POSTER ABSTRACTS

Society of Toxicology (MASOT)

www.masot.org

Fall 2015 Scientific Meeting

October 13th, 2015

Abstract 01 Tracking Inflammatory Macrophage Accumulation in the Lung during Ozone-induced Lung Injury in Mice

M Francis, M Mandal, C. Sun, H Choi, JD Laskin, DL Laskin

Rutgers University, Piscataway, NJ

Ozone induced lung injury is associated with an accumulation of pro- and antiinflammatory macrophages (MP) in the lung which have been implicated in tissue injury and repair. In these studies, we used in vivo tracking techniques to investigate the origin of cell. Initially we generated bone marrow (BM) chimeric mice by adoptive transfer of BM cells from GFP+ mice into irradiated C57BL/6 mice. After 4 weeks, mice were exposed to air or ozone (0.8 ppm, 3 h). Macrophages were isolated from lungs 24-72 h later, stained with fluorescent labeled antibodies, and analyzed by flow cytometry. Approximately 98% of BM cells were found to be GFP+ while only 5% were GFP+ in control lungs. Ozone exposure resulted in a marked increase in infiltrating mature GFP+CD11b+F4/80+ MP into the lung. Two populations, Ly6CHi proinflammatory and Ly6CLo antiinflammatory were identified. Proinflammatory GFP+Ly6CHi MP increased rapidly after ozone and remained elevated, increases in antiinflammatory GFP+Ly6CLo were transient. To assess potential mechanisms mediating the accumulation of these MP subpopulations in the lung, we used mice lacking Ccr2, a chemokine receptor involved in proinflammatory MP trafficking. Loss of Ccr2 resulted in decreased numbers of infiltrating CD11b+ MP in the lung. This was due to a selective reduction in proinflammatory Ly6CHi MP. Loss of Ccr2 also resulted in an increased expression of the fractalkine receptor Cx3CR1; this was correlated with an increase in Ly6CLo anti-inflammatory MP in the lung and reduced toxicity. To further characterize these cells, we used CX3CR1 GFP/+ mice. After ozone exposure, CX3CR1 GFP+ cells infiltrated into the lung; these cells were F4/80+ and expressed CD206, a marker of antiinflammatory MP. Taken together, these results demonstrate that following ozone exposure, inflammatory cells enter the lung from the bone marrow. Additionally, while Ccr2 plays a key role in proinflammatory MP trafficking to the lung, CX3CR1 is involved in antiinflammatory MP migration. Supported by NIH Grants ES004738, CA132624, AR055073, ES007148 and ES005022.

Abstract 02 Pharmacological Modulation of GRM1 Activation by Riluzole Results in a Reduction in Exosome Levels in Melanoma

Allison L. Isola1,2, Yvonne Wen3, James Goydos3 and Suzie Chen1,2,3

1 Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers, the State University, Piscataway, NJ, 08854; 2 Joint Graduate Program of Toxicology, Rutgers, the State University, Piscataway, NJ, 08854; 3 Rutgers, the Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ, 08903

Exosomes are naturally occurring small membrane enclosed nanovesicles generated and released constitutively by various cell types, and are more frequently released by tumor cells. The normal functions of exosomes include transporting proteins, nucleic acids and may facilitate communication between cells within the local microenvironment. Recently, several reports postulated the involvement of exosomes in tumor cell metastases including melanoma. Metabotropic glutamate receptor 1 (GRM1), a neuronal receptor, when ectopically expressed in melanocytes, is sufficient to induce melanocyte transformation in vitro and spontaneous malignant melanoma development in vivo in a transgenic mouse model. GRM1-transformed melanocytes exhibited abundant level of exosomes compared to normal melanocytes evaluated by electron microscopy, CD-63, a protein marker of exosomes, and Nanosight. Similar results were observed in GRM1-expressing human melanoma cells. Modulation of GRM1 expression levels by genetic (via silencing RNA) or pharmacological (inhibitor of GRM1-mediated glutamate signaling) means led to a reduction in cell growth in vitro and tumor progression in vivo. Furthermore, concurrent reduction in exosomal production/secretion was observed, while an increase in GRM1 expression by exogenous GRM1 cDNA resulted in parallel enhancement in exosomal production/secretion. Possible involvement of exosomes in promoting the metastatic phenotypes detected in our melanoma prone transgenic mice provides opportunities to explore this notion directly. Whether GRM1 regulates exosome production directly, via its signaling cascade, or by another route, is currently being investigated.

Abstract 03 Down-Regulation of the Human Placental BCRP/ABCG2 Transporter in Response to Hypoxia

Ludwik Gorczyca1,2, Jianyao Du4, Lissa Francois, Kristin Bircsak1,2, Xia Wen1, Lauren Aleksunes1,3

1Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ; 2Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ; 3Enviornmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, NJ; 4China Pharmaceutical University, Nanjing, Jiangsu, People’s Republic of China

The Breast Cancer Resistance Protein (BCRP/ABCG2) is expressed on the apical membrane of placental syncytiotrophoblasts and serves an important role in protecting the fetus from in utero chemical and drug exposure. BCRP actively limits the passage of drugs and xenobiotics across the placenta by expelling them back into maternal circulation. Recent studies have shown conflicting data in terms of BCRP transporter expression in response to altered oxygen tension. The partial oxygen pressure in the first and second trimester human placenta is approximately 20 mmHg (~3% O2) and 60 mmHg (~8% O2), respectively, and continues to increase to 20% throughout pregnancy. To assess how hypoxia affects the expression and regulation of the BCRP/ABCG2 transporter, BeWo choriocarcinoma cells were exposed to 3%, 8%, and 20% oxygen for 24 hours. mRNA and protein expression of BCRP and potential transcription factors involved in its regulation were assessed by qPCR and western blot. Hypoxia increased the mRNA expression of two HIF-1a prototypical target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1), confirming the activation of this signaling pathway. Placental BeWo cells, exposed to low oxygen tension, exhibited a dose-dependent down-regulation of BCRP mRNA and protein (~25-35%) when compared to normoxic conditions (20% O2). Screening of potential regulators of BCRP mRNA during hypoxia revealed a significant down-regulation of nuclear receptors PPARg, RXRa as well as transcription factors AhR and NRF2, compared to normoxia. On the other hand, in response to 3% and 8% O2 tension the expression of the nuclear receptor ERa was induced 6 and 2 fold, respectively. Together these data suggest that placental expression of the BCRP efflux transporter is regulated by oxygen tension, and that the fetus may be at higher risk of exposure to BCRP substrates during early pregnancy.

