the Cu level in the liver. AAS analyses of Cu contents in other organs in both CuO and CuD animals are currently in progress.

The long-term effects of early life PCB exposure on the liver

Bryana Braxton1, Kelsey Klinefelter1, Anna Lang2, Heather Clair2, Matt Cave2 and Christine Perdan Curran1

1.) Northern Kentucky University Department of Biological Sciences, 2.) University of Louisville School of Medicine Department of Pharmacology and Toxicology

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate and biomagnify in the food chain. PCBs can also be found in old electrical equipment, caulks and wood treatments of older homes, and as a byproduct of paint production. PCBs have been linked with learning, memory and motor deficits in children exposed during pregnancy and lactation. Our previous studies in mice identified two genes associated with increased susceptibility to developmental PCB neurotoxicity. We also discovered that PCBs have adverse effects on metabolism as animals reached old age. This study was designed to determine if these aged animals also suffer liver damage. Preserved livers were embedded in paraffin, cut into 4m sections on a microtome, and stained with hematoxylin and eosin. PCB-treated mice from three different genotypes were compared with corn oil-treated control animals. We also analyzed gene expression using multiplex real-time qPCR and assessed glycogen deposits using a Periodic acid-Schiff (PAS) stain. We found striking differences in the expression of CYP2B10 and PNPLA3 based on sex and treatment although histological findings were not remarkable different across groups.

Prion Protein Does Not Play a Major Role in Metal Uptake into the Brain: Implications of PrPC Natural Function

Erin Kay, Dan Cholger, Xue Fu, Wei Zheng

School of Health Sciences, Purdue University, West Lafayette IN 47907, United States

The prion protein (PrP) is known to play a critical pathogenic role in the prion disease, an infectious spongiform encephalopathy typically seen in the “mad cow” disease or prion protein scrapie (PrPSC). The natural function of cellular prion protein (PrPC) is hypothesized to be a Cu-binding protein with a potential role in cellular Cu transport and uptake. While this protein is known to be located ubiquitously throughout the regions of the brain, the presence and relative abundance of this protein in the blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier (BCB) are unknown. This study was designed to test the hypothesis that PrPC may play roles in metal uptake, therefore we would expect to find a higher PrPC expression in the BCB over a neuronal cell type. A Z310 cellular model (BCB) and a N27 cellular model (dopaminergic neuron) were chosen to directly compare PrPC

expression between a brain barrier structure and a neuronal cell type. We employed qPCR analysis following a 24 hour exposure to 250 M MnCl2 treated cells of both types and controls of both types. Our data shows that there is a two-fold greater expression of PrPC in N27 cells (Cq: 0.050+0.011) compared to Z310 cells (Cq: 0.021+0.006) in untreated control groups. Manganese treatment did not affect PrPC expression in either cell type and are statistically the same as their appropriate control. These findings suggest that PrPC does not play a major role in metal uptake into the brain, but rather suggests that it may serve a protective function for neuronal cells against metal induced toxicity.

Master’s Degree Poster Abstracts

COMPARATIVE DOPAMINERGIC NEUROTOXICITY OF HETEROCYCLIC AMINES IN PRIMARY MIDBRAIN CULTURES

Cruz-Hernandez A1, Agim ZS2, Montenegro P2, Rochet JC2, Cannon JR1

1.School of Health Sciences, Purdue University, West Lafayette, IN, USA.2.Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West

Lafayette, IN, USAThere are >20 known heterocyclic amines (HCAs) that are components of the diet. HCAs are primarily formed during high-temperature meat cooking. HCAs have been primarily investigated as mutagens. However, emerging data suggests they may be neurotoxic. For example, our lab has found that the most abundant HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is selectively neurotoxic to dopaminergic (DA) neurons in rat primary midbrain neuronal cell culture. The aim of this study is to determine the toxicity of additional prominent dietary HCAs. We tested the following dietary HCAs that represented multiple HCA subclasses (imidazoazaarenes and ß-carbolines): 3-methyl-3H-imidazo[4,5-f]quinolin-2-amine (IQ), 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-Amino-3,4-dimethyl-3H-imidazo[4,5-f]quinolone (MeIQ), 3,4,8-trimethylimidazo[4,5-f]quinoxalin-2-amine (4-8 DiMeIQx), and 2-Amino-9H-pyrido[2,3-b]indole (A alpha C). Primary midbrain neuronal cultures were prepared using E17 rat embryos. This system was used to test DA neurotoxicity at a range of HCA doses (100nM – 5µM). Cells were stained for tyrosine hydroxylase (TH, marker for DA neurons) and microtubule-associated protein (MAP2, pan-neuronal). Viability was assessed by countingMAP2- and TH-immunoreactive primary neurons in a blinded manner. One-way ANOVA with Dunnett’s post-hoc test was used for statistical analysis. A statistically significant decrease in the percentage of DA neurons emerged (lowest dose where selective toxicity was detected) at the following concentrations: MeIQ ≥ 200 nM (n=6); A alpha C ≥ 200 nM (n=6); PhIP ≥1 µM (n = 3); harman ≥1µM (n = 6); and IQ ≥ 5µM (n = 6). 4-8DiMeIQx, norharman and MeIQx did not produce statistically significant toxicity up to 5 µM. Thus, our data shows that multiple HCAs produce selective DA neuron toxicity in primary midbrain culture and that compounds from multiple HCA subclasses were able to produce such neurotoxicity. Further, the dose required to induce detectable cell loss varies by HCA, with MeIQ and A alpha C being the most potent HCAs tested. Mechanistic studies are underway to determine how structural differences influence mechanisms of HCA-induced neurotoxicity and to learn more or how HCA exposure may possibly be important in PD.

Effect of CRISPR-mediated genetic edits of the hs1,2 enhancer on ε IGH expression in U266 Cells

Siham Abdulla, Andrew Snyder, Sulentic Courtney

Wright State University, 3640 colonel glenn HWY HEALTH SCIENCE ROOM #206

The 3’ regulatory region (3’IghRR) within the Mouse Immunoglobulin heavy chain locus (Igh) is a significant target for a number of toxins such as TCDD which can induce marked effects on Ig expression. This regulatory region has enhancer activity and consists of four enhancers (hs3a, hs1,2, hs3b, hs4) in mice, while in humans it is duplicated and has only three enhancers (hs3, hs1,2, hs4). In addition, the human hs1,2 enhancer includes a 53 bp invariant sequence which is polymorphic and can be repeated up to 4 times, and also has several transcription factor binding sites that could be a target for different toxins. Murine hs1,2 lacks the invariant sequence and is not polymorphic. Polymorphisms within the hs1,2 enhancer have been correlated to a number of autoimmune diseases such as systemic lupus erythematosus and celiac disease. Previous work has shown the mouse 3’IghRR helps drive Ig expression and regulates class switch recombination. Also, supportive data on rodent and human cells has shown the hs1,2 enhancer has the strongest enhancer activity, while other studies identified the hs4 enhancer as a strong enhancer as well. Since no study has determined the role of the human hs1,2 enhancer polymorphism on Ig expression and secretion in a plasma cell line, in this study we are targeting the human 3’IGHRR in the multiple myeloma plasma cell line U266 by using CRISPR/CAS9 to induce genetic editing within the hs1,2 enhancer and test the impact of this mutation on ε functional transcript and IgE secretion. Previous and ongoing gene editing studies in our lab on mature B cells demonstrated so far a direct association between the number of invariant sequences within the hs1,2 enhancer and levels of secreted IgG and IgM. The U266 cells have been transfected with a CRISPR/Cas9 plasmid and were sorted for GFP expression. GFP-positive cells have been grown into clonal populations by limiting dilution and genotyping of those clones is underway. So far, a number of clones have been successfully edited, where the repeated sequence (53 bp invariant sequence) has been reduced to different number of repeats, one, two, and three repeats. The next step we are going to do is evaluating the functional impacts of these genetic edits on ε functional transcript and IgE secretion, in addition to assessing the U266 growth rate. So, at the end of this study, we will determine if the hs1,2 enhancer in humans is a key transcriptional regulator in a plasma cell line.

Elucidating the Physiological and Toxicological Role of the AhR in Human Ig Expression Using CRISPR/Cas9 Gene Editing.

Nicole Panstingel, Bassam Kashgari, and Courtney Sulentic

Boonshoft School of Medicine - Wright State University, 3640 Colonel Glenn Hwy Dayton, OH 45435

The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. AhR has been shown to mediate the toxic effects of the environmental toxin 2,3,7,8-tetrachlorododibenzo-p-dioxin (TCDD). In various animal models, TCDD inhibits the differentiation of B-cells into antibody secreting cells and the expression of immunoglobulin (Ig). Within the immunoglobulin heavy chain locus (IGH) are 3’ regulatory regions (3’RR). The TCDD-induced inhibition occurs in an AhR dependent manner that is partially regulated by the 3’RR. In humans, the effect of TCDD on the immunoglobulin heavy locus (IGH) is still unclear; however in human derived cell lines, a TCDD-induced suppression of IgG has been shown. Because of the growing number of auto-immune disorders linked to the polymorphism within the hs1,2 region of the 3’RR in IGH, determining the role of AhR in the IGH expression would provide valuable insight into possible health risks and aid in the development of possible treatments.

Previous model systems utilized to explore the role of AhR in suppression of human IGH by TCDD employed the use of short hairpin loop RNA (shRNA) interference to alter the expression of AhR in a CL-01 cell line isolated from a Burkitt’s lymphoma. However, there were limitations to this model, in that overtime the modifications could be lost in the subsequent clonal populations, modifications affected both alleles making it difficult to interpret the results and where the modifications were made could not be controlled making it impossible to attribute effects to AhR alone. To overcome these limitations, CRISPR/Cas 9 genetic editing technology was utilized to disrupt the endogenous AhR expression in a CL-01 cell line. The CRISPR plasmid was designed to specifically bind to the transcription start site of AhR, where subsequently a cut was made, which could ultimately result in a frameshift mutation. The advantages of CRISPR are: the modifications are permanent and mono-allelic targeting, making it clearer to attribute effects seen to AhR function.

Knockdown of AhR was verified by western blot. Immunoglobulin secretion will be measured using ELISA and a relative quantification of transcripts of the human IGH will be measured using RT-PCR. The results obtained from previous ELISA experiments show a decrease in AhR expression inhibits the overall secretion of IgG independent of TCDD exposure. From RT-PCR, it was shown that a decrease in AhR expression inhibits the germline transcripts of the γ1 region of the IGH. The results obtained suggest a significant physiological role of AhR on IgG expression and that a functional AhR may be necessary for TCDD induced inhibition of other classes of immunoglobulins. From this newly developed model, it is hopeful these effects will be seen.

Role of the AhR Antagonist in Human Immunoglobulins Expression in Influence Interactions of the AhR with other Cytosolic Signaling Proteins

Nasser Alhamdan, Kaulini Burra and Courtney Sulentic

Wright State University, Dayton, Ohio

The immunoglobulin heavy chain (Igh) locus is expressed only in B-lymphocytes and encodes the heavy chain protein that is an essential component of antibodies. The Igh gene has a large transcriptional regulatory region (i.e. 3’IghRR) containing enhancers that assist the B cell to produce different Ig isotypes. In humans, there are two regulatory regions and each has three enhancers (hs3, hs1.2, and hs4) whereas mice (the primary model used to study Igh gene expression) have only one regulatory region but four enhancers (hs3A, hs1.2, hs3B, and hs4). The human hs1.2 enhancer is known to be polymorphic and is associated with multiple autoimmune disorders. The ligand-activated transcription factor, aryl hydrocarbon receptor (AhR), has been shown to inhibit mouse 3’IghRR activity when activated by the high affinity environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In animal models, inhibition of the 3’IghRR appears to mediate TCDD-induced inhibition of B-cell differentiation into antibody-secreting cell. In order to elucidate the role of the AhR in human Ig expression and isotype switching, we used an AhR antagonist (AhRA). Recent studies in our lab using a mouse B-cell line demonstrated that AhRA inhibits AhR-mediated signaling and reverses the TCDD-induced inhibition of antibody secretion. In contrast, treatment of a human B-cell line with AhRA induced an unexpected increase in IgG secretion. Therefore, we hypothesized that the AhRA, which prevents the AhR from translocating to the nucleus and binding DNA, could influence interactions of the AhR with other cytosolic signaling proteins . The objective of the current study was to evaluate the effect of the AhRA on the activation (i.e. phosphorylation and activation of downstream transcription factors) of signaling proteins by Western blot analysis and by luciferase reporter plasmids. Our results to date suggest that the AhRA has no effect on the phosphorylation of STAT3, Src, or Erk proteins, and no effect on NFκB transcriptional activity. Ongoing studies are focused on analyzing more signaling proteins, such as other STAT proteins and AP-1 transcriptional activity. Determining the role of the AhR in human Ig expression could provide new insight into potential environmental triggers of immune disorders and provide new targets for drug development.

