South Central Chapter of the

Society of Toxicology 2021 Annual Virtual Meeting

Toxicology: from basic science to regulatory assessment

Friday, November 12, 2021

Virtual Meeting via Zoom

Hosted by University of Arkansas for Medical Sciences (UAMS)

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South Central Chapter of the Society of Toxicology 2021 Annual Virtual Meeting

Agenda

8:30 AM - SCC 2021 Regional meeting begins.

8:35 AM - Welcome remarks by SCC President and Vice President.

8:40 AM – Keynote Lecture - M. Ian Gilmour, PhD (US EPA). COMPARATIVE TOXICITY OF SMOKE FROM DIFFERENT FUELS AND COMBUSTION CONDITIONS

Platform Session 1 - Graduate Students & Non-faculty/non-students

9:40 AM - Oliver McGehee, University of Louisiana at Monroe. CHROMATOGRAPHIC IDENTIFICATION OF ECHINACEA CONSTITUENTS THAT MEDIATE FREE RADICAL QUENCHING UTILIZING CHEMOMETRIC ANALYSIS

10:00 AM - Julie D. Tobin, LSU Health Sciences Center, Shreveport. THE ROLE OF SODIUM-DEPENDENT DICARBOXYLATE TRANSPORTERS IN THE TOXICITY OF DIGLYCOLIC ACID

10:20 - 10:40 AM - BREAK

10:40 AM - Shilpa Thota, Southern University and A&M College. MELATONIN MITIGATES CIGARETTE SMOKE EXTRACT-INDUCED INFLAMMATION: IMPACT ON AUTOPHAGY AND CHAPERONES

11:00 AM - Rakeysha Pinkston, Southern University and A&M College/Louisiana State University. FOURTH-GENERATION DISPOSABLE ELECTRONIC NICOTINE DELIVERY SYSTEM (ENDS) AEROSOLS INDUCE INCREASED CELLULAR TOXICITY IN MURINE MACROPHAGES EXPOSED AT THE AIR-LIQUID INTERFACE COMPARED TO AEROSOLS PRODUCED BY A THIRD-GENERATION DEVICE

11:20 AM - Rashmi Pathak, Louisiana State University. GROWTH INHIBITORY AND IMMUNOMODULATORY EFFECTS OF BLUEBERRIES IN BREAST CANCER CELL LINES

11:40 AM - Alexandra Denys, LSU Health Sciences Center, New Orleans. CHRONIC BINGE ALCOHOL AND OVARIECTOMY INDEPENDENTLY AND DIFFERENTIALLY AFFECT BONE IN SIV-INFECTED MONKEYS

12:00 - 1:30 PM - LUNCH BREAK & POSTER VIEWING

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South Central Chapter of the Society of Toxicology 2021 Annual Virtual Meeting

Agenda (continued)

1:30 - 2:30 PM – Keynote Lecture - Shraddha Thakkar, PhD (US FDA). SAFETAI INITIATIVE: AI BASED PREDICTION INITIATIVE TO ASSIST REVIEWERS WITH PREDICTING TOXICITY END POINT

Platform Session 2 - Graduate Students & Non-faculty/non-students

2:30 PM - Chandra Mohan Reddy Muthumula, University of Louisiana at Monroe. IDENTIFICATION OF CONSTITUENTS OF HYDROETHANOLIC ECHINACEA EXTRACTS ACTIVE IN FREE RADICAL QUENCHING BY N-HEXANE PARTITIONING AND CHEMOMETRIC ANALYSES

2:50 PM - Amelia Craze, University of Mississippi. EXTRACTION TECHNIQUE COMPARISON AND SEASONAL DIFFERENCES IN OXIDATIVE POTENTIAL ANALYSIS OF FINE PARTICULATE MATTER (PM2.5)

3:10 - 3:15 PM - Closing remarks.

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KEYNOTE SPEAKER M. Ian Gilmour, PhD

Dr. M. Ian Gilmour is a Principal Investigator and Chief of the Cardiopulmonary and Immunotoxicology Branch at the U.S Environmental Protection Agency (EPA) in Research Triangle Park (RTP). He received an Honors degree in Microbiology from the University of Glasgow, and a Doctorate in Veterinary Science from the University of Bristol. After post-doctoral work at the Johns Hopkins School of Public Health and the University of North Carolina, he joined the EPA in 2000. He holds adjunct faculty positions and teaches at the UNC School of Public Health and the Curriculum in Toxicology. He has published over 120 journal articles and reviews in the field of pulmonary immunobiology where his research focuses on the effect of air pollution chemistry on the development of infectious and allergic lung disease. Most recently he has studied how different substrates and combustion conditions affect the chemistry and toxicity of smoke from biomass and other burns. He has received numerous awards for his work, has served as elected councilor and president of the SOT Inhalation and Respiratory Specialty Section and is a member of several external scientific advisory committees and journal editorial boards.

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KEYNOTE SPEAKER Shraddha Thakkar, PhD

Dr. Thakkar received a master's and Ph.D. in bioinformatics from the University of Arkansas at Little Rock/University of Arkansas Medical Sciences, Joint Bioinformatics program. Before joining CDER/OTS/OCS, she worked at FDA's National Center for Toxicological Research. She specialized in macromolecular crystallography, chemo-informatics, toxico-informatics, toxico-genomics, structural biology, and AI-driven predictive modeling for drug development and safety assessments. Before that, Dr. Thakkar received an MSc. in Biotechnology from Bangalore University, India. At this date, she has over 24 peer-reviewed publications, two book chapters, 2 approved U.S. patents and 13 U.S. and other

patent applications, as well as over 80 scientific presentations to her credit in the area of drug development for radiation protection, predictive toxicology, leukemia, and atherosclerosis.

Dr. Thakkar's primary research interest is to apply bioinformatics and chemoinformatics, Artificial Intelligence (AI) and Machine Learning, and data analytics for biomarker discovery, drug safety, pharmacogenomics/toxicogenomics, and precision medicine. Her current research work is in applying bioinformatics and cheminformatics, and AI-driven methods to study toxicity and drug development with a specific interest in drug-induced liver injury. Dr. Thakkar is leading the research efforts in developing a liver toxicity knowledge base (LTKB) for drug safety, updating the classification with an extended drug list, and developing various predictive models to enable early prediction of toxicity liabilities. Upon joining the computational sciences office, Dr. Thakkar utilized the project management skillset to lead the various collaborative projects such as Smart Template System and Statistical Programming Support. Programs supported and develop under this support were also used for the COVID-19 for both vaccine emergency authorization analysis by CBER reviewers.

Dr. Thakkar has adjunct appointments at both the University of Arkansas for Medical Sciences and the University of Arkansas at Little Rock (Assistant Professor). Dr. Thakkar has received multiple research and leadership awards regionally and nationally, and within the FDA. Dr. Thakkar has had a leading role in many national and international societies. Dr. Thakkar was elected as Board member of the Mid-South Computational Biology and Bioinformatics Society (MCBIOS) in 2014 and served as President of the Society from 2016-2017. She is also the Founder and Chair of Personalized Medicine Community (2018-19) and served on the Awards Committee at the American Association of Pharmaceutical Scientist (AAPS) (2016-20). For her contribution to AAPS, she is elected to the AAPS board of directors for term 2020-22. Dr. Thakkar is also serving as a leader on the Bioinformatics Working and Cross Training Working Group Global Coalition for Regulatory Science Research.

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South Central Chapter of the Society of Toxicology 2021 Annual Virtual Meeting

Posters

1. COMPARISON BETWEEN BLACK CARBON AND OXIDATIVE POTENTIAL OF FINE PARTICULATE MATTER (PM2.5) FROM FOUR LOCATIONS IN TENNESSEE *Voke Tonia Aminone, Courtney Roper. BioMolecular sciences Department, University of Mississippi, Mississippi, United states.