Abstract 04 Nitrogen Mustard Induces DNA Damage and Structural Changes in Mouse Skin Hair Follicles

G. Composto1, S. Kim1, D. Heck2, J. Laskin1, D. Laskin1 and L. Joseph1

1Rutgers University, Piscataway, NJ and 2New York Medical College, Valhalla, NY

Nitrogen mustard (NM) is a potent skin vesicant. Evidence suggests that the pilosebaceous unit in the skin is a target for NM. To assess this, we analyzed changes in hair follicles and sebaceous glands in mouse skin following NM exposure. Dorsal skin of female CD-1 mice was exposed to 77 ng NM for 6 min, and harvested 1-5 d later. Control (CTL) skin contained sebaceous glands expressing fatty acid synthase (FAS), a marker for sebum production. Proliferating cell nuclear antigen (PCNA, marker of cellular division) and keratin-10 (K-10, marker of cellular differentiation) were expressed in the hair outer root sheath (ORS) and infundibulum of CTL skin. One d post NM, FAS, PCNA and K-10 expression was decreased in sebaceous glands, ORS, and infundibulum, respectively, while phospho-H2A.X, a marker for double strand DNA breaks, was evident in the ORS and basal epidermis. By 2 d, an eschar formed; there were reduced numbers of pilosebaceous units within the eschar and this was associated with increased expression of phospho-H2A.X in hyperplastic interfollicular epidermis (IFE). Three days post-NM, low levels of FAS, PCNA, and K-10 were expressed in the pilosebaceous units at the wound edge. By 4 d, small pilosebaceous units within the neo-epidermis expressed increased levels of FAS, PCNA and K-10, while decreased levels of phospho-H2A.X were observed within the IFE. By 5 d, wound healing was evident with increased FAS and K-10 expression in the sebaceous glands and infundibulum, respectively. PCNA was also increased in the ORS, while phospho-H2A.X was decreased. These data indicate that the pilosebaceous unit is an important target contributing to NM-induced skin injury. Support: NIH AR055073.

Abstract 05 Genistein Reduces Glyburide Efflux by the Human Placental BCRP Transporter: Transcriptional Regulation and Direct Inhibition

Kristin M Bircsak1,2, Yixin Lin1, Lauren M Aleksunes1

1Dept of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ and 2Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ

The hypoglycemic drug glyburide is routinely used in the treatment of gestational diabetes. Fetal exposure to glyburide is typically low because the BCRP/ABCG2 transporter on placental syncytiotrophoblasts prevents its maternal-to-fetal transfer. The purpose of this study was to determine the effect of the dietary soy isoflavone genistein on the direct inhibition and transcriptional regulation of BCRP. To measure the direct inhibition of BCRP by genistein, HEK cells overexpressing the wild-type (WT) or functionally-reduced BCRP variant (C421A) and human choriocarcinoma BeWo placental cells were used. In the presence of genistein, BCRP function was determined by substrate (3H-glyburide) accumulation. For the regulation studies, human choriocarcinoma BeWo placental cells were incubated with genistein (0-10 µM) for 48 h. The direct inhibition studies demonstrated up to a 600% increase in the accumulation of 3H-glyburide in both the WT- (IC50=4.7 ± 0.15 µM) and C421A-BCRP (IC50=3.9 ± 0.73 µM) cells compared to those incubated without genistein. Additionally, genistein directly increased the accumulation of 3H-glyburide in placental BeWo cells at concentrations relevant to human exposure (IC50=0.18 ± 0.11 µM). After 48 h exposure to genistein, placental BeWo cells exhibited a 40% reduction in BCRP mRNA and protein. Down-regulation of BCRP led to a 40% increase in the accumulation of 3H-glyburide in BeWo cells treated with genistein. Taken together, pregnant women expressing the common loss-of-function C421A-BCRP variant and/or consume genistein through a soy diet may be at an elevated risk for fetal exposure to glyburide and subsequent neonatal hypoglycemia. Support: ES020522, ES005022, ES007148, AFPE and PhRMA Predoctoral Fellowships.

Abstract 06 Targeting Heme in Cytochrome P450 to Inhibit Mixed Function Oxidase Reactions

Szilagyi, John T.1, 2; Mishin, Vladimir2; Heck, Diane E.4; Jan, Yi-Hua3; Richardson, Jason R.3; Laskin, Debra L.1, 2; Laskin, Jeffrey D.1, 3

1. Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ, United States; 2. Pharmacology & Toxicology, Rutgers University, Piscataway, NJ, United States; 3. Environmental & Occupational Medicine, Rutgers University Robert Wood Johnson Medical School, Piscataway, NJ, United States; 4. Environmental Health Science, New York Medical College, Valhalla, NY, United States

As key mediators of mixed-function oxidation reactions, cytochrome P450 enzymes are critical for drug metabolism. A number of cytochrome P450 enzyme inhibitors have been described. However, their mechanisms of action are often not well understood. The aromatic heterocyclic cation, diphenyleneiodonium (DPI), has been shown to be a flavoenzyme inhibitor that targets NADPH cytochrome P450 reductase. We report an alternative site of action of DPI in the cytochrome P450 system. We found that DPI was effective in inhibiting a number of cytochrome P450-related monooxygenase reactions including 7-methoxyresorufin O-deethylation, dibenzylfluorescein O-debenzylation, and 7-ethoxycoumarin O-deethylation as well as cytochrome P450 related NADPH oxidase , a reaction that generates hydrogen peroxide in the absence of metabolizing substrates. Enzyme inhibition was time- and concentration-dependent with IC50's ranging from 0.5-1.0μM. High (10-20μM) but not low (0.5-1.0μM) concentrations of DPI inhibited electron flow via NADPH-cytochrome P450 reductase as measured by its oxidase activity and redox cycling reactions and its ability to reduce cytochrome c and ferricyanide. Difference spectra using rat liver microsomes, recombinant cytochrome P450's and purified cytochrome P450's demonstrated that DPI affected the interaction between the cytochrome P450 heme and carbon monoxide. In the presence of NADPH, DPI treatment of microsomes yielded a type II spectral shift. These data indicate that, in addition to interacting with NADPH cytochrome P450 reductase, DPI can react directly with the cytochrome P450 enzymes. Supported by NIH AR055073, NS079249 and ES005022.

Abstract 07 Pharmacological Activation of Fxr Attenuates Pregnancy-Induced Repression of Hepatic and Ileal Bile Acid Transporters in Mice

Jamie E Moscovitz, Bo Kong, Grace L Guo, Lauren M Aleksunes

Department of Pharmacology and Toxicology, Rutgers University Ernest Mario School of Pharmacy, 170 Frelinghuysen Rd, Piscataway, NJ 08854

The Farnesoid X Receptor (Fxr) controls bile acid homeostasis by coordinately regulating the expression of bile acid synthesis enzymes (Cyp7a1) and transporters in the intestine (Asbt, Ostβ) and liver (Ntcp, Oatp1a4, Bsep, Mrp3, Ostβ), as well as endocrine (Fgf15) and transcription (Shp) factors. We have demonstrated that expression of bile acid synthesis enzymes and transporters is dysregulated in pregnant mice. To determine the role of Fxr in the adaptive gene responses of pregnancy, mRNA and protein expression were quantified in livers and intestines from wild-type and Fxr-null mice, as well as wild-type mice administered the Fxr agonist GW4064 (GW). In response to pregnancy, ileal Fgf15, Asbt and Ostβ mRNAs decreased by 25-57% in wild-type mice. Expression of these genes was also constitutively reduced 40 to 99% in Fxr-null mice, although unchanged by pregnancy. In the liver, pregnancy-mediated repression of Bsep mRNA was observed in wild-type, but not Fxr-null mice. Similar to wild-types, pregnancy further reduced hepatic Shp, Ntcp, Oatp1a4 and Mrp3 mRNA levels of Fxr-null mice. Treatment of pregnant mice with GW induced hepatic Shp, Ntcp, Bsep, Mrp3 and Ostβ, and ileal Fgf15 and Ostβ mRNA levels by 20-300%. GW also enhanced protein levels of Ntcp and Bsep in pregnant mice. Likewise, the induction of Cyp7a1 in pregnant mice was reduced by GW to levels lower than virgin controls. Collectively, these data implicate both intestinal and hepatic Fxr signaling in pregnancy-related procholestatic gene changes, and suggest that Fxr activation may restore bile acid homeostasis in maternal cholestasis. Supported by AFPE, R01ES020522, GM104037 and T32ES007148.

