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2021 Virtual Student & Postdoctoral Researchers’ Summer MeetingHosted by: The Department of Pharmacology and Toxicology, University of Louisville School of Medicine

Wednesday July 28th, 2021 | 9:00 AM – 1:30 PM ET | Hosted via WebEx

AGENDA

9:00 – 9:05 Opening Remarks9:05 – 9:50 KEYNOTE SPEAKER, Dr. Anita Corbett

“Scientific Communication–Know Your Audience”9:50 – 10:00 Q&A with Dr. Corbett10:00 – 11:00 PhD Platform Presentations11:00 – 12:00 PostDoc Platform Presentations12:00 – 12:05 Break 12:05 – 12:10 Undergraduate 3-Minute Thesis12:10 – 12:30 Masters 3-Minute Thesis12:30 – 1:00 PhD 3-Minute Thesis1:00 – 1:15 Trivia Game1:15 – 1:20 Award Announcements1:20 – 1:30 Closing Remarks

Welcome! On behalf of the Executive Committee of the Society of Toxicology Ohio Valley Regional Chapter, we’d like to thank you for attending the 2021 Student & Postdoctoral Researchers’ Meeting. Each summer we host this event as an opportunity for students and postdoctoral trainees to share their research to peers, practice presenting and fielding questions, and networking within the region. All undergraduate, graduate, and postdoctoral researchers studying toxicology and tox-related fields are welcome to participate. Thank you to the 21 presenters who will be sharing their exciting findings with us.

This year’s theme is Scientific Communication. We are excited to welcome Dr. Anita Corbett, with the keynote talk, “Scientific Communication—Know

Your Audience.” Effective scientific communication is critical for successful articles and reports, winning grants and fellowships, engaging oral presentations, and a compelling biosketch. Our hope is that attendees take away strategies to improve their communication skills and gain confidence and interest in expanding the reach of their research.

Thank you again and we hope to see you this fall at the OVSOT Annual Meeting at Wright State University!

Sincerely,Jennifer Toyoda, Graduate Student Representative Brandon Lewis, PhD, Postdoctoral Representative

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KEYNOTE SPEAKERDr. Anita Corbett

“Scientific Communication—Know Your Audience”

Dr. Anita Corbett is a professor in the Department of Biology at Emory College. She received her undergraduate degree in Chemistry at Colgate University, earned her PhD in Biochemistry at Vanderbilt University, and then moved to Harvard Medical School for her postdoctoral research. In 1997 Dr. Corbett entered Emory School of Medicine as an assistant professor in the Department of Biochemistry and in 2003 she became the

first woman to receive tenure in that department. Currently, Dr. Corbett’s lab studies RNA-binding proteins implicated in tissue-specific diseases using many genetic model organisms and highly interdisciplinary research strategies. Dr. Corbett is dedicated to mentoring and training as well as increasing diversity and inclusion in science. In 2018 she won the Nature Award for Mentoring in Science. Her excellence in mentorship was further recognized in 2019 with the selection of her protégé as the American Society for Biochemistry and Molecular Biology Young Investigator Award and again by the same society in 2020 with her receipt of the Mid-Career Leadership Award. Today’s talk reflects Dr. Corbett’s passion for scientific communication education.

ABSTRACTS

UNDERGRADUATE STUDENTS

Comparing mercury concentrations in the feathers of nestling tree swallows (Tachycineta bicolor) by site and yearChrisula Stone1, Rebecka Brasso2, and Lindsey Walters1

1Northern Kentucky University, Nunn Drive, Highland Heights, KY 41099 2Weber State University, 3848 Harrison Blvd., Ogden, UT 84408Beginning in the industrial age, anthropogenic activities have contributed to increased levels of mercury (Hg) in the environment, which can assimilate into the tissues of living organisms as the potent neurotoxin methylmercury (MeHg). The tree swallow (Tachycineta bicolor) is an abundant North American passerine that has been studied often for its ability to accumulate mercury in its tissues, as it occupies a high trophic level and breeds near bodies of water. For this project, we sought to establish whether measurable and significant differences in total mercury (THg) could be detected via atomic absorption spectroscopy in body feathers collected from 12 to14-day-old nestlings across different locations approximately 20 km apart in the same year, and between different years (2019 and 2020) in

the same location. We sampled body feathers from 12 to 14-day-old nestlings and analyzed them using thermal decomposition, gold amalgamation and atomic absorption for THg concentrations specific to each nestling sampled. Concentration of THg in feathers is representative of methylmercury, as >95% of Hg in body tissues of birds is MeHg. Measurable differences in feather THg were detected to 0.001 ppm among individual nestlings, averaging 5.384 ppm in 2019, and 0.403 ppm in 2020. A linear mixed effects model using nest as a random effect found no significant difference in THg between sites (df=1, 51, F= 1.679561, p= 0.2008). A significant difference was found between years at the same site, with approximately 10 times higher THg concentrations in 2019 than 2020 (df=1, 51, F= 409.8195, p<0.0001). The ability to collect numerous samples humanely, coupled with the reliability of AAS, shows promise in developing a system that uses tree swallow nestling feathers as reliable bioindicators of environmental mercury contamination.

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MASTER’S CANDIDATES

The diabetogenic potential of the loop-diuretic bumetanide in miceRana Abdelgawad1, Yaksh Rathod1, Christian Hübner2 and Mauricio Di Fulvio1

1Department of Pharmacology and Toxicology, Wright State University, SoM, Dayton OH. 2Institut für Humangenetik, Universitätsklinikum Jena, GermanyBumetanide (BTD), one of the most prescribed diuretics in the clinic to treat edematous conditions has well-known adverse and toxic effects, some of them not related to its diuretic action. Among them, BTD has been shown to reduce insulin secretion in vitro and to provoke glucose and insulin intolerance in vivo in mice. In susceptible humans, BTD and other diuretics were linked to new onset diabetes. However, it remains unknown one of the pharmacological targets of BTD i.e., the Na+K+2Cl– cotransporter-1 (Nkcc1) expressed in insulin-secreting β-cells of the islets of Langerhans in the pancreas play a role in glucose homeostasis. Address that gap in our knowledge, we used the Cre/LoxP strategy to create mice lacking Nkcc1 exclusively in β-cells (Nkcc1βKO) and subjected them to static and dynamic tests of glucose homeostasis at different ages. Our results indicate that by using the Cre/LoxP strategy. Our results demonstrate impaired insulin responses to glucose in islets purified from Nkcc1βKO mice and that those responses are inhibited by BTD in WT islets but not in those lacking Nkcc1. In vivo, 10w old Nkcc1βKO mice fed ad libitum are hyperlipidemic and hyperglycemic, but hypoinsulinemic indicating deficient insulin responses to food. Yet, these mice are normotolerant to exogenous glucose and insulin indicating compensated fuel homeostasis. However, older Nkcc1βKO mice showed increased body weight gain, hyperinsulinemia and hyperglycemia accompanied by glucose and insulin intolerance and signs of adipose tissue inflammation and steatohepatitis, all potential risk factors for the development of diabetes and other metabolically associated conditions. Therefore, our results suggest that elimination of the BTD-sensitive Nkcc1 from insulin-secreting β-cells associates with age-dependent deterioration of metabolic health.