2. INHALATION OF COMBUSTION-DERIVED ENVIRONMENTALLY PERSISTENT FREE RADICALS CAUSES VASCULAR ENDOTHELIAL INJURY MEDIATED VIA AHR ACTIVATION IN ALVEOLAR TYPE-2 PNEUMOCYTE *Ankit Aryala, Ashlyn C. Harmona, Lavrent Khachatryanb, Pratiti Chowdhurya, Alexandra Noëla, Arthur Penna, Stephania Cormiera, Kurt Varnerc and Tammy R. Dugasa. aDepartment of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, bDepartment of Chemistry, Louisiana State University, cDepartment of Pharmacology and Experimental Therapeutics, School of Medicine, LSU Health Sciences Center.

3. ROLE OF DANGER-ASSOCIATED MOLECULAR PATTERNS IN VAPING-INDUCED INFLAMMATION R. Begum*1, S. Thota1, D. Kambiranda2 and S. Batra1. Laboratory of Pulmonary Immuno-toxicology, 1Department of Environmental Toxicology & 2Southern University Agriculture Research and Extension Center, Southern University and A&M College.

4. DIESEL PARTICULATE EXPOSURE: CROSS-TALK BETWEEN IL-17 FAMILY AND APOPTOTIC PATHWAY GENES N. Bidarimath, A. Abdulkadir, S. Batra. Laboratory of Pulmonary Immunotoxicology, Department of Environmental Toxicology, Southern University and A&M College.

5. DOES ACETATE MEDIATE SKELETAL BONE TOXICITY OF ALCOHOL? *C. Del Valle–Ponce de Leon, H. Hang, A. Denys, K. B. Pedersen, M. J. Ronis. Louisiana State University Health Sciences Center in New Orleans.

6. EFFECT OF UCP2 OVEREXPRESSION ON GLUCOSE METABOLISM THROUGH WNT SIGNALING *N.A. Jacquet, D. Frierson, and Y. Zhao. Department of Pharmacology, Toxicology and Neuroscience, LSU Health Sciences Center in Shreveport, Shreveport, Louisiana.

7. SOURCES AND FACTORS EFFECTING THE ENVIRONMENTAL BEHAVIOR OF BLACK CARBON AT SAMPLING SITES IN MISSISSIPPI BETWEEN SEPTEMBER 2013 AND DECEMBER 2014 *H.T. Nguyen and C. Roper. Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University, Mississippi.

8. PROTEOMICS REVEALS THE PROGNOSTIC VALUE OF LACTATE DEHYDROGENASE IN ACETAMINOPHEN-INDUCE ACUTE LIVER FAILURE PATIENTS J. Vazquez1,2, S. Kennon-McGill2, S.D. Byrum3, S.G. Mackintosh3, D.K. Williams4, W.M. Lee5, J.A. Dranoff6, M.R. McGill1,2.

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1Dept. of Pharmacology and Toxicology, College of Medicine; 2Dept. of Environmental and Occupational Health, Fay W. Boozman, College of Public Heallth; 3Dept. of Biochemistry, College of Medicine; 4Dept. of Biostatistics, College of Medicine; University of Arkansas for Medical Sciences. 5Dept. of Internal Medicine, University of Texas – Southwestern. 6Yale University. 9. IN VITRO MODEL TO STUDY THE EFFECTS OF CANNABINOIDS ON CELL POPULATIONS AND CYTOKINE PRODUCTION *O. Cresswell and B.L.F. Kaplan. Center for Environmental Health Sciences, Department of Comparative Biomedical Sciences, Mississippi State University.

10. EARLY DEVELOPMENTAL EXPOSURE TO Δ9-TETRAHYDROCANNABINOL CAUSES HYPERACTIVITY IN LARVAL ZEBRAFISH THAT PERSISTS INTO ADULTHOOD *V. L. Jackson, Z. Pandelides, C. E. Thornton, K. L. Willett. University of Mississippi.

11. MOLECULAR RESPONSES TO 3-MONTH OF EXPOSURE TO ELECTRONIC-CIGARETTE AEROSOL COMPOSED SOLELY OF HUMECTANTS IN LUNGS OF FEMALE MICE A. Jones1, B. Holliday2, M. Schexnayder3, Z. Perveen2, A. Noël2. Departments of 1Biological Sciences, 2Comparative Biomedical Sciences, Louisiana State University. 3Lincoln Memorial University.

12. CARBOXYLESTERASE 1 RELEASES OXYLIPINS FROM OXIDIZED TRIACYLGLYCEROLS: AN INITIAL EXAMINATION OF ITS SUBSTRATE SELECTIVITY Maggie Philips, A. Borazjani, M.K. Ross. Department of Comparative Biomedical Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University.

13. PRELIMINARY INVESTIGATION INTO EFFECTS OF LEACHATES FROM 3D-PRINTED OBJECTS ON ZEBRAFISH SPERM QUALITY *MJ Schwinga, Y Liua,b, JA Belgoderea, WT Monroea, TR Tierschb, A Abdelmoneima. aLouisiana State University, bAquatic Germplasm and Genetic Resources Center, Louisiana State University Agricultural Center.

14. SPLITTING FINE PARTICULATE MATTER (PM2.5) FILTERS: COMPARISON OF CHEMICAL COMPOSITION AND OXIDATIVE POTENTIAL BETWEEN FILTER PIECES *A.M. Sidwell, C.L. Roper. University of Mississippi

15. LUNGS OF FEMALE MICE EXPOSED IN UTERO TO COOL MINT-FLAVORED PUFF BAR AEROSOL HAVE INCREASED SUSCEPTIBILITY TO TH2-MEDIATED IMMUNE RESPONSES AND GREATER FUNCTIONAL DECLINE FOLLOWING HOUSE-DUST MITE EXPOSURES *H. Stewart1, M. Schexnayder2, A. Penn1, A. Noël1. 1Louisiana State University, 2Lincoln Memorial University.

16. BAP INDUCED MULTIGENERATIONAL GENE EXPRESSION AND DNA METHYLATION CHANGES ARE PRIMARILY INFLUENCED BY PATERNAL GENOTYPE *M. C. Sturgis, Z. Pandelides1, C. Thornton1, M. Patel1, N. Aluru2, K. L. Willett1. 1University of Mississippi, University, MS; 2Woods Hole Oceanographic Institution, Woods Hole, MA.

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Meeting Abstracts Platform

Platform 1. CHROMATOGRAPHIC IDENTIFICATION OF ECHINACEA CONSTITUENTS THAT MEDIATE FREE RADICAL QUENCHING UTILIZING CHEMOMETRIC ANALYSIS O. C. McGehee1, C. Muthumula1, S. Nagumalli1, J. D. Carstens2, C. V. Landingham3, and S. A. Meyer1. 1University of Louisiana at Monroe, Monroe, LA; 2North Central Regional Plant Introduction Station, USDA-ARS, Ames, IA; and 3Ramboll Environ US Corporation, Monroe, LA. A continuing challenge of natural products research is to identify constituents that contribute a given bioactivity in situ to source material. Many studies have shown that Echinacea contains phenolic compounds that possess strong antioxidant properties. However, little is known on which constituents are contributing the most or if any synergism between the constituents might be promoting these effects. Previous studies in our lab using a predictive modeling approach have directed us towards the aromatic region of 1HNMR as good quenchers of free radicals. Hence, we optimized an RP-HPLC method for separating and identifying polyphenols in situ so we could measure which specific polyphenols were contributing most to the quenching. RP-HPLC chromatograms were obtained for reagent grade standard caffeic acid derivatives and for three separate extractions across 11 different accessions of Echinacea. Using area under the curve data obtained from chromatograms as well as free-radical quenching activity obtained from electron paramagnetic resonance (EPR) spectrometry, we were able to identify peaks in the chromatogram that contribute most to this increase in activity. The predictive model has directed us to certain polyphenols that best correlate towards the quenching activity present in species of Echinacea in situ. Further studies will explore factors of the mixtures that alter constituent specific activities.