Abstract 08 The Selective mGluR Group I Agonist, 2-chloro-5-hydroxyphenylglycine, Increases BDNF and Myelin Proteins following Cuprizone-Induced Demyelination

Kyle Saitta1,2, Yangyang Huang2, and Cheryl F. Dreyfus2

1 Department of Pharmacology & Toxicology and Joint Graduate Program in Toxicology, Rutgers University, Piscataway, New Jersey, 08854; 2 Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey, 08854

Brain-derived neurotrophic factor (BDNF) is an important neurotrophin involved in the development and survival of oligodendrocyte lineage cells. While it is known that BDNF increases DNA synthesis in cultured oligodendrocyte progenitors and promotes the differentiation of oligodendrocytes to myelin-producing cells, less is known about the role of endogenous BDNF in demyelinating diseases such as multiple sclerosis. In fact, patients with relapsing-remitting multiple sclerosis have decreased levels of BDNF. However, administration of exogenous proteins such as BDNF to the central nervous system may prove difficult as a form of treatment. Therefore, an intriguing therapeutic approach for these types of demyelinating diseases may be to enhance the endogenous source of BDNF. We have previously demonstrated that the Group I and Group II mGluR agonist, ACPD, was able to enhance the expression of BDNF and myelin proteins when injected directly into the corpus callosum of mice following a cuprizone-induced lesion. The aim of this study was to expand this concept to the selective mGluR Group I agonist, 2-chloro-5-hydroxyphenylglycine (CHPG), by using the more clinically relevant approach of a peripheral injection. Male C57BL/6 mice were fed either 0.2% cuprizone milled into mouse feed or identically processed control feed for 4 weeks starting at 8 weeks of age. Cuprizone is a copper chelator used to induce demyelination in the corpus callosum. CHPG or saline vehicle was injected intraperitoneally after 4 weeks of continuous treatment, and the levels of BDNF, myelin proteins, and progenitor cell markers were analyzed by Western blot. 4 weeks of cuprizone treatment caused a decrease in BDNF and myelin proteins within the corpus callosum. Cuprizone also increased oligodendrocyte progenitor cell markers NG2 and PDGFRα compared to mice on control feed. However, intraperitoneal injections of CHPG (20 or 40 mg/kg) to the curprizone-treated mice reversed cuprizone effects and increased levels of BDNF and myelin proteins while also decreasing oligodendrocyte progenitor markers. Importantly, CHPG did not alter these proteins when given to mice receiving control feed. Taken together, these data suggest that selective mGluR Group I agonists such as CHPG may be a therapeutic approach for treating demyelinating diseases by increasing the levels of BDNF and myelin proteins while promoting differentiation of oligodendrocyte progenitor cells. (Supp: NIH NS036647; HD023315; T32ES007148 and NMSS RG 4257B4/1)

Abstract 09 Role of Fibroblast Growth Factor 15 in the Development of High Fat Diet Induced NASH

Justin D. Schumacher1, Bo Kong1, Yang Pan2, Le Zhan1, Runbin Sun2, Jiye Aa2, Jason R. Richardson1, Debra Laskin1, Grace L. Guo1

1. Toxicology, Rutgers University, Piscataway NJ, United States; 2. Key Laboratory of Drug Metabolism and Pharmacokinetics, Chinese Pharmaceutical University, Nanjing, China.

With the rise in obesity in Western civilizations, the prevalence of non-alcoholic steatohepatitis (NASH) is increasing with an estimated 5-10% of the population affected. The primary features of NASH include steatosis, inflammation, and fibrosis often accompanied with metabolic syndrome. Fibroblast growth factor 15 (Fgf15) , an endocrine factor mainly produced in the distal part of small intestine, emerges to be a critical factor in regulating bile acid homeostasis, energy metabolism, and liver regeneration. In order to investigate the effects of Fgf15 on the development of each the listed features of NASH, Fgf15-/- mice were bred into a 75% A129 and 25% C57BL/6 background. Four-week old Fgf15-/- and wild-type (WT) control mice were fed either a high fat diet (HFD) or control diet for six months. Body weight changes and food consumption were recorded regularly and a glucose tolerance test was administered during the fifth month. At the end of feeding, liver, intestine, and blood samples were collected for gene expression analysis, determination of serum and tissue lipid composition, identification of biomarker concentrations, and histology. The results showed that HFD-fed Fgf15-/- mice had metabolic syndrome presenting with increased body weight, decreased insulin sensitivity and increased basal total serum cholesterol levels. Knockout animals had altered expression of lipid metabolic enzymes with basal Mtp down-regulated and Acss2 up-regulated while on a HFD compared to controls. Fgf15 deficiency had no observed effects on steatosis as WT and Fgf15-/- mice were found to have comparable hepatic levels of triglycerides and total cholesterol. However, the Fgf15-/- mice were protected from the development of hepatic fibrosis revealed by histology analysis and expression of the genes involved in fibrosis. No changes were observed in hepatic expression of inflammatory genes such as Tnf-α or Icam. Lastly, Fgf15-/- mice fed the HFD had increased bile acid pools and up-regulated gene expression of ileal Ibabp and hepatic Cyp7a1, Cyp8b1, and Bsep compared to WT mice. In summary, during NASH development, Fgf15 deficiency was found to have no effects on liver steatosis or inflammation, however, led to decreased insulin tolerance, increased basal serum total cholesterol levels, altered expression of lipid metabolic enzymes, disrupted bile homeostasis, and decreased liver fibrosis.