Exploring the role of PTEN in non-small cell lung cancer: Implications in therapeutic approachesRawan Alojair, Zaid Sirhan ,Anita Thyagarajan, Ravi P. SahuDepartment of Pharmacology and Toxicology, Boonshoft School of Medicine at Wright State University, Dayton OH 45345

Lung cancer remains one of the major human malignancies affecting both men and women worldwide with non-small cell lung cancer (NSCLC) being the most prevalent type. Of several factors, smoking remains one of the major risk factors, and that multiple mechanisms have been identified which favor tumor growth as well as impede the efficacy of therapeutic regimens in lung cancer patients. Among various signaling cascades, tumor suppressor genes play critical roles in regulating cancer growth. Phosphatase and tensin homolog (PTEN) is one of important family members of tumor suppressor genes, which has been shown to regulate several activities of tumor cells including cell proliferation, migration, angiogenesis, metastasis, and apoptosis via targeting multiple mechanisms. Notably, clinical studies have also documented that lung tumors having impaired or loss of PTEN were associated with a low survival rate or high tumor recurrence rate. To that end, PTEN has been explored as a promising target for several anti-cancer drugs, and new approaches have also been proposed to device more effective therapies. In this review, we discussed the significance of PTEN and its implications in therapeutic approaches against lung cancer.

UVB-Induced Microvesicle Particle Release in Human Skin in vivo is Diminished Following Oral Vitamin C and E Antioxidant AdministrationBenjamin Schmeusser MS41, Cameron McGlone MD1, Jeffrey B. Travers MD, PhD1,2

1Departments of Pharmacology and Toxicology and 2Dermatology, Boonshoft School of Medicine at Wright State University, Dayton, OH 45435An important question in photobiology and toxicology asks how Ultraviolet B (UVB, 290 – 320 nm) radiation, which mostly absorbs in the outer epidermis of skin, can generate a systemic response such as immunosuppression. Previous in vitro and ex vivo studies demonstrate UVB-dependent release of bioactive molecule-containing microvesicle particles (MVPs) from keratinocytes. Furthermore, MVP has been shown to be diminished upon antioxidant administration in human skin explants. The purpose of this study is to examine UVB-induced MVP release and antioxidant response in vivo. In this study, 8 male participants with Fitzpatrick type I or II skin were treated with 1000 J/m2 UVB irradiation to a 5 by 5 mm area of volar forearm skin. 4 hours later, punch biopsies and erythema measurements were performed. This procedure was repeated 8 days later following a course of oral antioxidants (vitamin C 2g/day, vitamin E 1000IU/day). Following sample collection, the biopsies underwent

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processing to quantify MVP concentration. On average, tissue MVP release increased 1.8-fold (+/- 0.31, P = 0.02) following UVB treatment. Following a course of oral antioxidants, the average UVB-induced tissue MVP release did not differ from the control (0.9-fold +/- 0.13, P = 0.23). There was no significant change in UVB-induced erythema between pre- and post-antioxidant administration. This data further supports UVB radiation causing MVP release from human keratinocytes in vivo. Administration of antioxidants vitamin C and E reduced MVP release. The prominent limitation of this study include its small, but still significant, sample size (N=8), negatively impacted by the COVID-19 pandemic halting human studies. Future studies may evaluate antioxidant effect on sunburn duration, identify the therapeutic photoprotective dose of antioxidants, or attempt to establish how much time is needed to provide photoprotection after antioxidant administration.

Exploring the Therapeutic efficacy of Tucatinib versus Tucatinib combination approaches in HER-2+ breast cancer patientsZaid Sirhan, Anita Thyagarajan, Ravi P. Sahu Department of Pharmacology and Toxicology, Boonshoft School of Medicine Wright State University,Dayton,OH 45435, USAThe overexpression of the human epidermal growth factor (HER2) has been identified in 15-20% of cases of breast cancer patients, which is linked with poorer prognosis and disease progression. Among various treatment options, the monoclonal antibodies (e.g., Trastuzumab) and tyrosine kinase inhibitors (TKIs) (e.g., Pyrotinib) have mostly been used in the last decades to treat HER2-positive breast cancer patients. While promising clinical outcomes were documented in both an advanced disease and neoadjuvant settings, the development of resistance to such approaches has been a major challenge in the continuous usage of these drugs. Besides, patients who experienced disease progression after treatments with multiple HER2-targeted therapies have limited treatment options. To that end, many drugs have been approved by the U.S. Food and Drug Administration (FDA) for such patients, which have shown promising therapeutic effects. The current review explores the therapeutic responses of the recently FDA-approved targeted therapy (i.e., Tucatinib) as a single agent versus its combination with immunotherapy and chemotherapeutic agents in HER2-positive breast cancer patients. Several studies have suggested that the combination of Tucatinib with Trastuzumab and Capecitabine exhibit

improved effectiveness in treating HER2+ breast cancer patients. As Tucatinib is a relatively new FDA-approved targeted therapy, its efficacy with other combinations should also be explored to test their overall effectiveness and compare with the published data. Importantly, few clinical trials are still ongoing and recruiting patients to evaluate Tucatinib combination with other anti-cancer agents.

AhR-mediated transcriptional regulation of the human immunoglobulin hs1.2 enhancerSydney White, Abdullah Freiwan, Andrew Snyder, Courtney SulenticWright State University, 3640 Colonel Glenn Hwy, Dayton, OH 45435The 3’IGHRR, an ~ 17kb transcriptional regulatory region within the human immunoglobulin heavy chain gene (IGH), is thought to be responsible for the transcription of the IGH locus, which is essential for antibody production. The 3’IGHRR contains the hs1.2 enhancer which is polymorphic in humans and consists of a 53 bp invariant sequence containing transcription factor binding sites, including a potential dioxin response element (DRE), that can be duplicated one to four times. Previous experiments have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce transcriptional activity of the human hs1.2 enhancer alleles via the aryl hydrocarbon receptor (AhR) signaling pathway. The objective of this study is to assess the role of the AhR transactivation domain (TAD) in TCDD-induced hs1.2 enhancer activity and the transcriptional impact of an increased number of invariant sequences. Luciferase reporter plasmids containing one of the four human polymorphic hs1.2 enhancers (α1A, α1B, α1C, or α1D corresponding to one, two, three, or four invariant sequence repeats, respectively) were transfected via electroporation into a human B-cell line (CL-01) expressing an AhR with either a functional ornonfunctional TAD as determined by the ability toinduce a reporter plasmid regulated by 6 DREs. In Bcells with a nonfunctional AhR TAD, TCDD activatedthe hs1.2 enhancer in a concentration-dependentmanner but the number of invariant sequences didnot impact TCDD-induced activation suggesting aTAD-independent activation of the hs1.2 enhancerby AhR ligands. Ongoing studies are evaluating theimpact of TCDD on the hs1.2 enhancer alleles in Bcells expressing a functional AhR TAD to determineif a functional TAD will increase the sensitivity ofhs1.2 enhancer alleles with a greater number ofinvariant sequences to TCDD-induced activation.Since the polymorphic hs1.2 enhancer has beenassociated with altered antibody levels and a numberof hypersensitivity and autoimmune diseases, these

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results will provide greater insight in assessing risk by exposure to environmental, dietary, and endogenous ligands of the AhR.