PhD Student Poster Abstracts____________________________________________________________________________

Functional characterization of the polymorphic hs1.2 enhancer using CRISPR/Cas9 gene editing and nanopore sequencing technology

C Buckner, A Snyder, S Abdulla, C Sulentic

Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, 206 Health Sciences Bldg., 3640 Colonel Glenn Hwy., Dayton,

Ohio 45435

The human immunoglobulin heavy chain gene locus (IGH) has two 3’ regulatory regions (3’IGHRR), each containing three enhancer regions (hs3, hs1.2, hs4). In animal models, the 3’IghRR regulates Igh expression and class switching (CSR) to different Ig isotypes. The 3’IGHRR hs1.2 enhancer in humans is polymorphic in that an invariant sequence (IS) can be repeated one to four times in tandem. The hs1.2 polymorphism is of interest due to its association with several human autoimmune disorders and its sensitivity to exogenous substances such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). In mouse models, TCDD inhibits the hs1.2 enhancer and 3’IghRR activation, which correlates with Ig inhibition. However, in humans TCDD activates the hs1.2 enhancer, decreases IgG secretion and increases IgE secretion, suggesting differences in hs1.2 activity and overall 3’IGHRR function in humans. Using a human B-cell line that can be induced to secrete antibodies and undergo CSR, we utilized CRISPR/Cas9 gene editing to target the hs1.2. Several clones exhibited a functional effect on IGH expression with a reduction in the number of hs1.2 IS repeats within one or both of the 3’IGHRRs. The exact gene editing has not been fully characterized and may likely involve insertions and deletions within the remaining IS, which complicates interpretation of the functional effects on IGH expression. To further characterize the gene edits within our clones, the current objective is to use a new sequencing technology involving nanopores and long sequence reads. Successful implementation of this technique should provide long-range sequencing data that is impossible to achieve with standard sequencing due to redundant sequences within and around the 3’IGHRR, and the identical nature of the 3’IGHRRs, excluding the number of IS repeats. Linking the functional effects to a specific genetic profile within the hs1.2 enhancer will be the first direct analysis of the role of the polymorphic hs1.2 enhancer in human Ig expression.

PCB exposure and diet alter the hepatic proteome in the progression of steatohepatitis

Josiah Hardesty, Banrida Wahlang, Heather Clair, Hongxue Shi, Daniel Wilkey, Cameron Falkner, Michael Merchant, Russell Prough, Matt Cave

University of Louisville, 505 S. Hancock St. Louisville, KY 40202

Poly-chlorinated biphenyl (PCB) exposure has been associated with an increased prevalence of liver disease and diabetes in epidemiological studies and worsens liver disease in an obesity animal model. PCBs are thought to be receptor based toxicants described as being either dioxin or phenobarbital like whose signaling is mediated by the Aryl hydrocarbon (Ah) - or Constitutive androstane receptors (CAR), respectively. Recent studies demonstrate that PCBs inhibit Epidermal Growth Factor Receptor (EGFR) signaling and diminished protein expression of downstream effector proteins. The effects of diet and PCB exposure on the hepatic proteome have not been previously characterized. This led us to explore the hypothesis that PCBs alter the hepatic proteome through receptor based mechanisms. Methods: Mice were fed either a control diet or high fat diet and treated with either corn oil or Aroclor1260 (20mg/kg) in corn oil by gavage. Livers were removed and prepped for LC/MS/MS analysis and western blot analysis of proteins of interest. Identified proteins were processed through searching the Mus protein databank by MASCOT. Peptide abundance normalized to total spectra and identified with a FDR of .05 were statistically compared by two-way ANOVA. MetaCore software was used to identify shared pathways of significantly different proteins. Results: Aroclor 1260 exposure diminished the protein expression of antioxidant proteins and proteins involved in NAD+ synthesis. Aroclor 1260 exposure diminished fatty acid oxidation which we propose contributes to the worsening of steatosis. Interestingly PCB exposure increased expression of proteins involved in inflammatory response and metabolism of linoleic metabolism to isoleukotoxins which we hypothesize worsens steatohepatitis. Transcription factors predicted to be downregulated by PCB exposure were HNF4, cMyc, and STAT3 which are downstream of EGFR. A decrease in HNF4 protein expression with Aroclor 1260 exposure was validated by western blot analysis. Conclusion: Aroclor 1260 exposure diminished expression of proteins most tightly associated with antioxidation pathways, fatty acid oxidation, and increased expression of proteins associated with inflammatory response and toxic metabolite formation. Future directions will be to establish how these proteomic alterations occur and if intervention can prevent the NASH pathologic features previously established with PCB exposure and a HFD.

Fibroblast Growth Factor 1 Ameliorates Diabetic Nephropathy Through an Anti-inflammatory Mechanism

Qian Lin, Lu Cai, Yi Tan

University of Louisville, 570S Preston St , Baxter research building suite 311, Louisville,KY,40202

A recent study demonstrated metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis (Nature. 2014; 513:436). But its strong mitogenic activity hampers its wide use in diabetes. We have recently engineered a FGF1 partial agonist carrying triple mutations (FGF1 HS△ ) that diminish the ability of FGF1 to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1 HS△ exhibited a major loss in proliferative potential, while preserving the full metabolic activity of wild-type FGF1 (FGF1WT). In the present study, we further tested the potential of FGF1 HS△ in treatment of diabetic nephropathy (DN). Treatment of db/db type 2 diabetes (T2D) with FGFWT and FGF1 HS△ (0.5 mg/kg) every other day for 2 months, both FGFWT and FGF1 HS△ significantly reduced blood glucose, urinary albumin, and prevented glomerular hypertrophy, mesangial matrix expansion and renal fibrosis. Similarly, treatment of streptozotocin (STZ)-induced type 1 diabetes (TID) with FGF1WT and FGF1 HS△ also significantly prevented diabetes-induced renal morphological and functional changes, but without significant effects on blood glucose levels. These results imply that the renal protection of FGFWT and FGF1 HS△ is independent of their insulin sensitizing activity. Since renal inflammation is a critical cause of DN, and FGF1 exhibits a potent anti-inflammatory property (Nature. 2014; 513:436), we hypothesized that the protection of FGF1 against DN is associated with its anti-inflammatory capability. As expected, both FGF1WT and FGF1 HS△ treatments significantly reduced the inflammatory cytokines (TNF-α and IL-6) expression and CD68+ macrophage infiltration in renal tissues in both T1D and T2D, which were accompanied by an inhibition of inflammatory signal (JNK/NF-κB) activation. Direct exposure of renal mesangial cell to high glucose or TNF-α induced similar inflammatory and fibrotic responses as observed in diabetes, which could be prevented by FGF1WT and FGF1 HS△ pre-treatment. More importantly, administration of FGF1 HS△ to 9-month-old db/db mice completely prevented the further development of DN with remarkable amelioration of renal inflammation and fibrosis. These results demonstrate that FGF1 prevents DN largely via inhibiting renal inflammation. FGF1 HS△

might be a therapeutic approach for the treatment of DN without promoting undesired tissue proliferation.

Induction of Cell Cycle Pathways In Human Keratinocytes At Early Stages Of Chronic Exposure To Low Arsenite

Laila Al-Eryani1, Sabine Waigel2, Samantha Jenkins1, Vennila Arumugam2, J. Christopher States1

Departments of 1Pharmacology and Toxicology, and 2Medicine, University of Louisville, Louisville, KY

Background: Arsenic is a metalloid that is naturally prevalent in the earth’s crust and widely distributed in air and water. The toxicity of long-term exposure to arsenic is associated with a broad range of health problems including cancer, especially skin cancer. Arsenic-induced carcinogenesis mechanisms are not yet clear and several mechanisms have been proposed including epigenetic alterations. Immortalized human keratinocytes (HaCaT) are an in vitro model for normal human keratinocytes and are commonly used to study epidermal carcinogenesis. Chronic exposure to low levels of arsenite malignantly transforms HaCaT cells in vitro. Expression profiling of mRNAs and small RNAs have not yet been performed in human keratinocytes during chronic arsenite exposure or in arsenite-transformed keratinocytes. Hypothesis: Arsenic causes exposure- and time-dependent mRNA and miRNA expression changes that drive carcinogenesis. Methods: HaCaT cells were exposed to 0 or 100 nM sodium arsenite for 3 and 7 weeks. Total RNA was purified from exposed/unexposed HaCaT cells as well as HaCaT transformed by the Waalkes laboratory (0/100 nM sodium arsenite for 28 weeks). Small RNA and mRNA expression was assayed using Affymetrix microarrays. Differential expression with fold change ≥1.5 and p-value ≤0.05 was determined using Partek Genomic Suite™ and pathway analysis using MetaCore™ software (FDR ≤0.05). Results: Forty-seven small RNAs were differentially expressed at 3 weeks; 116, at 7 weeks. Two miRNAs, miR-548a-3p and miR-1254, differentially expressed at both time points are reported to be dysregulated in breast and early stages of non-small cell lung cancers respectively. Fourteen mRNAs were differentially expressed at 3 weeks; 723, at 7 weeks. Of the 14 mRNAs, only SAA1 and SAA2 populated a pathway (Immune response_IL-6-induced acute-phase response in hepatocytes). These genes are induced in renal and lung cancers. Several of the 723 mRNAs populated mostly cell cycle/cell cycle regulation pathways but also cytoskeleton remodeling, apoptosis and survival, proteolysis and epithelial to mesenchymal transition regulation pathways. Genes populating multiple pathways include CDC25A, CDK2, Cdt1, cyclin D, cyclin D3, DP1, E2F1, MCM2, MCM4 MCM5, RING-box protein 1, SKP1 and SKP2. Conclusions: Our data provide strong evidence of time-dependent changes in small RNA and gene expression profiles in human keratinocytes chronically exposed to arsenite associated with carcinogenesis in other systems. The dramatic change in the number of differentially expressed small RNAs and mRNAs suggest that the cells are still able to adapt to stress at 3 weeks while the transformation related changes are evident after 7 weeks exposure. Supported in part by NIH grant R21ES 023627.

The Role of Optineurin in Golgi Fragmentation during Parkinson’s Disease

John Pierce Wise, Jr., Jason R. Cannon

School of Health Sciences, Purdue University, West Lafayette, IN, United States, 550 Stadium Mall Dr, West Lafayette, IN 47907-2051

Fragmentation of the Golgi body is an event observed in many neurodegenerative diseases including Alzheimer’s, ALS, and Parkinson’s diseases (PD). This event is observed early in disease pathogenesis, typically occurring prior to formation of protein aggregates; yet the mechanisms and purposes of Golgi fragmentation are largely unknown. Optineurin (OPTN) is genetically linked to ALS and glaucoma, and is known to have numerous cellular functions including maintenance of Golgi structure; depletion or overexpression both induce Golgi fragmentation. Given that PD patients have an elevated risk of developing glaucoma and the OPTNM98K variant was reported to increase risk of developing PD, we predict OPTN has an important role in PD pathogenesis. We previously demonstrated OPTN expression and colocalization in dopaminergic neurons of the substantia nigra. OPTN colocalization with LC3 and α-synuclein increases in rotenone rat models of PD, prior to cell loss; these data indicated increased OPTN expression modulation may be related PD pathogenesis. Given these data and OPTN’s known roles in Golgi structure maintenance, we hypothesized OPTN served a role in Golgi fragmentation. Here, through immunofluorescence and confocal microscopy, we show the presence of Golgi fragmentation in different stages of an in vivo rotenone model of PD. Further, the Golgi fragments colocalized with OPTN. While fragmentation appears to be present in all stages of our model, colocalization of OPTN to Golgi fragments diminishes in end-stage models, likely around the time of neurotoxicant-induced cell death. Future work will consider OPTN’s role in the relationship between Golgi fragmentation and autophagic dysfunction and whether these pathways may serve as therapeutic targets.