Abstract 10 Multiplex Quantification of Novel Urinary Biomarkers for Subclinical Nephrotoxicity in Oncology Patients Treated with Cisplatin

Blessy George1,2,, Xia Wen2, Lucas Ellison3, Madeleine Gomez3, Nickie Johnston3, Melanie S. Joy3, Lauren M. Aleksunes2

1 Rutgers University, Joint Graduate Program in Toxicology, Piscataway, NJ; 2 Rutgers University, Ernest Mario School of Pharmacy, Dept of Pharmacology & Toxicology, Piscataway NJ; 3 University of Colorado, Dept of Pharmaceutical Sciences, Aurora CO

Acute kidney injury can be observed in up to a third of oncology patients treated with cisplatin using traditional clinical markers, including serum creatinine and urinary albumin excretion. However, these indicators of nephrotoxicity are considered insensitive since a significant degree of tubular damage is needed in order to increase their levels. Preclinical studies support detection of novel proteins in urine as sensitive biomarkers of cisplatin-induced injury. Therefore, we sought to characterize urinary levels of several novel biomarkers including kidney injury molecule-1 (KIM-1), calbindin, clusterin, glutathione S-transferase (GST-π), interleukin-18 (IL-18), monocyte chemoattractant protein-1 (MCP-1), albumin, beta-2 microglobulin (B2M), cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), osteopontin, trefoil factor 3 (TFF3). Forty-two patients scheduled for outpatient chemotherapy for solid tumors with IV cisplatin ≥ 25 mg/m2 were recruited. Urine was obtained at baseline as well as 3 and 10 days post infusion and analyzed using a multiplex protein assay for the initial tracked dose. Urinary KIM-1 protein concentrations were also measured using an ELISA assay. Serum creatinine levels did not differ post-infusion compared to baseline. However, urinary levels of several novel biomarkers including KIM-1, calbindin, clusterin, MCP-1, albumin, and TFF3, were significantly elevated at least ~2-fold by day 10. B2M, cystatin C, and TFF3 were significantly elevated at least ~1.5-fold by day 3. These data suggest that novel urinary protein biomarkers may be more sensitive than traditional biomarkers for the detection of subclinical nephrotoxicity in patients treated with cisplatin. Supported by an AFPE Fellowship, DK080744, DK093903, and CA072720.

Abstract 11

δ-T and γ-T Tocopherols Inhibited PhIP/DSS-induced Colon Carcinogenesis in CYP1A-humanized Mice

Jayson X. Chen1,2, Anna Liu1, Hong Wang1, Mao-Jung Lee1 , Guangxun Li1, Siyuan Yu1, Eric Chi1, Kenneth Reuhl3, Nanjoo Suh1,4, and Chung S. Yang1,4

1Department of Chemical Biology, Ernest Mario School of Pharmacy; 2Joint Graduate Program in Toxicology; 3Department of Pharmacology and Toxicology, Rutgers, The State University of New Jersey, Piscataway, NJ; 4Cancer Institute of New Jersey, New Brunswick, NJ

Tocopherols (T), the major forms of vitamin E, are a family of fat-soluble phenolic compounds and important dietary antioxidants. Previous animal studies and human intervention trials with the α-T have yielded disappointing results, particularly in relation to colorectal cancer. However, several recent studies demonstrated that δ-T and γ-T as well as a γ-T-rich mixture of tocopherols (γ-TmT) are more effective than α-T in inhibiting chemical-induced rodent colon carcinogenesis models. In the present study, we investigated the effect of purified δ-T, γ-T and α-T on a novel colon carcinogenesis model induced by the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and promoted by dextran sodium sulfate (DSS) in CYP1A-humanized (hCYP1A) mice. The hCYP1A mice, at 5-7 weeks of age, were fed the AIN93M control diet or diet supplemented with 0.2% δ-T, γ-T, or α-T, starting 1 week before PhIP administration (2x 100mg/kg b.w., 3 days apart). DSS (1.5%) was given in drinking water at 1 week after 1st PhIP administration for 4 days. At week 10 after PhIP/DSS treatments, significant reduction in colon tumors multiplicity was found in the male mice by the δ-T and γ-T diet groups, but not the α-T diet group. In the female mice, no significant difference in tumor multiplicity was observed between the control and tocopherol-supplemented groups. Further investigations in male mice indicate that δ-T and γ-T are more potent than α-T in inhibiting oxidative and nitrosative stress (i.e., 8-oxo-dG and nitrotyrosine) and suppressing pro-inflammatory mediators (i.e., NFκB and p-STAT3) in the colon tissues. In addition, dietary δ-T and γ-T significantly reduced PhIP-induced DNA damage, oxidative and nitrosative stress, and enhanced apoptosis markers in the early stage (i.e., days 3, 7 and 14 after PhIP administration) of colon carcinogenesis. Altogether, we demonstrated the superior inhibitory effects of δ-T and γ-T in a dietary carcinogen-induced, colitis-promoted colon carcinogenesis model. The inhibition is likely due to the antioxidative, anti-inflammatory, and early chemopreventive activities of δ-T and γ-T. (Supported by NIH grant (RO1CA133021), NIH fellowship grant (F31CA168333), and NIEHS training grant (32ES007148) as well as shared facilities funded by CA72720 and ES05022)

Abstract 12 Autophagy In Hyperoxia-Compromised Macrophage Function Of Phagocytosis

Michelle Zur1 and Lin Mantell1&2

1Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY, USA; 2Center for Heart and Lung Research, The Feinstein Institute for Medical Research, North Shore-LIJ Health System, Manhasset, NY, USA

Mechanical ventilation with hyperoxia is routinely used to treat patients with respiratory distress. However, prolonged exposure to hyperoxia compromises macrophages’ ability to phagocytose and clear bacteria, leading to increased susceptibility of pulmonary infections. We and others have shown that hyperoxia can induce the accumulation of nuclear protein high mobility group box-1 (HMGB1) both in the airways of patients with ventilator–associated pneumonia or cystitic fibrosis and in the extracellular milieu of cultured macrophages. Extracellular HMGB1 can directly impair macrophage phagocytosis. Autophagy is a cellular process necessary for recycling old or damaged proteins and organelles. Although autophagy has been implicated in macrophage phagocytosis, its role in hyperoxia-compromised macrophage function is unclear. In this study we investigated whether autophagy is involved in the pathway of HMGB1-compromised macrophage functions under hyperoxic conditions, using macrophage-like RAW 264.7 cells exposed to 95% O2 and Western Blot analysis for the ratio of LC3-II/LC3-I. Significant autophagy activation was observed in these cells following hyperoxic exposure for 24 and 48 h. Interestingly, the onset of autophagy activation directly correlated with significantly reduced macrophage phagocytic activity in hyperoxia. Furthermore, at 10ug/ml, not only did treatment of macrophages with recombinant HMGB1 (rHMGB1) significantly induce autophagy activation, it also reduced macrophage phagocytic activity. To further confirm the role of autophagy in dysfunction of phagocytosis, RAW cells were treated with rapamycin or glucose starvation. Autophagy activated by either rapamycin or starvation exhibited significantly reduced phagocytic activity. Taken together, these data suggest that autophagy is the downstream mechanism of HMGB1 in hyperoxia-compromised macrophage function of phagocytosis.