PHD CANDIDATES

Susceptibility to Nanoparticle Inhalation associated with Metabolic SyndromeSaeed Alqahtani, Li Xia, Jonathan ShannahanSchool of Health Sciences, Purdue University, West Lafayette INMetabolic Syndrome (MetS) is increasingly prevalent within our society affecting approximately 30% of the U.S. population. Growing evidence suggests that individuals with MetS and other chronic diseases respond more robustly to exposures, exhibiting enhanced inflammation and toxicity. Inflammation is regulated via numerous lipids involved in both initiation and resolution processes. MetS is associated with dyslipidemia which may alter the regulation of inflammation following exposures resulting in exacerbated toxicity. Therefore, we hypothesize that MetS-associated dysregulation of lipids contributes to exacerbated inflammatory responses following exposures due to alterations in resolution mediators. Our current study, exposes healthy and MetS mouse models to silver nanoparticles (50ug) via oropharyngeal aspiration and evaluates acute pulmonary inflammation 24h post-exposure. To examine specific resolution mediators, 30min prior to nanoparticle exposure mice were treated with vehicle, 14HDHA, 17HDHA, or 18HEPE (1ug) via i.p. injection. Disease was verified by evaluation of body weight, total cholesterol, HDL, LDL, triglycerides, insulin, and A1C. MetS mice demonstrated exacerbated NP-induced pulmonary inflammation compared to healthy mice. Specifically, elevations in bronchoalveolar lavage fluid neutrophils and cytokine/chemokine (MCP-1, MIP-2, and IL-6) levels were observed. Although, elevated in both models in response to nanoparticles, no differences in IL-1β or CXCL1 were determined due to MetS. Inflammatory alterations corresponded with decreases in multiple resolution mediators (resolvin E series, resolvin D series, maresin 1, etc.) in only MetS mice. Supplementation with 14HDHA and 17HDHA but not 18HEPE inhibited the MetS-associated exacerbated inflammatory response to levels observed in the healthy model in response to NP exposure. Targeted mass spectrometry demonstrated precursor treatments elevated distinct resolution mediators in the lung. A western blot assessment of resolution receptors demonstrated reduced levels in the MetS mouse model and reductions due to

nanoparticle exposures. Additionally, through the use of hyperspectral microscope. Together, our findings demonstrate exacerbated inflammation in MetS that is mediated via a disruption of resolution mediator signaling. A detailed understanding of common subpopulation susceptibility to exposures is necessary for the development of comprehensive public health policy and understanding of disease progression following environmental exposures.

Potential Physiological role of AhR in Antibody ProductionMili Bhakta, Clayton Allex-Buckner, Sydney White, Mia Williams Burnett and Courtney SulenticDepartment of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State UniversityThe aryl hydrocarbon receptor (AhR) mediates the immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in murine B cells. The effects of AhR activation on the regulation of expression of human immunoglobulin (Ig) isotypes (μ, γ1-4, α1-2 and ε) and Ig secretion is unclear. Our previous results using the CL-01 cell-line originating from a Burkitt’s lymphoma patient, demonstrated an inhibitory effect of TCDD on IgG expression but a surprising and marked loss of IgG secretion when the AhR was knocked out by siRNA or CRISPR/Cas9 gene editing. To determine if the AhR is a critical mediator of IgG expression, the current study is focused on characterizing IgG expression in another human B-cell line (SKW 6.4 or SKW WT) originating from a different, non-related Burkitt’s lymphoma. We confirmed that SKW WT cells do not have endogenous expression of AhR using PCR analysis and Western blotting. We also demonstrated that SKW 6.4 cells can be stimulated in-vitro using CD40L and IL-4 to produce more IgM antibodies as detected by ELISA assays. Further, we demonstrate that total IgG secretion induced by CD40L and IL-4 stimulation is severely impaired in SKW WT cells. Conversely, the qRT-PCR studies show that the expression of ε, γ2-4 transcripts that code for IgE and IgG2-4 respectively is significantly increased with stimulation as compared to un-stimulated SKW cell lines. The expression of γ1 was low in naïve as well as stimulated SKW WT cells. The α1-2 transcripts coding for IgA1-2 respectively are not expressed at all in SKW cells regardless of stimulation. To further investigate, we used CL-01 AhRTA cells that express AhR with functional transactivation domain (TAD), to compare the expression of different isotypes. We found that the expression of γ1-4 and ε transcripts was significantly higher in AhR expressing CL-01 AhRTA cells as compared to the AhR-deficient SKW WT cells. Our observations imply that the AhR plays

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a critical role in the expression of the Ig heavy chain (IGH) gene.

Evaluation of physiologically relevant aryl hydrocarbon receptor ligands on antibody expression in a human B-cell line model.Mia L. Williams Burnett, Valerie Benedict, David Cool, and Courtney E. W. SulenticDepartment of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OHThe aryl hydrocarbon receptor (AhR) is a transcription factor that affects immune cell differentiation and function in animal models. AhR may serve as an environmental sensor since it not only binds to toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but also to endogenous, dietary and bacterial ligands such as 6-formylindolo[3,2-b]carbazole (FICZ) and indole. Indole is a human specific AhR ligand that is relatively abundant in the gut. In animal studies, different AhR ligands have produced different and even opposite functional effects on T cell subtypes, which may be due to transient vs. persistent activation of the AhR. The objective of this study was to determine if AhR ligands differentially modulate antibody expression and secretion in a human B-cell line (CL-01). Previous results have demonstrated inhibition of IgG but not IgM secretion by TCDD (30 nM). Current preliminary results suggest that higher concentrations of FICZ (100 nM) and indole (100 µM) inhibit both IgM and IgG secretion. These results suggest that antibody expression may be less sensitive to endogenous ligands than TCDD and different antibody isotypes may be differentially affected by ligands. Ongoing studies will determine the sensitivity of transcriptional expression of individual antibody isotypes (i.e. IgM, IgG1-4, IgA1-2, IgE) to AhR ligands and whether physiologically relevant AhR ligands induce different antibody isotype profiles.