In vivo Visual Reporter System for Endocrine Disrupting Chemicals using Transgenic See-through Japanese medaka Oryzias latipes

Ahmed Abdel-moneim, Cecon T. Mahapatra and Maria S. Sepúlveda

Department of Forestry and Natural Resources, Purdue University, 195 Marsteller Street, Forestry Building. West Lafayette, IN 47907

Water bodies receive large amounts of urban, industrial and agricultural wastes, holding numerous endocrine disrupting chemicals (EDCs). Exposure to these chemicals, particularly during sexual differentiation in fish, can cause gonadal intersex to develop. Thus, there is an urgent need to develop tools for screening and testing of ~ 87,000 chemicals in the environment for possible endocrine disrupting effects. We hypothesize that a reliable hormone responsive gene in an animal model that could be sexed non-invasively very early during development can be used as a biomarker to develop a robust in vivo screening system for EDCs. The main objective of this study is to develop a novel in vivo visual reporter system for rapid detection of EDCs. First, we identified a gene responsive to EDC exposure in an animal model that could be sexed non-invasively very early during development (Japanese medaka SK2 line). Sex is chromosomally determined in medaka and thus genetic sex can be known very early during embryo development (7 dpf). Ovarian structural protein 1 (osp1) has a strong female-specific expression starting at 12 dpf (days post fertilization) and its expression is down-regulated in females upon exposure to synthetic androgens. Next, we built a pOSP1-AcGFP (promoterOSP1-Aequorea coerulescens green florescence protein) Japanese medaka transgenic line with osp1 promoter region driving the expression of a reporter protein, AcGFP. Finally, we are in the process of implementing the use of this transgenic line in an in vivo visual reporter system for identifying putative EDCs in a sensitive, non-lethal and cost-effective way. Overall, our results support the hypothesis that molecular biomarkers are sensitive tools that can be used for early prediction of the effects of EDCs on fish and are ideal endpoints in wide-scale EDC screening assays.

Decreased human NAT1 or rat NAT2 activity elevates cellular acetyl-coenzyme A levels

Marcus Stepp, Mark Doll, and David Hein

Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky

Background/introductionRecent work within our lab has confirmed a novel role of human arylamine N-acetyltransferase 1 (NAT1) to catalyze hydrolysis of acetyl-coenzyme A (AcCoA) in a folate dependent manner. This hydrolytic activity has been confirmed with rat arylamine N-acetyltransferase 2 (NAT2), the rat ortholog of human NAT1. In this current study, we investigated the effects of altered levels of human NAT1 or rat NAT2 activity on cellular AcCoA levels.

Hypothesis/ObjectiveCells with decreased human NAT1 or rat NAT2 activity will have increased AcCoA levels.

MethodsRecombinant human NAT1 and rat NAT2 were generated and assayed for folate-dependent AcCoA hydrolysis. Rat NAT2 enzymatic activity and endogenous AcCoA levels were measured in tissue samples and embryonic fibroblasts isolated from rapid and slow acetylator rat NAT2 congenic rats. Human breast cancer cell lines (MDA-MB-231 and MCF7) were modified by CRISPR/Cas9 to knockout human NAT1. The parental and modified cell lines were characterized for enzymatic activity and endogenous AcCoA levels.

ResultsRecombinant human NAT1 and rat NAT2 catalyzed AcCoA hydrolysis in a folate-dependent manner. Recombinant rat rapid-NAT2, as well as tissue samples from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent AcCoA hydrolysis at higher rates than rat slow-NAT2 (all comparisons, p<0.01). Rat embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than embryonic fibroblasts derived from slow NAT2 rats (p<0.01). The human NAT1 knock out cell lines had no detectable p-aminobenzoic acid acetylation activity compared to their parental cell lines. Human NAT1 knockout MDA-MB-231 and MCF7 breast cancer cell lines had elevated cellular AcCoA levels compared to the parental cell lines, (p<0.001 and p<0.05, respectively).

Conclusion:The results support a role for human NAT1 and rat NAT2 in regulating endogenous levels of AcCoA.

Grant support: This study was partially supported by USPHS grant T32-ES011564.

The Role of Aryl Hydrocarbon Receptor in PCB-induced Fatty Liver Disease in Mice

Hongxue Shi1, Heather B. Clair2, Josiah E. Hardesty2, Jian Jin1, K. Cameron Falkner3, Russell A. Prough2, Ming Song3, Matthew C. Cave1, 2, 3

1Department of Pharmacology and Toxicology, 2Department of Biochemistry and Molecular Genetics. 3Department of Medicine Division of Gastroenterology, Hepatology and Nutrition, University of Louisville, School of Medicine, Louisville, KY 40202, 505 S. Hancock St., Rm 542C

BackgroundIn epidemiology studies, exposure to the pollutants polychlorinated biphenyls (PCBs) have been associated with steatohepatitis and diabetes with insulin insufficiency. In terms of their structure and toxicity, PCBs are divided into dioxin-like (DL) and nondioxin-like (NDL) PCB classes. Interaction of planar DL PCB with the aryl hydrocarbon receptor (AhR) has been shown to be involved in fatty liver disease.Hypothesis and Objective We hypothesized that AhR plays a critical role in fatty liver disease of mice treated with a clinically relevant PCB (DL/NDL) mixtures. MethodsMale C57BL6/J mice (8-10 weeks) on TekLab TD06416 diet were treated by one-time gavage with Aroclor 1260 (20 mg/kg), PCB126 (20 µg/kg), and Aroclor 1260 with 0.1% PCB 126 added in corn oil for 2 weeks. Fasting blood glucose was measured using glucometer. After euthanasia, tissues were obtained for measurements of gene expression by real-time PCR and hematoxylin–eosin (H&E) staining was performed to observe histological changes. Hepatic triglycerides and free fatty acid contents were measured using InfinityTM Triglycerides kit and Free Fatty Acids, half micro test kit, respectively. ResultsPCB exposure exhibited a trend toward increased liver weight and decreased epididymal adipose tissue weight. Mice treated with Ar1260 increased hepatic constitutive androstane receptor (CAR) target genes, cytochrome Cyp2b10 expression, and increased hepatic Pnpla3, a gene expression previously shown to act against NASH. Scavenger receptor CD36 was upregulated by either Ar1260 or PCB126, but downregulated by Ar1260/PCB126 treatment. PCB126- and Ar1260/PCB126-treated mice exhibited increased AhR target gene Cyp1a2 expression, but not by Ar1260 alone. Pnpla3 expression was decreased under condition involving PCB126. PCB126-treated mice demonstrated increased hepatic triglycerides and free fatty acid content associated with hepatic steatosis. The SCD1 gene was downregulated in all groups. The liver derived hepatokines were also altered. The insulin-like growth factor 1(IGF1) was increased, and fibroblast growth factor 21 (FGF21) was decreased after treatment with either Ar1260 or PCB126, while Ar1260/PCB126 exposure attenuated expression of both IGF1 and FGF21 expression. The hepatic fatty acid oxidation genes, including Cpt1 and Cyp4a10 were upregulated by PCB126 administration. The fasting blood glucose was decreased by PCB126, but this condition resulted in increased levels of gluconeogenic enzyme Pck1.ConclusionsThe DL PCB and NDL PCB exhibits differential effects on fatty liver disease due to activation of several different transcriptional factors. There is a complex interaction between DL PCB and NDL PCB in the mixture when compared to each alone. Therefore, AhR is essential for clinically relevant PCB mixture-induced fatty liver disease. AcknowledgementsSupported by grant 1 R01 ES021375 from the National Institute of Environmental Health.

Critical role of mammalian target of rapamycin (mTOR) in liver damage caused by VC metabolites in mice.

Anna L Lang1, Brenna R Kaelin1, Heegook Yeo1, Cierra N Sharp1, Gavin E Arteel1, Juliane I Beier1

1Department of Pharmacology and Toxicology, Louisville, KY 40206, USA.

Background. Vinyl chloride (VC) is a prevalent environmental toxicant that causes liver injury at high, acute exposures. However, we have shown that experimental VC metabolites at concentrations that are not overtly hepatotoxic sensitized the liver to hepatotoxicity caused by lipopolysaccharide. These effects correlated with changes in hepatic energy metabolism. The kinase mTOR is a key regulator between pathways that control the balance of cellular energy metabolism and autophagy in response to stress. Therefore the role of mTOR was determined in this model via inhibition pharmacologically with rapamycin.

Methods. C57Bl/6J mice received chloroethanol (CE, 50 mg/kg, i.g.), a major VC metabolite, followed by rapamycin (1 mg/kg, i.p.) one hour later; 24 hours after CE, lipopolysaccharide was administered (LPS, 10 mg/kg, i.p., t=0). Animals were sacrificed at t=0, 4, and 24 hours. Plasma and liver samples were collected for determination of inflammation, histology, and liver damage.

Results. Livers of control mice (vehicle, CE or CE + rapamycin) showed no signs of liver injury, inflammation, or increased cell death. CE significantly enhanced all indices of LPS induced liver damage and inflammation characterized by a significant increase in plasma transaminase levels, pro-inflammatory markers (PAI-1, TNF-α), neutrophil accumulation, and necro-inflammatory foci. However, the inhibition of mTOR by rapamycin significantly attenuated hepatic damage caused by LPS + CE. Transaminase levels were attenuated, neutrophil infiltration was decreased, and inflammatory markers were inhibited.

Conclusion. Taken together, these results suggest that mTOR is critical in mediating CE (as a surrogate for VC exposure) and LPS-induced liver injury. Previously, over activation of mTOR has been shown to inhibit autophagy resulting in increased liver damage. Therefore, the results here also support the hypothesis that autophagy may play a critical role in the development of liver injury due to VC exposure.

Developmental toxicity after embryonic trichloroethylene (TCE) exposure in zebrafish (Danio rerio)

Katharine A. Horzmann and Jennifer L. Freeman

School of Health Sciences, Purdue University, West Lafayette, IN, School of Health Sciences Purdue University, 550 Stadium Mall Dr., Hampton Hall of Civil Engineering, West

Lafayette, IN 47907

Trichloroethylene (TCE), an industrial degreaser and solvent, is a significant environmental toxicant that contaminates over half of all Superfund sites in the United States and is one of the three most commonly identified chemicals of concern in cancer cluster outbreak investigations. While TCE is recognized as a known human carcinogen by the International Agency for Research on Cancer, TCE has also been linked to congenital cardiac defects, immune and reproductive system dysfunction, and neurodegenerative disease. Environmental exposure to TCE typically occurs through ingestion and inhalation of contaminated ground water sources and the US Environmental Protection Agency has a maximum contaminant level of 5 ppb. However, ground water levels of TCE have been reported at over 10,000 ppb at Superfund sites. The developmental toxicity of TCE near regulatory levels needs further characterization in order to better assess the risk of this environmental toxicant. In this study, the zebrafish model was used to evaluate the acute developmental toxicity of near regulatory level concentrations of TCE by monitoring survivability, percent hatching, morphological measurements, and neurobehavior. Zebrafish embryos were dosed immediately after fertilization with 0, 5, 10, 50, or 500 parts per billion (ppb; µg/L) TCE, or 0.5 ppb 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), a TCE metabolite. Embryos were exposed to the chemical through 72 hours post fertilization (hpf; the end of embryogenesis), rinsed, and kept in filtered aquaria water until 120 hpf. The percent survival and hatching were not significantly different between treatment groups (p > 0.05). Minor morphological measurements indicated that the 5 ppb and 10 ppb TCE treatment groups as well as the 0.5 ppb TaClo groups had significantly shorter head lengths compared to the other treatments (p < 0.05). The 10, 50, and 500 ppb treatment groups had a significantly smaller head width compared to the other treatments (p < 0.05). No significant differences were observed between TCE treatment groups during the evaluation of neurobehavior in the visual motor response assay (p > 0.05). The morphologic alterations suggest that developmental TCE toxicity is still a concern near regulatory concentrations and that TCE should remain a priority environmental toxicant.