Abstract 13 Ascorbic Acid Attenuates Hyperoxia-Compromised Host Defense Against Pulmonary Bacterial Infection

Vivek S. Patela, Vaishali Sampata, Ravikumar Sitaparaa, Haichao Wangb, Michael Graham Espeyc, Xiaojing Yanga, Retina Kundua, Charles R. Ashby Jr.a Douglas D. Thomasd, and Lin L. Mantella,b,e

aDepartment of Pharmaceutical sciences, St. John’s University College of Pharmacy and Health Sciences, Queens, NY; bThe Feinstein Institute for Medical Research, North Shore-LIJ Health Science, Manhasset, NY; cNational Cancer Institute, Bethesda, MD; dDepartment of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial infections, causing ventilator-associated pneumonia (VAP). The phagocytic activity of macrophages is impaired by hyperoxia-induced increases in the levels of reactive oxygen species (ROS) and extracellular high mobility group protein B1 (HMGB1). Ascorbic acid (AA), an essential nutrient and antioxidant, has been shown to be beneficial in various animal models of ROS-mediated diseases. The aim of this study was to determine whether AA could attenuate hyperoxia-compromised host defense and improve macrophage functions. C57BL/6 male mice were exposed to hyperoxia (≥98% O2, 48 h), followed by intratracheal inoculation with Pseudomonas aeruginosa, and simultaneous intraperitoneal administration of AA. AA (50 mg/kg) significantly increased bacterial clearance in the lungs and airways, and significantly reduced the accumulation of HMGB1 in the airways. The incubation of RAW 264.7 cells (a macrophage like cell line) with AA (0-1000 µM) prior to hyperoxic exposure (95% O2) stabilized the phagocytic activity of macrophages in a concentration dependent manner. Furthermore, supplementation of media with AA significantly decreased the production of intracellular ROS in RAW 264.7 cells and the accumulation of extracellular HMGB1. Our study suggests that AA supplementation may prevent or attenuate VAP in patients receiving oxygen support.

Abstract 14 Oral Exposure to E-Waste Leachate Alters Chromatin Integrity and Morphology of Mouse Sperm in a Dose-dependent Manner

Costa P1, Igbo, J.2 Blum JL1, Sisco, M1, Doherty-Lyons S1, Kaur, K1, Zelikoff JT1

NYU School of Medicine 1Dept of Environmental Medicine, Tuxedo, NY 10987; Department of Biological Oceanography2, Nigerian Institute of Oceanography and Marine Research, 3 Wilmot Point Road, Victoria Island, Lagos, Nigeria.

Exposure of adult male mice to chemical mixtures can lead to diminished fertility through changes in gametogenesis, Sertoli or Leydig cell function, or endocrine disruption to yield reduced sperm numbers, loss of motility, or increasing morphological defects. Contaminants in electronic waste (e-waste) include toxic metals (e.g., Pb, Hg, As, Cd,) and persistent organic pollutants (e.g., PBDE, PCBs, PVCs and PAHs). In the Alaba International Market (Lagos, Nigeria), electronics are manually disassembled at riverbank sites near residential quarters, and discarded in open-fire dumpsites and landfills. E-waste leaches into soil, rivers, groundwater, and vegetation to toxic levels. The present animal study investigated the potential reproductive toxicity of e-waste contaminated leachate collected from sites near the Alaba Market.

In a pilot study, three groups (n = 3) of 12 wk-old B6C3F1 male mice were exposed (5 d/wk for 3 wk) to e-waste leachate via oral gavage (10ml/kg body weight) at 10%, 25% and 50% concentrations (v/v; leachate: distilled water); the control group (n = 2) received de-ionized water. Following euthanasia, half of each thymus, spleen, liver, and kidneys were fixed in 3% formalin; left testis and epididymis were fixed in Bouin’s solution for later histological analysis. The remaining half organs, heart, seminal vesicles, and prostate, were frozen in liquid nitrogen for later gene expression analyses. Sperm from the right cauda were excised into M16 solution for counting and determination of sperm viability (trypan blue exclusion), chromatin damage (toluidine blue staining), and morphology (Coomassie Blue); 400 sperm/animal/group were examined at 100X by bright light microscopy.

E-waste exposure groups demonstrated significant (p < 0.05) and dose-dependent chromatin damage (4.8%, 5.5%, and 6.9% damage of total sperm counted for the 10%, 25%, and 50% treatment dilution groups, respectively) compared to control (4.1%). In addition, total percentages of sperm abnormalities (10%, 12.8%, and 15.3% for the 10%, 25%, and 50% groups, respectively) were significantly elevated compared to control (9%). Sperm abnormalities included: amorphous or missing heads; misfolded, doubled, or missing tails; and cytoplasmic droplets. In addition, prostate weight in the highest treatment group was significantly increased (0.71mg) compared to 0.57, 0.53, and 0.39 mg in 10%, 25%, and control, respectively. No significant differences in the remaining organ weights, sperm count, or viability were observed.

Results from this study demonstrate that ingestion of e-waste leachate by adult male mice adversely affects sperm quality. Increases in chromatin damage suggest that e-waste leachate exposure injures sperm DNA. Morphological parameters provide a direct measurement of sperm production quality; e.g. abnormally shaped sperm heads may be due to induced point mutations in spermatogonia at pre-meiotic stages of spermatogenesis. Concentrations of As, Cd, Cu, Fe, and Hg in the tested samples were higher than WHO standards and likely contribute to the adverse effects. Implications are significant for Nigeria and other countries where e-waste dumpsite leachate can contaminate drinking, ground water, and soil. Safer practices, community education, and enforcement of regulations are crucial to ameliorate human e-waste exposures. Funding by NYU Global Institute of Public Health & NYU NIEHS Center.

Abstract 15 Inhibition of Cyclosporine A Induced Renal Dysfunction and Fibrosis by Chrysin and Apigenin

Rohan Nagavally and Sue Ford

St. John’s University, Toxicology Program, Queens, NY

Cyclosporine-A (CsA) is the drug of choice for immunosuppression in organ transplantation. It acts by impeding T-cell activation by means of inhibiting calciurenin. Since the discovery of CsA’s immunosuppressive properties in 1970s, its usage made a dramatic improvement in success rate of solid organ allografts. However, the major drawback of CsA is its nephrotoxicity. CsA-induced nephrotoxicity manifests by an extreme decrease in glomerular filtration rate (GFR), a sharp increase in the serum creatinine, along with a decline in creatinine clearance. CsA induced nephrotoxicity manifests histologically with tubular atrophy, mononuclear cell infiltration, tubular dilation, tubular cast formation, interstitial collagen deposition and fibrosis.

One of the mechanisms contributing to fibrosis due to nephrotoxicants such as CsA and cadmium is epithelial mesenchymal transition (EMT). Inhibition of EMT has been shown to prevent kidney fibrosis in several studies. EMT involves a shift from the epithelial to the mesenchymal phenotype subsequent to tissue injury. EMT occurs via loss of epithelial markers like E-cadherin and acquisition of mesenchymal markers including α-smooth muscle actin (α-SMA), vimentin and extracellular matrix proteins such as collagen, resulting in increased motility of cells. Our previous work showed that chrysin inhibits EMT in LLC-PK1 cells by modulation of the Akt signaling. Chrysin's potential to inhibit EMT of epithelial cells suggests a possible protective effect on CsA induced nephrotoxicity.