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Platform 2. THE ROLE OF SODIUM-DEPENDENT DICARBOXYLATE TRANSPORTERS IN THE TOXICITY OF DIGLYCOLIC ACID *J. Tobin, C. Jamison, C. Robinson, K. McMartin. Department of Pharmacology, Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA. Diethylene glycol (DEG) is an industrial solvent, generally found in brake fluid and lubricants, implicated in mass poisonings worldwide. The predominant metabolites of DEG are 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA). Exposure to DEG causes proximal tubular necrosis, as well as hepatotoxicity and a delayed peripheral neuropathy. DGA has been shown to accumulate in the organs, which implicates it as the metabolite responsible for the renal and hepatic toxicity seen during these poisonings. Due to this accumulation, transport of DGA into kidney cells was relevant. DGA is structurally comparable to succinate, making it a possible substrate for the sodium dependent dicarboxylate transporters (NADCs). Pharmacological inhibition of NADCs showed reduced DGA accumulation. In a genetic knockdown model, NADC1 and NADC3 activities were targeted via nucleofection with siRNA. The cells were grown on membrane inserts and dosed with DGA. Cell toxicity was measured using lactate dehydrogenase (LDH) release. Knockdown of NADC1 reduced the cytotoxicity of DGA, suggesting decreased uptake into cells. This corroborated the inhibitor studies and suggested that NADC1 is a possible therapeutic target in DGA toxicity. In a toxicity study of DEG in rats, only about half of the animals given the same toxic dose developed kidney damage. It was of interest to determine whether a difference in NADC transporters in the kidneys might explain a differential susceptibility to DGA uptake and toxicity. Kidney RNA was extracted and mRNA levels of NADC1 were confirmed via qRT-PCR. There was a difference found between the mRNA levels of the sick animals compared to their matched controls, which suggests that the reason for only some animals getting sick may be related to the amount of NADC1 and hence of DGA uptake. (Support: American Chemistry Council)

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Platform 3. MELATONIN MITIGATES CIGARETTE SMOKE EXTRACT-INDUCED INFLAMMATION: IMPACT ON AUTOPHAGY AND CHAPERONES S. Thota1*, R. Begum1, D. Kambiranda1,2, S. Batra1. 1Laboratory of Pulmonary Immunotoxicology, Department of Environmental Toxicology, 2Southern University agriculture and extension center, Southern University and A&M College, Baton Rouge, Louisiana-70813, USA. Cigarette smoking and other nicotine-based products cause chronic lung inflammation. Despite improved therapeutic options, the prognosis of cigarette smoke (CS) induced inflammation and associated pathologies has not improved significantly. Diet supplementation strategies are alluring in this regard because the implementation is relatively simple in populations with limited income and resources. Though N-acetyl-5-methoxy-tryptamine (melatonin) is produced by the pineal gland and other human tissues, its role as a dietary supplement is being extensively studied in various inflammatory models. We therefore hypothesize that supplementation of melatonin can rescue CS-induced inflammation. To prove our hypothesis, we first examined the impact of CS-extract (CSE-250 ng/ml)-on cytokine/chemokine production and the activation of transcription factors in human lung adenocarcinoma cells (A549) with type II epithelial characteristics. A significant increase in the production/transcription of chemokines (CXCL8, CCL2 and CCL5) and pro-inflammatory cytokines (IL-1b and IL-6) was observed in CSE-challenged A549 cells. Our results showed important role of transcription factors-NF-kB in CSE-induced inflammation. Besides regulating pro-inflammatory mediators, NF-kB has also been shown to regulate autophagy process in response to a wide variety of stimulants. Our results showed a CSE-mediated increase in the expression of several autophagy related proteins (LC3B, Beclin-1, ATG5, and ATG16). Interestingly, pre-treatment with melatonin rescued CSE-induced activation of NF-kB and abrogated the expression of autophagy genes. Further, CSE-mediated increased expression of heat shock protein (HSP)90 and TLR-4 demonstrate their role in inflammatory process. Interestingly, Beclin-1 has been shown to regulate TLR-4 mediated autophagy by directly binding to TLR-4 signaling complex, while Hsp90 maintains Beclin-1 homeostasis and participates in TLR-mediated autophagy. Further, studies are in progress to delineate the effect of melatonin on these molecular mechanisms regulated/induced by CSE.

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Platform 4. FOURTH-GENERATION DISPOSABLE ELECTRONIC NICOTINE DELIVERY SYSTEM (ENDS) AEROSOLS INDUCE INCREASED CELLULAR TOXICITY IN MURINE MACROPHAGES EXPOSED AT THE AIR-LIQUID INTERFACE COMPARED TO AEROSOLS PRODUCED BY A THIRD-GENERATION DEVICE *R. Pinkston1,2, A. L. Penn2, and A. Noël2. 1Department of Environmental Toxicology, Southern University and A &M College, Baton Rouge, LA, 70813; 2Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, 1909 Skip Bertman Drive, Baton Rouge, LA, 70803, USA. Background: Electronic nicotine delivery systems (ENDS), including electronic-cigarettes (e-cigs) and JUUL-like devices, are now considered the most commonly used form of tobacco products among youth in the United States. The prevalence of ENDS usage is currently more than 2 million among middle and high school students, fueling concerns over the adverse health effects related to ENDS usage. New disposable ENDS devices contain high levels of nicotine salt (60 mg/mL), whose cellular and molecular effects on immune cells are currently unknown. Here, we used a physiologically-relevant in vitro air-liquid interface (ALI) exposure model to assess the toxicity of two distinct ENDS, a 3rd-generation electronic-cigarette (e-cig) and a 4th-generation disposable device (Posh Plus). Methods: ENDS aerosols were generated using a peristaltic pump (Posh Plus) or a third-generation e-cig device (Scireq). Applying standard vaping topography profiles, murine macrophages (RAW 264.7) were exposed at the ALI to crème brûlée-flavored (CB) aerosols generated from those two devices for 1-hour per day for 1 or 3 consecutive days. Medical grade compressed air was used as a control. Cellular and molecular toxicity was evaluated 24 hours post-exposure. Results: Posh CB-flavored aerosols displayed significant cytotoxicity--decreased cell viability (≥ 50%) and increased lactate dehydrogenase (LDH) levels (≥ 150%)--after 1- and 3-day exposures, while the 3rd-generation e-cig device only displayed significant cytotoxicity after 3 days. Further, both Posh and e-cig CB-aerosols elicited ≥ 50% increase in reactive oxygen species (ROS) plus high levels of 8-isoprostane (≥150pg/ml) after 1 and 3 days, indicating increased oxidative stress (OS). Posh and e-cig CB-aerosols elicited ≥ 50% reduction in nitric oxide (NO) levels after one day but elicited ≥ 30% increase in NO after 3 days. Genes in common dysregulated by both devices after 1 day included α7nAChR, Cyp1a1, Ahr, Mmp12, and iNos. At 3 days, Posh aerosol dysregulated 29 genes associated with allergy and asthma, compared to 9 genes for the e-cig aerosol. Conclusion: Our results suggest that ENDS CB-flavored aerosol exposures from both 3rd- and 4th-generation ENDS devices are cytotoxic to macrophages and cause OS, which can translate into macrophage dysfunction. Although 4th-generation disposable ENDS devices have no adjustable operational settings, the cellular toxicity of their aerosols may be more harmful than that of aerosols produced by previous generations of ENDS. Thus, this study provides scientific evidence for regulation of nicotine salt-based disposable ENDS products. (Support: LSU-SVM Start-up funds (AN), NIH NHLBI K01 (AN))