CRISPR/Cas9 knockout of human arylamine N-acetyltransferase 1 leads to an altered bioenergetics profile in MDA-MB-231 breast cancer cells

Samantha M. Carlisle1, Carolyn M. Klinge2, David W. Hein1

1Department of Pharmacology and Toxicology, University of Louisville School of Medicine. 2Department of Biochemistry and Molecular Genetics, University of Louisville School of Medicine, Kosair Charities Clinical and Translational Research Building 505 South Hancock Street, RM 352A Louisville, KY 40202

Background/Introduction: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. More recently, NAT1 has also been shown to hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate using folate as a co-factor. NAT1 expression varies inter-individually and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. To date however, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. Objective:We further evaluated the role of NAT1 in breast cancer risk and progression by determining the effect of NAT1 overexpression and knockout on the cellular bioenergetics of MDA-MB-231 triple negative breast cancer cells which endogenously express mid-level NAT1 compared to other breast cancer cell lines.

Methods: MDA-MB-231 cells, expressing parental, increased, and knockout levels of NAT1 were metabolically profiled in real-time using a Seahorse Biosciences XF24 Extracellular Flux Analyzer. First, 40,000 cells/well were plated in a 24 well Seahorse plate and allowed to grow for 24 hours. Next, basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured followed by programmed sequential injection of Oligomycin (ATP synthase inhibitor), FCCP (ETC uncoupler), Antimycin A (Complex III inhibitor) and Rotenone (Complex I inhibitor) to evaluate mitochondrial function.

Results, Conclusions:OCR, ECAR, reserve capacity, coupling efficiency, proton leak, and non-mitochondrial respiration were measured. Results show an approximate 5-fold increase in reserve capacity in the NAT1 knockout cell lines compared to cell lines expressing parental and increased NAT1. Increased reserve capacity may reflect several changes including enhanced oxidative capacity, mitochondrial biosynthesis, or increased substrate provision. Further investigation is ongoing.

Keywords: NAT1, Breast Cancer, Bioenergetics

Support: This study was partially supported by USPHS grant T32-ES011564

Cardiopulmonary Effects of Acute Exposure to Electronic Cigarette and Formaldehyde in Mice

Jordan Finch1, Gary Hoyle2, Connie Schlueter2, Ian McKinley3, Aruni Bhatnagar1,3, Daniel J. Conklin1,2,3

1Department of Pharmacology & Toxicology, 2Environmental & Occupational Health Sciences, 3School of Medicine, University of Louisville, 580 S. Preston Street, Delia Baxter

Bldg, Rm. 427, Louisville, KY 40202

Tobacco smoking is the most significant modifiable risk factor for cardiopulmonary disease. Although electronic cigarettes (e-cigs) have been promoted as a safer alternative to tobacco cigarettes, the health effects of these devices are largely unknown. E-cigs use e-liquids with humectants (propylene glycol, glycerin) that decompose during heating to form aldehydes (acetaldehyde, formaldehyde, and acrolein) that are abundant in tobacco smoke and are linked with cardiopulmonary toxicity. Thus, we examined whether acute exposure (6h/day, 4 d) of mice to bluPlus+™ e-cig aerosol or formaldehyde altered cardiopulmonary function. By FlexiVent testing, e-cig exposure had no effect on tissue resistance or compliance in the lungs, central airways, or the alveoli. Methacholine altered resistance and compliance to a similar degree in air control and e-cig-exposed mice. Similarly, aortic responses ex vivo to phenylephrine (PE), acetylcholine (ACh), or sodium nitroprusside (SNP) were unaltered in e-cig-exposed mice. Because formaldehyde is a major aldehyde of e-cig aerosol, we examined cardiopulmonary responses following formaldehyde (1 ppm) exposure. Formaldehyde induced a significant decrease in alveolar resistance compared with air control. Methacholine increased airway resistance similarly in control and formaldehyde-exposed mice. Formaldehyde exposure had no effect on aortic reactivity, while formaldehyde (200 µM) directly relaxed PE-contracted naïve aorta in an eNOS- and formate-independent manner. Systemically, e-cig exposure decreased total white blood cell count yet increased red blood cell hemoglobin, while formaldehyde exposure increased plasma albumin and ALT levels. Formaldehyde exposure did not recapitulate systemic effects of e-cig exposure yet diminished pulmonary tissue resistance and plasma albumin. Thus, effects of e-cigs are likely due either to other aldehydes (e.g., acetaldehyde, acrolein) or nicotine or other constituents. Moreover, while inhaled formaldehyde had no effect on aortic reactivity ex vivo (similar to e-cigs), direct formaldehyde (not formate) was anti-contractile. Overall, these data implicate formaldehyde metabolism as protective against cardiopulmonary toxicity of formaldehyde and perhaps e-cigs.

Beneficial effect of Carnosine on motor function in the Thy1-aSyn mouse model of Parkinson’s Disease

Mei-Ling Bermudez and Mary Beth Genter

University of Cincinnati

Parkinson's disease (PD) is the second leading neurodegenerative disease, which affects millions of people worldwide. No cure exists for this devastating disease. PD is characterized by several motor and prior onset non-motor deficits, including gait instability and decreased olfactory function. Molecular hallmarks of PD include protein aggregates and oxidative stress. Our studies evaluated a novel mechanism-based treatment for PD, using the Thy1-aSyn mouse model of PD. Carnosine, an endogenous dipeptide abundant in muscle, brain and the olfactory system, declines with age and pathological conditions. Recent, in vivo studies indicate that carnosine reduces protein aggregation and protects against oxidative stress, two features of PD. Therefore, we hypothesize that intranasal (IN) administration of carnosine will significantly reduce disease progression in the Thyl-aSyn mouse model of PD. Wild-type and Thy1-aSyn mice were treated IN with 2 mg/day carnosine or sterile water (as control) for 2 months. Immunohistochemistry, buried food pellet, and the challenging beam traversal (CBT) tests were used to evaluate tissue structure, and sensorimotor functions at the beginning and end of treatment. Olfactory function and structure were preserved, and alpha-synuclein (aSyn) positive inclusions were notably lower in the olfactory epithelium of carnosine treated Thy1-aSyn mice compared to controls. Strikingly, in the CBT test, the number of errors per step was lower in the carnosine treated Thy1-aSyn group compared to the untreated Thy1-aSyn group (0.5 vs 0.7, p<0.05). Our novel findings suggest that carnosine prevents the progression of motor deficits and aSyn aggregation in the Thy1-aSyn model of PD.

Mechanisms of PHIP-induced dopaminergic neurotoxicity in primary midbrain cultures

Zeynep Sena Agim1, Aurélie Jacquet2, Jean-Christophe Rochet2, Jason R. Cannon1

1School of Health Sciences, Purdue University, Lilly Hall of Life Sciences, 915 W. State Street, West Lafayette, IN 47907, United States, 2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Heine Pharmacy Building, 575 Stadium Mall

Drive, West Lafayette, IN 47907, United States

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine produced during high temperature cooking of meat products, classified as a group 2B carcinogen and primarily investigated as a mutagen. We recently showed that PhIP is selectively neurotoxic to dopamine (DA) neurons in primary neuronal cell culture. This culture system offers an excellent opportunity to preliminarily test neurotoxicity and identify important mechanisms of action that may be important in Parkinson’s disease (PD). The primary objective in this study was to use primary midbrain neuronal cell cultures to investigate mechanisms of selective DA neurotoxicity. Here, E17 primary midbrain neuronal cultures containing DA and non-DA neurons, and glial cells were used to test mechanisms of neurotoxicity of PhIP. A neuronal cell line (SH-SY5Y) was also utilized for some mechanistic studies. Whether PhIP induced antioxidant-response activation, proteasome inhibition or DNA damage was evaluated. Further, whether PhIP exerts neurotoxicity through interactions with cytosolic dopamine was also investigated. Anti-oxidant response element activation and proteasome inhibition were examined using eGFP or GFPu reporter systems, respectively. Further, effects of free cytosolic DA amount on PhIP-induced toxicity were assessed using nontoxic dose of VMAT2 inhibitor, reserpine, and subtoxic dose of PhIP. Our data showed that PhIP treatment at doses up to 10 μM, did not induce Nrf2 activation in primary midbrain cultures. Preliminary data suggests that proteasome inhibition may be important, however, PhIP-induced effects failed to reach statistical significance. A nontoxic dose of PhIP (200 nM) induced DA toxicity in primary cultures when co-treated with reserpine.

Increasing evidence in the literature suggests that there is a strong association between environmental factors and PD. Here, we examined potential mechanisms of PhIP neurotoxicity. Nrf2 activation and proteasome inhibition did not seem to be important in mediating toxicity. However, VMAT inhibition, a strategy to increase free unpackaged DA, increased neurotoxicity. Unpackaged DA has been repeatedly shown to be important in DA cell death. Thus, this mechanism should be further investigated as a key mechanism of heterocyclic amine-induced neurotoxicity. DNA damage and mitochondrial dysfunction will be investigated further in relation with PhIP dopaminergic neurotoxicity.

Comparison of the Morphological and Behavioral Effects of Developmental Exposure to Three Perfluorocarboxylic Acids in Zebrafish

Kathryn M. Thomson, Jennifer L. Freeman

School of Health Sciences, Purdue University, School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, IN 47907

Perfluorooctanoic Acid (PFOA; CF3(CF2)6COOH), a fully fluorinated eight-carbon carboxylic acid chain used in industry, is emerging as a persistent environmental contaminant. PFOA is ubiquitously detected worldwide in the environment as well as the general human population. PFOA has been detected in human cord samples and breast milk, indicating probable maternal transfer to child. Since May 2016, the EPA has reduced the health advisory for drinking water of PFOA from 0.4 ppb to 0.07 ppb. Several companies have voluntarily chosen to eliminate their use of PFOA. Shorter chain derivatives have been proposed as replacement compounds due to evidence of decreased acute toxicity. However, the amount of literature covering the toxicity of these shorter perfluorocarboxylic acids such as the six-carbon derivative Perfluorohexanoic Acid (PFHxA) and four-carbon derivative Perfluorobutanoic Acid (PFBA) is considerably lacking. Additionally, these shorter compounds are becoming more predominant in the environment. The knowledge of the neurobehavioral effects of developmental exposure to these perfluorocarboxylic acids is limited. The purpose of this study was to compare the morphological and behavioral effects of PFOA exposure with exposure to PFHxA or PFBA in the zebrafish model. We hypothesized that developmental exposure to PFOA would cause a change in morphology and behavior in zebrafish larvae and that PFHxA and PFBA exposure would not have any significant effect. Zebrafish were exposed to control or 4, 40, or 400 ppb of PFOA, PFHxA, or PFBA throughout embryonic development from 1-72 hours post fertilization (hpf). Morphology measurements of body length, head length, and head width were recorded of zebrafish larvae, and significant differences were found to occur for each chemical (p<0.05). Behavior analysis was conducted at 120 hpf. Distance and velocity were significantly increased in zebrafish larvae exposed to 4000 ppb PFOA (p<0.05). No significance was found in the behavior of larvae exposed to PFHxA. In contrast, the amount of time spent moving was significantly lower in the larvae exposed to 4000 ppb PFBA (p<0.05). This difference in behavior among the PFOA, PFHxA, and PFBA exposed groups suggests that this family of perfluorocarboxylic acids behave and act through different pathways in vivo despite similarities in structure. Additionally, although shorter chains such as PFBA have shown to be overall less toxic compared to PFOA, findings from this study indicate that some exceptions may exist and that further study is required over the shorter compounds.

Combating malignant melanoma with the multifaceted, soy-derived peptide Lunasin

Chris Shidal, Kavitha Yaddanapudi, and Keith Davis

Indiana University, Biotechnology Program, University of Louisville, Department of Pharmacology and Toxicology, 212 S Hawthorne Dr., Bloomington, IN 47401, Simon Hall

Rm. 213A

Lunasin is a 43-44 amino acid peptide derived from the soybean seed that has been shown to have cancer chemopreventative and chemotherapeutic properties. In this study, we investigated the potential utility of lunasin as a chemotherapeutic in a melanoma model. Initial studies showed that lunasin has little activity against established melanoma cell lines in vitro using adherent culture methods; however, lunasin’s in vitro activity was significantly higher in non-adherent colony-forming assays in soft agar and oncosphere assays. These results led us to investigate whether lunasin has selective effects on cancer stem cells (CSCs) that are known to be present in these melanoma cell lines. We found that lunasin treatment did selectively inhibit the proliferation of high-ALDH expressing melanoma-initiating-cells (MICs) in vitro, and had the striking effect of preventing oncosphere formation under non-adherent culture conditions. These in vitro results were extended into mouse xenograft studies using both bulk melanoma cells and isolated MICs. Lunasin significantly inhibited tumor growth in both cases, with the highest inhibition being observed in tumors initiated by MICs while achieving an excellent safety profile as assessed by blood, liver, and renal toxicity. Lunasin substantially reduced the invasive potential of melanoma cell lines in both in vitro invasion assays and in an in vivo experimental metastasis model. Mechanistic studies identify that lunasin disrupts integrin signal transduction by inhibiting phosphorylation patterns of the intracellular kinase FAK as well as altering the PI3K/AKT axis. Additionally, we demonstrate that histone acetylation patterns of H3 and H4 core histone are significantly altered in melanomas treated with lunasin. While histone acetylation is certainly involved in the anticancer activity of lunasin, the effects seen in our studies are mainly integrin-driven. These studies demonstrate for the first time that lunasin has activity against putative CICs, and that lunasin has potential utility as a therapeutic agent for the treatment of malignant melanomas.