Male Sprague Dawley rats were treated with CsA alone (25 mg/kg/day) or with the flavonoids chrysin (50 and 10 mg/kg/day) or apigenin (10 mg/kg/day) for 28 days. Co-treatment with chrysin or apigenin inhibited CsA induced renal dysfunction in rats as measured by BUN, serum creatinine and creatinine clearance. Treatment with CsA caused a 20% increase in BUN at 28 days of treatment, 30% and 50% increase in serum creatinine at 14 and 28 days of treatment respectively and 15% and 20% decrease in creatinine clearance at 14 and 28 days of treatment respectively when compared to the controls. Co-treatment with chrysin and apigenin abolished these changes caused by CsA. Histological studies of kidney sections stained with H&E showed that the flavonoids inhibited CsA-induced dilation and atrophy of the tubules, cast formation within the tubules and mononuclear cell infiltration. The flavonoids were found to inhibit the fibrosis caused by CsA as evaluated by Sirius red assay for collagen content of the kidneys and histology by Sirius red staining. Finally, treatment with the flavonoids also inhibited the expression of α-SMA induced by CsA. Our study shows that the flavonoids chrysin and apigenin inhibit CsA-induced renal dysfunction, kidney

damage, and fibrosis. The protection by chrysin and apigenin against both CsA-induced nephropathy and the expression of mesenchymal markers may be related to its ability to inhibit TGF-β1-induced EMT in LLC-PK1 cells.

Abstract 16 MDR1 Transporter Protects Against Paraquat-Induced Dopaminergic Neurodegeneration

Dahea You1, Christopher J. Gibson1, Muhammad Hossain2, Xia Wen2,3, Ill Yang2, Brian Buckley2, Jason R. Richardson4, Lauren M. Aleksunes2,3

1Joint Graduate Program in Toxicology, Graduate School of Biomedical Sciences, Rutgers University; 2Environmental and Occupational Health Sciences Institute and Department of Environmental and Occupational Medicine, Robert Wood Johnson Medical School, Rutgers University; 3Ernest Mario School of Pharmacy, Rutgers University; 4Northeast Ohio Medical University

Parkinson’s disease (PD) is a chronic, neurodegenerative disorder affecting around seven million people worldwide. However, its etiology has not been fully understood. The interaction of genetic and environmental factors including the exposure to pesticides such as paraquat may contribute to the pathogenesis of PD. Prior studies have observed a loss-of-function genetic polymorphism and overall reduction in the expression and function of the multidrug resistance protein 1 (MDR1, ABCB1) transporter in PD patients. Therefore, MDR1 may be a key element in the pathogenesis of PD. In this study, we evaluate the role of MDR1 in the transport of neurotoxicants and determine whether loss of Mdr1 function in mice altered the susceptibility to neurotoxicity. In a human brain capillary endothelial cell line (hCMEC/D3) which endogenously expresses transporters, the reduction of MDR1 transport using the antagonists PSC833 or siRNA transfection resulted in up to 200% greater accumulation of paraquat. In vivo studies assessed the accumulation and toxicity of paraquat in the midbrains of wild-type and Mdr1a/1b knockout mice. The knockout mice showed increased susceptibility to paraquat-induced neurotoxicity. One week after a single dose of paraquat (10mg/kg i.p.), Mdr1a/1b knockout mice had a 40% reduction in tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra pars compacta as compared to wild-type mice, which had similar staining as vehicle-treated controls. In addition, the Mdr1a/1b knockout mice treated with paraquat also experienced a more profound reduction in the expression of the dopamine transporter (DAT) and greater accumulation of alpha-synuclein compared to the wild-type mice. DAT and the organic cation transporter 3 are uptake transporters responsible for the entry of paraquat into cells; there were no basal differences in their expression between the genotypes. Collectively, these results suggest that the MDR1 transporter plays an important role in the efflux of paraquat and protection against paraquat-induced neurotoxicity. Funded by R01 ES021800, T32 ES007148, P30 ES005022.

Abstract 17 Knockout of POLD3 Compromise Somatic Hypermutation in Human Burkett’s Lymphoma Cells

Wenjun Wu, Zhihui Xiao, Igor Ban, V. Poltoratsky

School of Pharmacy and Health Sciences, St.John’s University, Jamaica, NY 11439

Somatic hypermutation (SHM) process diversifies immunoglobulin gene and increases the affinity of B cell receptors to antigens. The molecular process of SHM is initiated with the conversion of cytosine to uracil by activation-induced cytidine deaminase. The formed uracil:guanine mismatch are then removed by Uracil DNA glycosylase and form an abasic site. While initial steps of formation of the lesion in SHM are well described, the later stages of error prone lesion repair are not yet well defined. Here we discuss the putative role of the third subunit of the DNA polymerase δ (POLD3) in mutagenesis in Raji cells.

POLD3 was originally described as one of the subunit of DNA polymerase δ (POLD3), which has been demonstrated not essential in yeast viability, but crucial for DNA damage repair in yeast. On the basis of previous studies, we hypothesized that POLD3 participated in SHM.

In order to test this hypothesis, we mutated POLD3 in human Raji cells using the CRISPR technology and investigated the function of mutant POLD3 in Raji cells’ (2C9 cells) SHM. The POLD3 expression was abrogated in 2C9 cells as it was confirmed by western-blot analysis. Since POLD3 is one of the enzymes responsible for DNA synthesis, we checked the cell cycle of both WT and 2C9 Raji cells using flow cytometry and found that 2C9 cells exhibited a high frequency of detaining in the S and G2/M phase. In addition, as POLD3 is involved in base excision- and mismatched- DNA repair pathways, we tested the sensitivity of both Raji WT and 2C9 cells to different genotoxic agents including methyl methanesulfonate, cisplatin and hydrogen peroxide. Compared with WT cells, the 2C9 cells were significantly more sensitive to the tested genotoxic agents. We further tested the effects of POLD3 mutation on SHM in Raji cells by checking the mutagenesis at the immunoglobulin gene V-region. Our results indicated that SHM function was significantly compromised in 2C9 cells.

Abstract 18 PARP-Inhibitor Increases Cell Survivability in High ROS States

Igor Ban, Uday Kiran Velagapudi, Tanaji Talele, Vladimir Poltoratsky

Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, New York 11439, United States

Reactive oxygen species have been well documented as of one the most potent factors in cell death and disease. ROS is linked to diseases such as cancer, diabetes mellitus, cardiovascular diseases, chronic inflammation, atherosclerosis, and aging. These negative effects are brought to fruition by ROS induced damage to genetic sequences which lead to cellular dysfunction.