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Platform 5. GROWTH INHIBITORY AND IMMUNOMODULATORY EFFECTS OF BLUEBERRIES IN BREAST CANCER CELL LINES *R. Pathaka, A. Kumarb,c, D.P. Singha, P.J. Ebenezera, Joseph Francisa. aComparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA. bDepartment of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA. cPennington Biomedical Research Center, Baton Rouge, Louisiana 70808, U.S.A. Breast cancer is one of the most common cancers among women worldwide. Immune evasion by cancer cells allows tumor progression and presents a major challenge in therapy. Natural products like blueberries are rich in anthocyanins and polyphenolic contents and have been shown to produce anti-carcinogenic effects. However, the effect of blueberries on the immune escape potential of breast cancer cells has not been studied extensively. The present study was therefore conducted to investigate the effects of blueberry extract (BBE) on pathways implicated in cancer cell proliferation (mTOR-AKT) and immune evasion (PDL1-STAT3) in MCF-7 cells. The survival of control (MCF-10A) and cancer (MCF-7) cells after treatment with BBE at various concentrations (0, 0.25, 0.5, 1, 2, 4, 8, 16 mg/mL) was determined using MTT assay which indicated that BBE exhibited higher toxicity at all concentrations towards MCF-7 compared to MCF-10A cells. These results were further corroborated by observed inhibitory effects and morphological changes induced by BBE in MCF-7 cells. Immunoblotting assays demonstrated that BBE significantly reduced the expression of activated AKT, mTOR and p21 in MCF-7 cells. BBE also inhibited MCF-7 cell migration as shown by wound healing assay. A marked decrease in PD-L1 levels upon treatment with BBE was also detected. As predicted, STAT-3 which regulates PD-L1 expression, was also reduced by treatment with BBE. Similar results were also obtained in other breast cancer cell lines (MDA-MB-231 and 4T1). Collectively, our findings suggest that blueberries can prevent the growth and immune evasion potential of breast cancer cells. Further research is warranted to test if blueberry intervention in combination with existing therapeutics can offer an important strategy for cancer prevention.

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Platform 6. CHRONIC BINGE ALCOHOL AND OVARIECTOMY INDEPENDENTLY AND DIFFERENTIALLY AFFECT BONE IN SIV-INFECTED MONKEYS *A. Denys1, L.J. Suva2, L. Simon1, P.E. Molina1, M.J. Ronis1. 1Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA. 2Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA. People living with HIV (PLWH) represent a high-risk population for several comorbidities, including Alcohol Use Disorders (AUDs) and osteopenia/osteoporosis. Diminishing bone health in this population is multifactorial, influenced by HIV infection, antiretroviral therapy (ART) and alcohol use. The current study in rhesus macaques was designed to assess the effect of each of these factors on several pathological processes, including inhibition of bone formation. Thirty-two female animals were randomized first to either chronic binge alcohol (CBA) or isocaloric sucrose (SUC) treated groups. Three months after initiating the CBA or SUC administration protocols, animals were infected with simian immunodeficiency virus (SIV). ART was initiated approximately two and a half months after SIV inoculation and continued throughout the study period. One month into ART, animals were further randomized into ovariectomized (OVX) or sham (Sham) groups, resulting in four SIV+/ART+ experimental groups (N=8 per group): SUC/Sham; SUC/OVX; CBA/Sham; and CBA/OVX. Serum was collected at six timepoints, and bones were collected at necropsy for histomorphometry analysis. The mean concentrations for osteocalcin, a marker of bone formation, were decreased in an alcohol- and time-dependent fashion, consistent with our previous clinical findings that osteocalcin is significantly suppressed in relationship to alcohol consumption in PLWH. Cortical porosity of the mid shaft and cortical thickness were very significantly (p < 0.001) increased and decreased, respectively, by ovariectomy, though not affected by CBA. Instead, trabecular parameters appeared to be negatively influenced by both CBA and ovariectomy, including trabecular bone volume (BV/TV), bone mineral density (vBMD), trabecular number (Tb.N) and spacing (Tb.Sp). We conclude that CBA and estrogen status each independently alter trabecular bone parameters, while only ovariectomy seems to have profound effects on intracortical remodeling in female rhesus macaques. Supported by: F30 AA028999 (A.D.), R37 AA018282 (M.R.), P60 AA009803 (P.M.).

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Platform 7. IDENTIFICATION OF CONSTITUENTS OF HYDROETHANOLIC ECHINACEA EXTRACTS ACTIVE IN FREE RADICAL QUENCHING BY N-HEXANE PARTITIONING AND CHEMOMETRIC ANALYSES *C.M.R. Muthumula1, S.K. Nagumalli1, O.C. Mcgehee1, J.D. Carstens2, C.V. Landingham3, K.A. ElSayed1, and S.A. Meyer1. 1School of Basic Pharm. and Toxicological Sciences, ULM, Monroe, LA; 2N. Central Regional Plant Introduction Station (NCRPIS), USDA-ARS, Ames IA; 3Ramboll Environ, Monroe, LA. Echinacea spp. contains numerous classes of chemical constituents known to possess free radical-quenching activity (FRQA), but the relative potencies of these constituents in situ are unknown. To address this issue, we used chemometric approach, where chemical shifts of the spectral areas from 1H-NMR (400 MHz) spectroscopy (Xij), were correlated with the sample’s DPPH• quenching activity (Yi) by Orthogonal-partial least square (OPLS) regression. 1H-NMR profiling of different Echinacea spp. was performed and relative areas of spectral regions (0.04 ppm bins; 0.00 – 10.00 ppm) were integrated. FRQA of 75% ethanolic and n-hexane washed ethanolic extracts of different Echinacea spp. against the free radical, DPPH•, was determined using EPR spectroscopy and expressed as Trolox-equivalents. OPLS-regression analyses of 1H-NMR data, using 75% ethanolic extracts, identified the chemical shift range of 5.10-7.70 ppm in the olefinic/aromatic region that best correlated with FRQA. We observed a small increase in the FRQA of the 75% ethanolic extracts after defatting with n-hexane. These data show enrichment of constituents responsible for the FRQA of Echinacea after defatting. In conclusion, this data supports the application of chemometrics as a tool to identify the constituent classes of bioactive chemicals within a complex mixture in situ.

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Platform 8. EXTRACTION TECHNIQUE COMPARISON AND SEASONAL DIFFERENCES IN OXIDATIVE POTENTIAL ANALYSIS OF FINE PARTICULATE MATTER (PM2.5) *A.M. Craze, C. Roper. Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University, Mississippi. Fine particulate matter (PM2.5) is a complex mixture of the solid and liquid portion of air pollution that is 2.5 microns or smaller in aerodynamic diameter and has been shown create harmful free radicals that can cause a variety of health impacts not limited to the respiratory system. To analyze these airborne particles, sampling can be performed using collection onto filters. At this time, there is no standardized method for extraction of PM2.5 from a filter surface, but studies have shown that the extraction method can impact both chemical and toxicological analysis. The dithiothreitol (DTT) assay is one acellular assay to determine the oxidative potential of a sample via colorimetric analysis of oxidized thiols, which simulate sulfur groups in biological organisms. The objective of this study was to compare the impact of filter extraction methods on oxidative potential analysis results across seasons from filters collected by the Arkansas Department of Environmental Quality for both urban and rural locations. Of the components of PM2.5, black carbon (BC) or soot, is commonly associated with vehicular traffic. BC analysis was performed using a SootScan Transmissometer on whole filters across seasons with averages ranging from 1.99 to 7.16 mg.m3, and BC concentration was positively correlated with PM2.5 concentrations. After BC analysis, filters were split into quadrants and each quadrant was extracted using a different solvent, but all other extraction steps remained identical. Across all locations and seasons, significant differences in DTT consumption were observed between DCM and all other solvents, for both whole particle and soluble fractions of PM2.5 filter extracts. To conclude, this project has demonstrated the need for a standardized extraction method to better analyze sampled PM2.5 to understand spatial and seasonal differences.

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Meeting Abstracts Posters

1. COMPARISON BETWEEN BLACK CARBON AND OXIDATIVE POTENTIAL OF FINE PARTICULATE MATTER (PM2.5) FROM FOUR LOCATIONS IN TENNESSEE Voke Tonia Aminone, Courtney Roper. BioMolecular sciences Department, University of Mississippi, Mississippi, United states.