Modeling binding kinetics of integrin mediators of EtOH-enhanced LPS liver injury

Shanice V. Hudson, Christine E. Dolin, Lauren G. Poole, Veronica L. Massey, Daniel W. Wilkey, Michael L. Merchant, Hermann B. Frieboes, Gavin E. Arteel

Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, 505 S. Hancock, Louisville, KY 40202, USA

Well before fibrosis, insults to the liver can alter the composition of the hepatic extracellular matrix (ECM). This transitional tissue remodeling can impact the hepatic response to injury. Indeed recent work by this group demonstrated that the hepatic ECM proteome (i.e., ‘mastrisome’) responds dynamically to alcohol and/or LPS exposure in mice. Importantly, several of the ECM proteins that were quantitatively/qualitatively changed bind to integrins in the liver. The purpose of the current study was to develop a computational model to predict changes in integrin signaling mediated by changes in the hepatic matrisome. Methods. Male mice were exposed to 6 wk ethanol diet and/or acute lipopolysaccharide (LPS). ECM proteins were quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS). A mathematical model for deterministic ECM-integrin binding kinetics that incorporates the divalence of the receptor and a simple aggregation scheme to represent clustering was employed to analyze the results. Results. Modeling of the quantitative proteomic results indicate that changes in the matrisome caused by alcohol and/or LPS impact ECM/integrin binding kinetics, mostly increasing the binding interactions; importantly, many of these changes validate established experimental phenotypes (e.g., integrin αvβ3) in inflammatory liver disease. Conclusions. A new computational model characterizing and predicting alterations in integrin signaling driven by changes in the hepatic mastrisome has been developed. This model profiling method can help distinguish patterns between homeostatic and diseased livers, is predictive of the predominant phenotype of integrin signaling, and may identify new key targets. This work was supported in part by NIH grants R01AA021978, R01AA021978S1.

Cyclic AMP-responsive element binding protein (CREB-1) is a common regulator of genes differentially transcribed with Aroclor 1260 exposure

Heather Clair1, Hardesty1, Falkner2, Kalbfleisch1, Prough1, Cave2,3

1Department of Biochemistry and Molecular Genetics, 2Division of Gastroenterology, Hepatology and Nutrition; University of Louisville School of Medicine, Louisville, KY 40202;

3Robley Rex Louisville VAMC, Louisville, KY 40206

Background: Exposure to polychlorinated biphenyls (PCB) is widespread and has been associated with the development of toxicant-associated steatohepatitis (TASH), and conditions comprising the metabolic syndrome (cardiovascular disease, obesity, and diabetes). The mechanism of PCB-related TASH appears to affect clinical biomarkers of liver injury in human populations: impeding diagnosis, and therefore delaying intervention. Assessment of steatohepatitis etiology and pathological mechanism may be further complicated by common comorbidities such as obesity and alcohol use, which may alter shared pathways. We used an RNAseq approach with subsequent in silico pathways analysis to explore transcriptional alterations in PCB-exposed primary hepatocytes and liver tissue from PCB-exposed mice. Methods: Primary murine hepatocytes were isolated by collagenase extraction and seeded onto collagen-coated plates. Cells were exposed to Aroclor 1260 (Ar1260) for 6h prior to collection and RNA extraction. Identity and fold-change (vs. DMSO-treated control) of differentially-transcribed genes was assessed by RNAseq and selectively validated by qPCR. Liver tissue from mice exposed to 20mg/kg or 200mg/kg Ar1260 (12 weeks exposure, post-gavage) was collected and RNA was isolated from fresh dissociated liver tissue. Identity and fold-change (vs. corn oil gavaged control) of differentially-transcribed genes was assessed by RNAseq and selectively validated by qPCR. Results: For both acutely exposed primary murine hepatocyte culture and liver tissue from chronically exposed mice, Metacore analysis identified cyclic AMP-responsive binding protein (CREB-1) as the primary direct regulator of differentially transcribed genes. Total differentially regulated direct transcriptional targets of CREB-1 increased from 39 in the primary hepatocyte experiment to 42 in the 20mg/kg chronic exposure to 353 in the 200mg/kg chronic exposure with control diet. Of these, only 4 genes were common to all three experiments, and 21 were shared in common between the moderate and high chronic exposures. Conclusions: CREB-1, a transcription factor which has been shown to play a role in multiple metabolic diseases, is a direct effector of many of the hepatic genes differentially transcribed after acute or chronic exposure to PCBs.

Effects of Ethanol and Lipopolysaccharide on the Renal Cortex Proteome and Transcriptome

Christine E. Dolin1, Lauren G. Poole1, Daniel W. Wilkey2, Gavin E. Arteel1, Eric C. Rouchka3, Michelle T. Barati2, Michael L. Merchant1,2

1Pharmacology & Toxicology, University of Louisville; 2Medicine, University of Louisville and 3Computer Engineering & Computer Science, University of Louisville

Background: Chronic heavy ethanol (EtOH) consumption is known to impact the kidney through hepato-renal syndrome. However, the direct renal effects of chronic moderate EtOH consumption and sensitization to secondary hits are unclear. We hypothesize that moderate EtOH consumption can alter renal transcriptomic/proteomic responses to acute lipopolysaccharide (LPS) exposure.

Methods: Mice were pair fed EtOH-containing Lieber-DeCarli diet for 6 weeks and/or injected i.p. with LPS 4h prior to sacrifice. Kidney cortex sections were isolated and snap frozen. Comparative Omics studies used (a) two-dimensional liquid chromatography-mass spectrometric analysis with an Orbitrap Elite and TMT labeling reagents and (b) mirVana™ isolation of total RNA, library preparation using the TruSeq Stranded Total RNA LT Sample Prep Kit-Set A with Ribo-Zero Gold and data collection using Illumina NextSeq 500/550 with the RNASeq protocol. Proteomic data were filtered by Benjamini-Hochberg (BH) corrected ANOVA p-value <0.05 and fold change (FC) ≥ 1.2. Transcriptomic data were filtered by q-value <0.05 and FC ≥ 2. Ingenuity Pathways Analysis (IPA) was used to identify pathways changed by EtOH, LPS, and/or the combination. Individual protein changes were validated with immunoblot and/or immunohistochemistry.

Results: 170 of 1863 proteins were significantly differentially abundant at FC ≥ 1.2 across groups. 853 of 47,719 transcripts were significantly differentially abundant at FC ≥ 2 across groups. The majority of these effects were LPS-derived. Expected increases in Fibrinogen A protein and transcript were observed in LPS-exposed samples. IPA results show EtOH preexposure attenuated LPS effects on LXR/RXR and Nrf2-mediated pathways. Western blot and immunohistochemistry of Nrf2 target proteins validate proteomic findings.

Conclusions: This study on the effects of EtOH and/or LPS on the renal transcriptome and proteome supported expected observations and revealed new changes in proteins, transcripts, and pathways. These changes provide insight into mechanisms by which EtOH affects the kidney and alters response to a second pathologic stimulus.

NIH, NIAAA (1R01AA021978-01 and P50AA024337-01)NIDDK (R01-01DK091584)KBRIN Bioinformatics Core: NIH/NIGMS (P20GM103436 and P30GM106396)

Immuno-cytochemical exploration of the interaction between the Na/K ATPase (NKA), Bcl-2 (BclXl) and other proteins as determinants of digitalis-NKA receptor

internalization

Joshua Stricker, Norma C Adragna, and Peter K Lauf

Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH, 45435

Background: Cardio-tonic steroids (CTS) such as ouabain, a cardiac glycoside binding stoichiometrically to its receptor, the Na/K ATPase (NKA), are utilized therapeutically to treat congestive heart failure due to their positive inotropic effects of strengthening heart contractions. Previous studies with a fluorescing derivative of ouabain, BODIPY fluorescent ouabain (BFO) binding to NKA, have shown fluorescence internalization of BFO (Alonso et.al 2013). These findings are corroborated by the observation of green fluorescent protein-labeled NKA’s cytosolic internalization into endosomes and lysosomes (Yosef et al 2014). However, it is not known whether BFO is internalized alone or in complex with NKA, or both, and the detailed mechanism of NKA permeation is currently not understood. Interestingly, NKA has been shown to interact with proteins such as the Bcl-2 protein BclXl (Lauf et al. 2015) and caveolin-1 (Pierre & Xie, 2006), whereas the tubulin polymerization rate to microtubules is affected by Bcl-2 proteins (Knipling & Wolf 2006), and tubulin interacts with NKA (Amaiden et al. 2010). The hypothesis of this presentation is that the mechanism of NKA internalization involves several proteins such as BCL2, caveolin, and tubulin. Thus, using BFO, and green and red-fluorescing antibodies as immuno-cytochemical tools, the possible complexation of NKA with BclXl, tubulin and caveolin1 in the cytoplasmic descent along a putative “microtubular escalator” was studied in cultured human B3 lens epithelial cells in the absence and presence of nano-micromolar BFO concentrations. Results: 1. BFO labeled diffusely green live B3 cells. Subsequent fixation and exposure to anti-NKA antibodies visualized with secondary red fluorescent antibodies yielded upon merger distinct yellow fluorescence indicating close proximity of the two markers on the NKA. 2. At micro > nano molar BFO concentrations, and in a time–dependent manner, the BFO-NKA complex was internalized as detected by green (BFO) and red fluorescence (NKA). 3. BFO-fluorescence, but not red anti-NKA labeling, was negligible in the presence of thousand-fold excess of non-labeled ouabain (NLO), and hence specific. 4. B3 cells exposed to micro-molar concentrations of BFO or NLO, or both, were larger as opposed to cells treated with nano-molar drug concentrations. 5. Confirming earlier results for another epithelial cell line (Lauf et al. 2015), BclXl showed bright co-localization with NKA, as did Caveolin-1, the latter preferentially in “caveolar” membrane regions; however, tubulin yielded the least overlap with NKA. Conclusion: These preliminary data suggest need of future elaboration of the interaction between NKA, Bcl-2 and caveolin as determinants of the ouabain-NKA receptor cytosolic internalization process, as they may be important to understand the transcriptional regulation of these factors during cell growth and death. Furthermore, in vivo NKA labeling by BFO might reveal cell volume increases at micro-molar drug concentrations consistent with complete inhibition of NKA and Na-loading. Support by WSU Foundation and Pharmacology & Toxicology Graduate Program.