Developmental toxicity characterization of a binary mixture of arsenic and lead using the zebrafishKeturah G. Kiper, Ellen Wells, Wei Zheng, and Jennifer L. FreemanSchool of Health Sciences, Purdue University, West Lafayette, Indiana, USAArsenic (As) and lead (Pb) are environmental pollutants, found in common sites, and linked to adverse health effects. This study evaluated As and Pb to determine if developmental toxicity significantly changes at lethal and sub-lethal mixture concentrations using the zebrafish model. The

concentration addition model was applied to survival data to determine if the interaction between Pb and As is additive. Metal exposures were from 1-hour post-fertilization (hpf) till 120 hpf. As concentrations were 0–10E6 µM. Pb concentrations were 0–480 µM. The LC25, LC50, and LC75 values at 120 hpf from the single metal exposures were used to determine mixture concentrations for observational data. The survival data indicated an additive effect occurred at lethal concentrations. The impact of the mixture on behavior and morphology were evaluated at sub-lethal concentrations of 10 and 100 ppb As (0.133, 1.33 µM) and Pb (0.048, 0.48 µM) individually or in mixtures. Data was analyzed with a repeated measures ANOVA (behavior) or an ANOVA (morphology) with the least significant difference test (α=0.05). Zebrafish larvae exposed to 10 ppb As exhibited hyperactivity in all dark phases for the distance moved, time moving, and velocity, while those exposed to 10 ppb Pb only showed an increase in distance moved and velocity in the first dark phase. The 10 ppb mixture was found to have an intermediate impact with increased time moving in all dark phases and increased distance moved and velocity only in the first dark phase. In contrast, hyperactivity was observed only in the 100 ppb mixture in the last two dark phases for time moving and in the last dark phase for the distance moved. No significant behavioral alterations occurred in the single 100 ppb treatments. A decrease in mean brain length and brain length ratio to the total length in the 10 ppb mixture was observed with no significant morphology changes observed for head length, head width, or total length.

Role of Lipid Metabolism in Particulate Matter-Induced Lung Inflammation and InjuryHannah Lovins1, Michael Yaeger1, Katelyn Dunigan Russell1, Sanjay Varikuti1, Grace Hutton1, Nabeeha Rahman1, Courtney Boyle1, Matthew Mears3, Drew Miller3, Matthew W. Gorr2,3, Loren E. Wold2,3, Kymberly M. Gowdy1

1Division of Pulmonary, Critical Care and SleepMedicine, OSU Wexner Medical Center, Columbus, OH2Department of Physiology and Cell Biology, The OhioState University, Columbus, OH 3. College of Nursing,The Ohio State University, Columbus, OHParticulate matter (PM) is a criteria air pollutant shown to increase morbidity and mortality related to chronic lung diseases. Acute PM exposure leads to lung injury and inflammation, whereas chronic exposure leads to lung remodeling. The biological mechanisms by which PM exposure induces these changes in the lung are currently unknown. PM exposure has been linked to changes

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in lipid metabolism, including upregulation of pro-inflammatory lipid mediators such as prostaglandins and leukotrienes. However, recent studies have identified a novel class of lipid mediators, termed specialized pro-resolving mediators (SPMs) that are generated to resolve inflammation and restore tissue homeostasis. Given that PM is known to increase pro-inflammatory lipid mediators, it is of interest to understand whether PM exposure causes an imbalance between pro-inflammatory lipid mediators and SPMs leading to lung alterations. From this, we hypothesize that pulmonary inflammation and injury following PM exposure is due to a reduction in SPM production resulting in a lack of SPM receptor signaling leading to lung remodeling.To test this hypothesis, 8-10 week-old C57BL/6J male mice were exposed to either filtered air (FA) or concentrated PM (mean daily PM2.5 exposure of 78.0 ± 11.1 μg/m3) 6 hours a day for 3 weeks. 24 hours post final exposure, the mice were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess lung inflammation/injury and lipidomics. Our data parallel previous work showing PM exposure leads to pulmonary injury/inflammation demonstrated by an increase in neutrophilia in the airspace and total BAL protein. Lipidomics analysis on lung tissue was largely unchanged between PM and FA except for a significant increase in the pro-inflammatory lipid mediators 9,10-DiHOME and 12,13-DiHOME in the PM exposed mice. To evaluate the macrophage specific lipid metabolism response to PM, airspace macrophages were isolated and expression of 12/15 and 5-lipoxygenases (12/15-LOX or ALOX5) and SPM receptor expression were evaluated by qPCR. These data revealed a macrophage-specific significant increase in ALOX5 expression, suggesting a change in lipid metabolism.Taken together, these data suggest that subchronic PM exposure induces lung injury and inflammation not associated with changes in total lung SPM production, but is associated with increased 9,10-DiHOME and 12,13-DiHOME production. Additionally, preliminary data indicate that there may be a macrophage specific change in lipid metabolism following PM exposure, which will be the focus of future studies.

Particulate Hexavalent Chromium [Cr(VI)] Exposure Alters miRNA Profiles and Targets miRNAs Involved in Pathways of Cr(VI) CarcinogenesisIdoia Meaza1, Rachel M. Speer1, Jennifer H. Toyoda1, Yuan Lu2, Qian Xu1, Ron Walter2, Maiying Kong1, and

John P. Wise, Sr.1

1University of Louisville, Louisville, KY; and 2Texas Tech University, San Marcos, TX.Lung cancer is the number one cause of cancer deaths and number two cause of all deaths. One potential culprit is metal pollution. Metals are widespread in the environment and are known to induce cancer. Particulate hexavalent chromium [Cr(VI)] is a well-established human lung carcinogen with environmental and occupational exposure risks. Although the mechanism of Cr(VI)-induced carcinogenesis is not fully understood, literature shows Cr(VI) induces genomic instability and alters global gene expression. At the moment, it is not clear how Cr(VI) causes global gene expression. One possibility, is that Cr(VI) alters miRNA expression levels resulting in altered transcription of mRNA and translation to protein. To investigate whether Cr(VI) alters miRNA expression profiles, we exposed human lung cells (WTHBF-6) to zinc chromate for varying time periods (24, 72 and 120 h) and concentrations (0.1, 0.2 and 0.3 ug/cm2). RNA was isolated, sequenced and bioinformatically processed. Heatmaps were generated through the Morpheous data visualization tool and KEGG pathway analysis was performed to investigate potential targets of Cr(VI)-altered miRNAs by DIANA mirPath v.3. We identified 958 unique miRNAs expressed across our samples and observed that particulate Cr(VI) altered miRNA profiles with concentration and time. Overall, we observed a global miRNA downregulation, for example, after 120 h exposure to 0.1 ug/cm2 zinc chromate, 35 miRNAs were upregulated whereas 110 miRNAs were downregulated. Additionally, the number of downregulated of miRNAs increased with exposure time and concentration, for example, after 0.1 ug/cm2 exposure 15, 70 and 110 miRNAs were downregulated after 24, 72 and 120 h exposure, respectively. Heatmaps of differentially expressed miRNAs showed differences in clustering across timepoints, showing that the 24h response to Cr(VI) is different that the 72 and 120 h responses. These results correlate with previous data from the lab, showing the difference in response between acute and prolonged exposures. Pathway analysis showed that particulate Cr(VI)-altered miRNAs predicted to target key pathways of Cr(VI) carcinogenesis. In conclusion, these data show particulate Cr(VI) induces global miRNA downregulation and the provides the first evidence that Cr(VI)-altered miRNA may play a key role in particulate Cr(VI)-induced carcinogenesis.Research reported in this publication was supported by the National Institute of Environmental Health

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Sciences [R01ES016893 to JPW] and [T32ES011564 to JPW, RMS, JHT], the Jewish Heritage Foundation for Excellence [JPW], and the University of Louisville Graduate School Dissertation Completion Award [RMS].