Air pollution is harmful to the health of humans and animals even at low concentrations. Fine particulate matter (PM2.5) is one of the most common air pollutants of concern as it causes and exacerbates cardiovascular, respiratory, endocrine co-morbid conditions and damages tissues and DNA. Black carbon is a good indicator of combustion related air pollution and it is associated with a variety of cardiovascular conditions. The most common toxicity mediated mechanism of PM2.5 is its ability to cause oxidative stress which causes and exacerbates black carbon and PM2.5 related health conditions. The aim of this research is to analyze the black carbon content and oxidative potential in PM2.5 from filters collected in 2014 by the Tennessee Department of Environment and Conservation (TDEC). Black carbon concentrations were determined for 4 locations on the same dates in November (n=8/location), these locations were selected to capture variation in PM2.5 concentrations with the locations with the two highest and two lowest average PM2.5 concentrations used for subsequent analyses. Filters were extracted via sonication in methanol for 60 minutes. Aliquots of the extracted solutions were removed for the dithiothreitol (DTT) assay to measure the oxidative potential of each sample at 412 nm. The monthly average black carbon concentrations for Loudon (2.47 ± 1.57 µg/m3) and Harriman (2.46 ± 1.59 µg/m3) represented the high PM2.5 concentration locations. While the low PM2.5 concentration locations were Dyersburg and Jackson, with average black carbon concentrations of 1.18 ± 0.51 and 1.11 ± 0.41 µg/m3, respectively. Oxidative potential for the low PM2.5 concentrations varied between days in November, even at the same location, indicating daily variability in these measurements. There was a significant positive correlation between black carbon and PM2.5 concentrations from the four locations. Strong trends were not observed between oxidative potential and PM2.5 or black carbon concentrations. This work highlights the black carbon and oxidative potential differences between and within sampling locations in a single month. Future work will explore if these trends continue across additional locations and months.

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2. INHALATION OF COMBUSTION-DERIVED ENVIRONMENTALLY PERSISTENT FREE RADICALS CAUSES VASCULAR ENDOTHELIAL INJURY MEDIATED VIA AHR ACTIVATION IN ALVEOLAR TYPE-2 PNEUMOCYTE *Ankit Aryala, Ashlyn C. Harmona, Lavrent Khachatryanb, Pratiti Chowdhurya, Alexandra Noëla, Arthur Penna, Stephania Cormiera, Kurt Varnerc and Tammy R. Dugasa. aDepartment of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, bDepartment of Chemistry, Louisiana State University, cDepartment of Pharmacology and Experimental Therapeutics, School of Medicine, LSU Health Sciences Center. Particulate matter containing environmentally persistent free radicals (EPFRs) is formed during the thermal remediation of organic pollutants when incomplete combustion of the waste occurs in the presence of transition metals. Initial studies used laboratory generated EPFRlo: (1.5e16 radicals/g particles) and EPFRhi: (1.0e18 radicals/g) at 250 µg/m3 to investigate their effects on vascular endothelial and pulmonary oxidative stress in C57BL/6 male mice. These studies demonstrated that exposure to EPFRs results in an increase in endothelin-1 (ET-1) and intracellular adhesion molecule-1 (ICAM-1) at high, but not low radical concentrations. In addition, genes associated with AhR activation (Cyp1a1/1b1), and oxidative stress (NQO1) were upregulated in both the lungs and the aorta. We also found that AhR activation was significantly increased in AT-2 pneumocytes during EPFR hi exposure. We hypothesized that inhalation of EPFRs leads to vascular endothelial dysfunction via activation of AhR in the AT-2 cells in a manner dependent upon radical concentration. To address our hypothesis, AhR was knocked-down in AT-2 pneumocytes in male and female mice using the Cre/lox recombinase system prior to exposure to filtered air (FA), EPFRlo, or EPFRhi for 4h/d for 5 days. Plasma ET-1 remained unchanged between KO and WT mice exposed to FA and EPFRlo; however, ET-1 was significantly decreased in AhR KO mice exposed to EPFRhi versus WT. We also identified a decrease in blood pressure in AhR KO mice compared to WT mice when exposed to EPFRhi, supporting our hypothesis that AhR deficient mice would be protected from endothelial injury after EPFRhi exposure. Together, these data suggest that EPFR exposure promotes AhR activation in AT-2 pneumocyte and this in turn results in vascular endothelial dysfunction, likely at the air-blood interface. (Support: LSU Superfund)

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3. ROLE OF DANGER-ASSOCIATED MOLECULAR PATTERNS IN VAPING-INDUCED INFLAMMATION R. Begum*1, S. Thota1, D. Kambiranda2 and S. Batra1. Laboratory of Pulmonary Immuno-toxicology, 1Department of Environmental Toxicology & 2Southern University Agriculture Research and Extension Center, Southern University and A&M College. Electronic cigarettes (e-cigs) and other tobacco-based products are addictive. However, due to their projection as less harmful alternatives, electronic smoking devices or e-cigs gained popularity in the US markets rapidly. Earlier reports suggest that danger-associated molecular patterns (DAMPs) including high-mobility group box (HMGB)1and heat shock protein (HSP)70 engage with toll-like receptors (TLRs) or receptors for advanced glycation end-products (RAGE) and activate the downstream signaling events during cigarette smoke exposure. Additionally, aerosols from electronic-cigarettes (e-cigs) have been reported to contain toxins that can cause inflammation/immunomodulatory effects similar to cigarette smoke. Considering this background, we hypothesized important role of HMGB1 and HSP70 in regulating TLR-4/RAGE-dependent inflammation in e-cig vapor condensate (tobacco flavor; TF-ECVC) challenged human lung alveolar epithelial cells (A549). Our experiments revealed TF-ECVC mediated increase in-a) transcription of TLR-4 and RAGE; b) expression and activation of transcription factors-NF-kB and STAT3; c) production of pro-inflammatory cytokines/chemokines; d) transcription and extracellular accumulation of HMGB1 and HSP70-in A549 cells. Importantly, antibody mediated neutralization of extracellular HSP70 mitigated TF-ECVC-induced transcription of HMGB1, TLR4, RAGE, chemokines, and NF-kB. Overall, our findings unveil specific role of DAMPs in ECVC-induced inflammation and further studies will provide better understanding about the associated molecular mechanisms.

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4. DIESEL PARTICULATE EXPOSURE: CROSS-TALK BETWEEN IL-17 FAMILY AND APOPTOTIC PATHWAY GENES N. Bidarimath, A. Abdulkadir, S. Batra. Laboratory of Pulmonary Immunotoxicology, Department of Environmental Toxicology, Southern University and A&M College. Pulmonary health is routinely challenged with a variety of environmental factors, that directly influence the viability and function of the lungs. One of the environmental factors is the emission from heavy diesel engine motors being used in robust mass transportation, railroads, heavy-duty combustion engines in construction, farmland, and mining industrial settings. The emission from these sources contain harmful chemicals such as benzene, chrysene, formaldehyde, nitrous oxide, and several other organic and inorganic substances. Diesel particulate matter/extract (DPM/DPE) can cause immunomodulatory effects, including inflammatory oxidative stress and apoptosis. However, the molecular mechanisms associated with DPE-induced responses are still not clear. While, apoptosis (programmed cell death) is essential for normal homeostasis, maturation of the immune system, and cellular defense; earlier studies demonstrate IL-17 mediated regulation apoptosis in pathological conditions. The current study is aimed at investigating the role of IL-17 in proliferation, apoptosis, and inflammation using human alveolar epithelial cells (A549) with type II characteristics. In brief, A549 cells were treated with various concentrations of DPE (1 mM, 10 mM, and 25 mM) for 24 and 48 hours and the samples were processed to determine IL-17A, IL-17F and IL-17R transcriptional levels and the apoptotic genes. Our preliminary studies demonstrate significant increase in the transcription of IL-17A and IL-17R in DPE-challenged A549 cells. Interestingly, we also observed increased transcription of-IL-1β and TNF-α; proapoptotic genes-BAX, BAK, FADD, FAS; and executioner/effector caspase-8 and caspase-3 in DPE exposed A549 cells. Further studies are in progress to evaluate-a) DPE-induced translational changes in cytokine/chemokine levels and apoptotic markers; and b) the impact of IL-17A/IL-17F neutralization on apoptotic markers in DPE exposed A549 cells.