Fibroblast Growth Factor 1 Ameliorates Diabetic Nephropathy Through an Anti-inflammatory Mechanism

Qian Lin1,2, Lu Cai1,2, Yi Tan1,2

1Department of Pharmacology & Toxicology, University of Louisville, Louisville, KY 40202, USA, 2Department of Pediatrics, University of Louisville, Louisville, KY 40202, USA

A recent study demonstrated metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis (Nature. 2014; 513:436). But its strong mitogenic activity hampers its wide use in diabetes. We have recently engineered a FGF1 partial agonist carrying triple mutations (FGF1△HS) that diminish the ability of FGF1 to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1△HS exhibited a major loss in proliferative potential, while preserving the full metabolic activity of wild-type FGF1 (FGF1WT). In the present study, we further tested the potential of FGF1△HS in treatment of diabetic nephropathy (DN). Treatment of db/db type 2 diabetes (T2D) with FGFWT and FGF1△HS (0.5 mg/kg) every other day for 2 months, both FGFWT and FGF1△HS significantly reduced blood glucose, urinary albumin, and prevented glomerular hypertrophy, mesangial matrix expansion and renal fibrosis. Similarly, treatment of streptozotocin (STZ)-induced type 1 diabetes (TID) with FGF1WT and FGF1△HS also significantly prevented diabetes-induced renal morphological and functional changes, but without significant effects on blood glucose levels. These results imply that the renal protection of FGFWT and FGF1△HS is independent of their insulin sensitizing activity. Since renal inflammation is a critical cause of DN, and FGF1 exhibits a potent anti-inflammatory property (Nature. 2014; 513:436), we hypothesized that the protection of FGF1 against DN is associated with its anti-inflammatory capability. As expected, both FGF1WT and FGF1△HS treatments significantly reduced the inflammatory cytokines (TNF-α and IL-6) expression and CD68+ macrophage infiltration in renal tissues in both T1D and T2D, which were accompanied by an inhibition of inflammatory signal (JNK/NF-κB) activation. Direct exposure of renal mesangial cell to high glucose or TNF-α induced similar inflammatory and fibrotic responses as observed in diabetes, which could be prevented by FGF1WT and FGF1△HS pre-treatment. More importantly, administration of FGF1△HS to 9-month-old db/db mice completely prevented the further development of DN with remarkable amelioration of renal inflammation and fibrosis. These results demonstrate that FGF1 prevents DN largely via inhibiting renal inflammation. FGF1△HS might be a therapeutic approach for the treatment of DN without promoting undesired tissue proliferation.

Internalization of silver nanoparticles alters K metabolism and hemoglobin oxidative state in human red blood cells

1Alla, P.K.; 1,2Lauf, P.K.; 4Sizemore, I.; 4Paluri, A.S.L.; 3Yaklic, J. and 1Adragna, N.C.

Depts. of 1Pharmacology & Toxicology, 2Pathology, and 3Obstetrics & Gynecology, Boonshoft School of Medicine and 4Department of Chemistry, College of Science and

Mathematics, Wright State University, Dayton, OH

Silver nanoparticles (AgNPs) are nanomaterials (NMs) that have been used extensively in industry and medicine. In spite of the numerous applications, knowledge about their toxicity has not developed at the same pace as their production and application. When NMs are used for in vivo diagnostic or therapeutic purposes, one of the main tissues they may become in contact with is blood and, especially, the main compartment, which is composed of red blood cells (RNCs). Some studies have investigated the effect of NMs on RBC morphology, deformation, aggregation, hemoglobin (Hb) heme’s oxidative states and other properties. The present study reports on the effect of AgNPs on ion transport in human RBCs. Rubidium influx and K concentration were determined in the presence and absence of negatively charged 10 nm AgNPs. Furthermore, the Hb spectrum under the same experimental conditions as the ion transport studies was also measured. Internalization of AgNPs in RBCs was determined by integration of enhanced dark field optical microscopy and hyperspectral imaging in a CytoViva® equipment. Our preliminary findings indicate that the tested internalized AgNPs inhibit Rb influx and deplete the cells from K in a time- and dose-dependent manner. In addition, the AgNPs altered the absorbance for the Hb peaks and showed an additional broad shoulder between 594.1 and 658.8 nm. The changes in the Hb spectrum in the presence of AgNPs could be interpreted as the AgNPs causing an increase in the oxidized form of Hb, i.e. MetHb. Our studies indicate that AgNPs alter K metabolism and Hb oxidative state in human RBCs. Funding Support: National Science Foundation.

Risk values and exposure guidance for ethylene glycol monopropyl ether, a high production volume chemical

A.H. Kyakulaga1 and J.C. Lipscomb1,2

1Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, and 2U.S. Environmental Protection Agency, Office of Research and Development, National

Homeland Security Research Center, Cincinnati, OH

Ethylene glycol monopropyl ether (EGPE; 2807-30-9) is a high production volume chemical used as an industrial solvent and found at many Superfund sites, without reference or guideline values for oral or inhalation exposures. Differential abilities to identify, characterize and restrict exposure of the population, differences in potential exposure durations, and the intended use of risk values substantially impact the evaluation of data and identification of effects and points of departure (POD). Reference Concentration (RfC) values are developed for the entire population, assuming a continuous, lifetime exposure. EPA’s National Homeland Security Research Center (NHSRC) developed the Provisional Advisory Levels (PALs) program to protect emergency responders, to estimate the type and number of effects seen following an unprotected exposure, and to guide decisions for re-entry of contaminated areas and re-use of infrastructure following emergency release incidents. The tiered system of PALs values is derived on the basis of a protocol adapted from Acute Exposure Guideline Level (AEGL) protocol for inhalation exposures, covering both the inhalation and oral routes, and exposures beyond eight hours.  Lacking human dose response data for EGPE, inhalation and oral data from rats, mice and rabbits for acute, gestational and subchronic exposures identified potential effects and points of departure for risk value development. EGPE produced toxicity characteristic of glycol ethers. Estimated oral PALs (mg/L of drinking water) for 24-hour, and 30-day durations are, respectively: PAL 1 = 2.0, 1.4; PAL 2 = 2.5, 2.5; PAL 3 = 7.5, 5.6.  Estimated inhalation PALs (ppm) for the same durations are: PAL 1 = 0.51, 0.37; PAL 2 = 0.67, 0.73; PAL 3 = 2.0, not recommended. Developmental and subchronic inhalation studies identified potential critical effects and POD values for estimating a chronic RfC of 0.0059 ppm. Values like these would be useful in establishing recommendations for exposures and in estimating the consequences from human exposures to EGPE. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the Agency. 

Arsenic Induces Functional Changes of ZRANB2 and Expression of hsa-miR-186

Collin Stocke1, Laila Al Eryani2, Camille Gordon3, Vanessa A.R. States2, Shesh N. Rai4, Susan Galandiuk5, and Ashok K. Giri6, & J. Christopher States2

Departments of 1Environmental and Occupational Health Sciences, 2Pharmacology and Toxicology, 3Medicine, 4Bioinformatics and Biostatistics, and 5Surgery, University of

Louisville, Louisville, KY 40292, USA, and 6Indian Institute of Chemical Biology, Kolkata, India

Background: Chronic exposure to arsenic is a worldwide issue that leads to the development of cancers. While the exact mechanism behind arsenic-induced malignant transformation of cells remains to be determined, proposed mechanisms of carcinogenesis include epigenetic dysregulation, such as changes in microRNA expression, and promotion of aneuploidy. We have collected microRNA expression data from human tissue samples of malignant squamous cell carcinomas (SCC) and pre-malignant hyperkeratosis (HK), hsa-miR-186 was increased in SCC tissue versus HK tissue. When immortalized human keratinocytes (HaCaT) cells were transfected with a vector constitutively expressing hsa-miR-186, the cells showed signs of increased chromosomal instability. Hsa-miR-186 is embedded in intron 9 of ZRANB2 which encodes a zinc finger protein involved in RNA alternative splicing. Zinc can be displaced by arsenite on these types of proteins and disrupt their function, suggesting a linkage between ZRANB2 function and hsa-miR-186 induction. Hypothesis: Chronic arsenic exposure disrupts ZRANB2 function leading to an increase in transcription of ZRANB2 and consequently an increase in hsa-mir-186. Methods: Quadruplicate HaCaT cell cultures were maintained for 32 weeks in either 0 or 100 nM NaAsO2. At 4-week time intervals starting with week 3, total RNA was purified from cell cultures. Analysis by RT-qPCR was used to determine expression of ZRANB2 mRNA and hsa-miR-186 and changes in the ratios of two alternatively spliced mRNAs produced from genes targeted by ZRANB2 (ACAP1, WDR78). Results: Changes in transcription levels of ZRANB2 mRNA exhibit a +/- 20% fold change difference between arsenic exposed and unexposed groups over the course of the first 23 weeks of exposure. However, levels of hsa-miR-186 did exhibit an initial slight increase at 3 weeks’ exposure in arsenic treated groups (1.6 fold increase) with a significantly increased expression starting at week 28 compared to earlier weeks (over 2 fold increase). Ratios of alternatively spliced mRNA targets of ZRANB2 are generally decreased in arsenic treated cultures and are also correlated with changes in ZRANB2 mRNA levels. Conclusions: Arsenic exposure does cause fluctuation in ZRANB2 mRNA levels. This exposure results in disrupted function of ZRANB2 in the form of significantly different alternative splicing patterns of mRNAs targeted for processing by ZRANB2. Additionally, by 28 weeks, arsenic exposure results in a significant increased expression of hsa-miR-186.

Beneficial effect of Carnosine on motor function in the Thy1-aSyn mouse model of Parkinson’s disease

Bermudez, Mei-Ling and Dr. Genter, Mary Beth

Parkinson's disease (PD) is the second leading neurodegenerative disease, which affects millions of people worldwide. No cure exists for this devastating disease. PD is characterized by several motor and prior onset non-motor deficits, including gait instability and decreased olfactory function. Molecular hallmarks of PD include protein aggregates and oxidative stress. Our studies evaluated a novel mechanism-based treatment for PD, using the Thy1-aSyn mouse model of PD. Carnosine, an endogenous dipeptide abundant in muscle, brain and the olfactory system, declines with age and pathological conditions. Recent, in vivo studies indicate that carnosine reduces protein aggregation and protects against oxidative stress, two features of PD. Therefore, we hypothesize that intranasal (IN) administration of carnosine will significantly reduce disease progression in the Thyl-aSyn mouse model of PD. Wild-type and Thy1-aSyn mice were treated IN with 2 mg/day carnosine or sterile water (as control) for 2 months. Immunohistochemistry, buried food pellet, and the challenging beam traversal (CBT) tests were used to evaluate tissue structure, and sensorimotor functions at the beginning and end of treatment. Olfactory function and structure were preserved, and alpha-synuclein (aSyn) positive inclusions were notably lower in the olfactory epithelium of carnosine treated Thy1-aSyn mice compared to controls. Strikingly, in the CBT test, the number of errors per step was lower in the carnosine treated Thy1-aSyn group compared to the untreated Thy1-aSyn group (0.5 vs 0.7, p<0.05). Our novel findings suggest that carnosine prevents the progression of motor deficits and aSyn aggregation in the Thy1-aSyn model of PD

.

Buffering of ceramide through glucosylceramide synthase is protective of cisplatin-induced acute kidney injury

Tess V. Dupre, M.S.1, Mark A. Doll, M.S.1, Parag P. Shah, Ph.D.2, Cierra N. Sharp, M.S.1, Deanna Siow, Ph.D.1, Judit Megyesi, M.D.3, James Shayman, M.D.4, Alicja Bielawska Ph.D.5, Jacek Bielawski, Ph.D.5, Levi J. Beverly, Ph.D.1,2,6, Leah J. Siskind, Ph.D.1,6*

1Department of Pharmacology and Toxicology, University of Louisville, 2Department of Medicine, University of Louisville, 3Department of Internal Medicine, Division of Nephrology,

University of Arkansas for Medical Sciences and Central Arkansas, Veterans Healthcare System, 4Department Internal Medicine, 5Department of Biochemistry and Molecular Biology, Lipidomics Shared Resources Medical University of South Carolina, 6James

Graham Brown Cancer Center, University of Louisville

Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Data indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pre-treatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine whether ceramides or hexosylceramide were contributing to kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramide to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. This indicates that ceramide is being further metabolized to the hexosylceramide glucosylceramide in cisplatin-induced AKI as C10 is a specific inhibitor of glucosylceramide synthesis, a subclass of hexosylceramides. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney injury, inflammation, cell stress, apoptosis, and mitochondrial homeostasis. Thus, under conditions of high ceramide generation, flux through the glucosylceramide synthesis pathway becomes important for buffering kidney ceramide levels in cisplatin-induced AKI.

In Vitro 3-Dimensional Modeling of Tumor Microenvironment Reveals Host/Tumor Influence on Progression in Non-Small Cell Lung Carcinoma

Saforo Douglas J.1, Shah Parag P.2, Beverly Levi J.2, and Siskind Leah J.1

1Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY, and 2Department of Medicine, University of Louisville, Louisville, KY

1. Introduction & BackgroundLung cancer is the leading cause of cancer related death in the United States and worldwide. Five-year survival rates for non-small cell lung carcinoma patients have not improved in decades, and a vast majority of patients succumb to metastasis. Identification of therapeutic targets for advanced stage disease is essential for the development of novel therapies to halt the progression of this devastating disease. However, current 2-dimensional (2D) in vitro models are not reflective of the tumor microenvironment and are inadequate in identifying these targets. We previously reported that lung carcinoma cells grown on a human lung fibroblast derived 3-dimensional (3D) extracellular matrix (ECM) display differences in cell morphology, proliferation, migration, and survival as compared to when they are grown in 2D. Our 3D in vitro model provides the capability to study the interaction of lung carcinoma cells with the tumor microenvironment and characterize host microenvironment effects on cancer progression.