Potential long-term effects of diuretics on feeding behavior, overweight and obesityYaksh Rathod1, Rana Abdelgawad1, Lisa Kelly1, Christian Hübner2 and Mauricio Di Fulvio1

1Department of Pharmacology and Toxicology, Wright State University, SoM, Dayton OH. 2Institut für Humangenetik, Universit tsklinikum Jena, Germany.Clinically relevant diuretics, including bumetanide (BTD), are used, and abused to reduce body weight gain. The mechanism of action of such an effect is naturally thought to be mediated by water and solute loss via inhibition of the renal Na+K+2Cl– cotransporter-2 (Nkcc2). However, BTD also targets Nkcc1, which has a wide expression pattern. Nkcc1 is involved in many physiological processes being one of them related to the modulation of insulin secretion, a hormone well-known to control fuel homeostasis and to limit food intake. Therefore, BTD may play and indirect role in the control of food intake and body weight gain independently of its known diuretic effects. To test that hypothesis, we generated mice lacking Nkcc1 exclusively in insulin secreting β-cells of the islets of Langerhans in the pancreas and studied their feeding behavior at different age-points to correlate satiation and satiety responses to ad libitum food intake to body weight gain. The feeding behavior was studied using our recently validated paradigm to measure meal size and intermeal intervals as behavioral surrogates of satiation and satiety, respectively. Our results suggest that 10w old mice lacking Nkcc1 in β-cells (Nkcc1βKO) are hyperphagic during the nocturnal photoperiod of the day due to reduced satiation responses to food. Notably, these Nkcc1βKO mice were not heavier than normal but became significantly overweight at 15-16 weeks of age and obese at 25-30w. Moreover,20w and 30w old Nkcc1βKO mice showed impairedsatiation and satiety responses to food. Therefore,our results suggest that elimination of the BTD targetNkcc1 from insulin-secreting β-cells impairs insulinsecretion in response to food deteriorating thesatiation and satiety responses aimed at controllingfood intake, thus resulting in nocturnal hyperphagia,overweight and obesity. Hence, long term treatmentwith BTD may indirectly result in hyperphagia,overweight and obesity independently of the diureticeffects of the drug.

Effect of Acute Tobacco-Derived Crotonaldehyde Exposure on Platelet-Leukocyte AggregationAndre Richardson1,2 and Daniel J. Conklin, Ph.D.1-4

Department of Pharmacology & Toxicology1, American Heart Association – Tobacco Regulation Center2, Diabetes and Obesity Center3, Division of Environmental Medicine4, University of LouisvilleConventional cigarette smoking remains the largest risk factor for cardiovascular disease. Research shows exposure to mainstream cigarette smoke (MCS) induces platelet-leukocyte aggregation in vivo, a biomarker of platelet activation and thrombosis, via damaging blood vessels, and thus, increasing the risk of blood clots that lead to myocardial infarctions and/or stroke. Our lab has published that acute exposure of mice to crotonaldehyde (CR), an α,β-unsaturated aldehyde and a harmful or potentially harmful constituent (HPHC) in MCS, causes changes in platelet-leukocyte aggregate (PLA) formation, indicating CR may contribute to MCS effects. Yet, the mechanism by which CR affects PLAs is not well understood. The purpose of this research was to compare MCS- and CR-induced PLA formation by examining PLA composition as including platelet-granulocyte aggregates (PGAs) and platelet-lymphocyte:monocyte aggregates (PLyMAs) via flow cytometry. Adult male C57BL/6 mice were exposed (6h/d, 4d) to filtered air (control), MCS (3R4F; 12 cigs/d, 4d), or CR (1 & 3 ppm, 6h/d,4d). After final exposure, mice were euthanized and PLA levels were measured in peripheral blood by flow cytometry as the number of double positive aggregates (CD41+, platelet marker, and CD45+, pan-leukocyte marker). IgG isotype negative controls were used to guide gating. Exposures induced significant changes in PLA levels in mice following MCS (2.0±0.8% vs. 3.7±1.0%), 1 ppm CR (6.0±1.0% vs. 3.0±0.0%), and 3 ppm CR (6.0±1.0% vs.12.0±2.0%) exposures compared with their respective air controls. In the MCS study, the PGAs on average (65.8%±11.0% of PLAs) made up a greater % of PLAs than the PLyMAs (34.2%±11.0% of PLAs). In the 1 ppm CR study, the PGAs on average (70.4%±13.5% of PLAs) made up a greater % of PLAs than the PLyMAs (29.6%±13.5% of PLAs). In the 3 ppm CR study, the PGAs on average made up a greater % of PLAs (62.9%±11.4% of PLAs) than the PLyMAs (37.1%±11.4% of PLAs). In each study, the proportions of PGAs and PLyMAs were unchanged by toxicant exposures. With PLAs increasing in both MCS and 3 ppm CR exposures, these findings suggest that high levels of CR, as present in MCS, can increase thrombotic risk and that tobacco-derived aerosols influence thrombotic

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activation. More studies are needed in order to determine their influence on sensitizing granulocyte, lymphocyte, and monocyte activation. This research will be of use to the FDA in regulating the levels of HPHCs in tobacco product aerosols, as the levels of CR should be less than 3 ppm. Our research will also aid in understanding the potential mechanisms of MCS- and CR-induced thrombogenesis by highlighting the sensitivity of leukocyte activation.

Synthesis, physico-chemical characterization and neurotoxic impacts of titanium dioxide nanotubesAthira Sairanthry Suku, P V Mohanan, Jennifer L FreemanDivision of Toxicology, SCTIMST (Govt. of India), Trivandrum, Kerala, India; School of Health Sciences, Purdue University, West Lafayette, Indiana, USATitanium dioxide (TiO2) nanostructures gained significant attention in the scientific community because of their unique nanosized features, good biocompatibility, intrinsic properties, and versatile fabrication techniques. Among them, TiO2 nanotubes (TNTs) are identified as desirable structures to be applied for various biomedical applications including targeted drug delivery, cancer treatment, intravascular stents, and surface coating of tissue implants for controlled drug release. Such widespread applicability increased safety concerns among biologists in recent years. In view of these concerns, the present study focused on safety assessment of TNTs using C6 glial cells; which highlights possible neurotoxic effects in vitro. The study was divided into three phases in which the first phase included synthesis of TNTs based on solvothermal principle via Kasuga method. TNTs were then physico-chemically characterized in the second phase using various techniques in terms of functional groups, surface morphology, hydrodynamic diameter, surface charge, crystallographic nature, and thermal stability. The third phase included toxicity analysis using C6 glial cells as an in vitro model. TNTs were prepared in DMEM F12 medium at various concentrations ranging from 5-320 µg/mL. Initial cell viability was evaluated using MTT and neutral red uptake assays after 24h cellular exposure. Intracellular oxidative stress was assessed using DCFH-DA and NO release assays. LDH release assay and caspase activity analysis was performed as markers of change in enzymatic titer. Alteration in cellular morphology was analyzed by coomassie brilliant blue and rhodamine phalloidin staining. Sub-cellular organelle toxicity was evaluated using acridine orange and JC1 staining methods. Nuclear integrity was evaluated by DAPI staining and agarose gel electrophoresis methods. Flow cytometry