In states of exposures to ROS, cells accumulate single stranded breaks in their genome. These breaks are rapidly marked for repair by Poly ADP ribose polymerase (PARP), using NAD+ as a substrate. The activation of PARP and polymerization of poly (ADP-Ribose) represent one of the first steps in a coordinated series of events following single and double strand DNA damage repair. Upon marking sequences with ADP, repair pathways such as base excision repair (BER) and non-homologous end-joining (NHEJ) repair the damaged DNA sequence. At times of extensive oxidative stress multiple DNA lesions cause PARP activation and NAD depletion. At times of high oxidative stress PARP over activation, due to extensive DNA damage, depletes the cell NAD and consequently leads to necrosis.

In this study we tested the effect of newly synthesized PARP-inhibitors (compound 64 and Compound 65) on viability of cells during oxidative stress. Commercially available PARP inhibitor (Veliparib) was used as a control. A549 cells treated with high doses of hydrogen peroxide in combination with a PARP inhibitor show greater viability when compared to controls. For further investigation we plan to determine the presence of PARP using immunofluorescence as well as DNA damage using Comet assays. Flow cytometry assay will also be employed to determine the amount of apoptotic and necrotic cells as well as cell cycle progression.

Abstract 19 Manganese Superoxide Dismutase (MnSOD) Attenuates Hyperoxia-induced Cell Death by Altering ERK Activity

Xiaojing Yang1, Ravikumar Sitapara1, Melendez, J Andres2, Charles Ashby Jr.1, Douglas D. Thomas3, Lin Mantell1

1Department of Pharmaceutical Sciences, St. John’s University College of Pharmacy and Health Sciences, Queens, NY, United States; 2 Center for Immunology & Microbial Disease, Albany Medical College, Albany, NY, United States. 3Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois

Supraphysiological concentrations of oxygen are often required to treat patients with respiratory failure. However, prolonged exposure to hyperoxia can result in severe lung injury and pulmonary cell death. Hyperoxia-induced lung injury is mediated by the generation of reactive oxygen species (ROS) in mitochondria, which damages the structure and function of pulmonary cells. Manganese-superoxide dismutase (MnSOD), which converts superoxide into the less toxic molecule, hydrogen peroxide (H2O2), is widely accepted as a significant antioxidant enzyme in many human cells, including pulmonary cells. Here, we tested whether overexpressing MnSOD in human lung epithelial cells would protect them against hyperoxic-mediated toxicity. Human lung epithelial cells (A549 cells), wild type or overexpressing MnSOD, and human lung fibroblasts (HT-1090 cells), wild type or overexpressing MnSOD were exposed to 95% O2 for up to three days. The percentage of live cells was determined by Trypan Blue Exclusion assay. Epithelial and fibroblast overexpressing MnSOD in mitochondria survived after exposure to three days of hyperoxia compared to vector controls. Cells overexpressing MnSOD had significantly higher basal levels of extracellular signal-regulated kinases (ERK) activation and survival rate following hyperoxic exposure. The incubation of cells with the ERK inhibitor, PD98059, significantly reduced ERK activity and cell survival in hyperoxia. Hydrogen peroxide levels were significantly increased in MnSOD overexpressing cells, as shown by flow cytometry. These data suggest that moderate overexpression of MnSOD in mitochondria significantly protects against hyperoxia-induced cell death. Intracellular H2O2 may play an important role in mediating ERK activation and mediating survival in response to hyperoxia.

Abstract 20 Inhibition of S-nitrosoglutathione Reductase Reduces Inflammatory Signaling in the Lung in Response to Bleomycin.

Taylor, S., Fett, D., Golden, T., Bohtello, D., Gow, AJ.

Toxicology, Rutgers University, Piscataway, NJ.

Nitric Oxide (NO), an important second messenger molecule, is both a target for inflammatory induction and a mediator of tissue injury. It can exert its effects through a variety of mechanisms including the modification of cysteine residues to form S-nitrosothiols (SNOs). S-nitrosylation of cysteine residues may occur by transnitrosylation from small molecules such as nitrosylated glutathione (GSNO) or directly by the interaction of NO with reduced thiol. The intracellular pool of SNOs is regulated by a variety of enzymes including thioredoxin reducatse and the alcohol dehydrogenase S-nitrosoglutathione Reductase (GSNOR), which specifically reduces GSNO to GSH and hydroxylamine. Under conditions of inflammation, there is increased production of Nitric Oxide through increased activity of inducible Nitric Oxide Synthetase (iNOS), which can lead to increased SNO formation. The intratracheal instillation of Bleomycin (ITB) is a well-studied model of acute pulmonary injury resulting in fibrosis within which iNOS function has been identified as being critical to both the initiation and resolution of the inflammatory process. In the absence of iNOS function, acute injury is reduced, but long-term fibrosis is exacerbated, presumably by a failure to resolve the initial insult. The role of iNOS derived NO in inflammatory initiation is known, however, we speculated that increased intracellular SNO formation was required to initiate the resolution process. A number of intracellular targets involved in the inflammatory process are inhibited by SNO formation, including NF-κB and GAPDH, while protective mechanisms, such as KEAP-1 are activated. Therefore, we hypothesized that increasing intracellular SNO formation by inhibiting GSNO degradation would decrease inflammatory activation and improve fibrotic outcome in the ITB model. To this end we have examined the effects of administration of the GSNOR inhibitor N6022 when given early and late in the ITB model.

8-12 week old, male C57BL6/J mice were administered 3 U/kg intratracehal bleomycin or saline control and administered N6022 daily by i.p. injection for 8 days. These preliminary experiments demonstrated that GSNOR inhibition reduced the severity of injury in this early phase. N6022 treated animals that received ITB had a median inflammatory score of 2.5 as compared to ITB-treated animals receiving vehicle control whose median inflammatory score was 3.5(p<0.05 by Wilcoxon Rank Sum Test). To examine the role of GSNO in the fibrotic process, the ITB-mediated injury was repeated in mice that received a high or low dose of N6022 (or saline control) to inhibit GSNOR at the peak of inflammation, from 8-21 days post-ITB. Analysis of lung function, histology, and markers of injury such as cell count, and protein concentration revealed no

reduction in fibrosis as a result of N6022 administration. RT-qPCR analysis of cells from broncho-alveolar lavage fluid revealed altered inflammatory signaling. 21 days post ITB there was increased expression of NOS2, ARG1, IL6, IL10, and IL1B genes; however, N6022 administration reduced expression with the exception of ARG1, which remained unchanged compared to saline treatment. Taken together, it appears that GSNOR activity affects the development of inflammation in response to ITB, but does not alter the consequent fibrosis.