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5. DOES ACETATE MEDIATE SKELETAL BONE TOXICITY OF ALCOHOL? *C. Del Valle–Ponce de Leon, H. Hang, A. Denys, K. B. Pedersen, M. J. Ronis. Louisiana State University Health Sciences Center in New Orleans Excessive alcohol consumption in humans leads to increased risk for osteopenia. Alcohol can affect the bone by disrupting chondroblast differentiation in the growth plate and interfering with osteoblast and osteoclast function, differentiation and bone turnover. Metabolism of alcohol proceeds with oxidation to acetate via acetaldehyde. Literature estimates the amount of acetate going to the blood stream is ten times less the alcohol administered (Carmichael et al., 1993). We hypothesize that alcohol toxicity in the bone growth plate is mediated by acetate. We compared the effects of alcohol and acetate on a chondroblast precursor cell line ATDC5 in vitro. In ATDC5 cells cultured with chondroblastogenic medium, both ethanol (50mM) and acetate (5mM) inhibited Alcian Blue staining of cartilage and significantly (P < 0.05) downregulated marker genes associated with chondroblast differentiation (Ihh), (Acan) and (Col1a2). In a subsequent pilot in vivo experiment with 4 mice per group, we further compared 4 days of binge alcohol (3, 3,4 and 4.5g/kg) to 4 days of binge acetate (0.3, 0.3,0.4 and 0.45 g/kg) in male C57Bl6/J mice., we isolated femur shaft RNA and performed qRT-PCR for genes involved in osteoblast function and osteoclast differentiation. While not reaching statistical significance, the data trend that both ethanol and acetate downregulate genes involved in osteoblast function. Ethanol significantly increased the mRNA concentration of the osteoclast marker Calcr (P<0.05) while acetate had no effect. This experiment will be replicated with a larger number of animals to increase statistical power. In conclusion, acetate appears to imitate the deleterious effects of alcohol on several genes involved in chondroblast and osteoblast differentiation and function. (MJJR-R37AA018282, R25 GM12119-01)

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6. EFFECT OF UCP2 OVEREXPRESSION ON GLUCOSE METABOLISM THROUGH WNT SIGNALING *N.A. Jacquet, D. Frierson, and Y. Zhao. Department of Pharmacology, Toxicology and Neuroscience, LSU Health Sciences Center in Shreveport, Shreveport, Louisiana. Uncoupling protein 2 (UPC2) is an inner mitochondrial membrane protein that acts as an anion/ion transporter. UPC2 may play two different roles in cancer; in normal cells, it acts as a protective mechanism, while overexpression in cancer cells may result in resistance to chemotherapy. JB6 is a cell line of mouse epidermal cells that were used for cell transformation. Upregulation of UCP2 within these cells resulted in activation of AKT signaling pathway and phosphofructokinase 2/fructose-2,6-bisphosphatase 2 (PFKFB2). UCP2 overexpression plays a key role in cellular metabolism during cell transformation. RT2 PCR array was performed using mRNA extracted from control and ucp2 overexpressed cells from the JB6 cell line. The array was performed to analyze glucose metabolism pathways. The array found that 19 genes were upregulated and 12 were downregulated. Several gene families were found to be over and/or under-expressed by the array including Frizzled family (FZ), Wnt family, and DVL gene families. All three of these gene families play roles in Wnt signaling pathways, the data suggest UCP2 may regulate glucose metabolisms through Wnt signaling.

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7. SOURCES AND FACTORS EFFECTING THE ENVIRONMENTAL BEHAVIOR OF BLACK CARBON AT SAMPLING SITES IN MISSISSIPPI BETWEEN SEPTEMBER 2013 AND DECEMBER 2014 *H.T. Nguyen and C. Roper. Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University, Mississippi. Black Carbon (BC) is a component of air pollution that has adverse effects in humans but little is known currently about the amount of BC that humans are exposed to, particularly in the Southern United States. BC was analyzed using fine particulate matter (PM2.5) ambient data measured from two urban residential sites of Jackson in Jackson city and Pascagoula in Pascagoula city and an urban background site of Grenada in Granada city in Mississippi, USA from September 2013 to December 2014. The highest mean concentration of BC computed at three sites during the entire measurement period was 2.04 ± 1.43 mg m-3 at Jackson, followed by 1.73 ± 0.88 mg m-3 at Pascagoula and the lowest value of 1.01 ± 0.72 mg m-3 at Grenada. The temporal patterns of BC were recognized by the relative prominence in spring/fall over summer. The result of applying conditional bivariate probability function (CBPF) indicated that the rise in BC pollution was a combination of local sources close to the sampling sites (e.g., vehicular emissions, residential areas) at low wind speeds. The back trajectories analysis also revealed dominant concentrations of BC were observed in air masses coming from land/continent, while the lowest BC concentration (in June) was in air masses coming from the ocean. In addition, the meteorological parameters such as temperature, wind speed, wind direction, and precipitation were major factors affecting the distribution of BC at the study sites. The outcome of the present study suggests that the distribution of atmospheric BC in these study sites is not only influenced by the variation of source processes but also by the environmental conditions surrounding it. This data will be useful for future studies exploring the health effects and toxicity of BC in Mississippi.

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8. PROTEOMICS REVEALS THE PROGNOSTIC VALUE OF LACTATE DEHYDROGENASE IN ACETAMINOPHEN-INDUCE ACUTE LIVER FAILURE PATIENTS J. Vazquez1,2, S. Kennon-McGill2, S.D. Byrum3, S.G. Mackintosh3, D.K. Williams4, W.M. Lee5, J.A. Dranoff6, M.R. McGill1,2. 1Dept. of Pharmacology and Toxicology, College of Medicine; 2Dept. of Environmental and Occupational Health, Fay W. Boozman, College of Public Heallth; 3Dept. of Biochemistry, College of Medicine; 4Dept. of Biostatistics, College of Medicine; University of Arkansas for Medical Sciences. 5Dept. of Internal Medicine, University of Texas – Southwestern. 6Yale University. Acute liver failure (ALF) is a rare yet serious condition with a high rate of mortality (~30%). Unfortunately, there are currently no biomarkers that have sufficient prognostic value to determine if a patient is unlikely to survive without a liver transplant. We obtained early and later serum samples (study days 1s and 3, respectively) from patients with Acetaminophen (APAP)-induced ALF in the Acute Liver Failure Study Group biorepository, divided into transplant-free survivor (n=28) and non-survivor (n=30) groups, and from volunteer controls. We then selected samples (n=10/group) from patients for whom ALT was lower on day 1 than day 3, indicating an early timepoint in injury, and ran untargeted proteomics on their day 1 samples. We could quantify 1,682 proteins across all 30 samples, with 79 of those proteins elevated ≥4-fold in ALF patients vs. control and 23 further elevated ≥4-fold in non-survivors vs. survivors. Among the latter 23, lactate dehydrogenase (LDH) appeared to have the highest prognostic value. We then tested LDH in all day 1, day 3, and control samples from our subjects. LDH was significantly elevated in non-survivors compared to survivors on both day 1 and day 3, and receiver operator characteristic (ROC) curve analysis showed that LDH performs similar to the model for end-stage liver disease (MELD) score, while a combination of LDH and MELD derived from multiple logistic regression outperforms either one alone. An upstream analysis of our proteomics data also revealed that LKB1-AMPK signaling is important in liver regeneration, which we confirmed using a mouse model of APAP overdose. We conclude that LDH can predict death in APAP-induced ALF, and that LKB1-AMPK signaling may be a valuable target for therapy in ALF patients.