was conducted for cell death identification pathway using Annexin V/PI. Overall results indicated that TNTs were non-toxic up to the concentration of 160 µg/mL without any discernible changes to the cellular functions. However, at 320 µg/mL TNTs induced some significant toxic responses with 24h exposure, which was evident in all the assays conducted. Also, flow cytometry indicated early and late apoptotic neuronal cell death at this concentration. The present study provides a piece of information regarding safety levels of TNTs, which is to be considered with utmost care before being applied for any medical procedures. Also, the study recommends some surface functionalization strategies for masking such toxic nature of the material.

Changes in insulin signaling, gluconeogenic gene expression, and glucose production in cryopreserved human hepatocytes following exposure to heterocyclic aminesKennedy M. Walls, Kyung U. Hong, David W. HeinDepartment of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USAHeterocyclic amines (HCAs) are carcinogenic chemicals generated when cooking meat at high temperatures. Metabolism of HCAs by CYP1A2 and NAT2 in the liver potentiates their carcinogenic actions. Interestingly, a recent epidemiological study documented associations of high HCA intake with increased prevalence of insulin resistance, which is one of the hallmarks of type II diabetes mellitus and metabolic syndrome. However, it is unknown if HCAs can directly induce insulin resistance. To investigate the effects of HCA exposure on insulin sensitivity, we treated cryopreserved primary human hepatocytes with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), two common HCAs found in cooked meats, and measured changes in insulin signaling, gluconeogenic gene expression, and glucose production. Cryopreserved human hepatocytes were treated with varying concentrations of MeIQ (0-50 μM), then treated with insulin and analyzed for changes in insulin signaling via p-AKT induction on Western blot. MeIQ treatment decreased insulin-induced p-AKT levels, suggesting that the cells become insulin resistant in the presence of MeIQ. Additionally, MeIQ or MeIQx treatment significantly upregulated the transcripts of genes involved in gluconeogenesis (e.g., G6PC and PCK1). These results suggested that MeIQ and MeIQx impair insulin signaling and potentially induce glucose production in human hepatocytes. We then tested if the gene expression changes induced by MeIQ or MeIQx leads to increases in glucose production.

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Human hepatocytes treated with MeIQx showed an increase in glucose production compared to control-treated cells, as expected, but no changes in glucose production were observed following treatment with MeIQ. Future studies will examine if MeIQ or MeIQx exposure alters the suppressive effects of insulin on gluconeogenic gene expression and glucose production. Additionally, we will incorporate additional HCAs (e.g., IQ, IQx, and PhIP) into our studies. The alterations in insulin signaling, gluconeogenic gene expression, and glucose production following MeIQ or MeIQx treatment in human hepatocytes indicate that MeIQ and MeIQx alter insulin sensitivity and implies that exposure to HCAs via the consumption of cooked meat may potentially induce hepatic insulin resistance. Partially supported by USPHS grant T32-ES011564.

Developmental Toxicity of Legacy and Emerging Perfluoroalkyl Substances Using Zebrafish ModelOla Wasel, Hanna King, Kathryn Thompson, and Jennifer FreemanSchool of Health Sciences, Purdue University, West LafayettePerfluoroalkyl substances (PFAS) are synthetic compounds that are used in food packaging products, firefighting materials, electronics, cookware, carpets, furniture, clothing, and many other applications. PFAS are composed of a fluorinated carbon chain. PFAS are persistent in environment and bioaccumulate in organisms. The concerns of PFAS toxicity led to voluntarily phasing out of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) by their manufacturer. PFOA and PFOS are both composed of an 8 carbon chain (C8). Shorter chain chemicals (such as perfluorobutyrate (PFBA, C4) and perfluorobutane sulfonate (PFBS, C4)) and compounds with chemical modifications (such as GenX, C6) were used as a replacement to the long chain PFAS (>C7 for COO- containing PFAS or >C6 for SO3- containing PFAS) in order to increase their degradation potential. There is limited information on the safety of these emerging PFAS. In this study, we compared toxicity of five PFAS in order to assess the role of chain length, functional group and chemical structure in their toxicity. We compared the toxicity of PFOS, PFOA, PFBS, PFBA and GenX using zebrafish (Danio rerio). To determine LC50 of each chemical, zebrafish embryos were exposed to a range of concentrations of each chemical within 1-hour post fertilization (hpf) through 120 hpf. The toxicity of these compounds was assessed by monitoring the survivability every 24 hours through 120 hpf. 120hpf-LC50 were determined using GraphPad 8.0 software.

In addition, behavioral analysis using a visual motor response test was performed. For behavioral analysis, we used concentrations of 0, 4, 40, 400, and 4000 part per billion (ppb). The exposure was terminated at 72 hpf and the test was done at 120 hpf. Results of 120hpf-LC50s that toxicity ranking was PFOS> PFOA> PFBS >GenX > PFBA. Based on these results, we can conclude that toxicity increases with increasing the chain length. Also, presence of sulfonate group increased toxicity for PFAS of a given chain length. Behavioral analysis showed that embryonic exposure to PFOS, PFBS, PFBA or GenX induced changes in the locomotor activities in larvae, while PFOA didn’t cause any changes. Future work will focus on identifying the mechanism behind the observed behavioral changes.

Inhibiting Lung Adenocarcinoma Growth by a Bacterial Quorum-Sensing MoleculeAaron Whitt1, Mark Vincent Carreon Dela Cerna2, Joseph Burlison3, Donghan Lee3, Chi Li1,3

1Dept. of Pharmacology and Toxicology, University of Louisville School of Medicine; 2Dept,. of Biochemistry and Molecular Genetics, University of Louisville School of Medicine; 3Brown Cancer Center, Dept. of Medicine, Louisville, KYA hallmark of human lung tumors is evading cell death through dysregulated apoptotic pathways. Therefore, a critical objective of lung cancer research is to develop novel treatments to thwart this resistance to apoptosis. One pro-survival mechanism employed by lung adenocarcinoma (LUAD) cells is the upregulation of the intracellular lactonase Paraoxonase 2 (PON2), which promotes resistance to traditional chemotherapeutics. Decreasing expression of PON2 sensitizes LUAD cells to apoptotic stimuli and impairs cellular proliferation. Our recent research demonstrates that the lactonase activity of PON2 sensitizes cells to the cytotoxic effects of the bacterial quorum-sensing molecule, N-(3-oxododecanoyl)-l-homoserine lactone (C12), which is produced by the opportunistic pathogen Pseudomonas aeruginosa. C12 elicits its cytotoxicity independent of the apoptotic Bcl2 proteins which are commonly dysregulated in human LUAD, which highlights its potential as a novel therapy against neoplastic growth in the lung. We sought to examine C12’s anti-tumor activity in patient samples and LUAD cell lines using traditional biochemical approaches, subcutaneous murine xenograft tumor models, and high-resolution nuclear magnetic resonance (NMR). The results of these experiments indicate that C12 impairs growth and induces apoptosis of LUAD in vitro and in vivo. The anti-tumor activity of C12 is robust and unaffected