2. Hypothesis/ObjectiveECM interactions with lung adenocarcinoma cells promote hallmarks of cancer metastasis in an in vitro model.

3. MethodsHuman WI-38 fibroblasts and mouse primary fibroblasts were maintained for 8 days and then decellularized to produce ECM. Human lung adenocarcinoma cell lines A549 and mouse KrasLSL-G12D/+;p53flox/flox lung adenocarcinoma cell lines were then grown on these matrices (3D) or plastic (2D). ECM to cell interactions were disrupted via inhibitors of ‘outside-in’ signaling including integrins, focal adhesion kinase, src family kinases, and cytoskeletal remodeling. Cancer cell growth was assessed by AlamarBlue and markers of cancer progression were assessed by western blot analysis. Migration and invasion was assessed by matrigel scratch wound assay.

4. ResultsCarcinoma cells grown on 3D matrices are significantly more invasive as compared to 2D. The increased invasion of A549 cells grown in 3D ECM was attenuated with inhibitors of Src family kinases and focal adhesion kinases. The altered phenotype observed when A549 cells are grown on 3D ECM was not specific to the source of the matrix as it was also observed when ECM was generated by primary fibroblasts derived from mice. Growth of cancer cells on mouse derived ECM altered expression of known markers of EMT.

5. Conclusions & Future DirectionsUsing our 3D ECM model, we demonstrate that we are able manipulate ECM-to-cell interactions in vitro in a way that is not possible for 2D models. Additionally, our model is expandable to primary fibroblasts derived from mice, which allows manipulation of the host microenvironment to study cancer progression. An

understanding of how remodeling of the tumor microenvironment is involved in late stage disease will allow development of preventative and curative therapeutic strategies to halt tumor progression and metastasis.

Recombinant ANAPC2 Displays Multiple Internal Start Sites and Compounds targeting ANAPC2 Decrease Proliferation of Lung Cancer Cells in vitro

J. Mason Hoffman1, Nathan Wainscott1, B. Frazier Taylor1, John O. Trent2 & J. Christopher States1

Departments of 1Pharmacology and Toxicology and 2Medicine, University of Louisville, Louisville, KY 40292, USA

Background: Mitosis-inhibiting chemotherapeutics are frequently used to treat cancer. Successful therapy is often limited by drug-resistant tumors, neurotoxicity, and limited Bioavailability. There is high demand for new drugs with diverse cellular targets. The anaphase promoting complex/ cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase that controls cell cycle progression by marking key proteins for degradation, offers great potential as a target for drug development. The catalytic core consists of an ANAPC2/ ANAPC11 dimer. Homology models of the ANAPC2/ ANAPC11 subunits were analyzed in silico to identify lead compounds predicted to bind the ANPAC11-binding domain of ANAPC2, and inhibit ANAPC11 association and APC/C function. Objective: We aim to demonstrate the anti-proliferative effect of these compounds on several lung cancer cell lines in vitro and to produce recombinant ANAPC2, for future investigation of compound binding. Methods: Alamar blue and colony forming assays were performed in A549 and H460 lung cancer cells to measure cell proliferation and viability. ANAPC2 cDNA was optimized for expression in E. coli, and protein was purified using the NEB IMPACT® system. Results: Results of AlamarBlue and colony forming assays indicate that lead compounds cause a decrease in cell proliferation. Full-length and multiple truncated versions of recombinant ANAPC2 were observed by both western blot and coomassie-stained SDS PAGE. Molecular weights of the truncated proteins correspond to molecular weights of proteins starting from several internal methionines in the ANAPC2 sequence. Summary: These results suggest that the APC/C is a promising target for development of novel chemotherapeutics.

The Bacterial Quorum Sensing Molecule N-(3-Oxo-Acyl Homoserine Lactone Inhibits Tumor Growth Independent of Bcl-2 Proteins

Aaron Neely

The gram-negative human pathogen Pseudomonas aeruginosa uses N-(3-oxododecanoyl)-L-homoserine lactone (C12) as a quorum-sensing signal for bacterial intercellular communication. Recent studies have shown that C12 is not only important in regulating bacterial virulence genes but also interacts with eukaryotic cells and trigger cell death. C12 has been demonstrated to cause apoptosis in a variety of cancer cells. However, the detailed apoptotic signaling of C12 remain unclear and whether C12 cytotoxicity in vitro is relevant to tumor growth in vivo has never been studied. As a lactone, C12 is known to be hydrolyzed into a carboxylic acid by the lactonase paraoxonase 2 (PON2); which belongs to a gene family with Ca2+-dependent lactonase and arylesterase activities. Importantly, PON2 expression is markedly elevated in several human non-small cell lung carcinoma (NSCLC) cell lines, which is associated with resistance to classical anticancer drugs like doxorubicin or cisplatin. In contrast, overexpression of PON2 promotes C12-induced apoptosis in MEFs and HEK293T cells. To gain insights into the mechanism of C12-evoked tumor cell apoptosis, we evaluated the cytotoxic effects of C12 on tumor cells in vitro, the inhibitory effects of C12 on tumor growth in vivo and the role of pro and anti-apoptotic Bcl-2 and PON2 proteins in C12-induced apoptosis was systematically examined. C12 inhibited the tumor growth through inducing apoptosis in a PON2-dependent but not Bcl-2 protein-dependent manner. Our study reveals a unique anti-tumor function of C12, which may lead to the development of new therapeutic agents for cancer.

EpCAM Positive Cancer Stem Cells Promote Acquired Chemoresistance in Hepatocellular Carcinoma

Harshul Pandit1,2, Yan Li2, Guozhen Cui2, Xuanyi Li2, Suping Li2, Kevin Jacob2, Robert C.G. Martin2,1

Department of Pharmacology & Toxicology1, Hiram C. Polk, Jr. MD Department of Surgery2, University of Louisville School of Medicine, Louisville, KY 40202

Introduction: Hepatocellular carcinoma (HCC) attains resistance to chemotherapy which accounts for most of the therapeutic failures, and is one of the major factors for poor prognosis and poor overall survival of HCC patients. Underlying mechanism(s) responsible for acquired chemoresistance has not been well defined in HCC. Cancer stem cells (CSCs) are a subpopulation of cells with stem cell like properties, and are believed to contribute in tumor initiation, acquired chemoresistance, and recurrence. Accumulating evidence indicated that aberrant Wnt/β-catenin signaling contribute to CSC activation. Hypothesis: We hypothesize that Wnt/β-catenin mediated CSC activation is responsible for acquired chemoresistance in HCC. We aimed to study aberrant Wnt/β-catenin signaling in Epithelial Cell Adhesion Molecule (EpCAM) expressing HCC-CSCs. Methods: In vitro CSC enrichment was achieved by treating murine (Hepa1-6) and human (HepG2, Hep3B) HCC cells in serum-free condition. The CSCs were identified by CSC biomarkers (EpCAM, CD90, CD44, CD133) using flow-cytometry and Immunocytochemistry, and functional markers using Aldeflour assay and Hoechst-33342 efflux. Drug resistance of CSCs was tested with treatments of Doxorubicin and Sorafenib, and cell viability was determined by MTT assay. Tumorigenic potential of sorted EpCAM+ CSCs was studied in immunocompetent orthotopic HCC mouse models. Dynamic in vivo tumor growth was monitored using high-frequency ultrasound, and confirmed by histology in the liver tissues using HE and IHC staining. To investigate CSC activation mechanism, Wnt/β-catenin signaling was studied by analyzing expression of β-catenin, GSK3β, p-GSK3β, EpCAM, ABCG2; and downstream targets i.e. C-Myc, Cyclin-D1, TCF1. SiRNA mediated knockdown of β-catenin confirmed effect of canonical Wnt signaling on activation of CSCs and chemoresistance. The results were further corroborated by complementary study using chemical inhibitors of Wnt pathways i.e. LiCl, XAV939, FH535. Human HCC specimens were obtained and analyzed to confirm our findings. Results: Spheroid forming HCC-CSCs showed significant higher EpCAM expression and significant increased chemoresistance compared with control HCC cells. ABCG2 expression (and its efflux activity) was found to be increased in CSCs, which further supports acquired chemoresistance. Silencing β-catenin caused loss of acquired chemoresistance. Sorted EpCAM+ CSCs showed significant increased tumor size and higher tumor proliferation rate compared with control in the mouse models. Human HCC specimens confirmed concomitant increase/decrease in EpCAM and β-catenin expression. Conclusions: Our findings suggest that subpopulation of EpCAM+ CSCs contributes to acquired chemoresistance in HCC through Wnt/β-catenin signaling. Sorted EpCAM+ CSCs showed very aggressive in vivo tumor initiation and growth in immunocompetent orthotopic mouse model, especially in the NASH microenvironment. This EpCAM+ CSCs population could potentially responsible for HCC recurrence and therapeutic failure.

The Effects of PCB Congeners and PCB mixtures on Pancreatic Function

Jian Jin1, Hongxue Shi1, K. Cameron Falkner2, Matthew C. Cave2

1Department of Pharmacology & Toxicology, 2Department of Medicine, Division of Gastroenterology, Hepatology & Nutrition, The University of Louisville School of Medicine,

Louisville, KY 40292

Background/Introduction

Polychlorinated biphenyls (PCBs) are endocrine and metabolism disrupting chemicals (EDCs/MDCs) that increase susceptibility to NASH and diabetes mellitus (DM) in exposed populations. Multiple epidemiological studies have revealed associations between PCBs and DM. The detailed mechanisms of dioxin-like (DL) and non-dioxin-like (NDL) PCB-induced DM are unclear.

Hypothesis/Objective

PCBs cause the disruption of pancreatic function, which is related to PCBs-induced DM.

Methods

C57BL6/J mice (8-10 weeks) on synthetic TekLab TD06416 diet (10% fat) were treated by gavage with Aroclor 1260 (20 mg/kg), PCB126 (20 µg/kg) and Aroclor 1260+0.1%PCB 126 added in corn oil for 2 weeks. After euthanasia, tissues were removed for measurement of gene expression and histology.

Results

Of the four groups (Control, Ar1260, PCB126, or Ar1260+PCB126), the decreased expression of serum insulin was seen in the Ar1260+PCB126 treated group. However, Ar1260- and to a lesser extent PCB126-treated groups showed increased expression of serum insulin levels. A significant decrease of the pancreatic transcription factor Nkx6-1 and its target gene NR4a-1 was seen in Ar1260/PCB treated group, and while there was an increased expression seen both in Ar1260- and PCB-treated groups. H&E and Trichrome Staining demonstrated decreased pancreas mass with acinar atrophy in the PCB treatment groups.

Conclusions.

In the pancreas, the PCB mixture synergistically down-regulated the expression of insulin and transcription factors regulating βcell identity (Nkx6-180 and its nuclear receptor target Nr4a). In addition to diabetes, PCBs may affect other islet cell functions in pancreas.