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9. IN VITRO MODEL TO STUDY THE EFFECTS OF CANNABINOIDS ON CELL POPULATIONS AND CYTOKINE PRODUCTION *O. Cresswell and B.L.F. Kaplan. Center for Environmental Health Sciences, Department of Comparative Biomedical Sciences, Mississippi State University, Mississippi State, MS. Myelin oligodendrocyte glycoprotein (MOG) peptide is a myelin protein used to induce experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis. Previous studies showed that marijuana compounds such as D9-tetrahydrocannabinol (THC) or cannabidiol (CBD) suppressed clinical disease in the EAE model due, in part, to their immunosuppressive effects. These studies are important, but are limited by costs, time, and animal use. Thus, we investigated whether the MOG peptide could induce cytokines in vitro and showed that both CBD and THC inhibited cytokine production. Flow cytometry was also used to identify the percentage of T cells, macrophages, and B cells. Day 4 MOG-treated splenocyte populations looked like a typical mouse spleen, but by day 7, CD4+ T cells and CD8+ T cells quantities increased and F4/80+ macrophages and CD19+ B cells decreased. These results support that MOG treatment caused T cell increases and B cell and macrophage decreases sometime between day 4 and 7. There was also a slight increase in MOG-specific T cells with positive tetramer, suggesting that at least a subpopulation of T cells recognized MOG. Preliminary studies suggest that IFN-γ and TNF-α are secreted specifically by CD4 and CD8 T cells. Together these data show that MOG peptide can induce cytokine production in vitro and can be used to evaluate peptide-specific immune responses. Additional characterization is necessary to use this model more broadly. Studies supported by MSU-CVM.

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10. EARLY DEVELOPMENTAL EXPOSURE TO Δ9-TETRAHYDROCANNABINOL CAUSES HYPERACTIVITY IN LARVAL ZEBRAFISH THAT PERSISTS INTO ADULTHOOD *V. L. Jackson1, Z. Pandelides1, C. E. Thornton1, K. L. Willett1. 1University of Mississippi, University, MS. Due to the increasing availability of Δ9-tetrahydrocannabinol (THC), evaluation of the potential adverse behavioral effects following exposure to this cannabinoid during early development is crucial. Following exposure to 0.08, 0.4, or 1 µM THC from 6-96 hpf, latent behavioral effects caused by THC were assessed at 120 hpf, 3 wpf (juvenile), 11 wpf (onset of sexual maturity) and 24 wpf (adult). Using the larval photomotor response test, hyperactivity in the dark phase was evident in zebrafish exposed to 0.4 µM THC. Open field tests (OFT) conducted at 3 and 11 weeks post fertilization indicated dose-dependent hyperactivity and increased thigmotaxis at the two highest THC concentrations. Adult zebrafish behavior in the OFT revealed significant differences between male and females and increased velocity amongst fish exposed to 0.4 or 1 µM THC of both sexes. Collectively, these results suggest that exposure to THC during early development can cause behavioral alterations that persist into adulthood. This work was supported by the National Institute on Drug Abuse R21DA044473-01.

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11. MOLECULAR RESPONSES TO 3-MONTH OF EXPOSURE TO ELECTRONIC-CIGARETTE AEROSOL COMPOSED SOLELY OF HUMECTANTS IN LUNGS OF FEMALE MICE A. Jones1, B. Holliday2, M. Schexnayder3, Z. Perveen2, A. Noël2. Departments of 1Biological Sciences, 2Comparative Biomedical Sciences, Louisiana State University. 3Lincoln Memorial University. Although electronic-cigarettes (e-cigs) are widely used, primarily among young people, there is still little known about the potential health effects resulting from their usage. In e-cig devices, a mixture of nicotine, propylene glycol (PG), vegetable glycerin (VG) and flavors, is aerosolized by heat to produce an aerosol that the users inhale. While e-cig devices have been on the U.S. market since 2007, there are very few studies on the pulmonary effects of the e-liquid humectants, including PG, VG and vitamin E acetate (VEA). VEA was used in cartridges of tetrahydrocannabinol (THC)-containing products and identified as one culprit of the 2019 e-cig or vaping associated lung injury (EVALI) outbreak. We hypothesized that mice expose to e-cig aerosols produce from a liquid mixture of PG, VG and VEA will have the most effects on the lungs, as evidenced at the molecular level by dysregulated gene expression. 6-week-old female BALB/C mice were exposed 2 hours/day for 3 months (5 days/week) to either filtered air, 100% PG e-cig aerosol, or 33% PG + 34% VG + 33% VEA e-cig aerosol. We collected the broncho-alveolar lavage fluid (BALF) and the lungs of the mice after this 3-month exposure. We extracted miRNA from the lung tissue and analyzed gene expression through qRT-PCR. The genes analyzed included: Cyp1a1, Cyp1b1, Cxcl5, Ccl8, Mmp12, and Muc5ac. The total particulate matter (TPM) concentration measured inside the 5 L exposure chamber was 8 µg/puff for the 100% PG e-cig aerosol, and 100 µg/puff for the 33% PG + 34% VG + 33% VEA e-cig aerosol. Although these two e-cig aerosols were generated under the same exposure conditions and using the same vaping topography profile, the TPM levels for those two e-cig aerosols were different. This is most likely due to the distinct physico-chemical characteristics of PG, VG and VEA, including density, viscosity and thermal degradation properties. In mice, while BALF cytology results showed that macrophages were the predominant cell type in all three groups, a moderate macrophage vacuolation score (foamy macrophages) was obtained for the mice from the 33% PG + 34% VG + 33% VEA e-cig aerosol group, compared to minimal-to-mild macrophage vacuolation scores, which were obtained for the air and 100% PG e-cig aerosol groups. Foamy or vacuolated macrophages have been observed in the BALF of EVALI patient. Also, the expression of lung genes associated with biotransformation, inflammation and airway remodeling responses, were assessed. Thus far, our study in female mice shows that sub-chronic exposures to e-cig aerosols composed solely of e-liquid humectants can affect the phenotype/morphology of alveolar macrophages.

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12. CARBOXYLESTERASE 1 RELEASES OXYLIPINS FROM OXIDIZED TRIACYLGLYCEROLS: AN INITIAL EXAMINATION OF ITS SUBSTRATE SELECTIVITY M. Philips, A. Borazjani, M.K. Ross. Department of Comparative Biomedical Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University. Triacylglycerols (TAGs) are the most quantitatively important storage form of fat and are found primarily in cytoplasmic lipid droplets within cells. TAGs are broken down to its component free fatty acids by lipolytic enzymes when fuel reserves are required. However, when TAGs possess large quantities of polyunsaturated fatty acids (PUFAs), they are prone to nonenzymatic oxidation reactions leading to formation of oxylipins (i.e., oxidized forms of fatty acids) that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. Although it is known that CES1 is a bona fide TAG hydrolase, it is unclear which specific fatty acids are preferentially released during lipolysis. Moreover, to better understand the biochemical function of CES1 in macrophages, we need to determine its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets. Thus, the goal of this study is to systematically identify those oxidized fatty acids that are liberated from oxTAGs by CES1, because their release activates signaling pathways important for the development of lipid-driven inflammation. Gaining this knowledge will fill data gaps that exist between CES1 and the lipid-sensing nuclear receptors, PPARg and LXRα, that are abundantly expressed in macrophages and are important drivers of lipid metabolism and inflammation. Oxidized forms of triarachidonoylglycerol (oxTAG C20:4) and trilinoleoylglycerol (oxTAG C18:2) were incubated with recombinant CES1, or Pseudomonas lipase (a positive control), to assess the release of oxylipins and non-oxidized arachidonic acid (AA) and linoleic acid (LA), which were quantified by LC-MS/MS. CES1 was shown to efficiently metabolize oxTAG C20:4 and oxTAG C18:2, yielding several regioisomers of hydroxyeicosatetraenoic acid (5-,12-, and 15-HETE) and hydroxyoctadecadienoic acid (9- and 12-HODE), respectively. The CES1-catalyzed cumulative release of HODEs was faster than that of HETEs from the respective oxTAGs (7 pmol HODEs/min vs. 1 pmol HETEs/min). Thus, for oxTAGs, hydroxy fatty acids derived from LA are preferred by CES1 to those derived from AA. A similar trend was noted for the release of non-oxidized PUFAs from the respective oxTAGs (470 pmol LA/min vs. 83 pmol AA/min). CES1 also preferentially liberated 5-HETE over 12-HETE and 15-HETE from oxTAG C20:4, whereas no differences were noted between 9-HODE and 13-HODE. This study indicates that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs. It further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators that regulate inflammatory activities of immune cells. [Supported by NIH R15 GM128206]