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by overexpression of the anti-apoptotic protein Bcl2, which confers resistance to other traditional chemotherapeutic agents. Furthermore, knockout of the pro-apoptotic Bcl2 proteins Bax and/or Bak fails to alter C12’s activity, but protects against other pro-apoptotic stimuli. Finally, we used high-resolution NMR to identify compounds unique to C12-treated cells in order to determine the compound(s) generated by PON2 to elicit such a distinct apoptotic response. The results of this analysis yielded a number of known and unknown metabolites distinct to C12-treated samples, which will be prioritized and validated in future experiments. Taken together, these observations highlight a unique mechanism by which C12 hinders LUAD growth in vitro and in vivo through the activity of PON2 that may be exploited to expand future lung cancer treatment options.

Modulation of Pulmonary Toxicity in Metabolic Syndrome (MetS) due to Variations in Nanoparticle- Biocorona CompositionLi Xia, Saeed Alqahtani, Jonathan ShannahanSchool of Health Sciences, Purdue University, West Lafayette IN 47907, United StatesFollowing introduction into a biological environment nanoparticles (NPs) interact with biomolecules forming a biocorona (BC) which alters cell interactions and toxicity. Metabolic syndrome (MetS) is a prevalent condition with approximately 1/3 of the U.S. population suffering from MetS. Individuals with MetS have demonstrated enhanced susceptibility to inhalation exposures, however, the mechanisms remain unelucidated. We hypothesize MetS-associated alterations in biomolecular profile result in unique NP-BC formation contributing to enhanced toxicity following NP inhalation. Bronchoalveolar lavage fluid (BALF) was collected from C57BL/6J mice receiving either a healthy diet (10% fat) or a high fat western diet (60% fat) for 16 weeks. BCs were formed by incubating 20 nm iron oxide (Fe3O4) NPs in water, 100% healthy BALF, or 100% MetS BALF for 8h at 10°. Fe3O4 NPs without or with BCs were characterized for hydrodynamic size and zeta potential. Protein and lipid components of the MetS and healthy BCs were evaluated via a proteomic/lipidomic approach. This assessment demonstrated the association of unique biomolecules and differential abundance of shared components. Specifically, there were 35 common proteins bonded with Fe3O4 NPs in healthy and MetS BALF. Proteins including serine protease inhibitor A3N, vitronectin, clusterin, complement C3, gelsolin, apolipoprotein A, and others were significantly increased in the MetS-BC compared to the healthy BC. Increased abundance of lipids such as PC(32:1),

DG16:0_16:1, PG(32:1), DG 16:1_16:0, and others were also determined in the MetS BC. There are 11 and 93 unique proteins associated with healthy-BC and MetS-BC, respectively. A mouse macrophage cell line was utilized to examine toxicity differences due to BCs. Exposures to 6.25, 12.5, 25, and 50 μg/ml of Fe3O4 NPs with BCs for 1 or 24h did not demonstrate overt cytotoxicity. Darkfield microscopy determined enhanced Fe3O4 NP internalization due to the MetS BC compared to healthy. Additionally, 1h or 24h exposure to Fe3O4 NPs with a MetS-BC at a concentration of 25 μg/mL enhanced inflammatory markers: CCL2, IL6, and IL-1β compared to Fe3O4 NPs with a healthy BC. To further elucidate inflammatory pathways, western blots are being utilized to examine activation of specific proteins within MAP kinase, Jak/Stat, and NF- κB signaling pathways. In conclusion, our assessment suggests that the formation of distinct NP-BCs occur following inhalation in MetS which contribute to exacerbated inflammatory effects. A thorough understanding of these susceptibility mechanisms is necessary for therapeutic strategies and the protection of susceptible groups.

POSTDOCTORAL RESEARCHERS

CD163 and downstream HO-1 signaling protects against O3-induced lungKatelyn Dunigan Russell1,Sky W. Reece2,Michael Yaeger1,Sanjay Varikuti1,Hannah Lovins1,Grace Hutton1,Nabeeha Rahman1,Elizabeth A. Browder2,Myles X. Hodge2,Bin Luo2,Brita J. Kilburg-Basnyat2, Hongmei Zhu3,Timothy J. McMahon3,Anupam Agarwal4,Robert MTighe3,Kymberly M. Gowdy1

1Division of Pulmonary, Critical Care and SleepMedicine, OSU Wexner Medical Center, Columbus,OH; 2Department of Pharmacology & Toxicology,Brody School of Medicine. ECU, Greenville, NC;3Department of Medicine, Duke , Durham, NC. 4Divisionof Nephology, UAB, Birmingham, AL.Epidemiological studies have reported that exposure to elevated ambient concentrations of ozone (O3) is associated with adverse pulmonary and cardiovascular outcomes. The underling biological mechanisms mediating O3 associated adverse health effects are unknown. O3 induces oxidative stress in the lung as well as pulmonary inflammation and injury. Pulmonary inflammation and injury take place in part via damage associated molecular patterns (DAMPs). Recently, we have found that O3 exposure is associated with increased levels of the DAMP, cell free hemoglobin (CFH). CFH is cleared by

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CD163, a scavenger receptor expressed exclusively on macrophages/monocytes; the uptake results in the up-regulation of heme oxygenase 1 (HO-1). HO-1 expression facilitates the metabolism of CFH into ferritin, bilirubin, and carbon monoxide, which are anti-inflammatory mediators. Therefore, we hypothesize that CD163 clearance of CFH is critical for inducing HO-1 expression and thereby protecting the lung from O3-induced inflammation/injury. To test our hypothesis we exposed C57BL/6 (WT) and CD163-/- male mice to either filtered air (FA) or 1ppm O3 for 3h or treated the mice with CFH for 6h and 24h. Macrophage-specific LysM HO-1 -/- and Hmox Floxed (fx/fx) male mice were also exposed to either FA or 1ppm O3. Lung tissue, and bronchoalveolar lavage (BAL) were collected 24hr post exposure and BAL cell counts, CFH analysis and total protein were measured. Real time PCR was used to measure pulmonary expression of HO-1, pro-inflammatory cytokines, and chemokines. Following O3 exposure, WT mice had a significant increase in CFH in the BAL which was augmented in O3 exposed CD163-/- mice. O3 exposed CD163-/- mice had greater pulmonary inflammation and injury compared to WT mice as evidenced by increased BAL neutrophils, macrophages, and total protein. CD163-/- mice exposed to O3 had significantly lower pulmonary expression of HO-1 when compared to WT. O3 exposed LysM HO-1-/- mice had greater pulmonary injury compared to fx/fx mice as evidenced by increased total protein but no differences in BAL cell differentials or pulmonary expression of pro-inflammatory cytokines and chemokines . Taken together, our findings suggest that the mitigation of O3-induced inflammation and injury in the lung is dependent on CD163 expression; as HO-1 expression in macrophages shows protection following O3-induced injury. Future studies will examine the underlying mechanisms behind the protective effects of CD163 O3-induced lung injury/inflammation, as well as the protective effects of macrophage specific HO-1 against O3-induced lung injury.