AcknowledgementsSupported by grant 1 R01 ES021375 from the National Institute of Environmental Health Sciences

Post-Doctoral Poster Presentations

The P53-specific inhibitor Pifithrin- protects from diabetic cardiomyopathy through attenuating the early-stage apoptosis and improving the late-stage defect of

glycolysis and angiogenesis

Junlian Gu1, Shudong Wang2, Yi Tan1, Lu Cai1

1KCHRI of the Department of Pediatrics, the University of Louisville, Louisville, USA, 2The Cardiovascular Center, The First Hospital of Jilin University, Changchun, China;

Cumulative evidence indicates that elevated tumor suppressor P53 expression is associated with several heart diseases, including diabetic cardiomyopathy (DCM). However, the mechanisms are unclear. We report that expression levels of P53 were increased in streptozotocin-induced type 1 diabetes and db/db mice, and were associated with increases in cardiac pathological changes and dysfunctions, compared to nondiabetic mice. One of the key early cardiac responses under diabetic conditions is apoptosis, which, however, occurs predominantly in the early stage of diabetes. It is known that the persistent reduction of glucose uptake and utilization and an inadequate neovascularization usually plays a critical role in the late development of DCM. The aim of this study was to determine whether and how inhibition of P53, using a specific inhibitor pifithrin-α, protects from cardiac pathological changes and dysfunction in diabetic mice. FVB mice were treated with five daily injections of 50 mg/kg streptozotocin to induce type 1 diabetes. Both hyperglycemic and age-matched control mice were treated with or without p53 inhibitor PFT-α at 1.1 mg/kg five times weekly for 2 months and then these mice were kept without PFT-α treatment until 6 months. The results showed that treatment of diabetic mice with PFT-α for two months could lead to improve diabetes-induced progressively cardiac remodeling and dysfunction (i.e.: DCM). Mechanistically we further found that during the early state of DM (0.5 month), inhibition of P53 reduces cardiac mitochondrial cell death while in the late state of DM (3 – 6 months), inhibition of P53 protects from diabetes-induced impairment in glucose metabolism and angiogenesis via increase in hypoxia-induced factor (HIF)-1α protein stability and upregulation of HIF-1α transcription of specific protective genes. These results demonstrate for the first time that inhibition of P53-mediated early-stage apoptosis and late-stage defective glycolysis and angiogenesis results in the significant prevention of diabetes-induced cardiac pathological change and dysfunction, suggesting that an intervention with P53 may be a therapeutic strategy for the prevention and treatment of DCM.

Post-Doctoral Platform Presentations

Aberrant Adult Neurogenesis in the Subventricular Zone (SVZ)-Rostral Migratory Stream (RMS)-Olfactory Bulb (OB) System Following Subchronic Manganese (Mn)

Exposure

Xue Fu1, Wendy Jiang1, Xiang Gao2, Xiaoting Wang2, Andrew Zeng1, Daniel Cholger1, Jason Cannon1, Jinhui Chen2, Wei Zheng1

1School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette IN 47907, United States; 2Spinal Cord and Brain Injury Research Group, Stark Neuroscience Research Institute, and Department of Neurological Surgery, Indiana University School of

Medicine, Indianapolis, IN 46202, United States

Adult neurogenesis occurs in the SVZ of brain lateral ventricle; newly produced neural stem/progenitor cells (NSPCs) migrate alongside RMS, providing the cells to the OB as well as nearby brain structure. Our recent studies reveal a significant reduction of Cu content in Mn-exposed adult SVZ accompanying with increased expressions of DMT1 and BrdU(+) cells in this neurogenic region. This study was designed to further determine whether Mn exposure disturbed the proliferation, migration, survival and differentiation of NSPCs from SVZ via RMS to OB. Adult rats were administered with a single ip dose of BrdU at the end of 4-wk subchronic Mn exposure to label the proliferating cells within SVZ. Immunostaining and cell counting data showed a total of 89.9±17.5 (mean+SD)×103 BrdU(+) cells in Mn-exposed SVZ, which was about 48% higher than that in the saline control (60.6±2.4×103) (p<0.05). The BrdU(+) proliferating cells were identified as a mixed population of GFAP-, Nestin- and DCX-positive cells. To investigate how these newly generated cells migrated, survived and differentiated, another group of adult rats received BrdU injections for 3 consecutive days prior to the 4-wk subchronic Mn exposure. The fates of these BrdU-labeled cells in the RMS and OB were traced during and after Mn treatment. After 2-wk Mn exposure, 95% of the BrdU(+) cells were found to migrate away from the SVZ and RMS, and finally distribute mainly in the granular cell layer (GCL) and mitral cell layer (MCL) of OB. Mn exposure significantly reduced the surviving BrdU/DCX/DAPI-labeled immature neurons in the OB (49% survival rate) as compared to the control (92% survival rate) (p<0.01). At the end of 4-wk treatment, most of the surviving BrdU(+) cells within the GCL and MCL were differentiated into NeuN(+) mature neurons. However, the survival rates of BrdU/NeuN/DAPI triple-labeled cells in OB were 33% and 64% in the 4-wk Mn-exposed and control animals, respectively (p<0.01). To study whether Cu had a direct effect on the cell proliferation in adult SVZ, an intra-cerebroventricular (i.c.v.) injection technique was used to deliver Cu into the lateral ventricle. Our data showed that high levels of Cu in the CSF significantly decreased the BrdU(+) proliferating cells in the SVZ. Taken together, these results suggest that Mn exposure initially enhances the cell proliferation in adult SVZ mainly by increasing large populations of astrocytic proliferating cells; these proliferating cells are capable of migrating along the RMS. In the OB, however, Mn exposure significantly reduces the surviving adult-born cells and markedly inhibits their differentiation into mature neurons, resulting in an overall decreased adult neurogenesis in the OB. (Supported by NIEHS ES008146)

Elevating CXCR7 improves angiogenic function of EPCs via Akt/GSK-3beta/Fyn-mediated Nrf2 activation in diabetic limb ischemia

Xiaozhen Dai, Xiaoqing Yan, Jun Zeng, Jing Chen, Jun Chen, Yan Li, Yi Tan

KCHRI, the Department of Pediatrics of the University of Louisville School of Medicine, Louisville, USA, 570 S Preston Baxter 1 Room 307, Louisville, KY

Background—Endothelial progenitor cells (EPCs) participate in vascular repair and regeneration. In diabetes low EPC number combined with EPC dysfunction impairs recovery from vascular injury. EPCs respond to SDF-1 through two SDF-1 receptors, CXCR7 and CXCR4. Here we report reduced expression of CXCR7 in diabetic EPCs and simply upregulating CXCR7 augments EPCs function in diabetes.

Methods and Results—In EPCs from db/db mice or in EPCs treated with ox-LDL, a damaging component of diabetic hyperlipidemia, expression of CXCR7, but not CXCR4 was reduced, which coincided with impaired angiogenic function. Treatment of EPCs with ox-LDL impaired tube formation and increased oxidative stress and apoptosis. The damaging effects of ox-LDL were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus (CXCR7-EPCs) but not in EPCs transduced with control lentivirus (Null-EPCs). Most importantly, CXCR7-EPCs were superior to Null-EPCs for transplantation therapy of ischemic limbs in db/db mice, as measured by VEGF expression, blood perfusion and angiogenesis. Mechanistic studies demonstrated that ox-LDL inhibited Akt and GSK-3β phosphorylation, nuclear export of Fyn and nuclear localization of Nrf2, resulting in reduced levels of downstream Nrf2 target genes HO-1, NQO-1 and catalase and a corresponding increase in EPC oxidative stress. This destructive cascade could be blocked by SDF-1 treatment in CXCR7-EPCs but not in Null-EPCs. Furthermore, specific inhibition of PI3K/Akt prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function both in vitro and in vivo.

Conclusions—Elevated expression of CXCR7 enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs for treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by the Akt/GSK-3β/Fyn pathway producing increased activity of Nrf2.

Benzene Exposure is Associated with Insulin Resistance in Humans and Mice

Wesley Abplanalp, Stacey Konkle, Daniel Riggs, Shesh Rai, Daniel Conklin, Sanjay Srivastava, Aruni Bhatnagar, Timothy O’Toole

Department of Medicine, University of Louisville, 580 S. Preston St., Louisville, KY 40202

Introduction: Benzene is a ubiquitous, volatile pollutant associated with hematotoxicity. Benzene metabolism is known to induce oxidative stress. Sustained oxidative stress spurs inflammation which can precipitate pre-diabetes and broader cardiometabolic effects, yet, little is known about benzene exposure and cardiometabolic disruption. Hypothesis: Inhaled, volatile benzene is metabolized by hepatic CYP2E1 and induces local and systemic oxidative stress, inflammation and insulin resistance.Methods:  The association between benzene (or urinary benzene metabolites) and pre-diabetes was appraised in two studies. The first study exposed male, 12wk old C57 mice to volatile benzene (50ppm x 6h/d x 2wk) at levels comparable to that found in the primary source of benzene exposure, cigarette smoke, and indices of oxidative stress, inflammation and pre-diabetes were measured. The second study involved a nationally representative human cohort with 1500 participants - the 2011/2012 National Health and Nutrition Examination Survey. Levels of benzene metabolites were regressed against glycemic indices to assess insulin sensitivity.Results:  In our murine study, fasting plasma glucose, fasting insulin, HOMA-IR levels, and glucose intolerance were significantly increased and while insulin tolerance decreased after benzene exposure. Insulin sensitivity (i.e. levels of Akt phosphorylation) was diminished in liver and skeletal muscle. These tissues demonstrated increased oxidative stress as evidenced by depleted glutathione levels and increased malondialdehyde concentrations.  Inflammation, as measured by NF-kB p65 phosphorylation in liver and skeletal muscle was found to be increased after benzene exposure.  Transcript levels of MIP1-a, a target of NF-kB, was upregulated along with SOCS1 expression.  SOCS1 competitively inhibits IRS-2 tyrosine phosphorylation, and this was diminished after benzene exposure. These effects on insulin sensitivity preceded the hallmark cytopenias typically seen after benzene exposure. Treatment with the anti-oxidant TEMPOL reversed the benzene-induced effects on glutathione levels, NF-kB p65 phosphorylation, cytokine and SOCS1 levels, IRS-2 tyrosine phosphorylation and fasting glycemic indices.

In the human cohort, benzene metabolites were positively associated with elevated fasting insulin, HOMA-IR scores.  After removing subjects exposed to tobacco smoke, the association between benzene metabolites and fasting insulin levels and HOMA-IR remained significant.  These associations with benzene metabolites occur in the absence of characteristic benzene-induced cytopenias.  Conclusions: Exposure to volatile benzene induces pre-diabetes in mice and elevated levels of benzene metabolites are highly associated with indices of pre-diabetes in humans.  These changes in insulin sensitivity precede incidence of cytopenias in the study populations, suggesting that insulin resistance may be an earlier, more sensitive indicator of benzene exposure than hematological disruption. Furthermore, these results suggest that persistent benzene exposure may be playing a role in the global diabetes epidemic.

Keynote Address:

Toxicology in the 21st century (Tox21)

Dr. Nicole Kleinstreuer is the deputy director of the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) within the NIEHS, in RTP, NC. She worked previously for Integrated Laboratory Systems as the head of the NICEATM computational toxicology group. She received degrees in mathematics and biomedical engineering from the University of North Carolina at Chapel Hill, her PhD in BioEngineering from the University of Canterbury in Christchurch, New Zealand, and completed her postdoctoral training with the U.S. EPA’s National Center for Computational Toxicology. Dr. Kleinstreuer maintains adjunct appointments at the Eshelman School of Pharmacy at the University of North Carolina at Chapel Hill, and in the NIEHS division of intramural research biostatistics and computational biology branch. Her research focuses on in vitro alternatives to animal testing, high throughput screening and multidimensional data analyses, and mathematical and computational modeling of biological systems and their susceptibility to environmental perturbations that may result in adverse outcomes.

Keynote Abstract

Toxicology in the 21st century (Tox21) has ushered in a scientific revolution focusing on novel alternative approaches to understanding chemical hazard and safety, with an emphasis on non-animal high-throughput screening (HTS) methods, adverse outcome pathways, and computational systems models to predict effects on human biology. The NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) is an office within the Division of the National Toxicology Program, National Institute of Environmental Health Sciences, and serves as a partner in the Tox21 high-throughput screening (HTS) program. NICEATM activities support the development and evaluation of new, revised, and alternative methods to identify potential hazards to human health and the environment, with a focus on replacing, reducing, or refining animal use. The NICEATM group is composed of toxicologists, chem- and bio-informaticists, computational biologists, validation specialists, literature review experts, and communications and administrative staff. NICEATM works with sixteen U.S. federal agencies with an interest in non-animal testing, via the Interagency Coordinating Committee on Validation of Alternative Methods (ICCVAM), and focuses on research efforts driven by agency priorities, both regulatory and scientific in nature. As a Tox21 partner, NICEATM applies computational toxicology techniques to analyze high-throughput screening data sets and build predictive models of chemical impacts on human health and disease pathways. Activities include the design and oversight of validation studies of novel in vitro assays for endpoints like skin sensitization, projects to implement alternative tests in regulatory policy such as the Endocrine Disruptor Screening Program, curation of high-quality data from stakeholders and the public literature, organization of scientific workshops, and education, communication, and training with national and international partners. Recent initiatives are focusing on the development of online tools in an “Integrated Chemical Environment” (ICE) to make NICEATM analysis workflows publically available and provide datasets that are transparent, computable and searchable for model-building and evaluation.