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13. PRELIMINARY INVESTIGATION INTO EFFECTS OF LEACHATES FROM 3D-PRINTED OBJECTS ON ZEBRAFISH SPERM QUALITY *MJ Schwinga, Y Liua,b, JA Belgoderea, WT Monroea, TR Tierschb, A Abdelmoneima. aLouisiana State University, Baton Rouge, LA, 70803. bAquatic Germplasm and Genetic Resources Center, Louisiana State University Agricultural Center, Baton Rouge, LA, 70820. The use of 3D printing in biomedical and environmental applications is gaining popularity, yet, the effects leachates from 3D-printed objects may have on biological systems are not well understood. In this study, we are investigating how exposure to leachates from 3D-printed objects fabricated with photocurable resin may adversely affect zebrafish (Danio rerio) sperm quality. We also evaluated the effects of variables such as resin type, leaching time, and time of exposure to sperm on sperm quality. 3-D printed objects were fabricated using an Anycubic Photon S masked-stereolithography (MSLA) printer and Anycubic Eco resins. Objects were washed and cured using the Anycubic Wash & Cure Machine 2.0 to remove residual uncured resin. Once processed, objects were incubated with Hanksʹ Balanced Salt solution (HBSS) for 1 minute or 20 hours. The leachates in HBSS were mixed with an equal volume of fresh zebrafish sperm (2 x 108 cells/mL). After contact periods of 1 or 30 minutes, mixtures were activated using deionized water, and sperm motility was evaluated using a computer assisted sperm analysis (CASA) system. We also evaluated possible effects on membrane integrity in each mixture using flow cytometry. All experiments were blinded. Our preliminary findings suggest possible adverse effects on sperm quality associated with exposure to leachates from 3D-printed objects. Additional replicates and exposure variables are currently being investigated.

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14. SPLITTING FINE PARTICULATE MATTER (PM2.5) FILTERS: COMPARISON OF CHEMICAL COMPOSITION AND OXIDATIVE POTENTIAL BETWEEN FILTER PIECES *A.M. Sidwell, C.L. Roper. University of Mississippi, University, MS, 38655. Fine particulate matter (PM2.5) is a complex mixture of particles and sorbed chemicals that poses serious, adverse effects on human health such as increasing cardiovascular and respiratory morbidity. To conduct analytical and toxicology studies of PM2.5, researchers often split filters into sections. This process allows multiple, often destructive, assays to be performed. Our previous research showed chemical composition differences across PM2.5 filters. The goal of our study was to determine the validity of splitting filters for use in multiple analyses by assessing differences in chemical composition and oxidative potential within the same filter. Six PM2.5 filter samples collected from urban and rural locations were used. Each filter was split into quadrants, resulting in a total of 24 pieces; laboratory and blank filters were also prepared in the same manner. Each filter piece was extracted, concentrated, and a whole particle suspension was prepared. The extracted suspensions were then analyzed with dithiothreitol (DTT) assay run in triplicate to determine oxidative potential. Inductively coupled plasma mass spectrometry (ICP-MS) was run on all samples and controls to compare chemical composition of the filter quadrants (n=30). Significant differences in oxidative potential between filter quadrants were not seen. However, stark differences in total elemental content were observed between quadrants of the same filter. Correlation analysis between oxidative potential and elements is underway. This work will provide information about the feasibility of splitting PM2.5 filters for multiple analyses on the same sample.

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15. LUNGS OF FEMALE MICE EXPOSED IN UTERO TO COOL MINT-FLAVORED PUFF BAR AEROSOL HAVE INCREASED SUSCEPTIBILITY TO TH2-MEDIATED IMMUNE RESPONSES AND GREATER FUNCTIONAL DECLINE FOLLOWING HOUSE-DUST MITE EXPOSURES *H. Stewart1, M. Schexnayder2, A. Penn1, A. Noël1. 1Louisiana State University, Baton Rouge, LA, 70803, USA, 2Lincoln Memorial University, Harrogate, TN, 37752, USA. While many people believe that electronic-cigarettes (e-cig) could be a safer alternative to cigarettes, the health effects of vaping during pregnancy are mostly unknown. The goal of this study was to assess the outcomes of pregnancy in mice exposed to cool mint-flavored Puff Bar aerosols and the respiratory health status of the offspring. Pregnant BALB/c mice (n = 8 per group) were exposed to either HEPA filtered air or cool mint-flavored Puff Bar aerosols from days 1 to 20 of gestation for 1 hour a day in a 5 L whole-body exposure chamber. The Puff Bar aerosol was generated by connecting the disposable Puff Bar e-cig device to a peristaltic pump. The offspring mice were exposed during fetal development to a total particulate matter of 0.52 ± 0.21 mg/puff of Cool Mint Puff Bar aerosol. The female offspring were sacrificed at 7 weeks of age following intranasal instillation of house dust mite (HDM) or saline once a week for three weeks to induce asthmatic responses. Lung function, broncho-alveolar lavage (BAL) cytology, and lung gene expression were assessed. Our data show that in utero exposure to cool mint flavored-Puff Bar aerosol plus HDM treatment alters lung function in 7-week-old female mouse offspring. At baseline, respiratory system compliance was significantly decrease, and following methacholine challenge, the maximum respiratory resistance as well as the Newtonian resistance were significantly increased in offspring exposed in utero to Puff Bar aerosol and that received HDM treatment compared to air controls. Additionally, the HDM treatment significantly increased the percentage of BAL neutrophils and decreased the percentage of macrophages in both the in utero air and Puff Bar exposed offspring compared to their respective saline controls. Ccl17, Ccl22, Il13, Il33, and Muc5ac were among the up-regulated genes in both the in utero Puff Bar plus saline and in utero Puff Bar plus HDM groups, when compared to the in utero air plus saline controls. Our data show that in utero exposures to Puff Bar aerosols may predispose the lungs 1) to Th2-mediated immune responses, and 2) following exposure to an allergen, to greater lung function decline in childhood. Thus, our data support the argument that e-cig use during pregnancy is not ‘safe’ for the respiratory health of the offspring.

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16. BAP INDUCED MULTIGENERATIONAL GENE EXPRESSION AND DNA METHYLATION CHANGES ARE PRIMARILY INFLUENCED BY PATERNAL GENOTYPE *M. C. Sturgis, Z. Pandelides1, C. Thornton1, M. Patel1, N. Aluru2, K. L. Willett1. 1University of Mississippi, University, MS; 2Woods Hole Oceanographic Institution, Woods Hole, MA. Benzo[a]pyrene (BaP) is implicated in numerous adverse outcomes in offspring of exposed parents, but the molecular mechanisms for the developmental and multigenerational effects associated with BaP exposures have yet to be fully elucidated. The objectives of this study were to determine the developmental and behavioral effects of preconceptional exposure to BaP. 5D zebrafish were fed 1% of their bodyweight of 708 ± 24 μg BaP/g food twice per day, resulting in 14 μg BaP/g fish/day for 21 days. Fish were then spawned using a crossover design, resulting in F1 cohorts of control and three crosses from BaP-exposed parents. Behavioral effects were measured at 96 hpf in the F1 fish using the light:dark assay. Significant hyperactivity was observed in both crosses from BaP-exposed males. Using an open field test, female F1 adult offspring from the BaP male x BaP female cross were also significantly hyperactive. Gene expression and DNA methylation changes in 10 hpf F1 embryos were measured using RNAseq and reduced representation bisulfite sequencing, respectively. When compared to the control, the highest number of differentially expressed genes (DEG) were in offspring of the BaP male x control female cross. Many of the DEGs were involved in large scale growth and developmental processes. Differentially methylated regions (DMRs), primarily hypomethylation, predominated in embryos from BaP-treated male parents. Gene ontology analysis revealed DMRs in or adjacent to genes important in development (e.g., neural crest cell differentiation, ossification and retinol metabolism). A parental BaP-mediated DMR in histone deacetylases coding regions may be causing further epigenetic-mediated effects. Collectively, these results demonstrate that parental dietary BaP exposure causes persistent adverse behavioral outcomes in offspring and suggests that males may contribute more to F1 effects. Research supported by NIEHS 1R21ES030154.