Human Bronchial Epithelial Cells Exhibit N-Acetyltransferase 2 ActivityJames T.F. Wise, Raul A. Salazar-González, Mark A. Doll, and David W. HeinDepartment of Pharmacology and Toxicology, School of Medicine, University of Louisville, Louisville, KY 40202Lung cancer is the leading cause of cancer deaths in the United States. Lung cancer is largely viewed as disease in tobacco smokers. N-acetyltransferase

2 (NAT2) is a xenobiotic enzyme that catalyzes both N- and O-acetylation of carcinogens (e.g., hydrazines, aromatic and heterocyclic amines) present in tobacco smoke and is a key driver of their genotoxicity. Many SNPs are reported for NAT2 that result in allele variants, which results in NAT2 slow, intermediate, and rapid acetylation phenotype. A recent meta-analysis reported NAT2 non-rapid (slow and intermediate) phenotypes had a significantly increased risk of lung cancer. Previous reports have indicated that human lung cells do not exhibit NAT2 activity. Given the importance of NAT2 in cancer and inhalation of various carcinogens directly into the lungs, we investigated NAT2 expression in human lung cell lines. We used two immortalized human lung cells. BEP2D and HBEC2 cells and carried out experiments with selective substrates for NAT1 (p-aminobenzoic acid; PABA) and NAT2 (sulfamethazine; SMZ) in the presence and absence of a specific NAT1 inhibitor. BEP2D cells have a NAT2 genotype of NAT2*4/*5B (intermediate acetylator) and HBEC2 cells have a NAT2 genotype of NAT2*4/*6A (intermediate acetylator). PABA ¬N-acetylation (NAT1 activity) in BEP2D cell protein lysates was 0.18 ± 0.04 nmol/min/mg protein but was undetectable in the presence of 25 µM NAT1 inhibitor. In contrast, SMZ ¬N-acetylation (NAT2 activity) in BEP2D cell protein lysates was 0.49 ± 0.16 and 0.43 ± 0.17 nmol/min/mg protein in the presence of 0 and 25 µM NAT1 inhibitor respectfully. PABA ¬N-acetylation (NAT1 activity) in HBEC2 cell protein lysates was 0.12 ± 0.03 nmol/min/mg protein but was undetectable in the presence of 25 µM NAT1 inhibitor. In contrast, SMZ ¬N-acetylation (NAT2 activity) in HBEC2 cell protein lysates was 0.24 ± 0.02 and 0.25 ± 0.03 nmol/min/mg protein, in the presence of 0 and 25 µM NAT1 inhibitor respectively. These data establish that NAT2 is expressed and functional in human lung cells and will help us further our understanding of NAT2 in lung cancer. Future studies are directed at investigating a mechanistic role in human lung of NAT2 during the exposure to carcinogens. This work was supported by USPHS grant T32-ES011564.

Two Sides of a Toxic Coin: Cr(VI) as a Gerontogen and Neurotoxic Effects with AgeJohn P. Wise, Jr., Lu CaiDept of Pharmacology and Toxicology, U. of Louisville School of Medicine; Pediatric Research Institute, the Dept of Pediatrics, U. of Louisville School of MedicineOur research takes a novel approach to considering aging and toxicology that we refer to as a toxic coin. On the heads side, we consider how neurotoxic effects of hexavalent chromium [Cr(VI)] changes with age (young vs. middle-aged vs. geriatric). On the tails

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side, we consider how Cr(VI) accelerates or induces biological aging processes in cells and tissues, i.e. how it acts as a gerontogen. Traditionally, thebrain was considered composed of mostly post-mitotic neurons and hence genotoxic agents wereconsidered less of a threat. Now, we know theopposite is true – most of the brain’s cells, the glia,are mitotic and play critical roles in protecting andsupporting neuronal health. Recent studies showthese glial cells can exhibit increased aneuploidyand chromosome instability with aging and early inneurodegenerative diseases. Hence, there is a criticalneed to understand how genotoxic chemicals affectbrain health and contribute to premature aging. Cr(VI)is a major environmental health concern that caninduce aging phenotypes and has the best definedclastogenic mechanism of metals. Cr(VI) also causesbrain damage that may be linked to a variety ofneurological symptoms. We will investigate the role ofCr(VI) in this aging paradigm, using young vs middle-aged vs geriatric rats exposed to Cr(VI) for 90 days

via drinking water. We hypothesize 1) young and aged individuals are more susceptible to Cr(VI)-induced neurotoxicity and 2) Cr(VI)-induces brain aging by causing aneuploidy and chromosome instability resulting in glial senescence. Here we present preliminary data from a rat study with zinc chromate suggesting neurodegeneration in the cerebellum and hippocampus, metal dyshomeostasis, and induction of DNA damage. Rats were given an oral aspiration of zinc chromate twice weekly at 0, 0.4, or 0.8 mg Cr/kg for 24 hours or 90 days. We also present data characterizing Cr(VI) toxicity to M059K and M059J cells (derived from a glioblastoma: compared to M059K, M059J lacks DNA double strand break repair response). Ongoing work is focused on developing further mechanistic insight into this pathway from Cr(VI)-induced DNA damage to cellular senescence in glia of the rat brains and cultured glioblastoma cell models. J.W. and L.C. were supported fully or in part by T32-ES011564 and P30-ES030283

THANK YOUA huge thanks to the OVSOT Executive Committee for their support in organizing this meeting and judging the presentations. Thank you to OVSOT members for supporting the awards and attendance of this meeting. Thank you to all the researchers who submitted abstracts and those who attended to support our trainees. Of course this wouldn’t be possible without the support of SOT and the expertise of our Component Group Specialist, Jessie Yuhaniak.

David MattieWalter (Bert) WatsonJonathan ShannahanSandra S. Wise

Executive CommitteePresident Vice President Vice President-Elect Secretary (2021-2023) Treasurer (2020-2022) Sherleen Xue-Fu AdamsonEducation and Outreach Liaison Eddie Sloter

Petra Haberzettl Christopher WingardKymberly GowdyLu CaiBrandon Lewis

3rd year Councilor 2nd year Councilor 1st year Councilor Past President (Councilor) Post-Doc Rep Graduate Student Rep Jennifer Toyoda