Abstract 21 The α7nAChR Agonist GTS-21 Attenuates Hyperoxia-Induced Acute Lung Injury by Decreasing Hyperacetylation and the Release of Nuclear HMGB1

Ravikumar A Sitapara1, Vivek S Patel1, Sergio Valdes-Ferrer2, Daniel J Antoine3, Kevin J Tracey, Valentin A Pavlov2, Lin L Mantell1,2

1Department of Pharmaceutical Sciences, St. John's University, College of Pharmacy and Health Sciences, Queens, NY, USA; 2 The Laboratory of Biomedical Sciences, The Feinstein Institute for Medical Research, North Shore-LIJ Health System, Manhasset, NY, USA; 3Medical Research Council Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool, UK

Oxygen therapy with supraphysiological concentrations of oxygen (hyperoxia) is routinely administered during mechanical ventilation (MV) for the management of severe respiratory distress, such as acute respiratory distress syndrome. Prolonged exposure to hyperoxia results in acute lung injury (ALI), accompanied with the accumulation of high mobility group box 1 (HMGB1) in the airways. Previously, we have shown that airway HMGB1 mediates hyperoxia-induced pro-inflammatory lung injury in a mouse model of ALI. The aim of this study was to determine whether GTS-21 [3-(2,4 dimethoxy-benzylidene)-anabaseine dihydrochloride], an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, could inhibit hyperoxia-induced HMGB1 accumulation in the airways, leading to alleviated lung injury. GTS-21 (0.04, 0.4 and 4 mg/kg) or saline was administered via intraperitoneal injection to mice that were exposed to hyperoxia (≥99% O2) for 3 days. We found that systemic administration of 4 mg/kg GTS-21 significantly decreased both the accumulation of airway HMGB1 and pro-inflammatory ALI, indicated by the decreased total protein content and neutrophil infiltration in the airways. Hyperoxic exposure leads to hyperacetylation of nuclear localization signals of HMGB1, resulting in its pronounced translocation to the cytoplasm and its subsequent release from lung cells into the airways. In addition to reduced accumulation of HMGB1 in the airways, hyperoxia-induced hyperacetylation of HMGB1 was inhibited in mice treated with 4 mg/kg of GTS-21. Our results indicate that GTS-21 is effective in attenuating hyperoxia-induced ALI via inhibiting the release of nuclear HMGB1 by blunting its hyperacetylation. Therefore, the α7nAChR represents a possible pharmacological target to provide treatment regimens for oxidative inflammatory lung injury in patients receiving mechanical ventilation.

Abstract 22 CNS Genetic Alterations In Mice Exposed To E-cigarettes During Early Life

DE Lauterstein, K Corbett, T Gordon, CB Klein, JT Zelikoff

The WHO reports that tobacco use causes more than 5 million deaths per year, with current estimates predicting a rise to >8 million deaths annually by 2030. Although cigarettes have been the major form of tobacco used in the past, alternative tobacco products and nicotine delivery systems are increasing in popularity. Among these products, e-cigarettes, battery-powered devices containing nicotine, glycerin, propylene glycol, flavorings, and other substances have had the most dramatic usage spike. E-cigarettes are currently unregulated by the FDA (unless specifically marked for therapeutic purposes), and toxicological data are extremely limited. The lack of regulations and safety data is a substantial concern, particularly since these products are often seen as “reduced harm” or cessation devices away from conventional cigarettes, and may appeal to vulnerable populations, including pregnant women.

While the developmental neurotoxic effects of nicotine alone are well known, little information on the same outcomes is available for other e-cigarette aerosol components. To evaluate the relative neurotoxicity of e-cigarette vapor exposure on CNS development and later life outcomes, pregnant 9-10-wk-old C57BL/6 mice (n=10 pregnant mice/treatment) were exposed daily (3 hr/d; 5 d/wk, via whole body exposure) throughout gestation and lactation (weaned at ~3-wk-of-age) to e-cigarette vapors produced from blu™ e-cigarettes with (13-16 mg/ml) and without nicotine. Urinary cotinine levels for each treatment group was analyzed bi-weekly using ELISA. RNA-sequencing was used to complete transcriptome analysis on frontal cortex samples from1-mo-old female and male offspring. Cortex from female offspring exposed early in life to e-cigarette vapor containing nicotine demonstrated that 1,393 genes were significantly changed (p<0.01) when compared to controls, while only 152 genes were significantly changed (p<0.01) in age-matched male offspring from the same treatment group. In contrast, frontal samples from juvenile females exposed pre- and post-natally to e-cigarette vapor with no nicotine demonstrated a change (p<0.01) in 2,630 genes when compared to controls, while 2,615 genes were altered (p<0.01) in age-matched males from the same treatment group. Following RNA-seq analysis, selected genes important for mental health (e.g., nerve growth factor receptor, choline o-acetyltransferase, brain derived neurotrophic factor, glial cell line derived neurotrophic factor, galanin, t-box brain gene 1, and alpha-1d adrenoreceptor) were examined in male frontal cortex samples by qPCR; results from RNA-seq and qPCR were similar. Data from RNA-seq was imputed into Ingenuity Pathway Analysis (IPA) where a comparison analysis between sex and treatment groups was done. Predicted downstream disease and biological function effects associated with the observed gene expression changes were identified and included alterations in cognition, learning, long term depression, and memory. Results from this study demonstrate that e-cigarette

exposure during early life alters CNS developments leading potentially to chronic neuropathology. With e-cigarette usage increasing exponentially on a global scale, more laboratory studies are urgently needed to protect public health and set appropriate policy. Supported by NYU NIEHS Center (5P30ES000260-50) pilot grant.

Abstract 23 Contradicting Mechanisms of Micronucleus Induction in Rodent and Human Cell Lines

Dan Roberts1,2 Laura Custer1 and Helmut Zarbl2

1Bristol-Myers Squibb, Drug Safety Evaluation; 2Joint Graduate Program of Toxicology, Rutgers University

Understanding mechanisms of genotoxicity is useful when evaluating the potential carcinogenic risk of xenobiotics. Agents with direct DNA damaging modes of action are generally considered unsafe at all doses, while those that induce genotoxicity due to indirect effects may be managed by controlling exposure levels. Using the in vitro micronucleus (MN) assay, the gold-standard for determining indirect mechanisms of MN formation is to evaluate MN for the presence of whole chromosomes and/or chromatids via fluorescent microscopy using anti-kinetochore/telomere antibodies or FISH techniques with centromeric probes. There is growing evidence that more rapid detection of these aneugenic events is feasible using rodent cell lines and flow cytometry. In this platform, hypodiploidy induction has been strongly correlated with mechanism of MN formation. To evaluate this across the entire dose-response curve, we purified human sera containing kinetochore/centromeric DNA reactive antibodies (protein G affinity chromatography) and conjugated the resulting IgG to AlexaFluor 633 (AF633). This new direct conjugate was then added to an existing 24-well plate flow cytometric CHO-WBL MN assay, where 5 clastogens and aneugens were used at concentrations that induced robust MN, and at least 500 MN were evaluated for the presence of AF633 in each culture. Aneugens induced MN concomitantly with hypodiploidy and increases in AF633+ MN were observed in a concentration dependent manner. Clastogens generally induced MN without increases in hypodiploidy or AF633+ MN. Interestingly, a human cell clastogen, cytosine arabinofuranoside (araC), induced a strong aneugenic signature in CHO-WBL cells, which was unexpected. This discrepancy could be due to a deficiency of the mitotic assembly checkpoint in rodents (and rodent derived cell lines), as when TK6 cells were treated with araC, there was no increase in AF633+ MN. Data from the entire validation set will be presented and future experiments to elucidate the cause of this discrepancy will be discussed.